CN110893238A

CN110893238A - Application of substance for inhibiting activity and expression quantity of vascular endothelial growth factor in preparation of product for inhibiting lymph node metastasis

Info

Publication number: CN110893238A
Application number: CN201811068323.9A
Authority: CN
Inventors: 李金萍; 王建浩; 王恩锋
Original assignee: Changzhou University
Current assignee: Changzhou University
Priority date: 2018-09-13
Filing date: 2018-09-13
Publication date: 2020-03-20

Abstract

The invention discloses an application of a substance for inhibiting the activity and expression quantity of a vascular endothelial growth factor in preparing a product for inhibiting lymph node metastasis. Experiments prove that the inhibition of the expression of the vascular endothelial growth factor can inhibit the generation of lymphatic vessels and inhibit the invasion of tumor cells on the lymphatic vessels, thereby having important application value in the inhibition of the lymph node metastasis of the tumor cells. Inhibiting the expression of vascular endothelial growth factor can prevent and/or treat cancer. The invention has important application value.

Description

Application of substance for inhibiting activity and expression quantity of vascular endothelial growth factor in preparation of product for inhibiting lymph node metastasis

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a substance for inhibiting the activity and the expression quantity of a vascular endothelial growth factor in preparing a product for inhibiting lymph node metastasis, in particular to application of a substance for inhibiting the activity and the expression quantity of the vascular endothelial growth factor in preparing a product for inhibiting lymph node metastasis of tumor cells.

Background

Vascular Endothelial Growth Factor (VEGF), a specific heparin-binding growth factor for vascular endothelial cells, induces angiogenesis in vivo. VEGF-C (vascular endothelial growth factor C) is one of the VEGF family members, which participates in the proliferation and migration of lymphatic endothelial cells and the generation of new lymphatic vessels by binding to tyrosine kinase receptors and non-tyrosine kinase receptors to trigger signal transduction pathways.

Endometrial Cancer (EC) is a common gynecological malignancy, with the first incidence of cancer in the reproductive system of highly populated women in many countries and the second incidence of malignancy in the reproductive system of women living in china. With the development of society and the improvement of economic conditions, the incidence of obesity and diabetes is increased year by year, which leads to the increase of the incidence of EC, and the EC can become reproductive tract tumor with the highest incidence of gynecological malignant tumor of Chinese people in the future. Therefore, the control and treatment of EC has become an important issue. Lymph node metastasis is the major diffuse pathway of EC. Late-stage EC patients with lymph node metastasis have much higher postoperative recurrence and mortality than early-stage EC patients. The inhibition of lymph node metastasis has great significance for reducing the mortality rate and postoperative recurrence rate of tumor patients.

Disclosure of Invention

The object of the present invention is to inhibit lymph node metastasis of tumor cells.

The invention firstly protects the application of the substance for inhibiting the activity and/or the expression quantity of the vascular endothelial growth factor in preparing the product; the function of the product may be at least one of the following a1) to a 5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) can be used for treating cancer.

The invention also protects the application of substances for inhibiting the activity and/or expression quantity of the vascular endothelial growth factor, which can be at least one of the following A1) to A5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) can be used for treating cancer.

The invention also protects S1) or S2) or S3).

S1) application of the vascular endothelial growth factor as a drug target in preparing products; the function of the product may be at least one of the following a1) to a 5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) can be used for treating cancer.

S2) application of the vascular endothelial growth factor in preparing products; the function of the product may be C1) or C2) or C3) as follows: C1) promoting lymph node metastasis of tumor cells; C2) promoting lymphatic angiogenesis; C3) promoting tumor cells to invade lymphatic vessels.

S3) application of the vascular endothelial growth factor, which can be C1) or C2) or C3) as follows: C1) promoting lymph node metastasis of tumor cells; C2) promoting lymphatic angiogenesis; C3) promoting tumor cells to invade lymphatic vessels.

In any of the above applications, the product may be a medicament.

The invention also protects a method for developing a lymph node metastasis medicament for inhibiting tumor cells, which can take the vascular endothelial growth factor as a medicament target, and a substance capable of inhibiting the medicament target is the lymph node metastasis medicament for inhibiting the tumor cells.

In the method, the inhibition of the drug target can be inhibition of the activity and/or expression level of the vascular endothelial growth factor.

The invention also protects product A or product B.

The product A may contain vascular endothelial growth factor; the function of the product A can be C1) or C2) or C3) as follows: C1) promoting lymph node metastasis of tumor cells; C2) promoting lymphatic angiogenesis; C3) promoting tumor cells to invade lymphatic vessels.

The product B can contain substances for inhibiting the activity and/or expression of the vascular endothelial growth factor; the function of the product B can be at least one of the following A1) to A5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) can be used for treating cancer.

The product A or product B can be a medicine.

Any of the tumor cells described above may be human endometrial cancer cells.

Any one of the human endometrial cancer cells described above can be Ishikawa cells or HEC-1B cells.

Any one of the aforementioned vascular endothelial growth factors may be vascular endothelial growth factor C.

Any one of the vascular endothelial growth factor C described above may be a1) or a2) or a3) or a 4):

a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table;

a4) protein which has 80 percent or more than 80 percent of identity with the amino acid sequence limited by the sequence 2 in the sequence table, is derived from human and has the same biological function.

The invention also protects any substance which can inhibit the activity and/or expression of the vascular endothelial growth factor.

Any one of the above-mentioned "substances inhibiting the activity and/or expression level of vascular endothelial growth factor" may be z1) or z 2):

z1) specific RNA; the specific RNA can be double-stranded RNA shown in a sequence 3 in a sequence table;

z2) shRNA synthesized by shRNA expression system using said specific RNA as target.

Z2) is a plasmid pSUPERIOR-retro-neo-GFP-VEGF-C-shRNA which is constructed by using a vector pSUPERIOR-retro-neo + GFP and taking double-stranded RNA shown as a sequence 3 in a sequence table as a target spot. The plasmid pSUPERIOR-retro-neo-GFP-VEGF-C-shRNA can be a double-stranded DNA molecule which is inserted between recognition sites of restriction enzymes BgLII and Hind III of the vector pSUPERIOR-retro-neo + GFP and is shown as a sequence 4 in a sequence table. The vector pSUPERIOR-retro-neo + GFP may be a product of OligoEngine, USA, under the catalog number VEC-IND-0014.

Experiments prove that the inhibition of the expression of the vascular endothelial growth factor can inhibit the generation of lymphatic vessels and inhibit the invasion of tumor cells on the lymphatic vessels, thereby having important application value in the inhibition of the lymph node metastasis of the tumor cells. Inhibiting the expression of vascular endothelial growth factor can prevent and/or treat cancer.

Drawings

FIG. 1 shows the experimental results of step 2 in example 1.

FIG. 2 shows the results of the experiment in step 3 of example 1.

FIG. 3 shows the results of the experiment in example 2.

FIG. 4 shows the experimental results of step one in example 3.

FIG. 5 shows the results of the experiment 2 in step two of example 3.

FIG. 6 shows the results of step two 3 of example 3.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

VEGF-C antibody dilutions: and diluting the human VEGF-C antibody to 75 times of volume by using Bond antibody diluent. The human VEGF-C antibody is a product of Invitrogen (zymed) Inc., catalog number 18-2255. Bond antibody dilutions were made by Leica under the catalog number AR 9352.

The antigen retrieval liquid is a product of Leica company, and the catalog number of the product is AR 9961. The DAB kit is a product of Leica company, and the catalog number of the product is DS 9800.

D2-40 antibody dilution: the D2-40 antibody was diluted to 100 volumes with 0.01M PBS buffer at pH 7.4. The D2-40 antibody is available from Covance, Inc. USA under the catalog number SIG 3730-100.

1640 medium, product catalog No. 10363083, from Fisher Scientific.

Tissue microarray paraffin sections (TMA) and endometrial Tissue specimens at proliferative stage were purchased from USBiomax Inc (Rockville, MD, usa) under product catalog numbers EMC961, EMC102 and EMC 1502. The tissue microarray paraffin section comprises 310 ECs, wherein the lymph node metastasis positive group comprises 24 cases, the clinical FIGO stage diagnosis is stage III C or stage IV B, and the lymph node metastasis positive group comprises more than 1 lymph node metastasis; 286 cases in the negative group, stage I, II, IIIA, IIIB or IVA of clinical FIGO, and no lymph node metastasis.

All providers of tissue samples were volunteers who informed and signed a consent form.

Example 1 correlation analysis of VEGF-C expression levels with EC lymph node metastasis

1. Cases and samples

Endometrial tissue samples positive for EC lymph node metastasis were taken in 10 cases and endometrial tissue samples in the proliferative phase in 15 cases. All tissue samples provide clinical information on tumor type, histological grade, and degree of metastasis.

2. Detection of relative expression amount of Gene encoding VEGF-C (hereinafter referred to as VEGF-C Gene)

Three replicates were performed and averaged, with each replicate performed as follows:

(1) total RNA was extracted from tissue samples (endometrial tissue samples positive for EC lymph node metastasis or endometrial tissue samples at the proliferative stage).

(2) And (2) taking the total RNA of the sample obtained in the step (1) as a template, and carrying out a first strand synthesis reaction on the cDNA to obtain the cDNA.

(3) And (3) detecting the relative expression quantity of the VEGF-C in the tissues by using the cDNA obtained in the step (2) as a template through fluorescent quantitative PCR (36B 4 gene is used as an internal reference gene). Primers for VEGF-C identification were 5'-AGGGTCAGGCAGCGAACAAGA-3' and 5'-CCTCCTGAGCCAGGCATCTG-3'. The primers for identifying the 36B4 gene are as follows: 5'-ATACAGCAGATCCGCATG-3' and: 5'-TCATGGTGTTCTTGCCCATCA-3' are provided.

The VEGF-C gene has a sequence shown as a sequence 1 in a sequence table, and expresses a protein shown as a sequence 2 in the sequence table.

The relative expression level of VEGF-C in endometrial tissue samples that are in the proliferative phase is taken as 1, and the relative expression level of VEGF-C in endometrial tissue samples that are positive for EC lymph node metastasis is shown in FIG. 1. The results show that the relative expression amount of VEGF-C in the endometrial tissue sample with positive EC lymph node metastasis is 1.8 times of the relative expression amount of VEGF-C in the endometrial tissue sample in the proliferative stage. Thus, VEGF-C may be associated with EC lymph node metastasis.

3. Detection of expression level of VEGF-C

Detecting the expression level of VEGF-C in a lymph node metastasis positive group and a lymph node metastasis negative group by adopting an immunohistochemical staining and chemical scoring method, and specifically comprising the following steps of:

(1) fixing the tissue microarray paraffin section on a glass slide, baking the glass slide for 2 hours at 60 ℃, sequentially soaking and dewaxing the glass slide by dimethylbenzene, gradient ethanol and water, and washing the glass slide 3 times for 3min by PBS (pH7.4).

(2) And (3) after the step (1) is finished, taking the tissue microarray paraffin section, placing the tissue microarray paraffin section in antigen repairing liquid, and incubating for 20min at 95 ℃.

(3) And (3) after the step (2) is finished, taking the tissue microarray paraffin section, adding VEGF-C antibody diluent, and incubating for 15min at room temperature.

(4) After the step (3) is finished, taking the tissue microarray paraffin section, adding a secondary antibody and a tertiary antibody, and then performing DAB color development according to the steps of the DAB kit; the secondary antibody and the tertiary antibody are components in the DAB kit.

(5) And (4) after the step (4) is finished, taking the tissue microarray paraffin section, washing and sealing.

(6) And (5) after the step (5) is finished, performing immunohistochemical staining evaluation by using a histopathological staining scoring system. The system evaluates the staining intensity of the section and the percentage of positive staining after quantitative assigning, wherein the staining intensity can be defined as 0 grade, 1 grade, 2 grade and 3 grade, which respectively correspond to staining negative, staining weak positive, staining medium positive and staining strong positive; the percentage of positive staining throughout the tumor tissue in terms of each intensity was defined as 1, 2, 3, 4, corresponding to a percentage of < 25%, a percentage of < 25% or less and < 49%, a percentage of < 50% or less and < 75%, and a percentage of > 75%, respectively. All immunohistochemically stained sections were evaluated in parallel by 3 specialized pathologists and no information on the sections was known a priori. If 3 pathologists had a discrepancy in the interpretation of the section, 3 pathologists would re-evaluate the section together until consensus was reached. The formula for calculating immunohistochemical staining score was: the composite score is 1 × percent weak positive staining +2 × percent medium positive staining +3 × percent strong positive staining. The expression levels of VEGF-C in the positive and negative lymph node metastasis groups were compared by a single ANOVA method (the higher the average composite score, the higher the expression level of VEGF-C), and the two groups were analyzed for differences by t-test (p <0.05 is significant).

The results of the experiment are shown in FIG. 2 (in FIG. 2, the arrows indicate whether the paraffin sections of the tissue microarray were stained with weak, medium or strong positive). The results showed that the expression level of VEGF-C was higher in the lymph node metastasis positive group than in the negative group, and therefore VEGF-C was likely to be associated with EC lymph node metastasis.

Example 2 correlation of Lymphatic Vascular Density (LVD) and/or level of Lymphatic Vascular Infiltration (LVI) with EC lymph node metastasis

The common protein markers for the angiogenesis condition of the new generation lymph are VEGFR-3, HA receptor-1 (LYVE-1) of lymphatic endothelial cells or Podoplanin, wherein LYVE-1 is a homolog of CD44 molecule of lymphatic endothelial cells which are involved in transporting hyaluronan protein, and Podoplanin is glomerular podocyte membrane glycoprotein highly expressed in small lymphatic vessels. The monoclonal antibody named D2-40, which is capable of specifically detecting Podoplanin, is widely used for immunohistochemical identification of lymphatic endothelial cells. In this example, lymphatic vascular neogenesis activity was observed by measuring Lymphatic Vascular Density (LVD); the degree of tumor cell invasion into lymphatic vessels was reflected by the detection of lymphatic vessel infiltration Level (LVI).

The specific steps for detecting the density and infiltration level of the lymphatic vessels are as follows:

1. taking a tissue microarray paraffin section, placing the section in an antigen repairing solution, and incubating for 20min at 95 ℃ to achieve antigen renaturation).

2. After step 1, the tissue microarray paraffin sections were taken, hydrogen peroxide and collagen were added, and incubation was performed for 30min at 37 ℃.

3. After completion of step 2, the tissue microarray paraffin sections were taken, and the D2-40 antibody dilution was added and incubated overnight at 4 ℃.

4. After completing step 3, the tissue microarray paraffin sections were taken, biotin-labeled anti-mouse IgG (product of Vector Labs, usa under the catalog number BA1000) was added as a secondary antibody, then staining detection was performed with ABC peroxidase (product of Vector Labs, usa under the catalog number PK6100), and the number of LVD (lymphatic vessel density) in each section was counted under a low power microscope. After calculating the mean value of the LVD number in 10 fields, the LVD difference between the positive and negative lymph node metastasis groups was compared by a single ANOVA method, and the two groups were analyzed for difference by t-test (p <0.05 is significant difference).

5. After completion of step 4, LV i (lymphatic vascular infiltration) was counted based on the LVD data. Differences in LV I were compared between positive and negative lymph node metastasis groups using a single ANOVA method, and the two groups were analyzed for differences by the t-test (significant differences were found when p < 0.05).

The results are shown in FIG. 3A (arrows indicate lymphatic endothelial intima, dotted line boundary left lower tumor tissue side, right upper tumor) and FIG. 3B (arrows indicate lymphatic endothelial intima, dotted line boundary left tumor tissue side, right tumor). The results indicate that Lymphatic Vessel Density (LVD) and/or lymphatic vessel infiltration level (LV I) may be associated with EC lymph node metastasis. VEGF-C mediates EC lymph node metastasis by promoting lymphatic angiogenesis and/or promoting tumor cell invasion of lymphatic vessels.

Example 3 role of VEGF-C in EC lymph node metastasis

Establishment of mouse EC in-situ model based on tumor block embedding inoculation method

1. Culturing HEC-1B cells in 1640 medium to obtain a concentration of 2 × 10⁶HEC-1B cell suspension in 100. mu.L/cell.

2. A6-week-old female SCID mouse (CB-17 SCID mouse, catalog number, available from Beijing Wintolite laboratory animal technology Co., Ltd.) was subcutaneously injected with 100. mu.L of HEC-1B cell suspension into the left back of the female mouse.

3. After the completion of the step 23-4 weeks, the product with a size of 1cm can be formed³The mass of (a); stripping the lump tissue and cutting into pieces of about 1mm³The tissue mass of (1).

4. Taking 12 SCID female mice (a product of Beijing Wittiulihua laboratory animal technology Co., Ltd., product catalog number is CB-17SCID mice) with the age of 8 weeks, and carrying out anesthesia by intraperitoneal injection of Ketamine/Xylazine (the injection dose is 120mg &10mg/1kg BW); an approximately 1cm long incision was then made in the lower right dorsal side of the SCID female mouse, exposing the right uterine horn; transplanting the tissue block prepared in the step 3 to 1/3 parts below the uterine horn, adhering an incision with Vet tissue mucilage (product of Fisher Scientific company, product catalog number is NC9906009), cleaning the periphery of an operation area with PBS buffer solution with pH7.4 and 10mM, then dipping a cotton stick with dry liquid, sewing the incision with 4/0 threads, coating antibiotic ointment, warming with a desk lamp, keeping warm, placing the mouse back into a cage after anaesthetizing and waking, and feeding the mouse conventionally.

5. And after the steps are finished for 48-10 weeks, stripping all the uterus, oviducts and periaortic lymph nodes, preparing paraffin sections (the uterus needs to be stored in 4% (volume percentage) of formaldehyde aqueous solution for 4 hours, then is soaked in 30% (m/m) of sucrose aqueous solution for one night, and then is embedded by OCT (optical coherence tomography) for quick cryopreservation, and if other organs with suspicious metastases are found, preparing the paraffin sections in the same way). All paraffin sections were subjected to hematoxylin-eosin staining and immunohistochemical analysis using antibodies against human mitochondrial protein (Thermo Fisher Scientific, Cat. MA 5-15880).

Staining results of mouse EC in situ model based on tumor block embedding inoculation showed tumor growth in uterine horn (a and C in fig. 4), enlargement of periaortic lymph nodes and tumor tissue (B and D in fig. 4); the results of immunohistochemical analysis indicated that the formed cancer cells were derived from human HEC-1B cells (E and F in FIG. 4). In FIG. 4, E and C are both continuous slices of A, and F and D are both continuous slices of B.

Mouse EC in situ models based on tumor block embedding vaccination can be modeled to present important features from EC growth to tumor cell metastasis to lymph nodes.

Second, establishment of system for inducing expression of VEGF-C shRNA by tetracycline

1. Discovery of siRNA sequence inhibiting expression level of VEGF-C

Ishikawa cells (5X 10 each) were transfected with 4 single siRNA nucleotides (product of Dharmacon, USA)⁵Approximately 20nM siRNA transfected into each Ishikawa cell) and then cultured to obtain recombinant cells. By detecting the level of VEGF-C mRNA in the recombinant cells (random siRNA is used as a control, and 36B4 gene or GAPDH gene is used as a reference gene), one siRNA can inhibit the expression level of VEGF-C, and the siRNA is double-stranded RNA shown in a sequence 3 in a sequence table.

And (3) sequence: 5'-CAACCGAGAAUUUGAUGAUGAAUU-3' are provided.

2. Screening for Ishikawa Stable clones expressing the tetracycline inhibitor (Tet-R)

(1) Plasmid PCDNA6/TR (Invitrogen corporation) was transfected into Ishikawa cells (1X 10 cells per cell)⁶Approximately 2. mu.g of plasmid PCDNA6/TR was transfected into each Ishikawa cell), and 11 clones were selected using blistic as a selection marker, and named C3, C5, C6, C7, C9, C10, C11, C12, C13, C14, and C17, respectively.

(2) Respectively inoculating the clone strains obtained in the step (1) into 0.1mL 1640 culture medium, and then adding doxycycline (Dox) to obtain a treatment system, wherein the concentration of the Dox in the treatment system is 3 mu g/mL. 37 ℃ and 5% CO₂After 24h of treatment in the incubator, the luciferase activity was measured.

According to the method, 3 mu g/mL in the step (2) is replaced by 6 mu g/mL, and other steps are not changed, so that the corresponding luciferase activity is obtained.

The above procedure was followed to replace 3. mu.g/mL in step (2) with 0. mu.g/mL (i.e., no doxycycline was added), and the other steps were unchanged to obtain the corresponding luciferase activity as a blank.

The results of the experiment are shown in FIG. 5. The results showed that the activity of the basic fluorescent enzymes of C5 and C12 was relatively low and the sensitivity to Dox was high in the 11 clones. C5 and C12 are selected Ishikawa stable clone strains, and the next experiment is carried out.

3. Establishment of tetracycline inducible expression VEGF-C shRNA system

The steps for establishing a tetracycline inducible expression VEGF-C shRNA system and detecting are as follows:

(1) plasmid pSUPERIOR-retro-neo-GFP-VEGF-C-shRNA was obtained by inserting a double-stranded DNA molecule represented by sequence 4 in the sequence table between recognition sites of restriction enzymes BgLII and Hind III of a vector pSUPERIOR-retro-neo + GFP (manufactured by OligoEngine, USA, catalog number VEC-IND-0014; the vector contains an RNA polymerase III H1 gene promoter controlled by tetracycline TetO 2).

(2) Transfection of plasmid pSUPERIOR-retro-neo-GFP-VEGF-C-shRNA into C5 or C12 obtained in step 2 (per 1X 10)⁵Approximately 1 mu g of plasmid pSUPERIOR-retro-neo-GFP-VEGF-C-shRNA is transferred to C5 or C12, neomycin (80 mu g/mL) and blestic idin (20 mu g/mL) are used as screening markers, 5 clones (named D4, D5, D6, D7 and D8 respectively) which stably express Tet-VEGF-C-shRNA are screened out and mutually combined under the control of tetracycline or Dox to release TetO 2. TetO2 acts upstream of the promoter region to trigger expression of the downstream gene (VEGF-C shRNA).

(3) According to the method of step (2), the plasmid pSUPERIOR-retro-neo-GFP-VEGF-C-shRNA was replaced with the vector pSUPERIOR-retro-neo + GFP, and all other steps were unchanged, and 3 clones were selected as blank controls (designated D1, D2 and D3, respectively).

(4) D1, D2, D3, D4, D5, D6, D7 or D8 were respectively inoculated into a medium containing 3mL 1640, and doxycycline (doxycycline, Dox) was added to obtain a treatment system such that the concentration of Dox in the treatment system was 3. mu.g/mL. 37 ℃ and 5% CO₂The cells were treated in an incubator for 36h, centrifuged, collected and total RNA extracted.

(5) And (5) taking the total RNA obtained in the step (4) as a template, and carrying out a first strand synthesis reaction on the cDNA to obtain the cDNA.

(6) And (3) detecting the relative expression quantity of VEGF-C (VEGF-C) by using the cDNA obtained in the step (5) as a template through fluorescent quantitative PCR (36B 4 gene is used as an internal reference gene). Primers for VEGF-C identification were 5'-AGGGTCAGGCAGCGAACAAGA-3' and 5'-CCTCCTGAGCCAGGCATCTG-3'. The primers for identifying the 36B4 gene are as follows: 5'-ATACAGCAGATCCGCATG-3' and: 5'-TCATGGTGTTCTTGCCCATCA-3' are provided.

According to the method, 3 mu g/mL in the step (4) is replaced by 6 mu g/mL, and other steps are not changed, so that the corresponding VEGF-C relative expression level is obtained.

The relative expression level of VEGF-C was obtained as a blank control by replacing 3. mu.g/mL in step (2) with 0. mu.g/mL (i.e., no doxycycline was added) in the same manner as above.

The relative expression of VEGF-C in the blank control was taken as 1, and the relative expression of VEGF-C after treatment with doxycycline at 3. mu.g/mL or 6. mu.g/mL is shown in FIG. 6. The results show that D6 and D7 are both clones sensitive to Dox, and VEGF-C-shRNA is expressed under the induction of Dox to cause the RNA level of VEGF-C to be reduced. None of D1, D2, and D3 had this effect.

Therefore, the invention successfully establishes a tetracycline-induced expression VEGF-C shRNA system and lays a solid foundation for discussing the function of endogenous VEGF-C in an animal model.

Third, VEGF-C role in EC lymph node metastasis

And establishing a tetracycline induced expression VEGF-C shRNA system by utilizing the second step, and determining whether the VEGF-C has the effect of increasing the newborn lymphatic vessels or enhancing the lymphatic invasion capacity of tumor cells on EC lymph node metastasis.

Stable cell line Ishikawa cell line C5-D6 and C5-D7 cell mixture (2X 10)⁶Cell number/100 μ l) suspension was injected subcutaneously into the left dorsal side of 6-week-old immunodeficient (SCID)2 mice. After 34 weeks, the tumor size was about 1cm³Left and right. Stripping the lump tissue and cutting into pieces of about 1mm³The tissue mass of (1). 90 SCID female mice 8 weeks old were intraperitoneally injected with Ketamine/Xylazine (injection dose 120 mg)&10mg/1kg body weight) and a long incision of about 1cm was made in the lower right dorsal aspect to expose the right uterine horn. Will be 1mm³The small tumor tissue is transplanted 1/3 below the uterine horn. The incision was made with a drop of Vet tissue glue, and the surgical field was cleaned with 10mM PBS buffer at pH7.4 and then blotted with a cotton swab. The incision was sutured with 4/0 thread, coated with antibiotic ointment,heating with a desk lamp, keeping warm, anesthetizing and keeping awake, and then putting the mice back into the cage for conventional breeding.

Based on the tumorigenic and metastatic rates in example 2, the inventors of the present invention used 90 immunodeficient (SCID) mice, 10 of which were not given DOX (for intermittent detection of tumor growth rate, two per week from the fifth week after injection), and 80 were randomly divided into two groups: DOX non-treated group (negative control group) and DOX-treated group (VEGF-C-inhibited group), 40 of which were included in each group. The DOX-treated mice were given 3mg/mL DOX drinking water daily.

Mice were given excess CO at 8-10 weeks post-operative termination of the experiment₂All uterus, oviducts and periaortic lymph nodes were dissected and fixed with formalin to prepare paraffin sections of tissues. If suspicious metastases of other organs are found, taking out, fixing and making paraffin sections. The uterus tumor tissue is firstly stored in 4 percent (volume percentage) of formaldehyde aqueous solution for 4 hours, then is immersed in 30 percent (mass percentage) of sucrose aqueous solution for overnight, and then is embedded by OCT for quick freezing storage. All paraffin sections were subjected to hematoxylin-eosin staining and immunohistochemical analysis using antibodies against human mitochondrial protein (ThermoFisher Scientific, Cat. MA 5-15880).

The primary tumor tumorigenesis rate and the lymph node metastasis rate in both groups were examined and the difference in primary tumor tumorigenesis rate and lymph node metastasis rate was determined for mice expressing different levels of VEGF-C using a variance test analysis. Detection of VEGF-C protein levels and enumeration of LVD/LVI in tumor tissue at the primary site the method of example 2 will be used. Discriminate method discriminant analysis will be used to determine the effect of VEGF-C expression on tumor metastasis tendency (JMP7 software). Low levels of VEGF-C were detected in primary tumor tissues in uterus in DOX-treated mice, confirming the reliability of the shRNA method as a research tool. In the DOX-treated group, a significant reduction in the number of mice with lymphatic metastases was found, and VEGF-C was seen to play a promoting role in the lymph node spread of tumor cells. The correlation between the results of LVD/LVI counting in tumor tissues of DOX treatment group and non-treatment group is judged by analysis of variance to judge the lymph node metastasis rate, thereby revealing that VEGF-C influences the metastatic capacity of primary tumor through LVD and/or LVI.

Sequence listing

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Claims

1. The application of the substance for inhibiting the activity and/or expression quantity of the vascular endothelial growth factor in preparing products; the function of the product is at least one of the following A1) to A5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) can be used for treating cancer.

2. The substance for inhibiting the activity and/or the expression amount of the vascular endothelial growth factor is at least one of the following substances A1) to A5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) can be used for treating cancer.

3. Use according to claim 1 or 2, characterized in that: the "substance inhibiting the activity and/or expression amount of the vascular endothelial growth factor" is z1) or z 2):

z1) specific RNA; the specific RNA is double-stranded RNA shown in a sequence 3 in a sequence table;

4, S1) or S2) or S3):

s1) application of the vascular endothelial growth factor as a drug target in preparing products; the function of the product is at least one of the following A1) to A5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) treating cancer;

s2) application of the vascular endothelial growth factor in preparing products; the function of the product is as follows C1) or C2) or C3): C1) promoting lymph node metastasis of tumor cells; C2) promoting lymphatic angiogenesis; C3) promoting tumor cells to invade lymphatic vessels;

s3) application of the vascular endothelial growth factor is C1) or C2) or C3) as follows: C1) promoting lymph node metastasis of tumor cells; C2) promoting lymphatic angiogenesis; C3) promoting tumor cells to invade lymphatic vessels.

5. A method for developing a lymph node metastasis medicament for inhibiting tumor cells takes a vascular endothelial growth factor as a medicament target, and a substance capable of inhibiting the medicament target is the lymph node metastasis medicament for inhibiting the tumor cells.

6. The method of claim 5, wherein: the drug target point is the activity and/or expression quantity of the vascular endothelial growth factor.

7. Product A or product B;

the product A contains vascular endothelial growth factor; the function of the product A is C1) or C2) or C3) as follows: C1) promoting lymph node metastasis of tumor cells; C2) promoting lymphatic angiogenesis; C3) promoting tumor cells to invade lymphatic vessels;

the product B contains substances for inhibiting the activity and/or expression of vascular endothelial growth factor; the function of the product B is at least one of the following A1) to A5): A1) inhibiting lymph node metastasis of tumor cells; A2) inhibiting lymphatic angiogenesis; A3) inhibiting tumor cells from invading lymphatic vessels; A4) preventing cancer; A5) can be used for treating cancer.

8. Use according to any one of claims 1 to 4, or a method according to claim 5 or 6, or a product A or a product B according to claim 7, characterized in that: the tumor cells are human endometrial cancer cells.

9. The use according to claim 8, or the method according to claim 8, or the product a or the product b according to claim 8, characterized in that: the human endometrial cancer cell is an Ishikawa cell or an HEC-1B cell.

10. The substance according to any one of claims 1 to 3, which inhibits the activity and/or expression of vascular endothelial growth factor.