A method for constructing a liver cancer cell line overexpressing or knocking down the S100A16 gene and its application

Abstract

The present invention belongs to the field of biomedicine technology, and in particular, relates to a method for constructing a liver cancer cell line that overexpresses or knocks down the S100A16 gene and its application. A liver cancer cell line that overexpresses or knocks down the S100A16 gene is constructed by a lentiviral construction and lentiviral infection method, and is applied to the preparation of drugs that inhibit the proliferation, invasion and migration of hepatocellular carcinoma. The present invention verifies that the liver cancer cell line that knocks down the S100A16 gene can promote the apoptosis of liver cancer cells, and the proliferation and migration ability of liver cancer cells are significantly inhibited, while the liver cancer cells that overexpress the S100A16 gene significantly promote the proliferation, invasion and migration of liver cancer cells. Therefore, S100A16 can be used as a new drug target, and the S100A16 gene plays an important role in the preparation of drugs that inhibit the proliferation, invasion and migration of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for constructing a liver cancer cell line that overexpresses or knocks down the S100A16 gene and an application thereof. Background Art

[0002] Liver cancer is one of the most common digestive tract malignancies, with high morbidity and mortality rates worldwide. Among them, hepatocellular carcinoma (HCC) is the main type of liver cancer, accounting for about 75% to 80% of total liver cancer. The latest national cancer statistics show that the mortality rate of liver cancer in my country ranks second among malignant tumors in the country. The mortality rate increases with age and decreases with the increase in birth cohorts. The morbidity and mortality of liver cancer continue to rise, and it is a major global public health burden. The incidence of HCC is mainly related to hepatitis B and C virus infection, drinking, aflatoxin, lipid metabolism disorders and other factors. Although many genes and pathways that affect the development of liver cancer have been discovered, the treatment and patient prognosis are still unsatisfactory. Therefore, in-depth research on the pathogenesis of HCC, mining key biomarker molecules for the occurrence and development of HCC, and studying HCC targeted drugs are key scientific issues that need to be urgently addressed in the medical community. Summary of the invention

[0003] The purpose of the present invention is to provide a method for constructing a liver cancer cell line that overexpresses or knocks down the S100A16 gene and its application, so as to solve the problems raised in the above background technology.

[0004] In order to achieve the above technical purpose, the technical solution of the present invention is:

[0005] A method for constructing a liver cancer cell line that overexpresses or knocks down the S100A16 gene comprises the following steps:

[0006] S1. Lentivirus Construction

[0007] The S100A16 overexpression or knockdown plasmid was constructed by homologous recombination method, and then the S100A16 overexpression or knockdown plasmid was packaged into S100A16 overexpression or knockdown lentivirus;

[0008] S2. Lentivirus infection

[0009] The liver cancer cell lines in good condition were inoculated into 24-well plates, and the cells were counted and counted at 5×10 4 Cells / well were plated at a density of 1:1. One day later, 5 μg / μL Polybrene-containing DMEM complete medium was added, and then the S100A16 overexpression or knockdown lentivirus was added to the DMEM complete medium to infect the liver cancer cell line, and fresh culture medium was replaced within 24 hours of virus infection;

[0010] S3. Subculture

[0011] After the cells were infected with the virus for 72 hours, they were screened with DMEM complete medium containing 5 μg / mL puromycin. The conditions were maintained and culture and subculture were continued to obtain liver cancer cell lines that overexpressed or knocked down the S100A16 gene.

[0012] Furthermore, in step S1, the S100A16 overexpression gene or S100A16 knockdown sh-RNA and a plasmid vector are recombined to construct a S100A16 overexpression or knockdown plasmid.

[0013] Furthermore, the overexpression S100A16 gene sequence is shown in SEQ ID NO: 1, the knockdown S100A16 sh-RNA sequence is Sh-S100A16-18 or Sh-S100A16-19 or Sh-S100A16-20, the Sh-S100A16-18 is shown in SEQ ID NO: 2, the Sh-S100A16-19 is shown in SEQ ID NO: 3, and the Sh-S100A16-20 is shown in SEQ ID NO: 4.

[0014] Further, in step S1, the plasmid vector used for the S100A16 overexpression plasmid was PSLenti-EF1-EGFP-P2A-puro-CMV-MCS-3×FLAG-WPRE, and the plasmid vector used for the S100A16 knockdown plasmid was pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE.

[0015] Furthermore, in step S2, the liver cancer cell line is MHCC97L or HepG2.

[0016] Furthermore, in step S2, the liver cancer cell line is infected with the lentivirus at MOI=80.

[0017] The present invention also provides the use of a liver cancer cell line with a knocked-down S100A16 gene in the preparation of a drug for inhibiting proliferation, invasion and migration of liver cancer.

[0018] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0019] The liver cancer cell lines overexpressing or knocking down the S100A16 gene provided by the present invention can promote the apoptosis of liver cancer cells, and the proliferation and migration abilities of liver cancer cells are significantly inhibited, while the liver cancer cell lines overexpressing the S100A16 gene significantly promote the proliferation, invasion and migration of liver cancer cells. Therefore, S100A16 can be used as a new drug target, and the liver cancer cell lines knocking down the S100A16 gene play an important role in the preparation of drugs for inhibiting the proliferation, invasion and migration of hepatocellular carcinoma.

[0020] The present invention shows through a nude mouse subcutaneous tumor-bearing experiment that a liver cancer cell line overexpressing the S100A16 gene promotes the formation of a nude mouse subcutaneous tumor of liver cancer cells and the formation of a lung metastasis, while a liver cancer cell line knocking down the S100A16 gene can inhibit the formation of a nude mouse subcutaneous tumor of liver cancer cells and the formation of a lung metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 : are the result graphs of QPCR detection and Western blot detection in Example 1, wherein A is the verification result of RNA level of HepG2 cell line overexpressing S100A16 gene by QPCR detection, B is the verification result of RNA level of HepG2 cell line knocking down S100A16 gene by QPCR detection, C is the verification result of RNA level of MHCC97L cell line overexpressing S100A16 gene by QPCR detection, D is the verification result of RNA level of MHCC97L cell line knocking down S100A16 gene by QPCR detection, E is the verification result of protein level after overexpression and knockdown of S100A16 in HepG2 and MHCC97L cell lines by Western Blot detection, F is the grayscale analysis result of protein level after overexpression and knockdown of S100A16 in HepG2 cell line by Western Blot detection, and G is the grayscale analysis result of protein level after overexpression and knockdown of S100A16 in MHCC97L cell line by Western Blot detection;

[0023] Figure 23 is the result of the CCK8 proliferation experiment in Example 3, wherein A is the absorbance result of HepG2 cells overexpressing the S100A16 gene, B is the absorbance result of HepG2 cells knocking down the S100A16 gene, C is the absorbance result of MHCC97L cells overexpressing the S100A16 gene, and D is the absorbance result of MHCC97L cells knocking down the S100A16 gene;

[0024] Figure 3 3 are the result graphs of the plate clone formation experiment in Example 3, wherein A is a graph showing the proliferation of liver cancer cells by HepG2 cells overexpressing and knocking down the S100A16 gene, B is a graph showing the proliferation of liver cancer cells by MHCC97L cells overexpressing and knocking down the S100A16 gene, C is a graph showing the clone formation rate of HepG2 cells overexpressing the S100A16 gene, D is a graph showing the clone formation rate of HepG2 cells knocking down the S100A16 gene, E is a graph showing the clone formation rate of MHCC97L cells overexpressing the S100A16 gene, and F is a graph showing the clone formation rate of MHCC97L cells knocking down the S100A16 gene;

[0025] Figure 4 are partial result graphs of the cell cycle experiment in Example 3, wherein A is a peak graph of the cell cycle of HepG2 cells overexpressing the S100A16 gene detected by flow cytometry, B is a peak graph of the cell cycle of HepG2 cells knocking down the S100A16 gene detected by flow cytometry, C is a peak graph of the cell cycle of MHCC97L cells overexpressing the S100A16 gene detected by flow cytometry, and D is a peak graph of the cell cycle of MHCC97L cells knocking down the S100A16 gene detected by flow cytometry;

[0026] Figure 5 are result graphs of the cell apoptosis experiment in Example 3, wherein A is a scatter plot of apoptosis of HepG2 cells overexpressing the S100A16 gene detected by flow cytometry, B is a scatter plot of apoptosis of HepG2 cells knocking down the S100A16 gene detected by flow cytometry, C is a statistical graph of apoptosis of HepG2 cells overexpressing the S100A16 gene detected by flow cytometry, and D is a statistical graph of apoptosis of HepG2 cells knocking down the S100A16 gene detected by flow cytometry;

[0027] Figure 6The results of the cell scratch experiment, Transwell experiment and Western blot experiment in Example 4, wherein A is a graph showing the cell migration of HepG2 cells overexpressing and knocking down the S100A16 gene detected by the scratch experiment, B is a graph showing the scratch repair rate of HepG2 cells overexpressing the S100A16 gene detected by the scratch experiment, C is a graph showing the scratch repair rate of HepG2 cells knocking down the S100A16 gene detected by the scratch experiment, D is a graph showing the cell migration of HepG2 cells overexpressing and knocking down the S100A16 gene detected by the Transwell experiment, E is a graph showing the number of cell migration detected by the Transwell experiment in HepG2 cells overexpressing the S100A16 gene, F is a graph showing the number of cell migration detected by the Transwell experiment in HepG2 cells knocking down the S100A16 gene, and G is a Western blot experiment showing the number of cell migration detected by the Transwell experiment in HepG2 cells knocking down the S100A16 gene, The results of Western blot detection of β-catenin, N-cadherin, and E-cadherin protein levels in HepG2 cells overexpressing S100A16 gene. H is the results of Western blot detection of β-catenin, N-cadherin, and E-cadherin protein levels in HepG2 cells knocking down S100A16 gene.

[0028] Figure 7 The results of the nude mouse subcutaneous tumor bearing experiment and lung tissue HE staining in Example 5, wherein A is a result diagram of tumor volume of subcutaneously transplanted HepG2 cells overexpressing the S100A16 gene, B is a result diagram of tumor volume of subcutaneously transplanted HepG2 cells with S100A16 gene knocked down, C is a nude mouse live image of subcutaneously transplanted HepG2 cells overexpressing and knocking down the S100A16 gene, D is an in vitro image of tumors subcutaneously transplanted HepG2 cells overexpressing and knocking down the S100A16 gene, E is a result diagram of tumor weight of subcutaneously transplanted HepG2 cells overexpressing the S100A16 gene, F is a result diagram of tumor weight of subcutaneously transplanted HepG2 cells with S100A16 gene knocked down, and G is a nude mouse live image of subcutaneously transplanted HepG2 cells overexpressing and knocking down the S100A16 gene. The results of tumor volume of MHCC97L cells expressing S100A16 gene were subcutaneously transplanted, H was the results of tumor volume of MHCC97L cells with S100A16 gene knocked down, I was the in vivo images of nude mice with MHCC97L cells overexpressing and knocking down S100A16 gene subcutaneously transplanted, J was the in vitro images of tumors of MHCC97L cells overexpressing and knocking down S100A16 gene subcutaneously transplanted, K was the results of tumor weight of MHCC97L cells overexpressing S100A16 gene subcutaneously transplanted, L was the results of tumor weight of MHCC97L cells with S100A16 gene knocked down, M was the results of lung tissue HE staining of nude mice lung metastasis experiment;

[0029] Figure 8 is the result of Western Blot detection in Example 2, wherein A is the expression change of S100 family molecules in HCC analyzed by Bulk RNA sequencing, B is the expression level of S100A16 in cancer and adjacent tissues of liver cancer patients statistically analyzed by GEPIA database, C is the correlation between S100A16 expression and disease-free survival (DFS) of liver cancer patients (n=182) statistically analyzed by GEPIA database, and D is the correlation between S100A16 expression and overall survival (OS) of liver cancer patients (n=182) statistically analyzed by GEPIA database;

[0030] Fig. 9 : are the results of QPCR detection and ELISA detection in Example 2, wherein E is the expression level of S100A16 protein in cancer and adjacent tissues of different liver cancer patients detected by Western Blot, F is the grayscale analysis bar graph of the expression level of S100A16 protein in cancer and adjacent tissues of different liver cancer patients detected by Western Blot, G is the expression level of S100A16 protein in cancer and adjacent tissues of different liver cancer patients detected by QPCR, H is the result of ELISA detection of serum S100A16 levels in healthy controls and hepatitis patients, I is the result of ELISA detection of serum S100A16 levels in healthy controls and liver cirrhosis patients, J is the result of ELISA detection of serum S100A16 levels in healthy controls and liver cancer patients, and K is the expression level of S100A16 protein in cancer and adjacent tissues of different liver cancer patients detected by immunohistochemistry;

[0031] Fig.10 3 are partial result graphs of the cell cycle experiment in Example 3, wherein E is a statistical graph of the cell cycle of HepG2 cells overexpressing the S100A16 gene detected by flow cytometry, F is a statistical graph of the cell cycle of HepG2 cells with S100A16 gene knocked down detected by flow cytometry, G is a statistical graph of the cell cycle of MHCC97L cells overexpressing the S100A16 gene detected by flow cytometry, and H is a statistical graph of the cell cycle of MHCC97L cells with S100A16 gene knocked down detected by flow cytometry. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Construction of a liver cancer cell line with knockdown of the S100A16 gene, the specific method is as follows:

[0038] S1. Lentivirus Construction

[0039] The plasmid vector pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE with CFP green fluorescence marker was selected, and the knockdown S100A16 sh-RNA and plasmid vector were recombined to construct the S100A16 knockdown plasmid by homologous recombination, and then the S100A16 knockdown plasmid was packaged into the S100A16 knockdown lentivirus;

[0040] S2. Lentivirus infection

[0041] The liver cancer cell lines in good condition were inoculated into 24-well plates, and the cells were counted and counted at 5×10 4 The density of cells / well was set to ensure that the fusion rate reached 30-40% when infected with virus on the next day.

[0042] On the second day, virus infection was performed by adding 5 μg / μL Polybrene-containing DMEM complete medium to the 24-well plate, and then adding S100A16 knockdown lentivirus at MOI=80 to infect the liver cancer cell line. After 12-20 hours of virus infection, the cell status was observed and fresh culture medium was replaced within 24 hours;

[0043] 72 hours after virus infection, the efficiency of lentivirus infection of liver cancer cell lines was determined under a fluorescence microscope;

[0044] S3. Cell line screening and subculture

[0045] Stable cell lines were selected using DMEM complete medium containing 5 μg / mL puromycin, and then subcultured. The cells were observed daily using an inverted fluorescence microscope. Between 3 and 10 days after the action of puromycin, almost all surviving cells could express GFP. After the cell construction was successful, QPCR and Western blot were performed on the constructed cells to verify the expression of the S100A16 gene.

[0046] In this embodiment, in step S1, the S100A16 knockdown sh-RNA sequence is Sh-S100A16-18 or Sh-S100A16-19 or Sh-S100A16-20, wherein Sh-S100A16-18 is shown in SEQ ID NO: 2, Sh-S100A16-19 is shown in SEQ ID NO: 3, and Sh-S100A16-20 is shown in SEQ ID NO: 4.

[0047] In step S2, the liver cancer cell lines are MHCC97L and HepG2.

[0048] The specific method for constructing the liver cancer cell line overexpressing the S100A16 gene is the same as the method for constructing the liver cancer cell line knocking down the S100A16 gene, except that the plasmid vector used for the S100A16 overexpression plasmid is PSLenti-EF1-EGFP-P2A-puro-CMV-MCS-3×FLAG-WPRE.

[0049] The HepG2 cell line overexpressing the S100A16 gene, the HepG2 cell line knocking down the S100A16 gene, the MHCC97L cell line overexpressing the S100A16 gene, and the MHCC97L cell line knocking down the S100A16 gene obtained in this example were respectively subjected to QPCR detection and Western blot detection to verify whether the construction of the liver cancer cell line overexpressing the S100A16 gene and the liver cancer cell line knocking down the S100A16 gene was successful.

[0050] like Figure 1 The results of the QPCR and Western blot tests in this example are shown in FIG. Figure 1 A in the figure is the result of QPCR detection of RNA level in HepG2 cell line overexpressing S100A16 gene. Figure 1 B in the figure is the result of QPCR detection of RNA level verification in HepG2 cell line with knockdown of S100A16 gene. Figure 1 C in the figure is the result of QPCR detection of RNA level in MHCC97L cell line overexpressing S100A16 gene. Figure 1 D in the figure is the result of RNA level verification by QPCR detection of MHCC97L cell line with knockdown of S100A16 gene. Figure 1 E in the figure is the verification result of protein level after overexpression and knockdown of S100A16 in HepG2 and MHCC97L cell lines by Western Blot. Figure 1 F in the figure is the grayscale analysis result of protein level after overexpression and knockdown of S100A16 in HepG2 cell line detected by Western Blot. Figure 1 G in the figure is the grayscale analysis result of protein level after overexpression and knockdown of S100A16 in MHCC97L cell line detected by Western Blot. Figure 1 It can be seen that both the liver cancer cell lines overexpressing the S100A16 gene and the liver cancer cell lines knocking down the S100A16 gene were successfully constructed.

[0051] Figure 1 In the experiment, LV-S100A16 group was a liver cancer cell group overexpressing S100A16 gene, LV-control group was a control group of liver cancer cells overexpressing S100A16 gene, Sh-S100A16-18 was a liver cancer cell group with knockdown of Sh-S100A16-18 gene, Sh-S100A16-19 was a liver cancer cell group with knockdown of Sh-S100A16-19 gene, and Sh-S100A16-20 was a liver cancer cell group with knockdown of Sh-S100A16-20 gene. Figure 1 It can be seen that in the liver cancer cell group with Sh-S100A16-18 gene knockdown, the relative expression level of S100A16 mRNA was the lowest, indicating that Sh-S100A16-18 is the best sh-RNA sequence for knocking down S100A16.

[0052] Example 2 Paired tissue and serum samples were collected from HCC patients. The serum samples were subjected to ELISA detection of serum S100A16, and the paired tissue samples were subjected to QPCR, Western blot, and immunohistochemistry experiments of S100A16.

[0053] ELISA was used to confirm that S100A16 was highly expressed in the serum of HCC patients. The specific steps are as follows:

[0054] In this experiment, human S100 calcium-binding protein A16 (S100A16) ELISA kit was used to detect S100A16 in the serum of HCC patients.

[0055] First, equilibrate the ELISA plate to room temperature, dilute the matching 20× washing solution with ultrapure water to 1× washing solution in advance, add standard solutions of different concentrations to the standard wells, add HCC patient serum as the sample to be tested to the sample wells, and add specific antibodies to the wells of the ELISA plate, shake evenly, seal the plate with a sealing film, and place the ELISA plate in a constant temperature box at 37°C for 60 minutes to ensure that the sample and the antibody are fully combined. After the incubation, pat all the liquids in the wells dry with absorbent paper, and add washing solution to soak Soak for 1 min, pat dry the liquid in all wells with absorbent paper again, repeat this step 5 times to ensure the removal of unbound substances and reduce background signals. After washing, add secondary antibodies to the wells of the ELISA plate, seal the plate and incubate at 37°C for 15 min. After the incubation, add the reaction stop solution to each well, use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm, draw a standard curve, and obtain the concentration of S100A16 in the serum of HCC patients, the sample to be tested.

[0056] RNA extraction and QPCR detection confirmed that S100A16 was highly expressed in HCC patient tissues. The specific steps are as follows:

[0057] The centrifuge was precooled at 4°C, the cancer tissue cells of HCC patients were washed twice with PBS, 1 mL of Trizol was added, and the mixture was mixed by blowing with a pipette tip, 100 μL of chloroform was added, the tube cover was tightly closed, and the mixture was shaken vigorously for 15 seconds. The mixture was allowed to stand at 4°C for 10 minutes in a centrifuge, and then centrifuged at 14,000 rpm for 15 minutes at 4°C. After centrifugation, the mixture was divided into three layers. The upper transparent aqueous layer was transferred to a 1.5 mL enzyme-free centrifuge tube, 1.5 mL of isopropanol was added, the mixture was mixed by inversion, and the mixture was allowed to stand at 4°C for 10 minutes. The mixture was then centrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant was discarded, and 1 mL of 75% ethanol, flick the precipitate, wash the precipitate once, centrifuge at 12000g for 5 minutes at 4°C, discard the supernatant, dry the precipitate on ice for 10 minutes to completely evaporate the ethanol, add 10~30μL of water according to the amount of precipitate to dissolve for 10 minutes, detect the RNA concentration with a UV spectrophotometer, and identify the RNA yield and purity. Configure the reverse transcription reaction system and set the PCR instrument program: 37°C, 15min, 85°C, 5s, 4°C, forever. Configure the QPCR reaction system and set the PCR instrument program: 50°C, 2min, 1 cycle, 95°C, 2min, 1 cycle, 95°C, 15s, 40cycles, 60°C, 1min, 40 cycles.

[0058] The primer sequences required for the target gene are as follows:

[0059] Human S100A16 Fwd primer: as shown in SEQ ID NO: 5;

[0060] Human S100A16 Rev primer: as shown in SEQ ID NO: 6;

[0061] Human GAPDH Fwd primer: as shown in SEQ ID NO: 7;

[0062] Human GAPDH Rev primer: as shown in SEQ ID NO:8.

[0063] Western Blot experiments confirmed that S100A16 was highly expressed in HCC patient tissues. The specific steps are as follows:

[0064] Cancerous tissue cells of HCC patients were collected and washed with PBS, centrifuged at 5000rpm for 5min, the supernatant was discarded, 200-500μL of the prepared RIPA lysis buffer was added, vortexed for 15s, and the cells were ultrasonically disrupted 3 times, each time for 15s, and centrifuged at 12000rpm for 10min at 4°C. All the supernatant was transferred to a new 1.5mL EP tube, 10μL of standard was added to a 96-well plate, and then 190μL of working solution was added and mixed, incubated in a 37°C incubator for 30min, cooled to room temperature, and the protein concentration was detected by an enzyme reader at 560nm, a standard curve was drawn, the protein concentration was calculated, and a protein solution was prepared. The specific method is the existing technology and will not be repeated here. The protein solution was vortexed, denatured at 99°C for 5min, and stored in a -80°C refrigerator for later use.

[0065] Select 15% SDS-PAGE precast gel, add 20μg denatured protein solution and 5μL protein marker to each well, run the gel at 150V constant voltage for 1h, transfer the membrane at 300mA for 1h, use PVDF membrane in the transfer process, and block the PVDF membrane with Biyuntian rapid blocking solution for 30min. After blocking, cut the membrane according to the size of the target protein and the position of the marker and mark it. Put the cut strips into the primary antibody and incubate overnight at 4℃ or on a shaker at room temperature for 2h. After the primary antibody is completed, wash the membrane three times with PBST and incubate with the secondary antibody for 1h. After the secondary antibody is completed, wash the membrane three times with PBST and add the color developing solution for exposure.

[0066] Through immunohistochemistry experiments, it was confirmed that S100A16 was highly expressed in the cancer tissues of HCC patients. The specific steps are as follows:

[0067] The prepared sections were placed in a 68°C oven, and after baking for 1.5 hours, they were dewaxed and rehydrated. Antigen repair was performed with citrate buffer (pH=6.0), and the sections were washed 3 times with PBST. 100 μL of endogenous peroxidase blocker was added to each section. The sections were washed 3 times with PBST, 100 μL of 5% goat blocking serum was added to each section, rabbit anti-S100A16 antibody was added at a dilution of 1:50, 100 μL of primary antibody diluent was added to each section, and the sections were incubated at room temperature for 2 hours or at 4°C overnight. The primary antibody was discarded, the sections were washed 3 times with PBST, 100 μL of secondary antibody was added to each section, and the sections were incubated at room temperature for 30 minutes. The secondary antibody was discarded, the sections were washed 3 times with PBST, DAB color developer was prepared, 100 μL of freshly prepared color developer was added to each section, the degree of coloration was judged by the naked eye, incubated at room temperature for 3-4 minutes, and then immediately immersed in distilled water. Wash to stop color development, rinse with running water for 2 minutes, discard the residual color developer, re-stain in hematoxylin staining solution for 2 minutes, then rinse with running water for 2-5 minutes, put the slices into 0.5% hydrochloric acid alcohol differentiation solution for differentiation for 2 seconds, rinse with running water for 2 minutes, blue the cell nucleus in the blueing solution for 2 minutes to make the cell nucleus color more obvious and facilitate the distinction between the cell nucleus and cytoplasm under the microscope, rinse with running water for 2 minutes, and then dehydrate the tissue. After dehydration, wait until the xylene on the slice is basically evaporated, drop neutral gum in the center of the tissue, and cover the slice with a coverslip.

[0068] The experimental results of this embodiment are as follows Figure 8-9 As shown, Figure 8 A in the figure is Bulk RNA sequencing analysis of the expression changes of S100 family molecules in HCC. Figure 8 B in the figure is the S100A16 expression level in the cancer and adjacent tissues of liver cancer patients statistically analyzed by the GEPIA database. Figure 8 C in the figure is the correlation between S100A16 expression and disease-free survival (DFS) of liver cancer patients (n=182) according to the GEPIA database. Figure 8 D in the figure is the correlation between S100A16 expression and overall survival (OS) of liver cancer patients (n=182) according to the GEPIA database. Fig. 9 E in the figure is the expression level of S100A16 protein in cancerous and adjacent tissues of different liver cancer patients detected by Western Blot. Fig. 9 F in the figure is a grayscale analysis bar graph of the S100A16 protein expression level in the cancer and adjacent tissues of different liver cancer patients detected by Western Blot. Fig. 9 The G in the figure is the expression level of S100A16 protein in the cancerous and adjacent tissues of different liver cancer patients detected by QPCR. Fig. 9 The H in the figure is the result of ELISA testing the serum S100A16 levels in healthy controls and hepatitis patients. Fig. 9 I in the figure is the result of ELISA detection of S100A16 levels in healthy controls and patients with liver cirrhosis. Fig. 9 J in the figure is the result of ELISA detection of serum S100A16 levels in healthy controls and liver cancer patients. Fig. 9 K in the figure represents the expression level of S100A16 protein in cancer and adjacent tissues of different liver cancer patients detected by immunohistochemistry.

[0069] The above experimental results show that S100A16 is expressed at elevated levels in the serum and cancer tissues of HCC patients, suggesting that S100A16 is closely related to the onset of HCC and is also a valuable biomarker for HCC.

[0070] Example 3 Through a plate clone formation experiment, it was confirmed that S100A16 overexpression promoted cell proliferation and S100A16 knockdown inhibited cell proliferation. The specific steps are as follows:

[0071] The liver cancer cell lines overexpressing S100A16 gene and the liver cancer cell lines knocking down S100A16 gene prepared in Example 1 were seeded in 6-well plates at a density of 300 cells per dish, wherein the liver cancer cell line knocking down S100A16 gene used the knocking down S100A16 sh-RNA sequence Sh-S100A16-18, and the cells were gently shaken to disperse evenly. The 6-well plates were placed at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator with saturated humidity for 10 days. When visible clones appeared in the culture dishes, the culture was terminated, and 3 mL of 4% PFA fixative was added to the 6-well plates to fix the cells for 15 min. The fixative was then removed, and 3 mL of crystal violet stain was added for staining for 20 min. The stain was slowly washed off with running water, and the plates were air-dried for 15 min. The clone formation rate was calculated, and a t-test was performed based on the colony number and clone formation rate.

[0072] Through CCK8 proliferation assay, it was confirmed that S100A16 overexpression promoted cell proliferation, while S100A16 knockdown inhibited cell proliferation. The specific steps are as follows:

[0073] The liver cancer cell lines overexpressing S100A16 gene and the liver cancer cell lines knocking down Sh-S100A16-18 prepared in Example 1 were inoculated into 96-well plates, respectively, wherein the inoculation amount of HepG2 cells was 1500 cells / well, and the inoculation amount of MHCC97L cells was 3000 cells / well. The prepared CCK8 solution was added in the dark at 1d, 2d, 3d, 4d, 5d, and 6d, respectively, and incubated at 37°C for 1 h. The absorbance was measured at 450nm with a microplate reader to draw a growth curve.

[0074] The cell cycle experiment was performed to confirm the effects of S100A16 overexpression and knockdown on the cell cycle. The specific steps are as follows:

[0075] Precool PBS and anhydrous ethanol in an ice bath in advance, place the liver cancer cell line overexpressing S100A16 gene and the liver cancer cell line knocking down Sh-S100A16-18 prepared in Example 1 in centrifuge tubes for standby use, add 5-10 mL PBS to the centrifuge tube to wash away the residual serum, then add 0.25% trypsin to digest the cells, centrifuge at 1000 g for 5 min, discard the supernatant, add 1 mL of ice-bath precooled PBS to the precipitated cells, gently blow to resuspend the cells, centrifuge the precipitated cells again, discard the supernatant, gently tap the bottom of the centrifuge tube to properly disperse the cells to avoid cell clumping, and then add 300 μL of PBS, gently blow and mix with the tip of a pipette, then slowly add 700 μL of ice-bath pre-cooled anhydrous ethanol along the wall of the tube, gently invert and mix, fix overnight at -20°C, then centrifuge the tube at 1000g for 5 min, discard the supernatant, add 1 mL of ice-bath pre-cooled PBS to the centrifuge tube, gently blow and resuspend the cells, centrifuge again, discard the supernatant, gently tap the bottom of the centrifuge tube to properly disperse the cells to avoid cell clumping, prepare propidium iodide staining solution (50 μg / mL PI, 100 μg / mL RNase A), add 0.5 mL of propidium iodide staining solution to each tube of cell sample, gently blow and resuspend the cell pellet, warm in the dark at 37°C for 30 min, filter the cell suspension with a 300-mesh filter, and then store in the dark at 4°C. Complete flow cytometry within 24 hours after staining.

[0076] Through apoptosis experiments, it was confirmed that S100A16 knockdown promoted apoptosis of liver cancer cells. The specific steps are as follows:

[0077] Take the Sh-S100A16-18 knockdown liver cancer cell line in the logarithmic growth phase prepared in Example 1, count the cells and adjust the concentration to about 2×10 5 / mL plate, add 100μmol / L H 2 O 2 Treat the cells, incubate for 1 hour, wash twice with pre-cooled PBS by centrifugation, resuspend the cells with 500μL 1×Binding Buffer, add 5μL Annexin V-APC and 10μL PI-perCP, pipette repeatedly to ensure uniform mixing, incubate for 5 minutes at room temperature and in a dark environment, and detect on flow cytometer within 1 hour. The first quadrant is early apoptosis, the fourth quadrant is late apoptosis, and the sum of the two quadrants is the cell apoptosis ratio.

[0078] like Figure 2 The results of the CCK8 proliferation experiment are shown in Figure 2. Figure 2 A in the figure is the absorbance result of HepG2 cells overexpressing S100A16 gene. Figure 2B in the figure is the absorbance result of HepG2 cells with S100A16 gene knocked down. Figure 2 C in the figure is the absorbance result of MHCC97L cells overexpressing S100A16 gene. Figure 2 D in the figure shows the absorbance results of MHCC97L cells with S100A16 gene knocked down.

[0079] like Figure 3 The results of the plate clone formation experiment are shown in Figure 2. Figure 3 A in the figure shows the proliferation of liver cancer cells in HepG2 cells with overexpression and knockdown of S100A16 gene. Figure 3 B in the figure shows the proliferation of liver cancer cells in MHCC97L cells with overexpression and knockdown of S100A16 gene. Figure 3 C in the figure is the result of HepG2 cell clone formation rate overexpressing S100A16 gene. Figure 3 D in the figure is the result of HepG2 cell clone formation rate after knocking down S100A16 gene. Figure 3 E in the figure is the result of clone formation rate of MHCC97L cells overexpressing S100A16 gene. Figure 3 F in the figure is the result graph of the clone formation rate of MHCC97L cells with S100A16 gene knockdown.

[0080] like Figure 4 and Fig.10 The results of the cell cycle experiment are shown in Figure 2. Figure 4 A in the figure is the peak graph of the cell cycle of HepG2 cells overexpressing the S100A16 gene detected by flow cytometry. Figure 4 B is a peak diagram of the cell cycle of HepG2 cells knocked down by S100A16 gene detected by flow cytometry. Figure 4 C in the figure is the peak graph of the cell cycle of MHCC97L cells overexpressing the S100A16 gene detected by flow cytometry. Figure 4 D in the figure is the peak diagram of the cell cycle of MHCC97L cells with knockdown of S100A16 gene detected by flow cytometry. Fig.10 E in the figure is a statistical diagram of the cell cycle of HepG2 cells overexpressing the S100A16 gene detected by flow cytometry. Fig.10 F in the figure is a statistical diagram of the cell cycle of HepG2 cells with S100A16 gene knockdown detected by flow cytometry. Fig.10 G in the figure is a statistical diagram of the cell cycle of MHCC97L cells overexpressing the S100A16 gene detected by flow cytometry. Fig.10 H in the figure is a statistical diagram of the cell cycle status of MHCC97L cells with S100A16 gene knockdown detected by flow cytometry.

[0081] like Figure 5 The results of the cell apoptosis experiment are shown in Figure 2. Figure 5 A in the figure is a scatter plot of apoptosis of HepG2 cells overexpressing S100A16 gene detected by flow cytometry. Figure 5 B is a scatter plot of apoptosis of HepG2 cells with S100A16 gene knockdown detected by flow cytometry. Figure 5 C in the figure is a statistical diagram of apoptosis of HepG2 cells overexpressing the S100A16 gene detected by flow cytometry. Figure 5 D in the figure is a statistical graph of cell apoptosis in HepG2 cells with S100A16 gene knockdown detected by flow cytometry.

[0082] This example verifies that overexpression of S100A16 can promote the proliferation of liver cancer cells, while knockdown of S100A16 can inhibit the proliferation of liver cancer cells and promote the apoptosis of liver cancer cells.

[0083] Example 4 Through cell scratch experiments, it was confirmed that S100A16 overexpression promoted cell migration and S100A16 knockdown inhibited cell migration. The specific steps were as follows:

[0084] The liver cancer cell line overexpressing S100A16 gene (LV-S100A16) and its control cell (LV-control) and the liver cancer cell line knocking down Sh-S100A16-18 (Sh-S100A16) and its control cell (Sh-control) prepared in Example 1 were inoculated on a 6-well plate at an inoculation volume of 1×10 6 cells / well. After 24 hours, the cells were starved overnight in serum-free medium, and then a sterile 200 μL pipette tip was used to scratch the cells in the layer. The progress of migration was photographed at 0 h, 24 h, and 48 h after the scratch, and the scratch repair rate was calculated, scratch repair rate = (0 h scratch width - 48 h scratch width) / 0 h scratch width × 100%.

[0085] Through Transwell experiments, it was confirmed that S100A16 overexpression promoted cell migration, while S100A16 knockdown inhibited cell migration. The specific steps are as follows:

[0086] The liver cancer cell line overexpressing S100A16 gene (LV-S100A16) and its control cell (LV-control) prepared in Example 1 and the liver cancer cell line knocking down Sh-S100A16-18 (Sh-S100A16) and its control cell (Sh-control) were inoculated in a 24-well plate chamber in serum-free medium and cultured at an inoculation size of 5×10 4cells / well, add 20% FBS DMEM culture medium to the 24-well plate at the bottom, culture for 24 hours, fix with 4% paraformaldehyde for 20 minutes, then stain with crystal violet for 15 minutes, rinse off the dye with running water, wipe off the cells inside the chamber with a cotton swab, and count the number of cells at the bottom of the chamber under a microscope.

[0087] Through Western blot experiments, it was confirmed that S100A16 overexpression promoted cell epithelial-mesenchymal transition, and the specific experimental method was based on existing technology.

[0088] The experimental results of this embodiment are as follows Figure 6 As shown, Figure 6 A in the figure is a scratch assay to detect the cell migration of HepG2 cells with overexpression and knockdown of S100A16 gene. Figure 6 B is the result of the scratch test to detect the scratch repair rate of HepG2 cells overexpressing the S100A16 gene. Figure 6 C in the figure is the result of scratch test to detect the scratch repair rate of HepG2 cells with knockdown of S100A16 gene. Figure 6 D in the figure is a Transwell experiment to detect the cell migration of HepG2 cells with overexpression and knockdown of S100A16 gene. Figure 6 E in the figure is the result of Transwell experiment to detect the number of cell migration of HepG2 cells overexpressing S100A16 gene. Figure 6 F in the figure is the result of Transwell experiment to detect the number of cell migration in HepG2 cells with knockdown of S100A16 gene. Figure 6 The G in the figure is the result of Western blot detection of β-catenin, N-cadherin, and E-cadherin protein levels in HepG2 cells overexpressing the S100A16 gene. Figure 6 H in the figure is the result of Western blot detection of β-catenin, N-cadherin, and E-cadherin protein levels in HepG2 cells with S100A16 gene knockdown.

[0089] This experiment verified that S100A16 overexpression can promote the migration of liver cancer cells, and S100A16 knockdown can inhibit the migration of liver cancer cells.

[0090] Example 5 Through the nude mouse subcutaneous tumor bearing experiment, it was confirmed that S100A16 overexpression promoted the formation of nude mouse subcutaneous tumor bearing, and S100A16 knockdown inhibited the formation of nude mouse subcutaneous tumor bearing. The specific steps are as follows:

[0091] Forty female athymic mice aged 4-6 weeks were selected and divided into eight groups. The HepG2 cell line overexpressing the S100A16 gene and its control cell line, the HepG2 cell line knocking down the S100A16 gene and its control cell line, the MHCC97L cell line overexpressing the S100A16 gene and its control cell line, and the MHCC97L cell line knocking down the S100A16 gene and its control cell line prepared in Example 1 were subcutaneously transplanted into the eight groups of mice, and the number of transplanted cells was 5×10 6 Tumors were measured every three days using a vernier caliper and the equation V = 1 / 2ab was used. 2 Tumor volume (V), where a and b represent length and width in millimeters, was calculated at 4 weeks after transplantation.

[0092] The nude mouse metastasis experiment and lung tissue HE staining were used to confirm the formation of nude mouse lung metastasis. The specific steps are as follows:

[0093] Sixteen female athymic mice aged 4-6 weeks were selected and divided into two groups. The liver cancer cell line overexpressing the S100A16 gene prepared in Example 1 and the control cells were injected into the two groups of mice through the tail vein, respectively, with an injection volume of 1.5×10 6 The nude mice were placed in an SPF environment to observe their behavior and health status. After 3 months of breeding, the nude mice were killed by euthanasia, and their lungs were separated for HE staining and observed using a slide scanner.

[0094] The results of this example are as follows Figure 7 As shown, Figure 7 A in the figure is the tumor volume result of subcutaneous transplantation of HepG2 cells overexpressing S100A16 gene. Figure 7 B in the figure is the tumor volume result of subcutaneous transplantation of HepG2 cells with knockdown of S100A16 gene. Figure 7 C in the figure is a live picture of nude mice with HepG2 cells overexpressing or knocking down the S100A16 gene transplanted subcutaneously. Figure 7 D in the figure is an in vitro picture of tumors transplanted subcutaneously with HepG2 cells overexpressing or knocking down the S100A16 gene. Figure 7 E in the figure is the tumor weight result of subcutaneous transplantation of HepG2 cells overexpressing S100A16 gene. Figure 7 F in the figure is the result of subcutaneous transplantation of HepG2 cells with S100A16 gene knockdown. Figure 7 G in the figure is the tumor volume result of subcutaneous transplantation of MHCC97L cells overexpressing S100A16 gene. Figure 7 H in the figure is the tumor volume result of subcutaneous transplantation of MHCC97L cells with knockdown of S100A16 gene. Figure 7Figure I is a live picture of nude mice with subcutaneous transplantation of MHCC97L cells overexpressing and knocking down the S100A16 gene. Figure 7 J in the figure is an in vitro picture of tumors transplanted subcutaneously with MHCC97L cells overexpressing or knocking down the S100A16 gene. Figure 7 K in the figure is the tumor weight result of subcutaneous transplantation of MHCC97L cells overexpressing S100A16 gene. Figure 7 The L in the figure is the weight of subcutaneous transplanted tumors of MHCC97L cells with knockdown of S100A16 gene. Figure 7 M in the figure is the HE staining result of lung tissue in the nude mouse lung metastasis experiment.

[0095] The experimental results of this example show that overexpression of S100A16 promotes the formation of subcutaneous tumors and lung metastases of liver cancer cells in nude mice, and knockdown of S100A16 inhibits the formation of subcutaneous tumors and lung metastases of liver cancer cells in nude mice.

Claims

1. A method for constructing a liver cancer cell line with knockdown of S100A16 gene, characterized in that: The following steps are involved: S1. Lentivirus Construction The S100A16 knockdown plasmid was constructed by homologous recombination method, and then the S100A16 knockdown plasmid was packaged into S100A16 knockdown lentivirus; S2. Lentivirus infection The liver cancer cell lines in good condition were inoculated into 24-well plates, and the cells were counted and the number of cells was 5×10 4 Cells / well were plated at a density of 1:

1. One day later, 5 μg / μL Polybrene-containing DMEM complete medium was added, and then the S100A16 knockdown lentivirus was added to the DMEM complete medium to infect the liver cancer cell line, and fresh culture medium was replaced within 24 hours of virus infection; S3. Subculture After the cells were infected with the virus for 72 hours, they were screened with DMEM complete medium containing 5 μg / mL puromycin. The conditions were maintained and culture and subculture were continued to obtain liver cancer cell lines with knockdown of the S100A16 gene. In step S1, the plasmid vector used for S100A16 knockdown plasmid was pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPRE; In step S2, the liver cancer cell line is MHCC97L or HepG2; In step S2, the liver cancer cell line was infected with lentivirus at MOI=80; In step S1, the knockdown S100A16 sh-RNA and the plasmid vector are recombined to construct the S100A16 knockdown plasmid; The S100A16 knockdown sh-RNA sequence is Sh-S100A16-18, and the Sh-S100A16-18 is shown in SEQ ID NO:

2.

2. Use of the liver cancer cell line with S100A16 gene knockdown obtained by the method for constructing the liver cancer cell line with S100A16 gene knockdown according to claim 1 in the preparation of drugs for inhibiting proliferation, invasion and migration of hepatocellular carcinoma.

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