CN110859967A

CN110859967A - Application of human CDC37L1 gene and protein coded by same in preparation of medicine for treating gastric cancer

Info

Publication number: CN110859967A
Application number: CN201911094267.0A
Authority: CN
Inventors: 李砚东; 高勇; 栗丽; 王光学; 李钦传
Original assignee: Shanghai East Hospital Tongji University Affiliated East Hospital
Current assignee: Shanghai East Hospital Tongji University Affiliated East Hospital
Priority date: 2019-11-11
Filing date: 2019-11-11
Publication date: 2020-03-06

Abstract

The invention provides an application of a human CDC37L1 gene and a protein coded by the same in preparing a medicine for treating gastric cancer, belongs to the technical field of tumor treatment, and relates to an application of a human CDC37L1 gene in preparing a medicine for treating gastric cancer. The invention obtains the effect of CDC37L1 in the generation and development of gastric cancer through in vivo and in vitro experiments, shows that the CDC37L1 has the function of inhibiting the proliferation and migration of gastric cancer cells, simultaneously researches the molecular mechanism of the CDC37L1 playing a role in inhibiting cancer by using Western Blot experiments, and discovers that the CDC37L1 inhibits the development and progress of the gastric cancer by inhibiting the expression of CDK 6. The invention provides a new target point for treating gastric cancer so as to improve the survival time of patients. Therefore, the research has stronger theoretical and practical significance.

Description

Application of human CDC37L1 gene and protein coded by same in preparation of medicine for treating gastric cancer

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to an application of a human CDC37L1 gene and a protein coded by the same in preparation of a medicine for treating gastric cancer.

Background

Malignant tumor (cancer) has become a main disease threatening human life, and the cancer morbidity and mortality are always in an ascending state in recent years, wherein gastric cancer is one of common malignant tumors, the second place among the cancer mortality is in the world, the morbidity of the cancer has obvious regional difference, and the morbidity of the cancer is more serious in east Asia, central Europe, east Europe, south America and the like. According to the cancer report of 2019 in China, the incidence rate of gastric cancer in Chinese malignant tumors is the second place, the mortality rate is the third place, and the incidence rate accounts for half of the incidence rate and the mortality rate of gastric cancer in the world, so that the prevention and control situation of the gastric cancer in China is very severe. The research on the gastric cancer is greatly advanced and broken through nowadays, the treatment scheme of the gastric cancer is also improved to a great extent, including surgical treatment, chemotherapy, targeted treatment, immunotherapy and the like, and the survival rate of the gastric cancer patients in the later period is only 5% -15% in five years. Therefore, the method continuously researches and explores the pathogenesis of the gastric cancer, develops a new treatment target point and has important significance for prolonging the life cycle of the gastric cancer patient.

The cell division cyclin CDC37L1 is an analogue of the helper chaperone CDC37, has 337 amino acids in total length, and has 31 percent of homology with the CDC37 protein. The molecular chaperone protein is widely present in prokaryotic and eukaryotic cells, regulates the physiological function of the cells under stress, and plays an important role in protein folding and positioning, maintenance of protein conformation and activity and the like. Studies have shown that both CDC37L1 and CDC37 interact with heat shock protein HSP90, facilitating binding of client proteins (client proteins) to HSP90 and assisting in the correct folding of these client proteins. HSP90 is the most important chaperone protein in cells, and accounts for about 1-2% of the total protein content of cells, and its client protein is the key protein component of intracellular signal transduction pathways, including various kinases, and is closely related to the generation and development of tumors. No report was found on the function of CDC37L1 in gastric cancer by search.

Disclosure of Invention

In view of the above, the present invention aims to provide the use of the human CDC37L1 gene and its encoded protein in the preparation of a medicament for the treatment of gastric cancer.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an application of a human CDC37L1 gene in preparing a medicine for treating gastric cancer.

The invention also provides application of the human CDC37L1 gene in preparing a medicament for inhibiting gastric cancer cell proliferation.

The invention also provides application of the human CDC37L1 gene in preparing a medicament for inhibiting gastric cancer cell migration.

The invention also provides application of the human CDC37L1 gene in preparing a medicine for weakening gastric cancer cell tumor formation.

The invention also provides application of the human CDC37L1 gene in preparing a medicament for inhibiting cyclin-dependent kinase 6.

The invention also provides application of the protein coded by the human CDC37L1 gene in the technical scheme in preparation of drugs for treating gastric cancer.

The invention also provides application of the protein coded by the human CDC37L1 gene in preparation of a medicine for inhibiting gastric cancer cell migration.

The invention also provides application of the protein coded by the human CDC37L1 gene in preparation of a medicine for inhibiting gastric cancer cell proliferation.

The invention also provides application of the protein coded by the human CDC37L1 gene in the technical scheme in preparation of a medicine for weakening gastric cancer cell tumor formation.

The invention also provides application of the protein coded by the human CDC37L1 gene in the technical scheme in preparation of drugs for inhibiting cyclin-dependent kinase 6.

The invention provides an application of a human CDC37L1 gene and a protein coded by the same in preparing a medicament for treating gastric cancer. In the invention, the human CDC37L1 gene can inhibit the migration and proliferation of gastric cancer cells and inhibit the tumor formation capability of the gastric cancer cells by inhibiting cyclin-dependent kinase 6, thereby achieving the purpose of treating gastric cancer.

Drawings

FIG. 1 shows the cDNA sequence of CDC37L1 gene and the amino acid sequence of the encoded protein, wherein A in FIG. 1 shows the cDNA sequence of CDC37L1 gene, and B in FIG. 1 shows the amino acid sequence of the encoded protein of CDC37L1 gene;

FIG. 2 shows the effects of overexpression plasmid and interfering small RNA of CDC37L and the effect of changing expression of CDC37L1 on gastric cancer cell proliferation, wherein A shows successful overexpression of gastric cancer cells after transfection of exogenous CDC37L1+3Flag plasmid, and Western Blot shows that expression of CDC37L1 by siRNA fragments is successfully down-regulated; b shows that the EdU experiment indicates that CDC37L1 has influence on the proliferation capacity of the gastric cancer MGC-803 cells;

FIG. 3 shows the effect of overexpression or silent expression of CDC37L1 on the growth of gastric cancer cells, and A shows that the plate clone formation experiment shows that CDC37L1 has an effect on the growth of gastric cancer cell strains MGC-803, BGC-823 and AGS cells; b shows that CCK8 experiments show that CDC37L1 has influence on the growth capacity of MGC-803, BGC-823 and SGC-7901 cells;

FIG. 4 shows that expression of CDC37L1 affects gastric cancer cell migration, and A shows the effect of down-regulated CDC37L1 on MGC-803, BGC-823 cell migration; b shows the effect of overexpressed CDC37L1 on BGC-823 and AGS cell migration;

FIG. 5 shows that the expression of CDC37L1 affects the ability of gastric cancer cells to form tumors subcutaneously in nude mice, and that the over-expression of CDC37L1 weakens the ability of gastric cancer MGC-803 and BGC-823 cells to form tumors subcutaneously in nude mice;

fig. 6 shows the effect of expression of CDC37L1 on cell cycle-related protein expression, showing that Western Blot experiment indicates that down-regulation of CDC37L1 in gastric cancer cells affects protein expression of CDK 6.

Detailed Description

The invention provides an application of a human CDC37L1 gene in preparing a medicine for treating gastric cancer. The dosage form of the medicament is not particularly limited, and a pharmaceutically acceptable dosage form can be adopted. The content of the human CDC37L1 gene in the medicine, the adopted auxiliary materials and the auxiliary material content are not specially limited, the auxiliary material types and the auxiliary material dosage used for preparing various dosage forms conventionally are adopted, and the content of active substances contained in various dosage forms conventionally is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

In the present invention, the cDNA sequence of the human CDC37L1 gene has accession number CCDS 6454.1.

The invention also provides application of the human CDC37L1 gene in preparing a medicament for inhibiting gastric cancer cell proliferation. The content of the human CDC37L1 gene in the medicine, the adopted auxiliary materials and the auxiliary material content are not specially limited, the auxiliary material types and the auxiliary material dosage used for preparing various dosage forms conventionally are adopted, and the content of active substances contained in various dosage forms conventionally is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention also provides application of the human CDC37L1 gene in preparing a medicament for inhibiting gastric cancer cell migration. The content of the human CDC37L1 gene in the medicine, the adopted auxiliary materials and the auxiliary material content are not specially limited, the auxiliary material types and the auxiliary material dosage used for preparing various dosage forms conventionally are adopted, and the content of active substances contained in various dosage forms conventionally is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention also provides application of the human CDC37L1 gene in preparing a medicine for weakening gastric cancer cell tumor formation. The content of the human CDC37L1 gene in the medicine, the adopted auxiliary materials and the auxiliary material content are not specially limited, the auxiliary material types and the auxiliary material dosage used for preparing various dosage forms conventionally are adopted, and the content of active substances contained in various dosage forms conventionally is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

In the present invention, the gastric cancer cells preferably include MGC-803, BGC-823, SGC-7901 and AGS gastric cancer cells. In the present invention, the source of the gastric cancer cells is not particularly limited, and a conventional source may be used.

Application of human CDC37L1 gene in preparing medicine for inhibiting cyclin dependent kinase 6. The content of the human CDC37L1 gene in the medicine, the adopted auxiliary materials and the auxiliary material content are not specially limited, the auxiliary material types and the auxiliary material dosage used for preparing various dosage forms conventionally are adopted, and the content of active substances contained in various dosage forms conventionally is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention also provides the application of the protein coded by the human CDC37L1 gene in preparing a medicament for treating gastric cancer. The content of the protein in the medicament, the adopted auxiliary materials and the auxiliary material content are not specially limited, the types and the use amounts of the auxiliary materials used for preparing various dosage forms by a conventional method can be adopted, and the content of active substances contained in various dosage forms by the conventional method can be adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

In the present invention, the amino acid sequence of the protein has accession number NP-060383.2.

The invention also provides application of the protein coded by the human CDC37L1 gene in preparation of a medicine for inhibiting gastric cancer cell migration. The content of the protein in the medicament, the adopted auxiliary materials and the auxiliary material content are not specially limited, the types and the use amounts of the auxiliary materials used for preparing various dosage forms by a conventional method can be adopted, and the content of active substances contained in various dosage forms by the conventional method can be adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention also provides application of the protein coded by the human CDC37L1 gene in preparation of a medicine for inhibiting gastric cancer cell proliferation. The content of the protein in the medicament, the adopted auxiliary materials and the auxiliary material content are not specially limited, the types and the use amounts of the auxiliary materials used for preparing various dosage forms by a conventional method can be adopted, and the content of active substances contained in various dosage forms by the conventional method can be adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention also provides application of the protein coded by the human CDC37L1 gene in the technical scheme in preparation of a medicine for weakening gastric cancer cell tumor formation. The content of the protein in the medicament, the adopted auxiliary materials and the auxiliary material content are not specially limited, the types and the use amounts of the auxiliary materials used for preparing various dosage forms by a conventional method can be adopted, and the content of active substances contained in various dosage forms by the conventional method can be adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention also provides application of the protein coded by the human CDC37L1 gene in the technical scheme in preparation of drugs for inhibiting cyclin-dependent kinase 6. The content of the protein in the medicament, the adopted auxiliary materials and the auxiliary material content are not specially limited, the types and the use amounts of the auxiliary materials used for preparing various dosage forms by a conventional method can be adopted, and the content of active substances contained in various dosage forms by the conventional method can be adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The test operations used in examples 1-5 of the present invention were as follows:

(1) culture of gastric cancer cell lines

MGC-803, BGC-823, SGC-7901 and AGS gastric cancer cells are from Shanghai cell Bank, Chinese academy of sciences. DMEM medium containing 10% fetal calf serum is adopted and placed at 37 ℃ with 5% CO₂Culturing gastric cancer cells in an incubator with saturated humidity. 0.25% trypsin (with EDTA) was used to digest the cells.

(2) Reagent material

1) Plasmid: the CDC37L1 fusion 3Flag expression plasmid was designed by Shanghai Jima Gene Co.

2) siRNA the sequence related to siRNA fragment of the human interference CDC37L1 gene (SEQ ID No.1) is designed and completed by Shanghai Jima company: siCDC37L1: 5'-agcagaggaagaagguuau-3'

3) Overexpression of lentivirus: LV6-CDC37L1 and LV6NC were synthesized by Shanghai Jima Gene Co

4) CDC37L1(16293-1-AP, Proteintech, China), β -actin (# 81178; Santa Cruz Biotechnology, Dallas, TX, USA), CDK6(14052-1-AP, Proteintech, China), CDK4(#12790, Cell Signaling Technology, USA), Cyclin E1(#20808, Cell Signaling Technology, USA), Cyclin D1(#2922, Cell Signaling Technology, USA).

(3) Transfection of gastric cancer cells

1) Cells were transfected when seeded to 70-90% confluence.

2) Dilution with Opti-MEM Medium

3000 reagent (2 tubes), mix well.

3) Dilute siNC or siCDC37L1 using Opti-MEM media and dilute each tube

3000 reagents were added diluted siNC or siCDC37L1(1:1 ratio).

4) Incubate at room temperature for 5 minutes and add dropwise to the cells.

5) Cells were incubated at 37 ℃ for 2-4 days and then transfected cells were analyzed.

(4) Construction of lentivirus packaging and CDC37L1 gastric cancer cell stable strain with down-regulated effect

1) 293T cells were cultured in DMEM medium containing 10% FBS, 100IU/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ with 5% CO₂And conventionally culturing in a saturated humidity incubator.

2) When the strain grows to about 80% density, adding the empty plasmid LV3-PGK-Puro, the recombinant plasmid LV3-hsa-shCDC37L1-PGK-Pure, the packaging plasmid psPA x 2 and pMD2.G into a centrifuge tube filled with 1.5mL of serum-free culture medium in proportion, and mixing uniformly. The transfection reagent

3000 to 1.5mL of serum-free medium, mixing the two tubes of reagents, standing at room temperature for 5min, adding to the cell culture solution, and culturing in a cell culture box for 4-6 h.

3) Removing the transfection solution, adding serum-containing DMEM medium, and continuing to culture for 48 h. Sucking cell supernatant into a centrifuge tube, centrifuging at low speed, filtering at 0.45 μm, centrifuging at 4 deg.C and 20000r/min at ultra speed for 2 hr, and collecting concentrated solution.

4) Construction of gastric cancer cell strain with CDC37L1 stably regulated down

The virus solution was added to infect the cells and Polybrene was added to a final concentration of 4. mu.g/ml. After 24h the virus-containing medium was aspirated, complete medium was added, and after 48h incubation in an incubator, the stably transduced cell lines were selected by changing the medium to Puromycin at a final concentration of 1. mu.g/ml (two to three days with one change).

(5) Determination of cell growth curves

1) Different kinds of gastric cancer cells are grown according to 3-5 × 10³The total amount of cells was calculated per 100. mu.l/well, and after digesting the cells sufficiently, the cells were diluted to the desired concentration and seeded in a 96-well plate. Three wells per group per day, cells were inoculated on a 5-7 day basis.

2) Color reaction was carried out with CCK-8 developer (Cell Counting Kit-8, DOJINDO, Japan) by adding 10. mu.l of CCK-8 to 100. mu.l of the culture medium at 37 ℃ with 5% CO₂And (5) placing the incubator for incubation for 1h 15min, measuring the absorbance at 450nm by using an enzyme-labeling instrument, and recording the actual initial density of the cells.

3) The cell morphology was observed under a microscope, measured at regular time intervals, and the cell growth was recorded.

4) Typically 5 to 7 days. And after the measurement is finished, collecting data, processing and drawing a chart.

(6) Cell clone formation assay

1) Stable stomach cancer stable strains BGC-823 and AGS which stably over-express CDC37L1 and are infected with LV-NC or LV-CDC37L1 lentivirus and MGC-803 cells which are infected with LV-shNC and LV-shCDC37L1 and stably down-regulate CDC37L1 are inoculated into a six-well plate according to a certain number.

2) Culturing for 2-3 weeks, and replacing fresh culture solution every 3-5 days until macroscopic cell clone is formed.

3) The medium in the dish was aspirated, the 6-well plate was placed on ice and rinsed 2 times with pre-cooled PBS (4 ℃). Fixation was performed with pre-cooled 100% methanol (4 ℃) for 10 min.

4) The cells were removed from the ice, equilibrated at room temperature, and stained by overlaying the cells with crystal violet staining solution. Incubate at room temperature for 10min, discard crystal violet stain, wash cells with distilled water several times until the stain no longer flows out.

5) Colony formation staining results were photographed and cell clones on each dish were counted according to the same criteria (cell clone size).

(7) EdU cell proliferation assay

1) The cells were cultured in a cell culture medium at 1000: 1, preparing a proper amount of 50 mu M EdU culture medium;

2) adding 100 mu L of 50 mu M EdU culture medium into each hole, incubating for 2 hours, and removing the culture medium;

3) washing the cells with PBS for 5 minutes 1-2 times;

4) adding 50 μ L of cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating at room temperature for 30min, and discarding the fixing solution;

5) adding 50 mu L of 2mg/mL glycine into each well, incubating for 5 minutes in a decolorization shaker, and discarding the glycine solution

6) Adding 100 mu L PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS;

7) add 100. mu.L of 1X per well

Incubating the dyeing reaction liquid for 30 minutes in a dark place at room temperature by using a decoloring shaker, and then discarding the dyeing reaction liquid;

8) adding 100 μ L of penetrating agent (0.5% TritonX-100 PBS) and decolorizing and washing with shaker for 2-3 times, each time for 10min, and discarding the penetrating agent;

9) preparing a proper amount of 1X Hoechst33342 reaction solution, and storing in dark place;

10) adding 100 mu L of 1X Hoechst33342 reaction solution into each hole, incubating for 30 minutes in a light-proof, room temperature and decolorizing shaker, and then discarding the dyeing reaction solution;

11) adding 100 mu L PBS to each hole for washing for 1-3 times;

12) immediately after the completion of the staining, the staining was observed with a fluorescence microscope, and the exposure time was adjusted to about 30 ms.

(8) Transwell cell in vitro migration experiment

1) Gastric cancer cells stably up-regulated and down-regulated by CDC37L1 were completely digested with trypsin, added 2mL of DMEM medium, and transferred to 1.5mLEP tubes.

2) Centrifuge at 1000rpm for 3min, and aspirate off DMEM medium.

3) 1mL of serum-free MEM medium was added to each tube, and the cells were pipetted to uniform density.

4) The cell concentration of each tube was calculated using a cell counting plate.

5) 3.5X 10 addition to the upper side of the Transwell cell⁴Cells were plated and supplemented with serum-free MEM to a total volume of 400. mu.l.

6) Each well of the 24-well plate was filled with 800. mu.l of DMEM medium.

7) The cells were placed in the wells and 24-well plates were placed at 37 ℃ with 5% CO₂And culturing in a constant temperature incubator for 24 hours.

8) The chamber was removed, the medium was discarded, and fixed with paraformaldehyde for 15 min.

9) The cell was stained in 0.05% crystal violet for 20 min.

10) The cells on the inside of the chamber were carefully scraped off with a cotton swab.

11) The cells were observed under a microscope and counted.

(9) Nude mouse tumorigenesis experiment

1) The mice used were male BLAB/c nude mice of 4 week age, purchased from Shanghai Spiker laboratory animals, Inc.

2) Cells of stable strains MGC-803 and BGC-823 over-expressing gastric cancer by using LV-NC or LV-CDC37L1 at a ratio of 1 × 10⁶The same number was injected subcutaneously into the abdomen of nude mice.

3) The growth of the tumor was observed, the mice were sacrificed about 3-4 weeks until the maximum tumor diameter reached 15mm, and the tumor was photographed and weighed.

(10) Protein extraction

1) Cells that had been transfected for 48h were aspirated off the medium with a vacuum pump.

2) After washing twice with PBS, 70. mu.L of cell lysate and 100 XPI mix were added to each well of the six-well plate.

3) After 5min the cells were scraped off with a cell scraper and transferred to a 1.5ml EP tube for lysis on ice for 20 min.

4) Centrifuge at 12000rpm at 4 ℃ for 15 min.

5) The supernatant was collected and stored at-20 ℃.

(11) Protein quantification

1) BSA standard solution was diluted with PBS according to the reagent instructions.

2) Before measurement, the mixture was mixed at a ratio of BCA Reagent to BCA Reagent B of 100:1 to prepare a working solution.

3) Preparation of BSA standard curve.

4) 100. mu.L of diluted BSA standard solution was added to a 96-well microplate, and 2 replicates were taken for each concentration.

5) After adding 100. mu.L of the working solution to each well, mix well immediately.

6) After reacting in a 37 ℃ water bath for 30min, cooling to room temperature.

7) The absorbance value at 562nm was determined using a spectrophotometer. All samples were tested as soon as possible within 20 min.

8) The absorbance values of the BSA standard solutions at each concentration were subtracted by the average of the Blank values, and a standard curve of the BSA standard solution was plotted.

9) 100 mul of sample diluent to be detected is respectively added into a 96-hole microporous plate, and 2 parallel samples are taken from each sample.

10) After adding 100. mu.L of the working solution to each well, mix well immediately.

11) After reacting in a 37 ℃ water bath for 30min, cooling to room temperature.

12) The absorbance value at 562nm was determined using a spectrophotometer. For the measurement, a 1mL cuvette was used and the water was used for zero calibration. All samples were tested as soon as possible within 20 min.

13) The absorbance values of the BSA standard solutions at each concentration were subtracted by the average of the Blank values, and a standard curve of the BSA standard solution was plotted.

(11) Western Blot experiment

1) After protein quantification. The remaining supernatant was mixed with 1/4 volumes of 5 XSDS-PAGE protein loading buffer, cooked in a metal bath at 100 ℃ for 10min to denature the proteins and loaded.

2) Electrophoresis, gel concentration voltage 80V and gel separation voltage 150V until bromophenol blue reaches the bottom of the gel.

3) And (3) finishing electrophoresis, soaking the gel in a membrane transferring buffer solution, sequentially paving a sponge, filter paper, gel, an NC membrane, filter paper and the sponge on a clamp, removing bubbles by using a glass rod, and carrying out constant-current wet transfer for 1h at 250 mA.

4) After the membrane conversion is finished, the nitrocellulose membrane is placed in a sealing solution and is gently oscillated for 1h at room temperature.

5) The membrane is cut according to the protein molecular weight of the primary antibody to be incubated.

6) The membrane was placed in primary antibody, diluted with PBST buffer, CDC37L1(1:500), Actin (1:10000), CDK4(1:1000), CDK6(1:500), Cyclin D1(1:1000), and Cyclin E1(1:1000), and incubated in wet box for 1h30min at room temperature.

7) After the primary stop, the membranes were washed 3 times with PBST by gentle shaking for 5min each time.

8) Membranes were placed in PBST buffer as diluent, 1:1000 dilutions of the secondary antibodies R800 and M800 were incubated in a wet box for 1h at room temperature.

9) After secondary anticaking, the membrane was washed 3 times with PBST buffer by gentle shaking for 5min each time, and then, the membrane was protected from light. Scanning was performed with an Odessey scanning membrane machine.

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

Silencing CDC37L1 expression promotes gastric cancer cell proliferation

The cDNA sequence CCDS6454.1 of CDC37L1 (A in FIG. 1) and the amino acid sequence NP-060383.2 encoded (B in FIG. 1) were retrieved by NCBI web search (https:// www.ncbi.nlm.nih.gov /).

Firstly, stomach cancer cells are transfected with exogenous CDC37L1+3Flag plasmids and control plasmids thereof, and a Western Blot experiment shows that CDC37L1 is successfully overexpressed; subsequently, gastric cancer cells are transfected with siCDC37L1 targeting CDC37L1 and control siRNA, the influence of small interfering RNA on the expression of gastric cancer cell CDC37L1 is tested by Western Blot, and the expression of CDC37L1 protein is obviously reduced (A in FIG. 2).

Gastric cancer MGC-803 cells in the logarithmic growth phase are inoculated into a 24-well plate, and the influence of CDC37L1 on the proliferation capacity of the gastric cancer cells is tested by an EdU experiment. By respectively up-regulating and down-regulating the expression of CDC37L1 in the gastric cancer cell MGC-803, the situation that the gastric cancer cell growth can be obviously inhibited after the CDC37L1 is over-expressed is observed through a fluorescence microscope, and vice versa (B in figure 2).

Example 2

CDC37L1 ability to inhibit growth of gastric cancer cells

The long-term and short-term CDC37L1 function on gastric cancer cell growth was examined with plate clonogenic and CCK8 growth curve experiments, respectively.

Respectively infecting gastric cancer cells (control groups LV-NC and LV-shNC) by using over-expressed lentivirus LV-CDC37L1 and knockdown lentivirus LV-shCDC37L1, screening by Puromycin to obtain stably expressed cell strains, taking cells in logarithmic growth phase, and taking 2 x 10 cells per well³After 2-3 weeks of culture, CDC37L1 is detected to influence on long-term growth of gastric cancer cells, and statistical results show that the reduction of CDC37L1 remarkably promotes the growth of gastric cancer cells (A in figure 3).

Transient transfection of gastric cancer cells with CDC37L1+3Flag overexpression plasmid and siRNA to obtain gastric cancer cells overexpressing CDC37L1 and knocking down CDC37L1 and their respective controls, and taking log-phase cells of the cells with the expression of 3 x 10³After being inoculated into a 96-well plate per well, the growth of the cells is detected by using a CCK8 kit for 5 days, and the experimental result shows that the CDC37L1 has the effect of inhibiting the growth of the gastric cancer cells (B in figure 3). Therefore, these cell function experiments all show that CDC37L1 can effectively inhibit the proliferation ability of gastric cancer cells to prevent the progression of gastric cancer.

Example 3

CDC37L1 overexpression obviously inhibits migration capability of gastric cancer cells

Gastric cancer cell lines overexpressing and downregulating CDC37L1 were detected by Transwell cell in vitro migration assay.

For infection of knockdown lentivirusGastric cancer cell line LV-shCDC37L1 stably expressing down-regulated CDC37L1 added 3.5X 10 to the top of the Transwell cell⁴After each cell was cultured for 24 hours, the cell line with low expression of CDC37L1 was increased in number compared with the control cell line passing through the filter, i.e., the low expression of CDC37L1 had the effect of promoting the migration of gastric cancer cells (A in FIG. 4).

Similarly, the Transwell cell migration experiment was performed on the gastric cancer cell lines BGC-823 and AGS stably overexpressing CDC37L1, and the same conclusion was also obtained (B in FIG. 4). Therefore, it was found that CDC37L1 significantly inhibited the migration ability of gastric cancer cells.

Example 4

CDC37L1 reduces gastric cancer cell tumor formation ability

The in vitro experiments show that CDC37L1 can obviously inhibit the proliferation and migration ability of gastric cancer cells. To further illustrate that the recombinant human gastric cancer cell has an important role in the occurrence and development of human gastric cancer, a tumor-bearing nude mouse model is used for researching the influence of the expression of CDC37L1 on the subcutaneous tumorigenicity of gastric cancer cells of nude mice.

Firstly, MGC-803 and BGC-823 cells are infected by lentiviruses LV-CDC37L1 and LV-NC respectively, and gastric cancer cell strains stably expressing CDC37L1 are constructed by Puromycin screening. Then, 1 × 10⁶The stably overexpressed CDC37L1 cells and control cells were injected subcutaneously into the abdomen of nude mice, the growth of tumor bodies was observed, the mice were sacrificed about 4-8 weeks until the maximum tumor diameter reached 15mm, and the tumor bodies were photographed and weighed. The result shows that the highly expressed CDC37L1 can obviously inhibit the tumor forming ability of gastric cancer cells in the nude mouse skin (A in figure 5).

Example 5

Downregulated CDC37L1 exerts an oncogenic effect by enhancing protein levels of CDK6

In vivo and in vitro experiments have shown that CDC37L1 exists as an anti-cancer gene in human gastric cancer, and influences the proliferation and migration capability of gastric cancer cells. To explore the potential molecular mechanisms of CDC37L1 in the role of cancer suppression in gastric cancer, the cyclin D1 and cyclin E1 members of the cyclin family, as well as the cyclin-dependent kinases CDK4 and CDK6, were examined using Western Blot experiments.

According to the method, gastric cancer cells MGC-803 and BGC-823 are selected as research objects of a molecular mechanism, knockdown lentivirus LV-shCDC37L1 or control LV-shNC is infected respectively to obtain a gastric cancer cell strain with CDC37L1 down-regulated, and a Western Blot experiment shows that the CDC37L1 expression in the gastric cancer cells is down-regulated, the CDK6 protein expression level is increased (figure 6), and other proteins are not obviously changed. CDK6 is a cyclin kinase that promotes cell proliferation by phosphorylating cyclins. Meanwhile, the prior literature also proves that the CDK6 protein promotes the movement and the metastasis of gastric cancer cells. Our experimental results indicate that CDK6 expression level is inhibited by CDC37L1, so that CDC37L1 functions to inhibit gastric cancer occurrence probably by inhibiting CDK 6.

Gastric cancer is one of high-incidence malignant tumors worldwide, and seriously threatens human health. The invention obtains the effect of CDC37L1 in the generation and development of gastric cancer through in vivo and in vitro experiments, shows that the CDC37L1 has the function of inhibiting the proliferation and migration of gastric cancer cells, simultaneously researches the molecular mechanism of the CDC37L1 playing a role in inhibiting cancer by using Western Blot experiments, and discovers that the CDC37L1 inhibits the development and progress of the gastric cancer by inhibiting the expression of CDK 6. The invention provides a new target point for treating gastric cancer so as to improve the survival time of patients. Therefore, the research has stronger theoretical and practical significance.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> east Hospital of Shanghai City

<120> application of human CDC37L1 gene and protein coded by same in preparation of drugs for treating gastric cancer

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

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agcagaggaa gaagguuau 19

Claims

1. Application of human CDC37L1 gene in preparing medicine for treating gastric cancer.

2. Application of human CDC37L1 gene in preparing medicine for inhibiting gastric cancer cell proliferation.

3. Application of human CDC37L1 gene in preparing medicine for inhibiting gastric cancer cell migration.

4. Application of human CDC37L1 gene in preparing medicine for reducing gastric cancer cell tumor formation.

5. Application of human CDC37L1 gene in preparing medicine for inhibiting cyclin dependent kinase 6.

6. Use of a protein encoded by the human CDC37L1 gene as claimed in claim 1 in the manufacture of a medicament for the treatment of gastric cancer.

7. Use of a protein encoded by the human CDC37L1 gene as claimed in claim 1 in the preparation of a medicament for inhibiting gastric cancer cell migration.

8. Use of a protein encoded by the human CDC37L1 gene as claimed in claim 1 in the preparation of a medicament for inhibiting gastric cancer cell proliferation.

9. Use of a protein encoded by the human CDC37L1 gene of claim 1 in the manufacture of a medicament for reducing gastric cancer cell neoplasia.

10. Use of a protein encoded by the human CDC37L1 gene as claimed in claim 1 in the manufacture of a medicament for inhibiting cyclin dependent kinase 6.