CN115327125A - Application of a protein INF2 in the preparation of liver cancer diagnostic markers - Google Patents

Abstract

The invention discloses the application of protein INF2 in the preparation of liver cancer diagnostic markers, and is characterized in that the application of protein INF2 in the preparation of liver cancer diagnostic markers or in the preparation of liver cancer molecular targeted drugs, furthermore the gene fragment or gene expression of protein INF2 Application of spliceosome expression product in the preparation of molecular targeted drug for liver cancer; application of protein antibody encoded by protein INF2 gene in the preparation of molecular targeted drug for liver cancer; protein antibody encoded by protein INF2 gene in the preparation of molecular targeted drug for liver cancer It has the advantages of being used for auxiliary diagnosis of liver cancer. It has the characteristics of high sensitivity, strong specificity and short cycle, which is beneficial to the early diagnosis and treatment of liver cancer. In addition, INF2 has become a new biomarker and an effective therapeutic target in HCC. point potential.

Description

Application of a protein INF2 in the preparation of liver cancer diagnostic markers

technical field

The invention belongs to the field of molecular biology, and in particular relates to the application of INF2 protein as a diagnostic marker for liver cancer.

Background technique

Primary liver cancer (PLC) is a malignant tumor with insidious onset and high fatality. According to the data released by GLOBOCAN 2020, liver cancer is the sixth most common cancer and the third leading cause of cancer-related death worldwide. Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is the most common pathological type of liver cancer. Hepatitis B has always been the main cause of HCC. In recent years, the incidence of hepatocellular carcinoma caused by metabolic factors such as nonalcoholic fatty liver disease (NAFLD) is increasing year by year. Surgical resection, liver transplantation, and local radiofrequency ablation are the first choice for patients with early HCC, while transcatheter arterial chemotherapy (TACE) is the first choice for patients with mid-stage HCC, and systemic therapy is preferred for patients with advanced HCC. The prognosis of HCC is closely related to the tumor stage. The 5-year survival rate of patients with early HCC after standard treatment exceeds 70%, while the median survival period of advanced HCC after systemic treatment is only 1-1.5 years. The global 5-year survival rate of liver cancer is 18%. %. my country is a big country with hepatitis B, and the annual new cases of HCC account for more than half of the world, while the 5-year survival rate is only about 12%. Liver cancer is a global health challenge and a serious medical problem. Clinical early diagnosis and early treatment are extremely important for improving prognosis. With the increase in the incidence of liver cancer and the in-depth research on the pathogenesis of HCC, more and more molecular markers have been found to be related to the diagnosis, treatment and prognosis of liver cancer. Early diagnosis, early treatment and prognosis assessment are of great significance. Mitochondria are dynamic organelles that do not break and fuse in cells to form a steady state, a process called mitochondrial dynamics. Emerging evidence shows that dysregulation of mitochondrial dynamics contributes to the occurrence and development of tumors. Different types of tumor cells exhibit mitochondrial fragmentation. transition, EMT) and migration.

Hepatocytes are rich in mitochondria. A large number of mitochondria are involved in key metabolic processes such as hepatic tricarboxylic acid cycle (tricarboxylic acid, TCA) and free fatty acid β-oxidation, and maintain liver homeostasis. Mitochondrial dysfunction can lead to a series of liver diseases. The continuous accumulation of damaged mitochondria is an important reason for the occurrence of liver cancer. Liver cancer has a strong ability to migrate and invade, and is prone to recurrence and metastasis, which is also inseparable from the disorder of mitochondrial dynamics. Studies have shown that mitochondrial fission is hyperactive in liver cancer tissues, and in highly metastatic liver cancer, mitochondrial hypertrophy and mitochondrial fragmentation are particularly evident.

Trans-formin 2 (inverted formin 2, INF2) is an atypical transparent-associated formin protein that plays an important role in the polymerization and depolymerization of actin. Existing studies have found that mitochondrial dysfunction mediated by INF2-mediated mitochondrial dynamics can affect tumorigenesis and development, and is associated with tumor prognosis. We mainly used immunohistochemistry to investigate the expression level of INF2 in HCC, and explored the possibility of INF2 as a marker of liver cancer. The effect of proliferation and migration will be helpful for the early diagnosis of liver cancer, the development of targeted anti-tumor drugs, and the assessment of prognosis, so as to realize precision medicine and individualized treatment. At present, there is no published research report on using INF2 as a diagnostic marker in liver cancer at home and abroad.

Contents of the invention

The technical problem to be solved by the present invention is to provide the application of a protein INF2 with high sensitivity and specificity and a positive correlation with the prevalence of liver cancer in the preparation of liver cancer diagnostic markers or liver cancer molecular targeting drugs.

The technical solution adopted by the present invention to solve the above technical problems is: the application of a protein INF2 in the preparation of liver cancer diagnostic markers. The protein INF2 is composed of 1240 amino acids, and the protein size is about 134.6 kDa.

An application of protein INF2 in the preparation of liver cancer molecular targeting drugs.

Further, the application of the expression product of the gene fragment or spliced body of the protein INF2 in the preparation of molecularly targeted drugs for liver cancer.

Further, the application of the protein antibody encoded by the gene of protein INF2 in the preparation of liver cancer molecular targeting drug.

Further, the application of the protein antibody encoded by the gene of the protein INF2 in the preparation of liver cancer molecular targeted drugs.

Compared with the prior art, the present invention has the advantage that: the present invention discloses for the first time the protein INF2 that can be used to detect liver cancer and its application in the preparation of liver cancer detection reagents or auxiliary diagnostic liver cancer reagents. The histochemical technology was completed, and the immunohistochemical staining pictures were analyzed, and the expression of INF2 protein in liver cancer patient samples and normal samples was calculated and compared by Image J software, so as to assist in the diagnosis of liver cancer. Compared with the traditional liver cancer detection technology, it has the characteristics of high sensitivity, strong specificity, and short cycle, which is beneficial to the early diagnosis and treatment of liver cancer, and INF2 has the potential to become a new biomarker and an effective therapeutic target in HCC.

Description of drawings

Figure 1 is the immunohistochemical verification of the expression of INF2 in human HCC tissues. (a) Three representative images of INF2 immunohistochemical staining in 50 pairs of human HCC tissues (n=50, scale bar: 10 μm); (b) Immunohistochemical staining results of 50 pairs of human HCC tissues and paired paracancerous tissues Stacked histogram of (n=50, ***P<0.001). (where "negative" means INF2 expression, "low positive" means INF2 low expression, "negative" means INF2 high expression);

Figure 2 is the screening of INF2 knockout HepG2 by Western blotting (KO: INF2 knockout HepG2);

Fig. 3 is the colony formation to verify the effect of INF2 expression level on the growth of HepG2 cells. (a) Colony formation assay to detect HepG2 colony formation ability; (b) Quantification of colony formation assay results (ns means no statistical difference, ***P<0.001). (Control: wild-type HepG2, INF2-OE: INF2 high expression, INF2-KO: INF2 knockout);

Figure 4 is a scratch experiment to verify the effect of INF2 expression level on the migration of HepG2 cells. (a) Migration experiments of HepG2 cells after INF2 was overexpressed or knocked out, using a 200 µL pipette tip to scratch the monolayer of HepG2 cells; (b) Quantification of cell migration results (ns means no statistical difference, ***P<0.001). (Control: wild-type HepG2, INF2-OE: INF2 high expression, INF2-KO: INF2 knockout);

Figure 5 is the Transwell migration experiment to verify the effect of INF2 expression level on HepG2 cell migration (a) Migration experiment of HepG2 cells after overexpression or knockout of INF2; (b) Quantification of cell migration experiment results (ns means no statistical difference, ** *P<0.001). (Control: wild-type HepG2, INF2-OE: INF2 high expression, INF2-KO: INF2 knockout).

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

1. Experimental method

1. Collection of liver cancer tissue samples

The 69 pairs of human liver cancer tissue samples included in the study were diagnosed as HCC from January 1, 2021 to December 31, 2021 by the Ningbo Clinical Pathological Diagnosis Center. Among them, 50 pairs of formalin-fixed human liver cancer tissue samples were obtained from Ningbo Clinical Pathological Diagnosis Center, and 19 pairs of human fresh liver cancer tissue samples were obtained from Li Huili Hospital of Ningbo Medical Center.

(1) Collection, processing and storage of fresh human liver cancer tissue specimens: under the premise of meeting the inclusion criteria and not affecting the pathological diagnosis, special personnel will collect liver cancer tissue and paracancerous tissue (adjacent to cancer tissue) within 30 minutes of specimen isolation. 1−3 cm), cut into small pieces (0.5×0.5×0.4 cm), wash the residual blood on the specimen with normal saline, put the specimen into the specimen bottle, tighten the bottle cap, and note the patient information and collection date, etc. If there is no liquid nitrogen in the laboratory, store it in a −80°C refrigerator.

(2) Collection, processing and storage of formalin-fixed liver cancer tissue specimens: formalin specimens are the remaining liver tissues after hepatectomy and pathological diagnosis, and put them in a sufficient amount of 10% neutral formalin solution at room temperature Fixed. Add an appropriate amount of 4% paraformaldehyde fixative solution to the 1.8 mL specimen tube before collecting the specimen, and a professional pathologist will take a piece of liver cancer and paracancerous tissue (0.5×0.5×1 cm) from each sample, and put them into 1.8 mL Specimen tube, submerge the specimen in the liquid, screw the cap tightly, store in the refrigerator at 4°C for later use, and replace the 4% paraformaldehyde fixative regularly.

Inclusion criteria: 1. The pathological diagnosis of the sample is clear, and the diagnosis is primary hepatocellular carcinoma; 2. The patient has not received preoperative radiotherapy and chemotherapy; 3. The patient has no previous malignant tumors of other systems. The exclusion criteria are: 1. Combined with other systemic malignancies; 2. Patients who have received radiofrequency ablation before surgery; 3. Patients with infectious diseases such as syphilis and AIDS. All patients were treated by hepatectomy, and the specimens were obtained after signed informed consent from the patients. All human tissue samples involved in this experiment were applied by the research group, and with the consent of the Human Ethics Committee of Ningbo University School of Medicine, they were limited to laboratory research.

2. Immunohistochemical staining of formalin-fixed tissue specimens

(1) Tissue fixation and embedding

a. Liver cancer tissue fixation: Clip the formalin-fixed liver cancer tissue sample on the spread plastic wrap with tweezers, cut it into 0.5×0.5×0.2 cm tissue pieces with a sterile blade, soak in 4% paraformaldehyde More than 24 hours in the solution;

b. Washing: String the tissue embedding boxes with white cotton thread of the same length, place the fixed tissue in the tissue embedding box with sterile forceps, cover the embedding box tightly, and mark the relevant information of the sample on the embedding box with a pencil , put the embedding cassette in a foam box, rinse it under running tap water for 5 minutes, and drain the water;

c. Dehydration: put the tissue embedding cassettes into glass cylinders with different concentrations of ethanol prepared in advance (regularly replace the ethanol in the glass cylinder) for gradient dehydration, and immediately cover the glass cylinder lid after the embedding cassettes are placed in the glass cylinder , The cotton thread is placed outside the glass cylinder to facilitate the cylinder change. Soak in 75% ethanol solution for 1 h×2 times; soak in 80% ethanol solution for 1 h×2 times; soak in 95% ethanol solution for 1 h×2 times; soak in 100% ethanol solution for 1 h×2 times, finally drain and embed ethanol solution on the box;

d. Transparent: soak the tissue embedding cassette in xylene solution for 40 min×2 times;

e. Wax immersion: Put the wax baths containing paraffin slices with a melting point of 58−60°C in a 60°C oven to melt in advance, put the tissue embedding cassettes into the three wax baths in the oven in turn, and soak at least 1 in each wax bath. h, the embedding cassette should be fully immersed in liquid paraffin;

f. Embedding: Preheat the embedding machine in advance to melt the paraffin in the machine. After the paraffin is completely melted, put the iron box for embedding into the wax tank on the left side of the embedding machine, and take out the embedding box from the wax tank in the oven. Put it into the wax bath on the right side of the embedding machine. Remove the embedding box, put the tissue in the middle of the embedding iron box, fix the tissue with tweezers, pour liquid paraffin into the embedding iron box to submerge the tissue block, remove the cover of the plastic embedding box, and gently cover it on the box. Embed in the iron box, place it on a 4°C freezer to cool into a solid wax block, discard the embedding iron box, put the wax block in a sealed bag, and store it in a 4°C refrigerator for later use.

(2) Immunohistochemical staining

a. Slicing: Use a paraffin microtome to cut the tissue roughly at 10 μm to remove tissue-free paraffin, and polish the surface of the wax block smooth. Cut the tissue wax block into 4 μm thin slices, put them in a water bath at 40°C to spread the slices, select the thin slices with complete tissue and regular shape, pick up the tissue slices with a positively charged glass slide, and attach them to the slide , Mark the relevant information of the specimen with a pencil at the end of the slide;

b. Bake slices: Place the slides with tissues on a preheated 65°C oven for at least 2 hours, or put them in an oven at 37°C overnight;

c. Dewaxing and rehydration: soak slide racks full of sections in xylene for 20 min; soak in absolute ethanol solution for 5 min x 2 times; soak in 95% ethanol solution for 5 min x 1 time; Soak in % ethanol solution for 5 min x 1 time; finally place the slide rack in deionized water and wash for 5 min;

d. Antigen restoration: dilute 50× sodium citrate antigen restoration solution with deionized water to 1× and shake evenly, pour 1× sodium citrate antigen restoration solution into the pressure cooker, close the lid, and wait until the liquid in the pot After boiling, place the slide rack with slices in the pressure cooker, completely submerge the slices in the sodium citrate antigen restoration solution, tighten the lid of the pot, and start timing after the gas valve of the pressure cooker starts to emit gas evenly, and turn off the power of the induction cooker after 8 minutes. End heating. Place the pressure cooker under flowing tap water to cool down, open the lid, and let the paraffin sections cool down to room temperature;

e. Washing: Firstly, wash the sections thoroughly with deionized water for 5 minutes, then wash the sections with PBS buffer for 3 minutes, and repeat the washing 3 times. ;

f. Block endogenous peroxidase: dilute 10% hydrogen peroxide aqueous solution with deionized water to a concentration of 3%, prepare it for each time, put the paraffin section in a wet box, and place the tissue on the glass slide Add an appropriate amount of 3% hydrogen peroxide solution dropwise, close the lid of the wet box, and seal it at 37°C for 10 min;

g. Washing: wash the section with PBS phosphate buffered saline for 3 min×3 times, and shake off the liquid on the slide;

h. Serum blocking: the washed slices are placed in a wet box, and an appropriate amount of 10% donkey serum blocking solution is added dropwise to each slice, and after blocking for 15 minutes at room temperature, shake off the blocking solution on the slices;

i. Primary antibody incubation: wipe off the blocking solution around the tissue, draw a circle around the tissue with a special hydrophobic pen for immunohistochemical staining, place the section in a wet box, add the appropriate concentration of INF2 antibody diluent dropwise, and immerse the antibody diluent tissue, close the lid of the wet box, and place in a 4°C refrigerator overnight;

j. Washing: wash the slices with PBS phosphate buffered saline for 3 min×3 times;

k. Secondary antibody incubation: wipe off the liquid around the tissue, drop an appropriate concentration of HRP-labeled donkey secondary antibody on the tissue to make the antibody completely cover the tissue, close the lid of the wet box, and incubate at 37°C for 1 h;

l. Washing: wash the slices with PBS phosphate buffered saline for 3 min×3 times;

m. DAB color development: add DAB color development solution to the position of the tissue on the slide, place the slide glass on an inverted microscope to observe the staining of the tissue, discard the color development solution when the staining intensity reaches the best, and use the Wash with deionized water for 3 min×3 times;

n. Hematoxylin counterstaining: drop an appropriate amount of modified Lillie-Mayer hematoxylin staining solution on the position of the tissue on the slide, stain for 2 minutes, shake off the staining solution, and wash the slide rack with slides under running tap water for 10 minutes. min to return blue;

o. Dehydration: Soak slide racks with slices in 75% ethanol solution for 5 min×1 time; soak in 95% ethanol solution for 5 min×1 time; soak in absolute ethanol solution for 5 min×3 times;

p. Transparent: Soak the slices in xylene solution for 5 min x 2 times;

q. Seal the slide: Add an appropriate amount of neutral gum to the position of the tissue on the slide, use tweezers to pick up a cover glass and gently cover it on the slide, and place it in a fume hood to dry to form a piece;

r. Scoring: Place the slices on an inverted microscope, and observe the tissue morphology, nuclear staining, and target protein staining under low-power and high-power lenses in turn.

3. Screening of INF2 knockout cell lines by western blotting and DNA sequencing

(1) Transfect HepG2 and C3A liver cancer cells with the CRISPR-cas9-INF2 plasmid, digest the cells into a uniform cell suspension after 48 hours, and count the cells according to the above method. Take a six-well plate, plant the cell suspension into the six-well plate at 1000 cells per well, and add medium to 2 mL;

(2) Put the six-well plate in a constant temperature incubator and cultivate it for 10 days. When cell colonies are visible to the naked eye, digest a single cell colony with trypsin and transfer it to a 24-well plate. Place in a constant temperature incubator to continue culturing, each well of monoclonal cells as a group;

(3) After the cells evenly cover the 24-well plate, transfer the cells of each group to the 12-well plate according to the above method, and transfer the cells to the 6-well plate in the same way after the cells are evenly covered on the well plate. When the cell density reaches 80%, transfer the cells to the 12-well plate Each group of cells in the six-well plate was subcultured to a new 6-well plate according to 1 passage and 3, and placed in a constant temperature incubator to continue culturing;

(4) When the density of monoclonal cells reached 80%, cells in one well of each group were digested into a homogeneous single cell suspension, centrifuged at 1,000 rpm for 4 min, the supernatant was discarded, washed twice with PBS buffer, and finally washed with 100 Resuspend the cell pellet in µL PBS buffer, and gently pipette until the cell suspension is uniform;

(5) Genomic DNA extraction of monoclonal cells: Add 100 µL of PBS Solution to the above cell suspension and mix well. Then add 20 µL Proteinase K and blow evenly. Then add 200 µL Buffer DL, vortex to mix, and place in a water bath at 56°C for 10 min. Seeing that the mixture becomes clear, it indicates that the lysis is complete. Add 200 µL absolute ethanol to the above centrifuge tube and shake well. Connect the adsorption column to the collection tube, transfer the mixture to the adsorption column, let it stand for 2 min, centrifuge at 10,000 rpm for 1 min, and discard the liquid in the collection tube. Add 500 μL of GW Solution to the adsorption column, centrifuge at 10,000 rpm for 0.5 min and discard the waste liquid. Add 700 μL of eluent to the adsorption column, centrifuge at 10,000 rpm for 0.5 min, discard the waste liquid, and empty for 2 min, open the cover of the adsorption column, and place it at room temperature for several minutes to completely dry the residual eluent. Put the adsorption column into a new 1.5 mL centrifuge tube, add 50 μL CE Buffer and let it stand for 3 minutes, then centrifuge at 12,000 rpm for 2 minutes at room temperature to collect the DNA solution of monoclonal cells;

(6) Genome-level identification of INF2 gene editing: Take the above-mentioned monoclonal cell DNA solution that has been extracted, and use PCR to amplify the INF2 target fragment from the cell genome DNA template. The products obtained by PCR were subjected to agarose gel electrophoresis, and the INF2 gene editing status of the monoclonal cells was preliminarily judged according to the band results. The PCR product of the cell clone INF2 with the possibility of homozygous deletion was selected, and the PCDH-CD513B-cas9 vector was ligated and cloned. The ligation product was transformed into competent DH5a, and cultured overnight on a kanamycin plate. Pick 5−6 colonies from each group, amplify and extract the recombinant plasmid PCDH-CD513B-cas9 vector, and detect the DNA sequence (send to Nanjing Qingke Biotechnology Co., Ltd.). The obtained 7 INF2 DNA sequencing sequences were compared with the STING gene template to determine the deletion of the recognition sequence of the INF2 gene sgRNA by CRISPR-Cas9;

(7) Western blotting to screen INF2 knockout cell lines: Take monoclonal cells in one well of a six-well plate, discard the complete medium, wash the adherent cells twice with PBS buffer, suck off the PBS thoroughly, and add 200 μL The cell lysate was shaken on a constant temperature shaker at 4°C for 20 min, the cell lysate was transferred to a 1.5 mL centrifuge tube, and placed in a −80°C refrigerator overnight. The cell samples were thawed on ice, and subsequent operations were performed according to the western blotting technique described above.

4. Cloning formation

(1) Take well-growing HepG2 liver cancer cells, INF2 knockout cell lines, and well-growing HepG2 liver cancer cells after transient transfection of CD513B-INF2 plasmid for 24 hours, and digest the adherent cells into a uniform cell suspension;

(2) Take 20 µL of the above uniform cell suspension and add it to the cell counting plate, measure its concentration with a cell counter, dilute the cell suspension according to the corresponding ratio, and pipette evenly;

(3) Take several 6-well cell plates, add 1×103 cells to each well, quantify 2 mL, set up 3 duplicate wells for each treatment group, shake well using the “8” method, place in a constant temperature incubator, Change the medium once every 5 days, and co-culture for 10−15 days;

(4) When cell colonies are visible to the naked eye in the six-well plate, discard the culture medium in the well, rinse with PBS buffer twice, add an appropriate amount of 4% paraformaldehyde to each well to fix the cells, and place for 30 min;

(5) Discard the fixative, rinse twice with PBS buffer, add 800 µL of 0.1% crystal violet dye to each well, place on a shaker for staining for 10 min, recover the dye, rinse three times with PBS buffer, and place Oven overnight at 37°C, take pictures and count the data after the six-well plate is dried.

5. Scratch test

(1) Take well-growing HepG2 liver cancer cells, INF2 knockout cell lines and well-growing HepG2 liver cancer cells after transient transfection of CD513B-INF2 plasmid for 24 hours, and digest the adherent cells into a uniform cell suspension;

(2) Take 20 µL of the above uniform cell suspension and add it to the cell counting plate, measure its concentration with a cell counter, dilute the cell suspension according to the corresponding ratio, and pipette evenly;

(3) Take two six-well cell plates, draw 5 horizontal lines at intervals of 0.5−1 cm on the back of the six-well plate with a marker, inoculate 300,000 cells/well, and culture in a constant temperature incubator;

(4) When the cell density reaches 80%−90%, use a 200 µL pipette tip to draw a vertical line according to the horizontal line on the back of the six-well plate, wash off the floating cells with PBS buffer, replace with serum-free DMEM, and place in the incubator continue to train;

(5) Observe the healing of scratches at 0, 24, and 48 h under a microscope, take pictures and store them for later use, and use Image J software to measure the healing area of scratches in each group.

6. Transwell migration experiment

(1) Take well-growing HepG2 liver cancer cells, INF2 knockout cell lines and well-growing HepG2 liver cancer cells after transient transfection of CD513B-INF2 plasmid for 24 hours, and digest the adherent cells into a uniform cell suspension;

(2) Collect the cell suspension into a 1.5 mL centrifuge tube and centrifuge at 1,000 rpm for 4 min;

(3) Discard the supernatant, resuspend the cell pellet with 1 mL PBS solution, centrifuge again according to the above method, resuspend the cells with 1 mL DMEM, take a cell counting plate for counting, and dilute with DMEM as needed;

(4) Pick up 3 Transwell chambers with sterile tweezers, put them into a new 24-well cell plate, add 150 µL of the above cell suspension to the upper chamber, quantify 5×104 cells, and add 500 µL complete medium to the lower chamber, After standing for 30 minutes, put the orifice plate into the constant temperature incubator;

(5) Discard the medium inside and outside the chamber after 48 hours, and rinse the chamber and the bottom of the well plate twice with PBS buffer;

(6) Add 500 µL of paraformaldehyde fixative solution to the chamber and let it stand for 30 min;

(7) Discard the fixative, rinse twice with PBS solution, add 500 µL of 0.1% crystal violet dye, and place the well plate on a shaker for 20 min;

(8) Recover the staining agent, rinse with PBS solution for 3 times, dry the chamber naturally, take pictures and make statistical data.

7. Result analysis

This experiment ensures that the experimental data and the collected original clinical data are fully inspected, proofread, and organized to ensure that the data are as complete, accurate, and error-free as possible. Then use Excel software to establish tissue and cell databases, group and summarize the relevant clinical raw data and enter them into the table. Finally, SPSS 26.0 statistical software was used to organize and analyze the final experimental data. All data statistical tests are based on two-sided probability, and the statistical test is carried out according to the test level of α=0.05. If P<0.05, the difference between the two groups is considered to be statistically significant, otherwise, the difference is considered to be not statistically significant. The drawing of experimental result graphs such as histograms and line graphs was completed by using GraphPad Prism 9.0 and SPSS 26.0 software.

2. Experimental results

In order to examine the expression of INF2 in liver cancer tissues, we collected 50 pairs of formalin-fixed human liver cancer tissue specimens, embedded and paraffin-sectioned, and detected the expression level of INF2 in clinical samples by immunohistochemical staining. The results of immunohistochemical staining showed that INF2 staining showed different shades of brownish yellow and was mainly located in the cytoplasm of liver cancer tissues and adjacent tissues, and a small amount was located on the cell membrane. Compared with liver cancer tissues, INF2 was expressed in corresponding adjacent tissues Weak or not expressed. Using Image J software to analyze the immunohistochemical staining pictures, the results showed that the expression level of INF2 in liver cancer tissues was concentrated in three grades: negative (1 point), low positive (2 points), and positive (3 points) (Figure 1a). Meanwhile, in HCC tissues, the positive expression rate of INF2 was 86% (43/50), while that in paracancerous tissues was only 46% (23/50), the difference was statistically significant (X2=17.825, P<0.05). The expression level of INF2 in liver cancer tissues was significantly higher than that in corresponding paracancerous tissues (P<0.001) (Fig. 1b).

In order to further verify the function of INF2 protein in subsequent cell function experiments, we screened INF2 knockout cell lines. We knocked out INF2 in HepG2 using CRISPR-Cas9 technology, selected INF2-knockout cell colonies, and used Western blot technology and DNA sequencing technology for further confirmation. The final results showed that cell colonies 2 and 6 were INF2 knockout cell lines, and DNA sequencing showed that INF2-KO6 was a liver cancer cell colony with correct INF2 knockout. Expanding the culture of INF2-KO6 and supplementing cell function experiments (Figure 2), the cell line can Stable knockout of endogenous INF2 in cells was compared with transiently transfected highly expressed INF2.

In further experiments to verify the effect of INF2 on the proliferation and migration of liver cancer cells, we used colony formation experiments, cell scratch experiments and Transwell migration experiments. Colony formation experiments showed that, compared with wild-type liver cancer cells, the number of cell colonies in the INF2 high expression group was significantly increased, while the INF2-KO6 group had no statistically significant difference compared with the Control group (Figure 3. a and b). It shows that INF2 has the ability to promote the proliferation of liver cancer cells.

The results of cell scratch experiments suggested that the upregulation of INF2 expression promoted the migration of HepG2 cells (Figure 4. a and b). Transwell migration assay showed that in HepG2 cells, the number of cells penetrating the Transwell chamber in the INF2 high expression group was significantly higher than that in the Control group (Figure 5. a and b). It shows that INF2 has the ability to promote the migration of liver cancer cells.

These results suggest that the detection of INF2 protein has a relatively high value in the diagnosis of liver cancer.

By detecting the expression of INF2 in liver cancer tissue and the effect of high or low expression of INF2 on the proliferation and migration of liver cancer cells, combined with statistical principles and modern biological techniques, the invention provides high sensitivity, strong specificity, short cycle and stable results. The detection method provides a scientific basis for the diagnosis and treatment of liver cancer patients, and provides the possibility for molecular targeted therapy.

The above description does not limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention shall also belong to the protection scope of the present invention.

Claims (5)

1. The application of a protein INF2 in the preparation of liver cancer diagnostic markers. 2. The application of a protein INF2 in the preparation of liver cancer molecular targeting drugs. 3. The application of a protein according to claim 2, characterized in that: the application of the expression product of the gene fragment or spliced body of the protein INF2 in the preparation of liver cancer molecular targeting drugs. 4. The application of a protein according to claim 2, characterized in that: the application of the protein antibody encoded by the gene of the protein INF2 in the preparation of molecular targeted drugs for liver cancer. 5. The application of a protein according to claim 2, characterized in that: the application of the protein antibody encoded by the gene of the protein INF2 in the preparation of molecular targeting drugs for liver cancer.

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