CN111948394B - Application of TSTA3 and LAMP2 as targets in esophageal squamous carcinoma cell metastasis detection and drug screening - Google Patents
Application of TSTA3 and LAMP2 as targets in esophageal squamous carcinoma cell metastasis detection and drug screening Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, firstly provides application of TSTA3 and LAMP2 as target objects in detection of esophageal squamous carcinoma cell metastasis, and secondly provides application of TSTA3 and LAMP2 as target objects in screening medicines for inhibiting or slowing down esophageal squamous carcinoma cell metastasis. The TSTA3 is over-expressed in esophageal squamous carcinoma cells, LAMP2 is over-expressed, core fucosylation of LAMP2 is modified, and the esophageal squamous carcinoma is finely migrated and invaded. The application of TSTA3 and LAMP2 as targets in screening medicaments for inhibiting or slowing down esophageal squamous cell metastasis. In esophageal squamous carcinoma, TSTA3 is taken as a speed-limiting enzyme in a GDP-L-fucose synthesis pathway to participate in fucosylation modification, and invasion and migration of esophageal squamous carcinoma are promoted by increasing core fucosylation modification of LAMP2, so that oncogene effect is exerted.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to application of TSTA3 and LAMP2 serving as target objects in esophageal squamous carcinoma cell metastasis detection and drug screening, in particular to application of TSTA3 and LAMP2 serving as target objects in detection of esophageal squamous carcinoma cell metastasis onset; the application of TSTA3 and LAMP2 as targets in screening medicaments for inhibiting or slowing down esophageal squamous cell metastasis.
Background
Esophageal cancer (Esophageal cancer, EC) is one of the most common causes of cancer death, with morbidity and mortality residing in the eighth and sixth of the various types of cancer worldwide. The histological type of the Chinese medicine mainly comprises esophageal squamous carcinoma (Esophageal squamous cell carcinoma, ESCC), which accounts for more than 90%, and the morbidity and mortality of the Chinese medicine are the third and fourth positions of malignant tumors. The symptoms of early esophageal squamous carcinoma are not obvious, no specific diagnosis marker exists, the diagnosis is carried out at middle and late stages, the current treatment mode of esophageal carcinoma is mainly surgery, the prognosis is poor, and the survival rate of 5 years is only 15% -25%. Thus, studying the pathogenesis of ESCC, defining early diagnostic markers of ESCC and its therapeutic targets is critical for improving patient prognosis.
Esophageal squamous carcinoma has strong invasion and metastasis capability. There are a number of mechanisms currently involved in metastasis of tumor cells, of which aberrant fucosylation is a marker of malignant cell transformation, and is one of the important mechanisms for promoting tumor invasion and metastasis. Fucosylation is one of the modification modes of glycosylation, and refers to a process of transferring fucose from GDP-L-fucose to a substrate including some proteins, N-and O-linked glycans, etc. in glycoproteins or glycolipids. The synthesis of GDP-L-fucose is an important link of fucosylation and is the only donor for fucosylation. The synthesis of GDP-L-fucose involves two pathways: de novo synthesis pathway and salvage synthesis pathway. 90% of GDP-L-fucose in humans is synthesized from the de novo synthesis pathway, which is the primary source of GDP-L-fucose biosynthesis. In the de novo synthesis pathway, GDP-D-mannose is converted to GDP-L-fucose by a three-step enzymatic reaction under the action of GDP-mannose-4, 6-dehydroatase (GMDS) and GDP-4-keto-6-deoxymannose-3, 5-isomerase-4-reductase (GDP-4-keto-6-deoxymannase-3, 5-epothilase-4-reduction, FX or TSTA 3), where TSTA3 is the rate-limiting enzyme in the GDP-L-fucose synthesis pathway. In the mouse model, following the TSTA3 gene knockout, the mice exhibit a defect in the de novo synthetic pathway followed by an almost complete defect in the overall fucosylation of the cells. TSTA3 thus plays an extremely important role in the fucosylation process.
In the salvage synthetic pathway: the enzymes involved in the reaction are L-fucose kinase and GDP fucose pyrophosphatase, and under the catalysis of the two enzymes, L-fucose is converted into GDP-L-fucose through two-step reaction. Only 10% of GDP fucose in mammalian cells is synthesized by the salvage pathway, and studies in HeLa cells have shown that more than 90% of GDP fucose in the cytoplasm is derived from the de novo pathway, and thus the de novo pathway plays a key role in GDP-L-fucose synthesis.
Glycosylation is a common post-translational modification of proteins that plays a key role in many physiological processes, including protein folding, rearrangement, stabilization, transport, secretion, immune response, signaling, and regulation of many cellular functions, such as adhesion, proliferation, migration, and the like. Fucosylation is one of the modification modes of glycosylation and plays an important role in ABO blood group antigens, host-microorganism interactions, selectin-mediated leukocyte extravasation, lymphocyte homing, and the like. The fucosyltransferase uses the nucleotide activated fucose form, i.e., GDP fucose, to provide fucose for the construction of fucosylated oligosaccharides. GDP-L-fucose, which is the main source of fucosylation, is synthesized in the cytoplasm and then transported into the endoplasmic reticulum or Golgi apparatus via GDP fucose transporter (GDP-L-fucose transporter, GDP-L-Fuc Tr), and participates in fucosylation by fucosyltransferase.
Aberrant fucosylation is prevalent in various tumors, GDP-L-fucose synthetases involved in the fucosylation process, fucosylation glycan structural enzymes, different subtypes of fucosyltransferase, and fucosylation antigens are closely related to tumor progression and prognosis. More and more studies have established the mechanism of aberrant glycosylation in tumor metastasis and recurrence progression. Inhibition of fucosylation not only inhibits tumor growth, but also promotes T cell activation by down-regulating PD-1 activity. Aberrant fucosylation of tumor cells is also involved in cell surface important growth factor signaling pathways such as tgfβ, EGFR, VEGFR and c-mot mediated signaling pathways, and also affects the activity/signaling of other plasma membrane proteins including β1-integrin, β -catenin and E-cadherin, and in addition apoptosis signaling pathways, notch receptor signaling pathways, etc., involved in cell proliferation, invasive metastasis, apoptosis, drug sensitivity, etc. in tumors. TSTA3, a key enzyme for the de novo synthesis of GDP-L-fucose, has been found to be more highly expressed in high-metastatic variants of colorectal cancer than in low-metastatic variants of the same origin; in breast cancer, wild-type TSTA3 plays a role in carcinogenesis, and high expression of TSTA3 in tumor tissue is closely related to clinical staging and poor prognosis; in hepatocellular carcinoma, TSTA 3-related network regulation also encompasses cell migration and invasion processes.
The modification of fucosylation exists in different tumors, so that abnormal fucosylation can be used as a marker for diagnosing and predicting tumor recurrence and metastasis, and a theoretical basis is provided for a potential treatment strategy. However, aberrant fucosylation presents a great heterogeneity, and fucosylation present in different tumors varies, even in the same tumor, due to differences in pathological type or stage of disease progression, the fucosylation modifications may differ. For example, in gastrointestinal tumors, three studies have shown reduced fucosylation in the tumor, while two have shown increased fucosylation modification of the tumor. In different tumor types, the transferases involved in the fucosylation process may also be different. Therefore, in tumors, the specific fucosylated proteins and the signal mechanisms regulated by the specific fucosylated proteins still need to be studied in depth, so that theoretical basis is provided for clinically improving diagnosis and treatment strategies of tumors by utilizing the mechanistic insights, and prognosis of patients is expected to be improved.
Disclosure of Invention
The invention firstly provides application of TSTA3 and LAMP2 as target objects in detection of esophageal squamous cell metastasis, and secondly provides application of TSTA3 and LAMP2 as target objects in screening medicines for inhibiting or slowing down esophageal squamous cell metastasis.
The invention is realized by the following technical scheme: the application of TSTA3 and LAMP2 as targets in esophageal squamous carcinoma cell metastasis detection and drug screening, wherein the TSTA3 is over-expressed in esophageal squamous carcinoma cells or LAMP2 is over-expressed, and core fucosylation of LAMP2 is modified, so that esophageal squamous carcinoma cells migrate and invade. When the overexpression of TSTA3 exceeds 1.6 times of a normal value, migration and invasion of esophageal squamous carcinoma cells can be obviously promoted.
The application of TSTA3 and LAMP2 as targets in screening medicaments for inhibiting or slowing down esophageal squamous cell metastasis.
Through screening the drugs for inhibiting the overexpression of TSTA3 and LAMP2, the core fucosylation modification of LAMP2 is inhibited, so that the migration and invasion of esophageal squamous carcinoma cells are inhibited or reduced.
The drug for inhibiting or slowing down esophageal squamous carcinoma cell metastasis is peracetylated 2-F-Fuc, and the concentration is 10-30.64 mu M.
Preferably, the concentration of peracrylated 2-F-Fuc is 10.71-30.64 μm.
Through immunohistochemical analysis of the tissue chip, the expression of TSTA3 in esophageal squamous carcinoma tissues and lymphatic metastatic tissues is obviously higher than that of other normal tissues, and the expression of TSTA3 in lymphatic metastatic tissues is also obviously higher than that of esophageal squamous carcinoma tissues, which is consistent with the previous research result, namely, the expression of TSTA3 is positively related to TNM stage and lymph node metastasis of esophageal squamous carcinoma patients. In the cell function experiment, compared with the control group, the TSTA3 can be over expressed in esophageal squamous carcinoma cells to promote invasion and migration of the cells, but has no influence on cell proliferation. It is suggested that the TSTA3 gene plays an oncogene role in esophageal squamous carcinoma.
By utilizing the characteristic that LCA lectin is combined with core fucose, the change of the core fucosylation modification level of ESCC cells before and after the overexpression of TSTA3 is detected by using the LCA lectin marked by fluorescein. The cell-bound fluorescein-labeled LCA of the KYSE150 and KYSE450 cell control group and the TSTA3 over-expression group is detected by a flow cytometer, and after the TSTA3 is over-expressed, the core fucosylation modification of esophageal squamous carcinoma cells can be enhanced.
Subsequent mass spectrometry identification by N-glycosylation histology modification and LCA lectin enrichment chromatography of the cell line revealed that the intersection genes present included IKBIP, LAMP2, LMNA. It is suggested that TSTA3 may play a role in esophageal squamous carcinoma by modifying the core fucosylation of these proteins. By consulting the literature, there is an increase in glycosylation modification of LAMP2 protein in choriocarcinoma, promoting migration and invasion of choriocarcinoma cells, suggesting that glycosylated LAMP2 protein may be key for TSTA3 to play a role in esophageal squamous carcinoma cells.
LAMP2 is a lysosomal associated membrane protein, one of the members of the LAMP family. The LAMP family is a family of glycosylated proteins present on lysosomal membranes. Lysosomes are single membrane acidic organelles of eukaryotic cytoplasm, which, in addition to degrading intracellular and extracellular macromolecules into cellular available building blocks, are important mediators of intracellular homeostasis and are involved in the occurrence of different diseases such as neurodegenerative diseases, cardiovascular diseases and tumors. During transformation and cancer progression, lysosomes change their localization, volume and composition and enhance tumor invasiveness by releasing related enzymes. For example, various lysosomal enzymes, including cathepsins, are highly expressed in various tumors such as breast cancer, prostate cancer and colon cancer, and their secretion is increased by exocytosis, degradation of extracellular matrix and angiogenesis are promoted, thereby promoting invasive metastasis of tumor cells, and their expression levels are of clinical prognostic significance.
Compared with the prior art, the verification of the invention proves that: overexpression of TSTA3 in esophageal squamous carcinoma cells can promote core fucosylation of LAMP2, while fucose can compete with core fucosylation LAMP2 for binding to LCA lectin, further demonstrating that TSTA3 can specifically modulate core fucosylation modification of LAMP2 in esophageal squamous carcinoma; and after the expression of the LAMP2 is interfered in the esophageal squamous carcinoma cells with the overexpression of the TSTA3, through a Transwell experiment, the fact that the migration and invasion of the esophageal squamous carcinoma cells caused by the overexpression of the TSTA3 can be reversed by the interference of the expression of the LAMP2 is found. Thus, the present invention suggests that TSTA3 may affect esophageal squamous carcinoma cell migration and invasion by modulating aberrant core fucosylation of LAMP 2.
In esophageal squamous carcinoma, TSTA3 is taken as a speed-limiting enzyme in a GDP-L-fucose synthesis pathway to participate in fucosylation modification, and invasion and migration of esophageal squamous carcinoma are promoted by increasing core fucosylation modification of LAMP2, so that oncogene effect is exerted.
Drawings
FIG. 1 shows the detection of the overexpression efficiency of mRNA and protein of TSTA3 in esophageal cancer cells;
FIG. 2 is a graph showing the ability of TSTA3 to promote migration and invasion of ESCC cells following overexpression; in the figure: a: using a Transwell experiment to respectively detect the influence of TSTA3 overexpression on the migration and invasion capacities of KYSE150 and KYSE450 cells; b: statistical analysis of changes in the migration and invasion capacity of KYSE150 and KYSE450 cells following TSTA3 overexpression (< 0.01);
FIG. 3 is a core fucosylation modification of an enhanced ESCC cell line following overexpression of TSTA 3; in the figure: a: detecting changes in core fucosylation modifications of KYSE150 and KYSE450 cells before and after TSTA3 overexpression using a flow cytometer; b: statistical analysis of changes in the core fucosylation modifications of KYSE150 and KYSE450 cells caused before and after TSTA3 overexpression (< 0.05;) P < 0.01);
FIG. 4 is a schematic illustration of TSTA3 targeting core fucosylation at LAMP 2; in the figure: a: the fucosylated proteins were enriched with LCA-agarose and subjected to SDS-PAGE analysis, and 4 protein bands of different expression (indicated by arrows) in LCA pellet samples were excised for mass spectrometry; b: n-glycosylation histology of control and TSTA3 overexpressing cells; c: intersection genes of mass spectrometry results and glycosylation chemistry results;
FIG. 5 shows that overexpression of TSTA3 in ESCC cells can enhance core fucosylation of LAMP2 protein; in the figure: a: detecting differences in core fucosylation of KYSE150 cell control group (NC) and TSTA3 over-expression group (TSTA 3-OE) LAMP2 by LCA enrichment chromatography experiment; b: detecting differences in core fucosylation of LAMP2 in a KYSE450 cell control group (NC) and a TSTA 3-overexpressing group (TSTA 3-OE) using LCA enrichment chromatography experiments (< 0.01, < 0.001, < P);
FIG. 6 is competition of fucose and core fucosylated LAMP2 for binding to LCA lectin; in the figure: a: the gradient concentration of fucose was used to compete with core fucosylated LAMP2 protein for binding to LCA lectin in KYSE150 cell TSTA3-OE group proteins; b: gradient concentrations of fucose were used in proteins of the KYSE450 cell TSTA3-OE group to compete with core fucosylated LAMP2 protein for binding to LCA lectin.
FIG. 7 is a graph showing that interfering LAMP2 can reverse the invasive migration promoted by TSTA3 over-expression to ESCC cells; in the figure: a: detecting the interference efficiency of LAMP2 in a control group of KYSE150 cells and a TSTA3-OE group by using Western Blot; b: using Transwell to detect the effect on ESCC cell migration and invasion after interference of LAMP2 expression by a control group and a TSTA3-OE group of KYSE150 cells; c: statistical analysis of changes that caused ESCC cell migration and invasion following interference with LAMP2 expression (< 0.05, < 0.01, < 0.001, < P);
FIG. 8 is the effect of TSTA3 on esophageal squamous carcinoma cells mediated through fucosylation of LAMP 2;
FIG. 9 is the chemical structure of peracrylated 2-F-Fuc;
FIG. 10 is a graph showing the effect of 2-F-Fuc treatment on the migration and invasive capacity of KYSE150 cells overexpressed by TSTA3 using a Transwell assay; in the figure: a:2-F-Fuc can obviously inhibit the invasion capacity of esophageal squamous carcinoma P <0.001 A) is provided; b, 2-F-Fuc obviously inhibits the migration capacity of esophageal squamous carcinomaP < 0.01);
FIG. 11 shows that peracrylated 2-F-Fuc treatment reduced core fucosylation of LAMP2 protein and protein expression of TSTA 3.
Detailed Description
The invention will now be further illustrated by means of specific examples in conjunction with the accompanying drawings without limiting the invention.
1. Study object: 45 ESCC patients were collected from surgically resected primary tumor tissue, paired paracancerous normal tissue and lymphatic metastasis tissue at the university of Shanxi medical department affiliated tumor hospital, paraffin embedded tissue and appropriate tissue preparation tissue chips were selected as subjects. The study was approved by the ethical committee of the affiliated oncology hospital of the university of shanxi medical science. Cases include 33 men and 12 women; TNM stage I-II 23 cases, III-IV 22 cases; there were 22 cases of lymphatic metastasis and 23 cases of no lymphatic metastasis. Specific information is shown in Table 1.
Table 1: clinical pathological characteristics of esophageal squamous carcinoma patient
2. Cell and culture conditions: the human esophageal squamous carcinoma cell lines KYSE180, KYSE450, KYSE150, KYSE410, KYSE510, ECA109 and TE-1 used in the experiment are all stored in the key laboratory of transformation medical center esophageal cancer pathogenesis and transformation research center of mountain Western medicine university. All human esophageal squamous carcinoma cell lines were cultured in complete medium containing 10% fetal bovine serum (Fetal bovine serum, FBS) in a cell incubator at 37 ℃ with 5% co2. After construction of KYSE150 and KYSE450 cell lines stably overexpressing TSTA3, they were cultured in RPMI-1640 medium containing 10% FBS and 5. Mu.g/mL puromycin.
3. The primers are shown in Table 2.
Primers and sequences used in Table 2
4. Reagents and materials are shown in table 3:
table 3: reagents and materials therefor
5. Preparation of common reagents
5.1 preparation of cell culture related reagents:
(1) 1 XPhosphate buffer (1 XPBS, pH 7.4): potassium chloride (KCl): 0.1g, sodium chloride (NaCl) 4g, disodium hydrogen phosphate (Na 2 HPO 4 ) 0.72g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 0.12g, three distilled water to constant volume of 500ml。
(2) Cell cryopreservation solution: fetal Bovine Serum (FBS) 900. Mu.L, dimethyl sulfoxide (DMSO) 100. Mu.L.
5.2 Preparation of Western blot related reagent
(1) 10% ammonium persulfate (10% AP, -20 ℃ C. Storage): ammonium Persulfate (AP) 2g, triple distilled water 20ml.
(2) 10% release gum (7 mL): three distilled water 2.8ml,30% acrylamide (30% Acr Bis 29:1) 2.31ml,1.5mol/L Tris-HCl (pH 8.8) 1.75ml,10% SDS 0.07ml,10% ammonium persulfate (10% AP) 0.07ml,TEMED 0.0028ml.
(3) 5% concentrated gum (3 mL): three distilled water 2.1ml,30% acrylamide (30% Acr Bis 29:1) 0.05ml,1.0mol/L Tris-HCl (pH 6.8) 0.38ml,10% SDS 0.03ml,10% ammonium persulfate (10% AP) 0.03ml,TEMED 0.003ml.
(4) 1 Xrunning buffer (1.5L): glycine (Glycine) 21.6g, tris base 5.545g, SDS 1.5g, triple distilled water to a final volume of 1L.
(5) 1×transfer buffer (1.5L): glycine (Glycine) 21.6g, tris base 5.545g, methanol 300ml, triple distilled water to a final volume of 1.5L.
(6) 4 x protein loading buffer (2 mL): 1M Tris-HCl (pH 6.8) 500. Mu.L, SDS 0.2g, bromophenol Blue (Bromphenol Blue) 2mg, distilled water 500. Mu.L, beta-Mercaptoethanol (beta-Mercaptoethanol) 600. Mu.L, glycerol (Glycerol) 400. Mu.L.
(7) 1 XTBST eluate (0.1% Tween): sodium chloride (NaCl) 1.21g, tris base 8.775g, triple distilled water to volume 1L, tween-20 1ml.
(8) 5% of blocking solution: skimmed milk powder 1.25g,1 XTBE eluent 25ml.
5.3 Preparation of immunohistochemical related reagent
10×pbs stock solution (1L): sodium dihydrogen phosphate (NaH) 2 PO 4 ) 2.645g, disodium hydrogen phosphate (Na 2 HPO 4 ) 28.9g of sodium chloride (NaCl) 8g, and distilled water to a volume of 1L.
6. The experimental method comprises the following steps:
A. immunohistochemical analysis ESCC tissue chip TSTA3 expression
(1) Preparing a tissue chip: and (3) picking paraffin samples of ESCC tissues, paired beside-cancer tissues and lymphatic metastasis tissues, selecting typical tumor parts, beside-cancer parts and lymphatic parts on the corresponding tissues, and marking the paraffin samples. And (3) extracting the required tissues on the marked wax block by adopting a needle with the same size as the corresponding pore diameter, putting the obtained tissues into the corresponding pores of the new wax block according to the sequence of arranging the tissues in advance, and heating to integrate the new wax block and the extracted tissues.
Conventional slicing is carried out on the prepared tissue chip wax block, the slicing thickness is about 4 mu m, the tissue chip wax block is stuck on a glass slide, the slicing is baked for 4 hours at 65 ℃, the sample slice is fixed on the glass slide, and the sample slice is preserved after being cooled;
(2) Dewaxing: placing the prepared immune tissue slice into a 70 ℃ incubator to bake the slice for 10 min, melting the paraffin of the specimen, and dewaxing sequentially according to the following sequence and time: xylene 10 min, absolute alcohol 2 min,80% alcohol 2 min,70% alcohol 2 min,3% hydrogen peroxide 20 min;
(3) And (3) film washing: washing the slices with three distilled water for 5 min, then washing the slices with PBS for 6min, and dividing the slices into 3 times for 2 min each time;
(4) Antigen exposure and antigen retrieval: and (5) performing high-pressure thermal restoration by using citrate buffer solution with pH of 6.0. Placing the box containing the citrate buffer solution with pH of 6.0 into an autoclave, boiling the autoclave in advance, completely immersing the tissue slices into the citrate buffer solution with pH of 6.0, repairing for 8 min at the high pressure of 4 min and about 120 ℃, adjusting the repairing conditions according to different antigens, and then placing the box containing the slices into tap water for natural cooling;
(5) And (3) film washing: PBST (0.01% Tween) was washed 3 times for 2 min each;
(6) Incubating primary antibodies: adding the primary antibody diluted according to a certain proportion into the slice, incubating overnight at 4 ℃, and placing the slice at room temperature for the next day for rewarming 1 h;
(7) And (3) film washing: PBST is washed for 3 times, each time for 10 min;
(8) Incubating a secondary antibody: adding secondary antibodies diluted according to a certain proportion into the slices, and incubating for 20 min at 37 ℃;
(9) Washing the PBST slices for 3 times, and 5 min each time;
(10) DAB color development: preparing a developing solution according to the instruction, dripping the developing solution on the sliced tissues, simultaneously observing the slices under a mirror for a plurality of minutes, and immediately stopping the developing reaction by tap water after cytoplasms become light yellow to avoid excessive dyeing;
(11) Hematoxylin counterstain: placing the slice on a shelf, placing the slice in hematoxylin for dyeing for 1 min, taking out the cell nuclei of the slice after the cell nuclei of the slice are dyed blue, placing the cell nuclei into tap water, and washing off the residual hematoxylin on the slice; observing the staining condition of the slice specimen under a mirror, and if the staining of the tissue cell nuclei is shallow, putting the slice specimen into ammonia water for blue returning.
(12) Dehydrating: immunohistochemical sections were dehydrated sequentially and in the following order and time: 70% alcohol 5 min,80% alcohol 5 min, 90% alcohol 5 min, absolute alcohol 10 min, xylene 10 min;
(13) Sealing piece: covering the sliced sample tissue with a suitable cover glass by using neutral resin, and sealing the slice;
(14) Analysis: immunohistochemical section scanning was performed by the pathology scanning system ScanScope AT of Aperio corporation, and the expression of TSTA3 protein on tissue samples was analyzed using image analysis software (Aperio Image Scope). And respectively scanning and identifying cell nuclei and cell plasma in the chip tissue sample subjected to immunohistochemical staining, and automatically calculating an H-score value according to the color depth of the staining, namely representing the expression level of TSTA3 protein.
B. Cell culture
(1) Cell resuscitation: taking out the frozen storage tube containing the cells to be recovered from the liquid nitrogen tank, immediately placing the frozen storage tube into a water bath kettle at 37 ℃, and quickly dissolving the liquid by light shaking; the thawed liquid was transferred to a centrifuge tube containing 2 mL of complete medium with 10% fbs, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; slowly resuspension fine with 4 mL complete mediumCell pellet, transfer to 25cm 2 The flask was placed in a cell incubator at 37℃with 5% CO2 for culturing.
(2) Cell passage: and when the cell density reaches 80-90%, the cells need to be passaged. Discarding old culture medium in the culture flask, adding 1×PBS solution which has been equilibrated at room temperature, slowly washing cells for 2 times, adding appropriate amount of 0.25% trypsin, and culturing in cell incubator with 5% CO2 at 37deg.C; observing cells under a microscope, immediately adding a complete culture medium containing 10% FBS in an amount equal to or excessive to trypsin to stop digestion after most cells are rounded and floated, transferring the cell suspension into a centrifuge tube, and centrifuging at 1000 rpm for 3 min; and (3) discarding the supernatant, adding a proper amount of complete culture medium to resuspend cell sediment, and slowly and uniformly blowing, and transferring to 2-3 new culture flasks on average for continuous culture.
(3) Cell cryopreservation: discarding old culture medium in the culture flask, adding 1×PBS solution which is balanced at room temperature, slowly cleaning cells for 2 times, adding appropriate amount of 0.25% trypsin, and culturing in incubator with 5% CO2 at 37deg.C; after observing that most cells of the cells are rounded and floated under a microscope, immediately adding a complete medium containing 10% FBS in an amount equal to or excessive to trypsin to stop digestion, transferring the cell suspension into a centrifuge tube, and centrifuging at 1000 rpm for 3min; sucking supernatant, gently blowing cell deposit, re-suspending, packaging in sterile freezing tube, standing at 4deg.C for 30 min, standing at-20deg.C for 2-h, standing at-80deg.C overnight, and standing in liquid nitrogen for long term storage.
C. Lentivirus transfection
(1) Cell plating: one day in advance in 96-well plates, 0.3X10 are added per well 4 Individual cells (KYSE 150, KYSE 450) were cultured in a cell incubator at 37℃in 5% CO2, with 0.2. 0.2 mL complete medium containing 10% FBS per well;
(2) Dilution of TSTA3 overexpressing lentiviruses: polybrene was added to 100. Mu.L of complete medium to a final concentration of 5. Mu.g/mL. The multiplicity of infection (Multiplicity of infection, MOI) values of TSTA3 lentivirus (LV 13 EF-1AF/lucifeare 05-puro) were set to three gradients of 10, 30, 100, and the volumes of lentivirus stocks (TSTA 3-NC, TSTA 3-OE) required for transfection were calculated from the values of viral MOI. Lentiviral stock volume= (number of cells at transfection x MOI)/lentiviral gradient;
(3) Removing old culture medium, adding diluted slow virus diluents with different MOI gradients, setting a control group, and culturing in a cell incubator with 5% CO2 at 37 ℃;
(4) After 12-24 hours, removing virus liquid of transfected cells, adding 0.2-mL complete culture medium, and continuously culturing in a cell incubator with 5% CO2 at 37 ℃;
(5) According to the cell state and the fusion degree, the cells are transferred to a 24-well plate in a digestion way, and meanwhile, a complete culture medium of puromycin with proper concentration is added for continuous culture;
(6) The overexpression efficiency of the transferred gene was detected by qPCR and Western Blot.
D. Total RNA extraction and real-time fluorescent quantitative PCR
Trizol method for extracting total RNA
(1) Fresh cell pellet was collected in sterile RNA-free EP tube, cell number was about 1X 10 7 Adding 1 mL of Trizol, repeatedly blowing with a pipetting gun until cell precipitation disappears, and standing at room temperature for 5 min;
(2) Adding 0.2 mL chloroform into the mixture, mixing the mixture for 4 to 6 times in an upside down way, standing the mixture at room temperature for 5 minutes, centrifuging the mixture at a speed of 12000 rpm and a temperature of 4 ℃ for 15 minutes;
(3) After centrifugation, the mixture was divided into three layers, in order: colorless liquid (upper layer), albumin layer (middle), pink organic phase (lower layer), the supernatant was carefully transferred to a new RNA-free EP tube, taking care of the middle layer which was not white; adding equal volume of isopropanol, mixing completely, standing at-20deg.C for 30 min or 1 h, centrifuging at 12000 rpm at 4deg.C for 10 min;
(4) Discarding useless supernatant, washing the precipitate with 1 ml of 75% ethanol (750 mu L of absolute ethanol plus 250 mu L of DEPC-H2O), centrifuging at 12000 rpm for 5 min at 4 ℃, uncovering, naturally drying at room temperature for 2-3 min, and adding 30-50 mu L RNase free water to dissolve the precipitate;
(5) The purity and concentration of RNA was detected using Nanodrop.
Reverse transcription: reverse transcription was performed using a PrimeScriptTM RT Master Mix (Perfect Real Time) kit to reverse transcribe RNA into cDNA, with a reaction system of 20. Mu.L. Reaction conditions: reverse transcription reaction: recognition inactivation of reverse transcriptase reaction at 37℃for 15 min: 5. 5 s at 85 ℃, store: 4 ℃.
Fluorescent quantitative PCR (qPCR): qPCR was performed using a TB Green cube Ex Taq (Tli RNaseH Plus) kit, corresponding reagents were added to the EP tube according to the following table, and after mixing, they were sequentially packed in 8 connecting tubes placed in order, three duplicate wells were set for each sample, and the reaction system was 25. Mu.L. Reaction conditions: pre-denaturation: 95 ℃ for 10 min; and (3) PCR reaction: 95 ℃ for 15 s and 60 ℃ for 1 min, and 40 PCR cycles are performed; melt Curve.
qPCR result judgment: the housekeeping gene GAPDH in the control group is used as an internal reference, and the method adopts △△ The Ct method calculates the relative expression level of the target gene.
E. Western Blot experiment (Western Blot experiment)
Extraction of total cell proteins: collecting fresh cell sediment, adding appropriate amount of RIPA lysate (containing protease inhibitor) into sterile EP tube, placing on ice for lysis for 1 hr, and shaking for 10 min; after lysis was completed, centrifugation was performed at 12000 rpm at 4℃for 20 min, and then the supernatant was transferred to a new sterile EP tube.
BCA assay for protein concentration: and (3) preparing a working solution: according to the instruction, using the solution A and the solution B in the BCA kit, preparing the required working solution according to the ratio of 50:1; dilution of protein standard solution: protein standard solution with the concentration of 2 mg/mL is diluted by precooled 1 XPBS buffer solution, and the concentration is 0.5 mug/mu L; diluted protein standards were added to the first column of the 96-well plate according to the instructions, 0, 1, 2, 4, 8, 12, 16, 20 μl, followed by 1×pbs buffer to 20 μl per well; repeating the first column for a second column; in the third column of the 96-well plate, 1 μl of protein sample to be tested is added to each well, and 1×PBS buffer is added to each well to 20 μl; repeating the third column in the fourth column; 200 mu L of working solution is added into each hole, and vibration is carried out for 30 min at 37 ℃; and detecting an Optical Density (OD) value of each hole at 570 nm wavelength by using an automatic enzyme-labeled instrument, drawing a standard curve according to the OD value and the loading amount of the standard protein, and obtaining the protein concentration of the sample to be detected according to the standard curve.
Protein denaturation and preparation of protein samples: according to 50 mug protein loading per well, 4 Xprotein loading buffer was added and all samples were made up to the same volume with RIPA lysate (containing protease inhibitors) and the mixture was boiled at 100℃for 10 min, -80℃for further use.
Polyacrylamide gel electrophoresis (SDS-PAGE): cleaning the glass plate, airing, and installing according to the instruction; preparing proper amounts of 10% separating gel and 5% concentrating gel according to instructions; adding a proper amount of 10% separating glue into the gap of the glass plate, and then adding 1 mL isopropanol to keep the surface of the separating glue horizontal; after the separation glue is solidified, pouring out isopropanol, and cleaning the plane of the separation glue for 2-3 times by using three distilled water; sucking the three distilled water remained on the surface of the glue with clean filter paper, adding a proper amount of 5% concentrated glue, and inserting a matched tooth comb; after the concentrated gel is solidified, removing the comb teeth, adding a proper amount of electrophoresis liquid, sequentially loading prepared protein samples in sequence, and adding 5 mu L of protein Marker in a specific lane, and carrying out constant-pressure 80V electrophoresis for 2-3 hours; preparing a PVDF membrane with proper size, soaking the membrane in methanol, and balancing the PVDF membrane with a membrane transferring solution; meanwhile, soaking a filter paper sponge by using a membrane transferring liquid, and sequentially placing a sponge-filter paper-SDS gel-PVDF membrane-filter paper-sponge in a membrane transferring clamp according to the sequence of a negative electrode to a positive electrode, wherein air bubbles are avoided between each two layers; correctly placing the prepared sandwich into a film transfer groove, and transferring the film at a constant pressure of 100V and a temperature of 4 ℃ to 2 h; after the transfer, washing the PVDF membrane once with TBST, and then sealing 1 h with 5% skimmed milk on a horizontal shaker at room temperature; discarding the sealing solution, adding the primary antibody diluted by the sealing solution according to a certain proportion into the PVDF membrane, and incubating overnight in a chromatography cabinet at 4 ℃; the next day, on the shaker, the membrane was washed 3 times with TBST for 10 min each time; discarding TBST, adding appropriate amount of HRP-labeled corresponding secondary antibody diluted with blocking solution according to a certain proportion, and incubating 2 h on a shaker at room temperature; washing the membrane with TBST on a shaker for 4 times and 10 min each time; developed with ECL chemiluminescence.
F. Cell function experiment
Transwell attack migration experiments: BD Matrigel gel was diluted 1:6 with basal medium 4 h in advance, 100 μl was added to each chamber; avoiding bubble generation, and placing in an incubator with the temperature of 37 ℃ and the CO2 content of 5 percent for standby; each group of cells was digested with 0.25% trypsin, after stopping the digestion and centrifugation, the cell pellet was resuspended in basal medium and counted, complete medium containing 10% FBS was added to the chamber of the 24-well plate, and 200. Mu.L of cell suspension containing 5X 10 was added to the upper chamber 4 The cells are cultured in an incubator with CO2 at 37 ℃ and 5 percent, avoiding generating bubbles; after culturing for 24-48 h, wiping the cells at the bottom of the inner part of the cell by using a cotton swab, putting the cell into 4% paraformaldehyde for fixing for 20 min, rinsing the cell in three distilled water, then dyeing the cell for 30 min by using crystal violet, observing the cells passing through the bottom membrane of the cell under a microscope, randomly selecting 5 visual fields, photographing and counting. In the migration experiment, BD Matrigel glue is not needed to be added into the small chamber, and other operations are consistent with those of the invasion experiment.
Flow-type fluorescent lectin assay: digesting each group of cells with 0.25% trypsin, stopping digestion, centrifuging, washing cell sediment with 1 XPBS buffer solution, adding different concentrations of Fluorescein Fluorescein labeled LCA into each group of cells, and incubating at room temperature in a dark place for 1 h; after centrifugation, the cell pellet was resuspended with 1% paraformaldehyde, gently mixed, and fixed for 20 min, and then the fluorescence intensity of each group of cells was measured on-machine.
Hyacinth bean lectin (Lens culinaris lectin, LCA) chromatography experiments: collecting fresh cell pellets of each group, adding a proper amount of RIPA lysate (containing protease inhibitor), performing ice lysis for 1 h, and vibrating once every 10 min; after lysis, the supernatant was transferred to a new sterile EP tube by centrifugation at 12000 rpm at 4℃for 20 min and protein concentration was measured by BCA; 1000. Mu.g of protein was added to 100. Mu.L of LCA lectin and incubated overnight in a rotary shaker at 4℃with 1 XPBS buffer containing 1 mM MnCl2, 1 mM MgCl2 and 1 mM CaCl2 supplemented to 1 mL; the next day, 60 μl of streptavidin agarose beads were taken, the beads were washed 3 times with PBST (0.1% tween) for 10 min each, then the overnight incubated mixture was added to the streptavidin agarose beads, and incubation was continued for 4 h; after the incubation, centrifuging at 2000 rpm and 4 ℃ for 2 min to obtain streptavidin agarose beads which are the protein-lectin-bead mixture, and washing the mixture with PBST (0.05% Tween) for 5 times for 10 min each time; adding a proper amount of 4 x protein loading buffer solution into the centrifuged protein-lectin-bead mixture, and boiling at 100 ℃ for 5 min; electrophoresis for 2-3 hours at constant voltage of 80V, film transfer for 2 hours at constant voltage of 100V, and sealing 1 h of 5% skim milk at room temperature; the primary antibody diluted in a certain proportion was incubated overnight at 4℃and washed 3 times with TBST, the HPR-labeled secondary antibody was incubated 2 h at room temperature and washed 4 times with TBST, ECL chemiluminescence.
Fucose competition experiments, fucose was added in a gradient of 0 mg, 1 mg, 5 mg, 10 mg, respectively, to TSTA3 overexpressed cellular protein, incubated with LCA overnight at 4 ℃, other experimental methods were consistent with the above.
siRNA transfection: (1) One day in advance, digesting each group of cells, fully culturing the cell sediment by re-suspending the medium, counting, and inoculating each group of cells in a 6-hole plate to ensure that the cell fusion degree of each hole reaches 40-60%; (2) mu.L of Lipofectamine 2000 was added to 250. Mu.L of Opti-MEM medium and allowed to stand at room temperature for 5 min; (3) Add 5. Mu.L LAMP2-siRNA to 250. Mu.L Opti-MEM medium and leave it stand for 5 min at room temperature; (4) Slowly dripping the mixed solution in the step (2) into the step (3), gently mixing, and standing at room temperature for 20 min; (5) Discarding the old culture medium of the 6-well plate, washing 2 times with 1 XPBS buffer solution, adding 1.5 mL complete culture medium, adding the mixed solution in step (4) into the 6-well plate dropwise and uniformly, and placing in an incubator with 5% CO2 at 37 ℃ for continuous culture of 48 h; (6) Cell pellet was collected and the interference efficiency of cells was detected by qPCR and Western Blot for subsequent experiments.
F. Statistical analysis: the experimental results are expressed as Mean ± standard deviation (Mean ± SD). ANOVA single factor analysis of variance was applied to comparisons of multiple sets of metrology data and T-test was applied to two sets of comparisons of metrology data. Statistical analysis was performed using SPSS19.0 software, with P < 0.05 indicating that the difference was statistically significant.
7. Experimental results
A. Effect of TSTA3 on esophageal squamous carcinoma cell migration and invasiveness: TSTA3 was overexpressed in KYSE150 and KYSE450 cells (fig. 1). Transwell experiments of esophageal squamous carcinoma cell lines KYSE150 and KYSE450 showed that: the number of migrating cells in the control group and the TSTA3-OE group of KYSE150 are (87+ -10) and (203+ -16), respectively, and the number of invading cells is (60+ -27) and (180+ -17), respectively; the number of migrating cells in the control and TSTA3-OE groups of KYSE450 was (37.+ -. 5) and (89.+ -. 3), respectively, and the number of invading cells was (37.+ -. 27) and (79.+ -. 6), respectively. Compared with the control group, the TSTA3-OE group migration and invasion cell number are obviously increased in the KYSE150 and KYSE450, and the difference is statistically significant (P < 0.01, figure 2), which suggests that the TSTA3 overexpression promotes the migration and invasion of the esophageal squamous carcinoma cell line.
B. Enhancement of core fucosylation modification of esophageal squamous carcinoma cells after TSTA3 overexpression: LCA is a core fucose-binding lectin that can specifically bind to core fucosylated proteins and has long been used as a specific probe.
In this study, we examined the fluorescence intensity of each group of cells by flow cytometry using the feature of LCA lectin binding to core fucose by incubating the fluorescein-labeled LCA lectin for the control group of KYSE150 and KYSE450 cells and their TSTA3-OE groups, respectively.
The results show that: fluorescence intensities of the control group and TSTA3-OE group of KYSE150 were (96.00.+ -. 4.55) and (121.03.+ -. 7.01), respectively; the fluorescence intensities of the control and TSTA3-OE groups for KYSE450 were (66.71.+ -. 3.58) and (93.62.+ -. 3.34), respectively. The fluorescence intensity of the TSTA3-OE group was significantly higher than that of the control group, and the differences were statistically significant (P < 0.05, FIG. 3), suggesting that the level of core fucosylation modification of cells could be enhanced after overexpression of TSTA3 in esophageal squamous carcinoma cells.
C. TSTA3 targets core fucosylation acting on LAMP 2: LCA chromatography experiments are carried out by utilizing the characteristic that LCA lectin can be combined with core fucosylation protein, carrying out LCA lectin enrichment chromatography on cell proteins of a KYSE450 control group and TSTA3-OE groups thereof, after SDSPAGE electrophoresis, dyeing SDS gel with Coomassie brilliant blue, and selecting 4 different protein bands (marked by red arrows in figures 2-7A) with different expressions between the control group and the TSTA3-OE groups for mass spectrometry.
We aligned the up-regulated 133 proteins from the mass spectrometry analysis with the N-glycosylation chemistries of the previous control and TSTA3-OE cells (fig. 4) and found 3 intersection genes of IKBIP, LAMP2, LMNA (fig. 4). By examining the literature, it was found that there was an increase in glycosylation modification of LAMP2 protein in choriocarcinoma, thereby promoting migration and invasion of choriocarcinoma cells, suggesting that our glycosylated LAMP2 protein may be key for TSTA3 to play a role in ESCC cells.
D. TSTA3 overexpression promotes core fucosylation of LAMP2: in LCA enrichment chromatography experiments, LAMP2 is used for imprinting and detecting LAMP2 expression and LCA enriched core fucosylation LAMP2 proteins in a KYSE150 control group and a KYSE450 control group and TSTA3-OE group cells thereof respectively, and the results show that the total LAMP2 protein amount in the control group and the TSTA3-OE group is not significantly changed, compared with the control group, the LCA enriched LAMP2 protein in the TSTA3-OE group is obviously increased, namely the core fucosylation modification of the LAMP2 protein in the TSTA3-OE group is increased, and the differences are statistically significant (P < 0.01, figure 5), so that the TSTA3 over-expression in esophageal squamous carcinoma cells can promote the core fucosylation of LAMP 2.
E. Fucose competes for binding to core fucosylated LAMP2: LCA enrichment chromatography experiments were performed on proteins of the TSTA3-OE groups of KYSE150 and KYSE450 cells, with different concentrations of fucose added, allowing fucose to compete with LAMP2 for binding to LCA lectin, and the results show that with increasing fucose concentration, LCA enriched core fucosylated LAMP2 gradually decreased (FIG. 6), binding to D, further suggesting that TSTA3 overexpression in esophageal squamous carcinoma cells can promote core fucosylation of LAMP 2.
F. Interfering LAMP2 can reverse the promotion of esophageal squamous carcinoma cell migration invasion by TSTA 3: to further clarify that TSTA3 plays a role in esophageal squamous carcinoma by regulating core fucosylation modification of downstream LAMP2 protein, we interfered with LAMP2 expression by transient transfection of LAMP2-siRNA and detected interference efficiency by Western Blot in a control group of KYSE150 cells and a TSTA3-OE group (fig. 7A), while using a Transwell experiment to detect the effect on the migratory invasiveness of TSTA3 overexpressing ESCC cells after interfering LAMP2 expression.
The results showed that the migration number and invasion number of each of the SiNC, LAMP2-si1, LAMP2-si2 groups in the control group were (146.+ -. 11), (86.+ -. 3), (92.+ -. 5) and (67.+ -. 6), (15.+ -. 2), (19.+ -. 6), respectively; in the TSTA3-OE group, the migration number and invasion number of each of the SiNC, LAMP2-si1 and LAMP2-si2 groups are (203+ -15), (113+ -13), (135+ -4) and (109+ -10), (71+ -12) and (64+ -8), respectively, and it is found that in the KYSE150 cells of the TSTA3-OE group, after interference with the expression of LAMP2, the migration and invasion number of the cells are significantly reduced, and the difference is statistically significant (P < 0.05, FIG. 7B), which suggests that LAMP2 can reverse the migration and invasion of esophageal squamous carcinoma cells caused by the overexpression of TSTA3, and the result is further verified in the KYSE450 cells.
According to the invention, through immunohistochemical analysis of a tissue chip, the expression of TSTA3 in esophageal squamous carcinoma tissue and lymphatic metastatic tissue is obviously higher than that of a paracancerous normal tissue, and the expression of TSTA3 in the lymphatic metastatic tissue is also obviously higher than that of the esophageal squamous carcinoma tissue, which is consistent with the previous research result, namely, the expression of TSTA3 is positively correlated with TNM stage and lymph node metastasis of esophageal squamous carcinoma patients.
In the cell function experiment, compared with the control group, the TSTA3 can be over expressed in esophageal squamous carcinoma cells to promote invasion and migration of the cells, but has no influence on cell proliferation. It is suggested that the TSTA3 gene plays an oncogene role in esophageal squamous carcinoma.
Protein glycosylation is an important functional protein modification and plays an important role in various cellular processes such as cell adhesion, receptor activation, tumor invasion, metastasis and inflammatory reaction. Of these, fucosylation, particularly core fucosylation, is the most common glycosylation modification, and is involved in the development of diseases such as tumors and inflammation. Lectins are proteins that play an important role in immunology and hematology, are specific probes for membrane glycoprotein structures, and are proteins capable of recognizing specific glycan moieties, and are widely used to selectively enrich glycosylated proteins.
LCA belongs to the family of leguminous lectins, a core fucose-binding lectin that specifically binds to core fucosylated proteins, and has long been used as a specific probe. In this study, changes in the level of modification of core fucosylation in ESCC cells were detected before and after TSTA3 overexpression using LCA lectin labeled LCA lectin, which was characteristic of binding to core fucose. The cell-bound fluorescein-labeled LCA of the KYSE150 and KYSE450 cell control group and the TSTA3 over-expression group is detected by a flow cytometer, and after the TSTA3 is over-expressed, the core fucosylation modification of esophageal squamous carcinoma cells can be enhanced. Subsequent mass spectrometry identification by N-glycosylation histology modification and LCA lectin enrichment chromatography of the cell line revealed that the intersection genes present included IKBIP, LAMP2, LMNA. It is suggested that TSTA3 may play a role in esophageal squamous carcinoma by modifying the core fucosylation of these proteins.
By consulting the literature, there is an increase in glycosylation modification of LAMP2 protein in choriocarcinoma, promoting migration and invasion of choriocarcinoma cells, suggesting that glycosylated LAMP2 protein may be key for TSTA3 to play a role in esophageal squamous carcinoma cells.
LAMP2 is a lysosomal associated membrane protein, one of the members of the LAMP family. The LAMP family is a family of glycosylated proteins present on lysosomal membranes. Lysosomes are single membrane acidic organelles of eukaryotic cytoplasm, which, in addition to degrading intracellular and extracellular macromolecules into cellular available building blocks, are important mediators of intracellular homeostasis and are involved in the occurrence of different diseases such as neurodegenerative diseases, cardiovascular diseases and tumors. During transformation and cancer progression, lysosomes change their localization, volume and composition and enhance tumor invasiveness by releasing related enzymes. For example, various lysosomal enzymes, including cathepsins, are highly expressed in various tumors such as breast cancer, prostate cancer and colon cancer, and their secretion is increased by exocytosis, degradation and angiogenesis of extracellular matrix are promoted, thereby promoting proliferation, invasion and metastasis of tumor cells, and their expression levels are of clinical prognostic significance.
The LAMP family includes LAMP1, LAMP2, LAMP3, LAMP4 and LAMP5, and is involved in various cellular biological processes including phagocytosis, autophagy, lipid transport, aging, etc., and plays an important role in tumors. LAMP2 is widely expressed in human tissues and cell lines, is a major component of lysosomal membranes, belongs to type I transmembrane proteins, and is structurally characterized by a highly glycosylated luminal region, a transmembrane region and a short C-terminal cytoplasmic domain that form a glycoprotein layer in the lysosomal lumen. There are many different mutations in the LAMP2 gene, which are responsible for Danon's disease, a serious disease characterized by skeletal myopathy and cardiomyopathy and cognitive dysfunction.
LAMP2 promotes invasion and metastasis by abnormal localization on the tumor cytoplasmic membrane in melanoma cells a2058, colon cancer cells CaCo-2 and fibrosarcoma cells HT 1080. LAMP2 is also highly expressed in bronchoalveolar lavage fluid of patients with poorly differentiated human gastric adenocarcinoma, hepatocellular carcinoma, salivary gland adenoid cystic carcinoma and lung adenocarcinoma, and can be used as a novel molecular marker for tumor diagnosis.
LAMP2, however, also has tumor suppression, and depletion of the VEGF-NRP2 axis in prostate cancer cells can induce cell death; deletion of the VEGF-NRP2 axis causes LAMP2 and WDFY1 upregulation, inhibits autophagy, and promotes cell death.
In esophageal squamous carcinoma tissue, there is a significant difference in the level of LAMP2 expression between the different TNM stages and the degree of tumor tissue differentiation, the level of LAMP2 expression being inversely related to the degree of tumor tissue differentiation and positively related to the TNM stages, the worse the histological differentiation, the higher the LAMP2 expression level. The expression of LAMP2 has been reported to be related to cell differentiation, possibly to glycosylation of LAMP 2; a weak positive correlation between LAMP2 expression and TNM phase may be associated with an enhancement of autophagy activity in tumor cells.
The LCA chromatography experimental result of the research shows that the overexpression of TSTA3 in esophageal squamous carcinoma cells can promote core fucosylation of LAMP2, and simultaneously fucose can compete with core fucosylation LAMP2 for binding to LCA lectin, and further shows that in esophageal squamous carcinoma, TSTA3 can specifically regulate core fucosylation modification of LAMP 2; and after the expression of the LAMP2 is interfered in the esophageal squamous carcinoma cells with the overexpression of the TSTA3, through a Transwell experiment, the fact that the migration and invasion of the esophageal squamous carcinoma cells caused by the overexpression of the TSTA3 can be reversed by the interference of the expression of the LAMP2 is found. Thus, this study suggests that TSTA3 may affect esophageal squamous carcinoma cell migration and invasion by modulating aberrant core fucosylation of LAMP 2.
In esophageal squamous carcinoma, TSTA3, which is the rate-limiting enzyme in the GDP-L-fucose synthesis pathway, is involved in fucosylation modification, and promotes invasion and migration of esophageal squamous carcinoma by increasing core fucosylation modification of LAMP2, exerting oncogene action (FIG. 8).
Experimental example 1: verification experiment of the invasive migration ability of the inhibitor peracetylated 2-F-Fuc of fucosyltransferase on esophageal cancer cells: peracrylated 2-F-Fuc inhibits the invasive migratory capacity of esophageal cancer cells by inhibiting core fucosylation of LAMP2 and reducing expression of TSTA 3.
The chemical structure of peracrylated 2-F-Fuc is shown in FIG. 9. The product is named as Peracetylated 2- fl uo 2-deoxy-L-fuse or 1,3, 4-Tri-O-acetyl-2-deoxy-2-fluoroo-L-fuse. It is a peracetylated analogue of fucose containing fluorine atom at C2 position, is an inhibitor of fucosyltransferase, and its peracetylation modification can raise cell permeability, and is favorable for molecules to enter cell.
Experimental materials: peracrylated 2-F-Fuc was purchased from Carbosynth (product number: MT 15919).
The experimental method comprises the following steps: the invasion and migration experiment, the LCA chromatography enrichment experiment and the Western blot experiment are the same as above.
Experimental results:
1. peracetylated 2-F-Fuc significantly inhibited migration and invasion ability of TSTA3 overexpressed esophageal squamous carcinoma cells: IC5O value is detected by an enzyme-labeled instrument, the concentration range of the proper peracrylated 2-F-Fuc is 10.71-30.64 mu M, and 2-F-Fuc with the concentration of 10 mu M and 20 mu M is selected for subsequent experiments.
The results of the Transwell experiments of the esophageal squamous carcinoma cell line KYSE150 overexpressing TSTA3 are shown in FIG. 10, and the results show that: after 24 and 36 hours, the number of invading and migrating cells was significantly less in the 10. Mu.M 2-F-Fuc, 20. Mu.M 2-F-Fuc treated groups than in the DMSO-treated control group.
2. peracetylated 2-F-Fuc treatment significantly inhibited the expression of TSTA3 and the core fucosylation level of LAMP 2: in the LCA enrichment chromatography experiment, after the peracetylated 2-F-Fuc treatment of TSTA3 over-expressed KYSE150 cells are respectively detected by LAMP2 blotting, the results of LAMP2 expression and LCA enrichment of core fucosylated LAMP2 protein are shown in FIG. 11, and the results show that the total LAMP2 protein amount in the control group and the treatment group is not significantly changed, compared with the control group, the peracetylated 2-F-Fuc treatment group LCA enriched LAMP2 protein is significantly reduced, namely, the core fucosylation modification of the LAMP2 protein in the treatment group is increased, and the TSTA3 protein expression amount in the peracetylated 2-F-Fuc treatment group is reduced.
In combination with the experimental results described above and the accompanying drawings, it is evident that: peracetylated 2-F-Fuc can obviously inhibit the expression of TSTA3 and the core fucosylation level of LAMP2, and can obviously inhibit the migration and invasion capacity of esophageal squamous carcinoma cells over-expressed by TSTA 3. peracetylated 2-F-Fuc can obviously inhibit migration and invasion capacity of esophageal squamous carcinoma cells over-expressed by TSTA3 within a concentration range of 10.71-30.64 mu m.
Claims (2)
- The application of TSTA3 and LAMP2 as target objects in preparing esophageal squamous carcinoma cell metastasis detection reagents and drug screening is characterized in that: the application of the TSTA3 and the LAMP2 serving as target objects in screening medicaments for inhibiting or slowing down esophageal squamous cell metastasis; TSTA3 is over-expressed in esophageal squamous carcinoma cells, LAMP2 is over-expressed, core fucosylation of LAMP2 is modified, and the esophageal squamous carcinoma cells migrate and invade; TSTA3 promotes invasion and migration of esophageal squamous carcinoma by increasing core fucosylation modification of LAMP2, and TSTA3 over-expression exceeds a normal value by 1.6 times, so that esophageal squamous carcinoma cells are obviously migrated and invaded; the drug for inhibiting or slowing down esophageal squamous carcinoma cell metastasis is peracetylated 2-F-Fuc, and the concentration is 10-30.64 mu M.
- 2. The use according to claim 1, characterized in that: the concentration of peracrylated 2-F-Fuc is 10.71-30.64 mu M.
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