CN115327125A

CN115327125A - Application of protein INF2 in preparation of liver cancer diagnosis marker

Info

Publication number: CN115327125A
Application number: CN202210825819.6A
Authority: CN
Inventors: 金加孛; 金晓锋; 王海彪; 汪洁; 李新铭; 林缦
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2022-07-14
Filing date: 2022-07-14
Publication date: 2022-11-11
Also published as: CN116735875A

Abstract

The invention discloses an application of protein INF2 in preparing a liver cancer diagnosis marker, which is characterized in that the protein INF2 is applied in preparing the liver cancer diagnosis marker or a liver cancer molecule targeted drug, and further the application of a gene fragment of the protein INF2 or a gene shear expression product in preparing the liver cancer molecule targeted drug; the application of protein antibody coded by protein INF2 gene in preparing liver cancer molecular targeted medicine; the application of the protein antibody of the gene coding of the protein INF2 in the preparation of liver cancer molecular targeted drugs has the advantages of being used for auxiliary diagnosis of liver cancer, having the characteristics of high sensitivity, strong specificity, short period and the like, being beneficial to early diagnosis and treatment of liver cancer, and having the potential of becoming a novel biomarker and an effective treatment target in HCC (liver cancer).

Description

Application of protein INF2 in preparation of liver cancer diagnosis marker

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to application of INF2 protein as a liver cancer diagnosis marker.

Background

Primary Liver Cancer (PLC) is an insidious, highly fatal malignant tumor. According to the data published by GLOBOCAN 2020, liver cancer is the sixth most common cancer and the third most common cause of cancer-related death worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer pathology. Hepatitis b has been a major cause of HCC, and in recent years, the incidence of hepatocellular carcinoma caused by metabolic factors such as nonalcoholic fatty liver disease (NAFLD) has been increasing year by year. Early HCC patients were first treated with surgical resection, liver transplantation and local radio frequency ablation, while middle patients were the first choice for hepatic artery embolization chemotherapy (TACE) and late HCC patients were first treated systemically. The prognosis of HCC is closely related to the stage of tumor, the 5-year survival rate of patients with early HCC exceeds 70 percent after standard treatment, the median survival rate of patients with late HCC after systemic treatment is only 1 to 1.5 years, and the 5-year survival rate of liver cancer is 18 percent all over the world. China is a big country with hepatitis B, new cases of HCC are more than half of the world, and the 5-year survival rate is only about 12%. Liver cancer is a global health challenge and a serious medical problem, and early diagnosis and treatment in clinical practice are extremely important for improving prognosis. With the increase of incidence of liver cancer, people deeply research the pathogenesis of HCC, more and more molecular markers are found to be related to the diagnosis, treatment and prognosis of liver cancer, and the research on the molecular mechanism and the biomarker related to liver cancer has important significance on the early diagnosis, early treatment and prognosis evaluation of liver cancer. Mitochondria are dynamic organelles that continuously fission and fuse within cells to eventually form a stable state, a process known as mitochondrial dynamics. New evidence suggests that dysregulation of mitochondrial dynamics contributes to the development and progression of tumors, that different types of tumor cells exhibit mitochondrial fragmentation, and that inhibition of mitochondrial division reduces various tumor cell growth, epithelial-mesenchymal transition (EMT), and migration.

The liver cells are rich in mitochondria, a large number of mitochondria are involved in key metabolic processes such as hepatic tricarboxylic acid (TCA) and beta oxidation of free fatty acid, and maintain hepatic homeostasis, and mitochondrial dysfunction can cause a series of liver diseases. The continuous accumulation of damaged mitochondria is an important reason for liver cancer occurrence, and liver cancer has strong migration and invasion capacity and is easy to relapse and transfer, which is also inseparable from mitochondrial dynamics disorder. Research shows that the liver cancer tissue has hyperfunction of mitochondria division, and in the highly metastatic liver cancer, the hyperfunction of mitochondria division and the fragmentation of mitochondria are particularly obvious.

trans-Formin protein 2 (INF 2) is an atypical, transparent related Formin protein that plays an important role in the polymerization and depolymerization of actin. The current research finds that the dysfunction of mitochondria caused by INF2 mediated mitochondrial dynamics can influence the tumorigenesis and development and is related to the tumor prognosis. The expression level of INF2 in HCC is mainly explored by an immunohistochemistry technology, the possibility that INF2 serves as a liver cancer marker is discussed, meanwhile, the influence of INF2 on proliferation and migration of the HCC is found in liver cancer cells by a clone formation and scratch experiment and a 2.2.19 Transwell migration experiment, and the method is beneficial to early diagnosis of liver cancer, research and development of targeted antitumor drugs, prognosis evaluation and the like, so that accurate medical treatment and individualized treatment are realized. At present, no relevant research report about INF2 as a diagnostic marker in liver cancer is published at home and abroad.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of protein INF2 which has high sensitivity and specificity and is positively correlated with the prevalence rate of liver cancer in the preparation of liver cancer diagnosis markers or liver cancer molecular targeted drugs.

The technical scheme adopted by the invention for solving the technical problems is as follows: an application of protein INF2 in preparing liver cancer diagnosis marker. The protein INF2 consists of 1240 amino acids and has a protein size of about 134.6 kDa.

An application of protein INF2 in preparing liver cancer molecule targeted medicine is provided.

Further, the gene fragment of the protein INF2 or the gene splice expression product is applied to the preparation of liver cancer molecular targeted drugs.

Further, the protein antibody coded by the gene of the protein INF2 is applied to the preparation of liver cancer molecular targeted drugs.

Further, the protein antibody coded by the gene of the protein INF2 is applied to preparing a liver cancer molecular targeted drug.

Compared with the prior art, the invention has the advantages that: the invention discloses protein INF2 related to liver cancer and application thereof in preparation of a reagent for detecting liver cancer or a reagent for assisting in diagnosis of liver cancer for the first time, wherein the detection is mainly completed through an immunohistochemical technology, immunohistochemical staining pictures are analyzed, and the expression of the INF2 protein in a liver cancer patient sample and a normal sample is calculated and compared through Image J software so as to assist in diagnosis of liver cancer. Compared with the traditional liver cancer detection technology, the kit has the characteristics of high sensitivity, strong specificity, short period and the like, is favorable for early diagnosis and treatment of liver cancer, and INF2 has the potential of becoming a novel biomarker and an effective treatment target in HCC.

Drawings

FIG. 1 shows immunohistochemical verification of INF2 expression 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) Stacked histograms of immunohistochemical staining of 50 pairs of human HCC tissues and paired paracarcinoma tissues (n =50, P < 0.001). (wherein "negative" indicates INF2 expression, "low positive" indicates INF2 low expression, and "negative" indicates INF2 high expression);

FIG. 2 is a Western blotting screening of INF2 knockout HepG2 (KO: INF2 knockout HepG 2);

FIG. 3 is a graph demonstrating the effect of INF2 expression levels on HepG2 cell growth by colony formation. (a) a clone formation assay to detect HepG2 colony forming ability; (b) Quantification of the colony formation assay results (ns stands for no statistical difference,. P < 0.001). (Control: wild-type HepG2, INF2-OE: high expression of INF2, INF2-KO: INF2 knock-out);

fig. 4 is a scratch experiment to verify the influence of INF2 expression level on HepG2 cell migration, (a) migration experiment of HepG2 cells after INF2 overexpression or knock-out, and a 200 μ L pipette tip was used to scratch a monolayer of HepG2 cells; (b) Cell migration assay results were quantified (ns represents no statistical difference,. P < 0.001). (Control: wild-type HepG2, INF2-OE: INF2 high-expression, INF2-KO: INF2 knock-out);

FIG. 5 is a Transwell migration experiment demonstrating the effect of INF2 expression levels on migration of HepG2 cells (a) migration experiment of HepG2 cells after overexpression or knock-out of INF 2; (b) Cell migration assay results were quantified (ns represents no statistical difference,. P < 0.001). (Control: wild-type HepG2, INF2-OE: high expression of INF2, INF2-KO: INF2 knock-out).

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

1. Experimental methods

1. Liver cancer tissue specimen collection

The 69 human liver cancer tissue samples included in the study were from the clinical pathology diagnosis center of Ningbo city, 2021, 01-2021, 31-01, and were confirmed to be HCC. Wherein 50 pairs of human liver cancer tissue specimens fixed by formalin are from Ningbo city clinical pathological diagnosis center, and 19 pairs of human fresh liver cancer tissue specimens are from Ningbo city medical center Lihuili hospital.

(1) Collecting, processing and preserving a fresh human liver cancer tissue specimen: under the premise of meeting inclusion standards and not influencing pathological diagnosis material taking, a person specially uses the method to take liver cancer tissues and tissues beside the cancer (within 1-3 cm beside the cancer tissues) within 30min in vitro of a specimen, cut the liver cancer tissues and the tissues beside the cancer into small blocks (0.5 multiplied by 0.4 cm), wash residual blood on the specimen with physiological saline, place the specimen into a specimen bottle, screw a bottle cap tightly, annotate information of patients, collect date and other information, immediately place the specimen bottle into liquid nitrogen for storage, and store the specimen bottle in a refrigerator at the temperature of-80 ℃ when no liquid nitrogen exists in a laboratory.

(2) Collecting, processing and preserving a formalin-fixed liver cancer tissue specimen: the formalin specimen is the residual liver tissue after hepatectomy and pathological diagnosis, and is fixed in 10% neutral formalin solution at normal temperature. Before collecting the specimen, adding a proper amount of 4% paraformaldehyde fixing solution into a 1.8 mL specimen tube, taking one specimen (0.5 multiplied by 1 cm) of liver cancer and tissues beside the cancer from each specimen by a professional pathologist, respectively placing the specimen tube into the 1.8 mL specimen tube, immersing the specimen in the solution, screwing a cover, placing the specimen tube into a refrigerator at 4 ℃ for storage for later use, and periodically replacing the 4% paraformaldehyde fixing solution.

Inclusion criteria were: 1. the pathological diagnosis of the sample is definite, and the primary hepatocellular carcinoma is confirmed; 2. the patient does not receive the treatment of radiotherapy and chemotherapy before operation; 3. the patient had no other systemic malignancies. Exclusion criteria were: 1. merging other systemic malignancies; 2. patients who had received radiofrequency ablation damage therapy before surgery; 3. patients with infectious diseases such as syphilis and AIDS. All patients were treated by hepatectomy, and specimens were obtained after patients signed informed consent. All the human tissue specimens related to the experiment are applied by subject groups, and only limited to laboratory research under the condition agreed by human ethics committees of Ningbo university medical college.

2. Immunohistochemical staining of formalin-fixed tissue specimens

(1) Tissue fixation and embedding

a. Liver cancer tissue fixation: clamping the formalin-fixed liver cancer tissue sample on a spread preservative film by using a pair of forceps, cutting the formalin-fixed liver cancer tissue sample into tissue blocks with the size of 0.5 multiplied by 0.2 cm by using a sterile blade, and soaking the tissue blocks in 4% paraformaldehyde solution for more than 24 hours;

b. washing: respectively stringing the tissue embedding boxes by using white cotton threads with consistent lengths, placing the fixed tissues in the tissue embedding boxes by using sterile forceps, covering the embedding boxes tightly, marking sample related information on the embedding boxes by using pencils, placing the embedding boxes in a foam box, placing the foam box in running tap water for washing for 5 min, and draining water;

c. and (3) dehydrating: the tissue embedding box is sequentially placed into glass jars which are prepared in advance and filled with ethanol with different concentrations (the ethanol in the glass jars is replaced periodically) for gradient dehydration, the cover of the glass jar is immediately covered after the embedding box is placed into the glass jar, and cotton threads are placed outside the glass jar so as to facilitate jar replacement. Soaking in 75% ethanol solution for 1 hr times 2 times; soaking in 80% ethanol solution for 1 hr times 2 times; soaking in 95% ethanol solution for 1 hr times 2 times; soaking in 100% ethanol solution for 1 hr times 2 times, and draining off ethanol solution on the embedding box;

d. and (3) transparency: placing the tissue embedding box into a dimethylbenzene solution to be soaked for 40 min multiplied by 2 times;

e. wax dipping: putting the wax cylinder filled with the paraffin with the melting point of 58-60 ℃ into a 60 ℃ drying oven for melting, sequentially putting the tissue embedding box into three wax cylinders in the drying oven, and soaking each wax cylinder for at least 1 h, wherein the embedding box needs to be fully immersed in liquid paraffin;

f. embedding: preheating the embedding machine in advance to melt the paraffin in the machine, putting the iron box for embedding into the left side wax cylinder of the embedding machine after the paraffin is completely melted, and taking out the embedding box in the wax cylinder of the oven and putting the embedding box into the right side wax cylinder of the embedding machine. Removing the embedding box, putting the tissue into the center of the embedding iron box, fixing the tissue by using forceps, pouring liquid paraffin into the embedding iron box to immerse the tissue block, taking down the cover of the plastic embedding box, slightly covering the plastic embedding box on the embedding iron box, placing the plastic embedding box on a 4 ℃ freezing table, cooling the plastic embedding box into a solid wax block, removing the embedding iron box, placing the wax block into a sealing bag, and storing the wax block in a 4 ℃ refrigerator for later use.

(2) Immunohistochemical staining

a. Slicing: the tissue was roughly cut at 10 μm using a paraffin microtome to remove paraffin without tissue, and the surface of the wax block was polished smooth. Cutting the tissue wax block into 4-micron slices, placing the slices in a water bath kettle at 40 ℃, selecting slices with complete tissues and regular shapes, taking out the tissue slices by using a glass slide with positive charges to be attached to the glass slide, and marking relevant information of the sample at the tail end of the glass slide by using a pencil;

b. baking slices: placing the glass slide with the tissue on a baking machine preheated to 65 ℃ for baking for at least 2 h, or placing the glass slide in a 37 ℃ baking oven overnight;

c. dewaxing and rehydration: putting the slide racks fully loaded with the slices into dimethylbenzene to be soaked for 20 min in sequence; soaking in absolute ethanol solution for 5 min × 2 times; soaking in 95% ethanol solution for 5 min × 1 times; soaking in 75% ethanol solution for 5 min × 1 times; finally, placing the slide frame in deionized water to wash for 5 min;

d. antigen retrieval: diluting 50 x sodium citrate antigen repairing liquid to 1 x by using deionized water, uniformly shaking, pouring the 1 x sodium citrate antigen repairing liquid into a pressure cooker, closing a cooker cover, boiling liquid in the cooker, putting a slide with slices in the pressure cooker, completely immersing the slices in the sodium citrate antigen repairing liquid, screwing the cooker cover, starting timing after an air valve of the pressure cooker starts uniform air bleeding, closing a power supply of an electromagnetic oven after 8 min, and ending heating. Cooling the pressure cooker in running tap water, opening the cooker cover, and cooling the paraffin slices to room temperature;

e. washing: the sections were first washed extensively with deionized water for 5 min, followed by 3 min in PBS buffer and repeated 3 times. (ii) a

f. Blocking endogenous peroxidase: diluting 10% hydrogen peroxide water solution with deionized water to 3% concentration, preparing for use each time, placing paraffin section in a wet box, dripping appropriate amount of 3% hydrogen peroxide water solution on the position of tissue on the glass slide, closing the cover of the wet box, and sealing at 37 deg.C for 10 min;

g. washing: washing the section with PBS phosphate buffer solution for 3 min × 3 times, and throwing off the liquid on the slide glass;

h. and (3) sealing serum: placing the washed slices in a wet box, dropwise adding a proper amount of 10% donkey serum confining liquid on each slice, sealing for 15 min at room temperature, and then throwing off the confining liquid on the slices;

i. primary antibody incubation: wiping off sealing liquid around the tissue, drawing a circle around the tissue by using a special hydrophobic pen for immunohistochemical staining, placing the section in a wet box, dropwise adding INF2 antibody diluent with proper concentration to ensure that the tissue is immersed by the antibody diluent, closing the cover of the wet box, and placing in a refrigerator at 4 ℃ for overnight;

j. washing: washing the slices with PBS phosphate buffer solution for 3 min × 3 times;

k. and (3) secondary antibody incubation: wiping liquid around the tissue, dripping HRP-labeled donkey secondary antibody with proper concentration on the tissue to enable the antibody to completely cover the tissue, closing a cover of a wet box, and incubating for 1 h at 37 ℃;

l, washing: washing the slices with PBS phosphate buffer solution for 3 min × 3 times;

m. DAB color development: dripping DAB color developing liquid to the position of the tissue on the glass slide, placing the glass slide on an inverted microscope to observe the tissue dyeing condition, throwing off the color developing liquid when the dyeing intensity reaches the optimum, and washing for 3 min multiplied by 3 times by using deionized water;

n. hematoxylin counterstaining: dropping a proper amount of improved Lillie-Mayer hematoxylin staining solution at the position of the tissue on the glass slide, staining for 2 min, throwing away the staining solution, and placing the glass slide with the glass slide under running tap water for washing for 10 min to turn blue;

o, dehydration: soaking the slide rack with the slices in 75% ethanol solution for 5 min × 1 time; soaking in 95% ethanol solution for 5 min × 1 times; soaking in absolute ethanol solution for 5 min × 3 times;

p. transparent: soaking the slices in xylene solution for 5 min × 2 times;

q. mounting: dropping a proper amount of neutral gum at the position of the tissue on the glass slide, clamping a cover glass by using forceps, slightly covering the cover glass on the glass slide, and airing the cover glass in a fume hood to form a sheet;

r. score: and (3) placing the section on an inverted microscope, and observing the tissue morphology, the cell nucleus staining, the target protein staining and the like under a low-power microscope and a high-power microscope in sequence.

3. Screening INF2 knockout cell strain by western blotting technology and DNA sequencing technology

(1) And (3) transfecting the CRISPR-cas9-INF2 plasmid into HepG2 and C3A liver cancer cells, digesting the cells into uniform cell suspension after 48 hours, and counting the cells according to the method. Taking 1 six-hole plate, planting the cell suspension into the six-hole plate according to 1000 cells per hole, and adding a culture medium to 2 mL;

(2) Placing the six-hole plate into a constant-temperature incubator for culturing for 10 days, digesting a single cell colony by pancreatin when the cell colony is formed by naked eyes, transferring the cell colony into a 24-hole plate, blowing the cell colony uniformly by a pipette gun, placing the cell colony in the constant-temperature incubator for continuous culture, and taking the monoclonal cells in each hole as a group;

(3) After the 24-pore plate is uniformly paved with the cells, transferring each group of cells to a 12-pore plate according to the method, after the cells are uniformly paved to the 6-pore plate, transferring each group of cells to a new 6-pore plate according to the same method, when the cell density reaches 80%, carrying out passage 1 and 3 on each group of cells in the six-pore plate, and placing the cells in a constant temperature incubator for continuous culture;

(4) When the density of the monoclonal cells reaches 80%, digesting one hole of cell in each group into uniform single cell suspension, centrifuging for 4 min at the rotating speed of 1,000 rpm, discarding the supernatant, washing for 2 times by using PBS (phosphate buffer solution), finally resuspending the cell precipitate by using 100 mu L of PBS, and lightly blowing and beating to obtain uniform cell suspension;

(5) Extracting the genome DNA of the monoclonal cell: 100 μ L of PBS Solution was added to the cell suspension and mixed well. Then 20. Mu.L of Proteinase K is added and blown evenly. And adding 200 mu L Buffer DL, shaking and mixing uniformly, placing in a 56 ℃ water bath for 10 min, and indicating that the mixed solution becomes clear to prompt complete cracking. And adding 200 muL of absolute ethyl alcohol into the centrifuge tube, and fully shaking up. Connecting the adsorption column with the collection tube, transferring the mixture to the adsorption column, standing for 2 min, centrifuging at 10,000 rpm for 1 min, and discarding the liquid in the collection tube. Add 500. Mu.L GW Solution to the adsorption column, centrifuge at 10,000 rpm for 0.5 min and discard the waste. Adding 700 μ L of the eluate into the adsorption column, centrifuging at 10,000 rpm for 0.5 min, discarding the waste liquid, air-separating for 2 min, opening the adsorption column cover, and standing at room temperature for several minutes to completely air-dry the residual eluate. Placing the adsorption column into a new 1.5 mL centrifuge tube, adding 50 μ L CE Buffer, standing for 3 min, centrifuging at 12,000 rpm for 2 min at normal temperature, and collecting the DNA solution of the monoclonal cell;

(6) Genomic level identification of INF2 gene editing: and taking the extracted monoclonal cell DNA solution, and amplifying INF2 target fragments from a cell genome DNA template by using PCR. And (3) carrying out agarose gel electrophoresis on the product obtained by the PCR, and preliminarily judging the INF2 gene editing condition of the monoclonal cell according to the band result. PCR products of the clone INF2, which is likely to be homozygously deleted, were selected and subjected to PCDH-CD513B-cas9 vector ligation cloning. Ligation products were transformed into competent DH5a and plated on kanamycin plates overnight. 5-6 colonies from each group were picked, amplified to extract recombinant plasmid PCDH-CD513B-cas9 vector, and DNA sequence was detected (Tokyo Biotech Co., ltd., mitsubishi). Performing multiple comparison on the obtained 7 INF2 DNA sequencing sequences and the STING gene template, and finally determining the deletion condition of the CRISPR-Cas9 on the recognition sequence of the sgRNA of the INF2 gene;

(7) Screening INF2 knockout cell strains by western blotting technology: taking a monoclonal cell in one hole of a six-hole plate, discarding a complete culture medium, washing adherent cells for 2 times by using a PBS buffer solution, completely absorbing PBS, adding 200 mu L of cell lysate, placing on a constant temperature shaking bed at 4 ℃, shaking for 20 min, transferring the cell lysate to a 1.5 mL centrifuge tube, and placing in a refrigerator at-80 ℃ overnight. The cell samples were thawed on ice and the subsequent manipulations were performed according to the western blot technique described above.

4. Clone formation

(1) Taking HepG2 liver cancer cells which grow well after the transient transfection of the CD513B-INF2 plasmid for 24 hours, knocking out an INF2 cell strain and the HepG2 liver cancer cells which grow well, and digesting adherent cells into a uniform cell suspension;

(2) Sucking 20 mu L of the uniform cell suspension, adding the uniform cell suspension into a cell counting plate, measuring the concentration of the uniform cell suspension by using a cell counting instrument, diluting the cell suspension according to a corresponding proportion, and uniformly blowing by using a liquid transfer gun;

(3) Taking a plurality of 6-hole cell plates, adding 1 × 103 cells into each hole, quantifying 2 mL, arranging 3 multiple holes in each treatment group, shaking uniformly by using an 8-shaped method, placing in a constant-temperature incubator, changing the liquid once every 5 days, and culturing for 10-15 days;

(4) When cell colonies can be seen by naked eyes in the six-hole plate, removing culture medium in the holes, rinsing with PBS buffer solution for 2 times, adding a proper amount of 4% paraformaldehyde into each hole to fix cells, and standing for 30 min;

(5) Discarding the fixing solution, rinsing with PBS buffer solution for 2 times, adding 800 muL of 0.1% crystal violet dye into each well, placing on a shaking table for dyeing for 10 min, recovering the dye, rinsing with PBS buffer solution for 3 times, placing in an oven at 37 ℃ overnight, photographing after the six-well plate is dried, and counting data.

5. Scratch test

(1) Taking HepG2 liver cancer cells which grow well after transient transfection of CD513B-INF2 plasmids for 24 hours, knocking out INF2 cell strains and HepG2 liver cancer cells which grow well, and digesting adherent cells into uniform cell suspension;

(3) Taking 2 six-hole cell plates, marking 5 transverse lines on the back surfaces of the six-hole plates at intervals of 0.5-1 cm by using a mark pen, inoculating 30 ten thousand cells/hole, and putting the six-hole cell plates into a constant-temperature incubator for culture;

(4) When the cell density reaches 80% -90%, vertically scribing by using a 200 mu L gun head according to a transverse line on the back of a six-hole plate, washing floating cells by using PBS buffer solution, replacing serum-free DMEM, and placing into a culture box for continuous culture;

(5) The scratch healing conditions of 0 h, 24 h and 48 h are observed under a microscope, pictures are stored for later use, and the scratch healing area of each group is measured by using Image J software.

6. Transwell migration experiment

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

(3) Discarding the supernatant, resuspending the cell pellet with 1 mL of PBS solution, centrifuging again according to the method, resuspending the cells with 1 mL of DMEM, taking a cell counting plate to count the cells, and diluting with DMEM as required;

(4) Clamping 3 Transwell chambers by using sterile forceps, putting a new 24-hole cell plate, adding 150 muL of the cell suspension into the upper chamber, quantifying 5 x 104 cells, adding 500 muL of complete culture medium into the lower chamber, and putting the hole plate into a constant-temperature incubator after standing for 30 min;

(5) After 48 hours, removing culture media inside and outside the small chamber, and rinsing the small chamber and the bottom of the pore plate for 2 times by using PBS buffer solution;

(6) Adding 500 mu L of paraformaldehyde fixing solution into the small chamber, and standing for 30 min;

(7) Abandoning the fixing solution, rinsing with PBS solution for 2 times, adding 500 muL of 0.1% crystal violet dye, and placing the hole plate on a shaking bed for dyeing for 20 min;

(8) And (5) recovering the coloring agent, rinsing with PBS solution for 3 times, naturally drying the chamber, photographing and counting data.

7. Analysis of results

The experiment ensures that the experimental data and the collected original clinical data are comprehensively checked, corrected and collated, and ensures that the data are complete, accurate and error-free as possible. Then, an organization database and a cell database are respectively established by using Excel software, and related clinical original data are grouped and summarized and then are recorded into a form. And finally, utilizing SPSS 26.0 statistical software to carry out arrangement and analysis on the final experimental data. All data statistical tests are carried out according to the test level of alpha =0.05, and if P is less than 0.05, the difference between the two groups is considered to have statistical significance, otherwise, the difference is considered to have no statistical significance. The plotting of the experimental result graphs such as bar graph and line graph is completed by using GraphPad Prism 9.0 and SPSS 26.0 software.

2. Results of the experiment

To examine the expression of INF2 in liver cancer tissues, 50 formalin-fixed human liver cancer tissue specimens were collected, embedded and paraffin sectioned, and the expression level of INF2 in clinical specimens was examined by immunohistochemical staining. Immunohistochemical staining results show that INF2 staining is brownish yellow with different shades and is mainly positioned in cytoplasm of liver cancer tissues and tissues beside the cancer, a small amount of INF2 is positioned on cell membranes, and compared with the liver cancer tissues, INF2 is weakly expressed or not expressed in the corresponding tissues beside the cancer. The immunohistochemical staining images were analyzed by using Image J software, and the results showed that the level of INF2 expression in the liver cancer tissues was concentrated on three levels, namely negative (score 1), low Positive (score 2) and Positive (score 3) (fig. 1 a). Meanwhile, in liver cancer tissues, the positive expression rate of INF2 is 86% (43/50), while that of para-carcinoma tissues is only 46% (23/50), and the difference is statistically significant (X2 =17.825, P < 0.05). The expression level of INF2 in liver cancer tissues was significantly higher than that of corresponding paracarcinoma tissues (P < 0.001) (FIG. 1 b).

In order to follow up cellular functional experiments in which we further validated the function of INF2 protein, we screened INF2 knockout cell lines. We knocked out INF2 in HepG2 using CRISPR-Cas9 technology, selected INF2 knocked out cell colonies, and further confirmed using western immunoblotting and DNA sequencing techniques. The final result shows that cell colonies 2 and 6 are INF2 knockout cell strains, DNA sequencing shows that INF2-KO6 is liver cancer cell colonies with correct INF2 knockout, INF2-KO6 is cultured in an enlarged mode and cell function experiments are supplemented (figure 2), and the cell strains can stably knockout the INF2 endogenous in the cells and form a control with the high-expression INF2 transfected in a transient state.

In experiments to further verify the influence of INF2 on the proliferation and migration capacity of liver cancer cells, a clone formation experiment, a cell scratch experiment and a Transwell migration experiment are adopted. The clone formation experiment shows that the number of cell colonies in the INF2 high expression group is obviously increased compared with that in the wild liver cancer cell, and the INF2-KO6 group has no statistical significance compared with the Control group (FIG. 3. A and b). Indicating that INF2 has the capability of promoting the proliferation of liver cancer cells.

Cell scratch test results suggest that up-regulation of INF2 expression promotes HepG2 cell migration (fig. 4. A and b). Transwell migration experiments showed that the number of cells penetrating the Transwell chamber in the INF2 high expressing group was significantly increased in HepG2 cells compared to the Control group (FIGS. 5. A and b). Indicating that INF2 has the capability of promoting the migration of liver cancer cells.

These results suggest that detection of INF2 protein is of high value relative to diagnosis of liver cancer.

The invention provides a detection method with high sensitivity, strong specificity, short period and stable result by detecting the expression of INF2 in liver cancer tissues and the influence of high expression and low expression of INF2 on proliferation and migration of liver cancer cells and combining with a statistical principle and a modern biological technology, thereby providing scientific basis for diagnosis and treatment of liver cancer patients and providing possibility for molecular targeted treatment.

The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Claims

1. An application of protein INF2 in preparing liver cancer diagnosis marker.

2. An application of protein INF2 in preparing liver cancer molecule targeted medicine is provided.

3. Use of a protein according to claim 2, characterized in that: the application of the gene fragment of the protein INF2 or the expression product of the gene splice in the preparation of liver cancer molecular targeted drugs.

4. Use of a protein according to claim 2, characterized in that: the protein antibody coded by the gene of the protein INF2 is applied to the preparation of liver cancer molecule targeted drugs.

5. Use of a protein according to claim 2, characterized in that: the protein antibody coded by the gene of the protein INF2 is applied to the preparation of liver cancer molecule targeted drugs.