CN115814089B

CN115814089B - Application of MT1G protein inhibitor as target in preparation of medicines for treating kidney cancer

Info

Publication number: CN115814089B
Application number: CN202211304971.6A
Authority: CN
Inventors: 王森; 王科鑫; 张斌
Original assignee: AFFILIATED HOSPITAL OF JINING MEDICAL UNIVERSITY
Current assignee: AFFILIATED HOSPITAL OF JINING MEDICAL UNIVERSITY
Priority date: 2022-10-24
Filing date: 2022-10-24
Publication date: 2024-05-28
Anticipated expiration: 2042-10-24
Also published as: CN115814089A

Abstract

The invention provides an application of an MT1G protein inhibitor in preparing a medicament for treating renal cancer, which belongs to the technical field of biological medicines, and results prove that after MT1G lentivirus overexpresses renal clear cell carcinoma 786-0 cells, proliferation and migration of the 786-0 cells are obviously increased relative to a control group, and sensitivity of sorafenib is obviously inhibited; after the MT1G gene lentivirus knockdown of 786-0 cells, 786-0 cell proliferation and migration were significantly inhibited and sorafenib drug sensitivity was significantly increased compared to control group cells. The invention provides and verifies MT1G protein as a gene therapy target of kidney cancer for the first time, has important significance for screening anti-kidney cancer drugs, and also provides a thinking for treating kidney cancer.

Description

Application of MT1G protein inhibitor as target in preparation of medicines for treating kidney cancer

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of an MT1G protein inhibitor serving as a new target spot in preparation of a medicine for treating renal cancer.

Background

Renal cancer is one of the most common malignant tumors of the urinary system, and in recent years there is a growing trend, with nearly half of patients with renal cancer having advanced or distant metastasis at the time of first visit, about 50% of patients having postoperative recurrence or metastasis. At present, the treatment of the kidney cancer is mainly performed by surgery, but the operation treatment can increase the pain of patients, and the radiotherapy and the chemotherapy have poor effects, so that the biological treatment gradually shows advantages, wherein the gene treatment has become a hot spot for the research of the kidney cancer in recent years along with the discovery of new kidney cancer related genes.

Gene therapy refers to a therapeutic method in which a gene that is not originally expressed by a specific target cell is expressed, or an abnormally expressed gene is turned off or inhibited in a specific manner, thereby achieving the objective of treating a disease. Since kidney gene expression is limited to the kidneys themselves, the synthesized protein products are mainly concentrated in the local parts of the kidneys, and thus the kidneys are ideal target organs for gene therapy.

Metallothionein (MTs) is a family of low molecular weight 6-7 kDa, cysteine-rich cytoplasmic proteins that play an important role in metal ion homeostasis and detoxification. Recently, many studies have shown that there are differences in MT expression in different tumors, suggesting that MTs may play an important role in the oncogenic process. Abnormal cell proliferation and apoptosis have been considered as major features of cancer, however MT1G does not perform consistently in different cancer types, e.g., in one study conducted in thyroid cancer, MT1G has been shown to inhibit proliferation, invasion or induce apoptosis, but promote differentiation and chemosensitivity of colorectal cancer. To date, the biological function of MT1G in renal clear cell carcinoma (RCC) has never been studied.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of MT1G protein inhibitor as a new target in preparing a medicament for treating renal cancer.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of an MT1G protein inhibitor in preparing a medicament for treating renal cancer.

Preferably, the MT1G protein inhibitor comprises one or more of a modulator that reduces MT1G expression, a protease that degrades MT1G product, a nuclease, and a modulator that reduces MT1G product.

Preferably, the agent that reduces expression of MT1G comprises an agent that knocks out or silences MT 1G.

Preferably, the modulator that reduces MT1G product comprises an MT1G antibody.

Preferably, the agent that knocks out or silences MT1G comprises siRNA, shRNA or miRNA.

Preferably, the functional sequence for knocking down in the shRNA plasmid is shown as SEQ ID NO. 1.

Preferably, the agent inhibits renal cancer cell migration and cell proliferation.

Preferably, the medicament comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is an MT1G protein inhibitor.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides an application of MT1G protein inhibitor in preparing a medicament for treating kidney cancer, and results prove that proliferation and migration of renal clear cell carcinoma 786-0 cells overexpressed by MT1G lentivirus are obviously increased relative to a control group, and sensitivity of sorafenib is obviously inhibited; compared with the cells of the control group, the MT1G gene lentivirus knockdown 786-0 cell proliferation and migration are obviously inhibited, and the sorafenib drug sensitivity is obviously increased. The invention provides and verifies MT1G protein as a new gene therapy target point of kidney cancer for the first time, has important significance for screening new medicines for resisting kidney cancer, and also provides a new thought for treating kidney cancer.

Drawings

FIG. 1 is a bright field and fluorescent image of the infection efficiency of MT1G gene RNA interference lentiviral particles on cells;

FIG. 2 shows that MT1G gene lentivirus knockdown 786-0 cells and control lentivirus 786-0 cells interfere with MT1G expression efficiency;

FIG. 3 is an immunoblot electrophoretogram of MT1G expression in MT1G gene lentivirus knockdown 786-0 cell groups and control lentivirus 786-0 cells;

FIG. 4 is a graph showing the effect of MT1G on renal clear cell carcinoma cell migration;

FIG. 5 is a graph showing the effect of MT 1G-overexpressing lentiviral infection 786-0 cells in the group and control 786-0 cells on proliferation of clear cell carcinoma cells;

FIG. 6 shows the effect of MT1G gene lentiviral knockdown 786-0 cells in the control 786-0 cells on proliferation of clear cell carcinoma cells;

FIG. 7 is the effect of MT 1G-overexpressing lentiviral infection 786-0 cells in the group and control 786-0 cells on sorafenib sensitivity;

FIG. 8 is a graph showing the effect of MT1G gene lentiviral knockdown 786-0 cells in the group and control 786-0 cells on sorafenib sensitivity.

Detailed Description

The invention researches renal clear cell carcinoma and discovers a new target MT1G protein for treating renal carcinoma, so the invention provides an application of an MT1G protein inhibitor in preparing a medicament for treating renal carcinoma.

The MT1G protein of the invention may be a human MT1G, more specifically the nucleotide sequence of MT1G may be or include the NCBI reference sequence: sequence of nm_005950. The amino acid sequence of MT1G may be or include, but is not limited to, the sequence referenced to NCBI: the nm_005950 sequence has an amino acid sequence with at least 80%, 85%, 90% or 95% identity, and an amino acid sequence with the properties or functions of MT 1G.

In the present invention, the kidney cancer cell is preferably a renal clear cell carcinoma. In the present invention, the MT1G protein inhibitor preferably includes one or more of a modulator that reduces MT1G expression, a protease that degrades MT1G product, a nuclease, and a modulator that reduces MT1G product, i.e., is capable of inhibiting MT1G activity or expression.

In the present invention, the agent that reduces MT1G expression preferably comprises an agent that knocks out or silences MT 1G. Further preferably, the agent for knocking out or silencing MT1G comprises siRNA, shRNA or miRNA, in the present invention, the siRNA refers to short double-stranded RNA, which is capable of inducing RNA interference by cleaving some mRNAs, the siRNA comprises a sense RNA strand having a sequence homologous to mRNAs of a target gene and an antisense RNA strand having a sequence complementary thereto, the siRNA can inhibit the expression of the target gene, and can be used for gene knockdown and gene therapy. In the present invention, the shRNA (short hairpin RNA) is a single-stranded RNA that includes a stem portion and a loop portion that form a double-stranded portion by hydrogen bonding, is converted into siRNA by protein processing such as Dicer, and performs the same function as siRNA. In the present invention, miRNA refers to 21 to 23 non-coding RNAs which regulate gene expression after transcription by promoting degradation of target RNA or by inhibiting translation thereof. In the present invention, the functional sequence for knockdown in shRNA is preferably GCTCCCAAGTACAAATAGAGT (SEQ ID NO: 1).

In the present invention, the modulator for reducing MT1G product preferably comprises an MT1G antibody.

In the present invention, an MT1G protein inhibitor is an inhibitor that reduces MT1G expression in cancer cells compared to cancer cells that have not been treated with an MT1G protein inhibitor. Reduced MT1G expression refers to reduced MT1G gene expression or reduced MT1G protein levels.

In the present invention, the drug inhibits migration and proliferation of renal cancer cells. The medicament preferably comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is an MT1G protein inhibitor. The carrier may include one or more of lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, ethylene glycol, water, saline and aqueous dextrose. The medicament also includes an antioxidant such as ascorbic acid, sodium sulfite or sodium bisulfate. The medicament further comprises excipients such as corn starch, wheat starch, methylcellulose, sodium carboxymethylcellulose or gelatin. The route of administration of the medicament of the present invention includes oral, intravenous, parenteral, intramuscular, subcutaneous, intraperitoneal, intranasal, rectal or topical administration. In the present invention, the dosage of the drug of the present invention may be determined by the type of the treatment disease, the severity of the disease, the administration route, the age, sex, health condition of the patient, etc., and for example, the dosage of the drug of the present invention may be 0.01 mug to 1000 mg/day per patient.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1.1 Establishment of MT1G Gene lentiviral knockdown 786-0 cell line:

The first day:

Preparing cells: cells were cultured until logarithmic growth phase, cells were counted in control and objective groups after pancreatin, and cells were cultured in 10cm cell culture dishes by resuspension of the same number of cells in cell culture broth. The inoculated amount of 786-0 cells can grow to 80% -90% fusion degree on the 3 rd day after infection.

The following day:

(1) Lentiviral particles, constructed MT1G gene RNA interference lentiviral vector particles (LV-MT 1G-RNAi), control lentiviral vector particles, were prepared, the amount of lentiviral particles required was calculated, lentiviral particles frozen at-80℃were removed, and ice-bath thawed, wherein the functional sequence MT1G-RNAi for knocking down MT1G was GCTCCCAAGTACAAATAGAGT (SEQ ID No. 1), and the control insert was TTCTCCGAACGTGTCACGT (SEQ ID No. 2).

(2) Infecting the target cells: taking out cells from the incubator, and observing the growth state and the cell fusion degree of the cells under a microscope; if the cell state is good, the experiment is started:

A. carefully sucking the old culture solution of the 10cm cell culture dish by a pipetting gun, and adding the new complete culture solution;

B. respectively adding calculated slow virus particle liquid into cells, flatly placing a culture plate on a workbench, and gently and uniformly mixing in an 8-shaped manner;

C. After mixing, the cell culture plates were placed in a 5% CO ₂ incubator at 37℃overnight.

Third day:

changing the culture solution: after 12 to 16 hours of infection, the culture medium containing lentiviral particles was aspirated, and the culture medium was again added with complete culture medium containing 10% serum 1640 to the culture plate, and the culture was continued.

Fourth day:

the cells were continuously cultured, and whether or not the cell state was abnormal was observed.

Fifth day:

Lentiviral particle infection efficiency was observed (assessed): the 10cm cell culture dish was covered, the outer wall of the culture plate was cleaned with 70% ethanol, fluorescence was observed in an inverted fluorescence microscope, photographed, and the infection efficiency of lentiviral particles on cells was estimated.

Sixth day: the number of fluorescent cells was found to be 50% or more, and positive cells were selected using puromycin (see FIG. 1 for results), resulting in MT1G gene lentivirus knockdown 786-0 cells, shMT G.

The results of FIG. 1 show that the fluorescent cells were over 50% in number, i.e., MT1G gene lentivirus successfully infected 786-0 cells, by observation of fluorescence with an inverted fluorescent microscope.

1.2 Determination of interference efficiency of MT1G Gene lentivirus knockdown 786-0 cells

1) RNA extraction and preparation of cDNA:

RNA was extracted using RNAfast200,200 total RNA extraction kit at high speed and RNA concentration was determined using NANODROP2000,2000.

Preparation of cDNA:

a removal of genomic DNA reaction:

The PCR reaction solution was prepared on ice according to Table 1, and the PCR was performed at 42℃for 2 minutes.

TABLE 1 PCR reaction System for removing genomic DNA

B reverse transcription reaction:

after the first step of reaction is finished, sequentially adding the components in table 2 into a PCR reaction tube, and setting a program of a PCR instrument at 37 ℃ for 15min;85 ℃,7s; preserving at 4 ℃. The reverse transcription reaction product was diluted 20-fold and placed in a-20℃refrigerator.

TABLE 2 reverse transcription PCR reaction System

2) RT-PCR detection

Quantitative primers were designed by primer 3.0 on-line software and analyzed for primer specificity using the primer blast of NCBI website, company synthesis (see Table 3 for sequence information). The extracted RNA was reverse transcribed into cDNA, and the reverse transcribed cDNA was diluted 10-fold as template, with three replicates per set of samples. Using SYBR PremixEx TaqTM kit, the amplification procedure was pre-deformed for 2min at 95 ℃; denaturation at 94℃for 30s, annealing at 60℃for 10s, extension at 72℃for 10s for 40 cycles. By usingThe results of quantitative PCR were analyzed by the method.

TABLE 3 RT-PCR primer sequences

As can be seen from FIG. 2, the MT1G gene expression level of shMT G was significantly reduced compared with shNC, and a MT1G gene lentiviral knockdown 786-0 cell line was successfully established.

1.3 Immunoblotting detection

The prepared protein samples were mixed with 5×loading buffer in a 10% SDS-PAGE gel, and water-bath at 100deg.C for 10min. Pre-stained protein marker, 5. Mu.L/lane, a suitable volume of protein sample was taken and added to the lane. Electrophoresis was stopped using a constant pressure of 80V for 30min, 120V until bromophenol blue reached the bottom of the gel. Taking out the gel, marking and transferring the gel, soaking the gel in a transfer buffer solution for balancing for 10min, shearing a PVDF membrane and filter paper according to the size of the gel, placing the PVDF membrane in the transfer buffer solution for balancing for 10min, and soaking the PVDF membrane in methanol for 5 seconds before using. The gel was placed on the negative side, the transfer "sandwiches" were assembled in sequence, one layer at a time, and the bubbles were gently removed with a glass rod. The transfer tank is placed in ice, a sandwich is placed according to the electrode, a transfer buffer solution is added, a constant current of 250V is added, and the transfer time is set according to the molecular weight of the target protein. After the transfer, the transferred PVDF membrane is placed in TBST buffer solution for standby. The membrane was washed with TBST buffer for 5min and slowly shaken. The membranes were placed in TBST with 5% skim milk and blocked overnight at 4 ℃. The membrane was washed 3 times with TBST for 5 min/time, placed in a hybridization bag, added with an appropriate amount of TBST diluted primary antibody, incubated with slow shaking at 37℃for 2h, or overnight at 4 ℃. TBST washed the membrane 3 times, 5 min/time. The membrane was placed in a hybridization bag, a suitable amount of TBST diluted secondary antibody was added, and incubated at 37℃for 1h. TBST washed the membrane 3 times, 15 min/time. Preparing developer according to the instruction of the luminous kit, entering a darkroom, placing a film in a hybridization bag, uniformly coating a proper amount of developer, closing a bag opening, removing bubbles in the bag, fixing the hybridization bag in a cassette, tightly attaching a film to the hybridization bag without leaving bubbles, closing the cassette, fumbling proper exposure time, scanning the film and analyzing images. The antibody dilution ratio is referred to the antibody instruction.

TABLE 4 immunoblotting and IF antibodies

As can be seen from FIG. 3, the MT1G protein expression level of shMT G was significantly reduced compared with shNC, and a MT1G gene lentivirus knockdown 786-0 cell line was successfully established.

1.4 Effect of MT1G on renal clear cell carcinoma cell migration

Transwell migration experiments: MT1G gene lentivirus knockdown 786-0 cells (shMT G) and control 786-0 cells (shNC) thereof, MT 1G-overexpressing lentivirus infection 786-0 cells (OE-MT 1G) and control 786-0 cells (OE-NC) thereof, stable cell lines were selected respectively, the cells were digested and collected, washed three times with PBS, resuspended in serum-free medium, counted under a microscope to prepare a single cell suspension with a final concentration of 1X 10 ⁵ cells/mL, 100. Mu.L of the single cell suspension was taken, added into the upper chamber of an invasive cell, and 600. Mu.L of PRMI-1640 medium containing 10% FBS was added into the lower chamber of the invasive cell. Each group is provided with 3 parallel cells. Cell culture: cells were cultured at 37℃under 5% CO ₂ for 48h. Taking out the cell, wiping off the cell on the upper side of the cell membrane by using a cotton swab, fixing the cell by using 4% paraformaldehyde for 30min, and then dyeing by using 5% crystal violet solution. And selecting more than 3 visual fields of 100 times of light, counting the number of the cells penetrating the membrane, and carrying out statistical analysis.

As can be seen from the results of fig. 4, the overexpression of MT1G significantly promoted the migration of renal clear cell carcinoma cells, and the knock-down of MT1G expression significantly inhibited the migration of renal clear cell carcinoma cells.

1.5 Effect of MT1G on proliferation of clear cell carcinoma cells of the kidney

Single cell suspensions (MT 1G gene lentiviral knockdown 786-0 cells (shMT G) and control 786-0 cells thereof (shNC), MT 1G-overexpressing lentiviral infection 786-0 cells (OE-MT 1G) and control 786-0 cells thereof (OE-NC)) were prepared separately, 5000 cells/well were inoculated with 96-well plates in each group, three replicates were set for each assay, 10. Mu. LCCK8 reagent was added per well for different time periods, incubated at 37℃for 1h in the absence of light, absorbance values were detected at 490nm wavelength using an enzyme-labeled instrument, and proliferation curves were made using GraphPadprism5 to detect cell proliferation for OE-NC and OE-MT1G groups at 0h, 24h, 48h, 72h, 96h, 110h, respectively (see FIG. 5). Cell proliferation was examined in groups shNC and shMT G at 0h, 24h, 48h, 72h, 96h, respectively (see figure 6). 6. Mu.M sorafenib was added to the OE-NC and OE-MT1G groups, respectively, and cell proliferation was examined at 0h, 24h, and 48h for the OE-NC and OE-MT1G groups (see FIG. 7). Each of the groups shNC and shMT G was added with 0. Mu.M, 6. Mu.M, and 12. Mu.M sorafenib, and the proliferation of the cells was examined after culturing for 24 hours (see FIG. 8).

From the results of fig. 5 and 6, it was shown that overexpression of MT1G promoted proliferation of renal clear cell carcinoma cells, while knockdown of MT1G expression significantly inhibited proliferation of renal clear cell carcinoma cells.

From the results of fig. 7 and 8, it was shown that sensitivity of sorafenib was significantly inhibited after MT1G was overexpressed. Whereas, upon knockdown of MT1G expression, sorafenib drug sensitivity was significantly increased.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

The application of the MT1G protein inhibitor in preparing medicaments for treating kidney cancer is characterized in that the MT1G protein inhibitor is shRNA shown in SEQ ID NO. 1.
2. The use according to claim 1, wherein the medicament inhibits renal cancer cell migration and cell proliferation.
3. The use according to claim 1, wherein the medicament comprises an active ingredient which is an MT1G protein inhibitor, and a pharmaceutically acceptable carrier.