Application of a non-therapeutic PTBP2 protein in regulating the proliferation and / or migration of liver cancer cells

Abstract

This invention discloses the application of PTBP2 protein for non-therapeutic purposes in regulating the proliferation and / or migration of hepatocellular carcinoma cells. The key feature is that upregulation of PTBP2 expression promotes the proliferation and / or migration of hepatocellular carcinoma cells. The forward amplification primer sequence used for PTBP2 expression upregulation is 5′-AGGACTGTATTGCCACCAGC-3′, and the reverse amplification primer sequence is 5′-GCTTCCTGTTGCAGCTGTTC-3′. The advantages include high sensitivity, strong specificity, and short cycle time, which is beneficial for the early diagnosis and treatment of hepatocellular carcinoma. Furthermore, it has the potential to become a novel biomarker and effective therapeutic target in hepatocellular carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to the application of PTBP2 protein for non-therapeutic purposes in regulating the proliferation and / or migration of liver cancer cells. Background Technology

[0002] Primary liver cancer mainly includes hepatocellular carcinoma (HCC) (75%-85% of cases) and intrahepatic cholangiocarcinoma (10%-15% of cases). my country is a high-risk area for HCC, with chronic hepatitis B virus infection and aflatoxin being the main risk factors. Statistics show that approximately 390,000 new cases of HCC are diagnosed annually in my country, with about 360,000 deaths, making it one of the third leading causes of cancer death in the country. In the early and middle stages, HCC patients can receive radical treatments such as surgical resection, liver transplantation, radiofrequency ablation, or transarterial chemoembolization. However, most patients are diagnosed with advanced cancer due to late-stage symptoms and can only receive systemic treatment. With the advancement of gene therapy, such as CRISPR-Cas9 targeting specific genes, its potential for cancer treatment has been demonstrated. Therefore, identifying new therapeutic targets and early diagnostic biomarkers for HCC is of great significance.

[0003] We identified PTBP2 (Polypyrimidine tract-binding protein 2), a potential candidate gene considered an important regulator of RNA splicing and neuronal differentiation. Furthermore, a recent study showed that PTBP2 treatment of mediastinal neuroblastoma cells regulates tumor cell proliferation. In glioma cells, decreased PTBP2 expression inhibits cell migration and enhances cell adhesion to fibronectin and vitreous connexins through selective splicing of RTN4. Although targeting RNA splicing holds promise for cancer treatment, the functional significance of RNA splicing and individual splicing factors in HCC remains poorly understood. Currently, there are no published studies, either domestically or internationally, on using PTBP2 protein as a diagnostic and therapeutic biomarker in hepatocellular carcinoma. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a non-therapeutic PTBP2 protein with high sensitivity and specificity and positive correlation with the incidence of hepatocellular carcinoma for the application in regulating the proliferation and / or migration of hepatocellular carcinoma cells.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: the application of a non-therapeutic PTBP2 protein in regulating the proliferation and / or migration of liver cancer cells.

[0006] Furthermore, upregulation of PTBP2 expression promotes the proliferation and / or migration of liver cancer cells.

[0007] Furthermore, the forward amplification primer sequence used for the upregulation of PTBP2 expression is 5′-AGGACTGTATTGCCACCAGC-3′, and the reverse amplification primer sequence is 5′-GCTTCCTGTTGCAGCTGTTC-3′.

[0008] Compared with existing technologies, the advantages of this invention are as follows: This invention discloses for the first time the application of non-therapeutic PTBP2 protein in regulating the proliferation and / or migration of hepatocellular carcinoma cells. The detection is mainly performed through immunohistochemistry. Analysis of immunohistochemical staining images is used, and ImageJ software is employed to calculate and compare the expression of PTBP2 protein in hepatocellular carcinoma patient samples and normal samples, in order to assist in the diagnosis of hepatocellular carcinoma. Compared with traditional hepatocellular carcinoma detection technologies, this invention has the characteristics of high sensitivity, high specificity, and short cycle, which is beneficial for the early diagnosis and treatment of hepatocellular carcinoma. Furthermore, PTBP2 has the potential to become a novel biomarker and effective therapeutic target in hepatocytes. Attached Figure Description

[0009] Figure 1 To validate the expression of PTBP2 in human hepatocellular carcinoma tissues / cells and normal liver tissues / cells using the CPTAC database and Western blotting. (A) Analysis of PTBP2 protein expression levels in 165 pairs of human hepatocellular carcinoma tissues and paired adjacent normal tissues (n=165, ***: p (a) Western blot analysis of protein immunoblotting in cancer and adjacent tissues of HCC patients (n=14); (b) Difference analysis of PTBP2 / GAPDH gray values ​​in cancer and adjacent tissues of HCC patients (*: p <0.05); (D) Western blot analysis of proteins in hepatocellular carcinoma cells (HepG2 and Huh7) and normal hepatocytes (LO2); (E) Differential analysis of PTBP2 / GAPDH gray values ​​in hepatocellular carcinoma cells (HepG2 and Huh7) and normal hepatocytes (LO2) (Huh7, ns: p >0.05;HepG2,***: p <0.001);

[0010] Figure 2 To verify the expression of PTBP2 in human hepatocellular carcinoma (HCC) tissues and paired adjacent normal tissues using immunohistochemistry. (A) Three representative images of PTBP2 immunohistochemical staining in 50 pairs of human HCC tissues and paired adjacent normal tissues (n=50); (B) Stacked bar chart of immunohistochemical staining results for 50 pairs of human HCC tissues and paired adjacent normal tissues (n=50, ***: p<0.001). (Where "Negative" indicates PTBP2 is not expressed, "Low positive" indicates low PTBP2 expression, and "High positive" indicates high PTBP2 expression);

[0011] Figure 3 The expression of PTBP2 in two hepatocellular carcinoma cell lines was detected by transfection, Western blotting, and RT-qPCR. In the experimental group, A was RT-qPCR and B was Western blotting. The experimental groups were PTBP2 overexpression, PTBP2 knockout, and control group.

[0012] Figure 4 To verify the effect of PTBP2 expression level on the growth of hepatocellular carcinoma cells using the CCK8 assay. (A) Effect of PTBP2 expression level on HepG2 cell growth using the CCK8 assay; (B) Effect of PTBP2 expression level on Huh7 cell growth using the CCK8 assay (***: p <0.001);

[0013] Figure 5 To validate the effect of PTBP2 expression level on the growth of hepatocellular carcinoma cells using clonogenic assays. (A) Clonogenic assay to detect the colony-forming ability of HepG2 and Huh7 cells after PTBP2 overexpression or knockout; (B) Quantification of clonogenic assay results (*: p <0.05, **: p <0.01, ***: p <0.001);

[0014] Figure 6 To verify the effect of PTBP2 expression level on hepatocellular carcinoma cell migration using Transwell migration assays: (A) Migration experiments of HepG2 and Huh7 cells after PTBP2 overexpression or knockout; (B) Quantification of cell migration assay results (**: p <0.01, ****: p <0.0001);

[0015] Figure 7 To verify the effect of PTBP2 expression level on the occurrence and development of liver cancer cells in a mouse subcutaneous tumorigenesis experiment: (A) Removal of subcutaneous tumor tissue from mice after PTBP2 overexpression or knockout; (B) Quantification of tumor tissue weight results (**: p <0.01, ***: p <0.001); (C) Quantification of tumor tissue volume results (ns: p >0.05, *: p <0.05). Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0017] I. Experimental Methods

[0018] 1. Collection of hepatocellular carcinoma tissue specimens

[0019] The 50 pairs of paraffin-embedded human liver cancer tissues and 14 pairs of fresh liver cancer tissues included in the study were collected from January 1, 2020 to December 31, 2023 at the Affiliated Li Huili Hospital of Ningbo University. All human tissue specimens involved in this experiment were used for laboratory research only, subject to the approval of the Ethics Committee of Ningbo University School of Medicine and upon the request of the research group.

[0020] 2. Immunohistochemical staining of specimens

[0021] a. Baking: Place the slides containing the tissue onto a preheated slide baking machine at 65°C and bake for 4 hours;

[0022] b. Dewaxing and rehydration: The slide holders fully loaded with slide sections were immersed in xylene for 10 min × 2 times; in anhydrous ethanol solution for 5 min × 2 times; in 95% ethanol solution for 5 min × 1 time; in 75% ethanol solution for 5 min × 1 time; and finally, the slide holders were washed in deionized water for 5 min.

[0023] c. Antigen retrieval: Dilute 50× pH 9.0 EDTA antigen retrieval solution with deionized water to 1× and shake well. Pour the 1× EDTA antigen retrieval solution into a pressure cooker, close the lid, and wait for the liquid in the cooker to boil. Place the slide holder containing the sections into the pressure cooker, ensuring the sections are completely submerged in the sodium citrate antigen retrieval solution. Tighten the lid and start timing once the pressure cooker valve begins to release steam evenly. After 10 minutes, turn off the induction cooker to stop heating. Place the pressure cooker under running tap water to cool it down, open the lid, and allow the paraffin sections to cool to room temperature.

[0024] d. Washing: First, wash the slides thoroughly with deionized water for 5 min, then wash the slides with PBST buffer for 3 min, and repeat the washing 3 times;

[0025] e. Blocking endogenous peroxidase: Dilute 30% hydrogen peroxide aqueous solution with deionized water to a concentration of 3%, prepare fresh each time, place paraffin sections in a humidified chamber, drop an appropriate amount of 3% hydrogen peroxide aqueous solution on the tissue location on the slide, close the lid of the humidified chamber, and block at 37°C for 20 min.

[0026] f. Washing: Wash the slides with PBST buffer for 3 min × 3 times, and shake off the liquid on the slides;

[0027] g. Serum blocking: After washing, the slides are placed in a humidified chamber. An appropriate amount of 10% donkey serum blocking solution is added to each slide. After blocking at 37°C for 15 minutes, the blocking solution on the slides is removed.

[0028] h. Primary antibody incubation: Wipe away the blocking solution around the tissue, draw a circle around the tissue with a hydrophobic pen, place the slide in a humidified chamber, add an appropriate concentration of PTBP2 antibody to submerge the tissue, close the lid of the humidified chamber, and place it in a 4°C refrigerator overnight;

[0029] i. Warming: Place the slices that have been stored at 4°C overnight in a 37°C refrigerator for 20 minutes to allow the antibodies to bind better to the tissue;

[0030] j. Washing: Wash the slides with PBST buffer for 3 min × 3 times;

[0031] k. Secondary antibody incubation: Wipe the liquid around the tissue dry, add an appropriate concentration of HRP-labeled donkey secondary antibody to the tissue to completely cover the tissue, close the humidifier lid, and incubate at 37°C for 1 h;

[0032] 1. Washing: Wash the sections with PBST buffer for 3 min × 3 times;

[0033] m. DAB staining: Add DAB staining solution to the tissue location on the slide, place the slide on an inverted microscope to observe the tissue staining, and when the staining intensity reaches the best, discard the staining solution and wash with deionized water for 3 min × 3 times.

[0034] n. Hematoxylin counterstaining: Add an appropriate amount of modified Lillie-Mayer hematoxylin staining solution to the tissue location on the slide, stain for 10 seconds, discard the staining solution, and place the slide holder containing the slide under running tap water for 1 minute to restore the blue color.

[0035] o. Dehydration: Immerse the slide holder containing the slides in 75% ethanol solution for 5 min × 1 time; immerse in 95% ethanol solution for 5 min × 1 time; and immerse in anhydrous ethanol solution for 5 min × 3 times in sequence.

[0036] p. Transparency: Immerse the sections in xylene solution for 5 min twice;

[0037] q. Mounting: Apply an appropriate amount of neutral resin to the tissue location on the slide, use tweezers to gently cover the slide with a coverslip, and place it in a fume hood to dry into a slide;

[0038] r. Scoring: All samples were reviewed by two independent pathologists experienced in IHC assessment who were unaware of the patients' clinical outcomes. We assessed the percentage and intensity of positively stained cells to semi-quantitatively determine PTBP2 expression. The percentage of positively stained cells was scored as follows: 0, <10%; 1, 10%–50%; 2, >50%. The intensity of staining was graded as follows: 0 (no or weak staining = pale yellow), 1 (moderate staining = yellowish-brown), and 2 (strong staining = brown). The total score for PTBP2 expression was the sum of the percentage of positively stained cells and the intensity of the staining score, ranging from 0 to 4. For statistical analysis, the final score was a combination of the independent scores assigned by the two pathologists reported in this study. Any discrepancies in scores were resolved through discussion between the two pathologists.

[0039] 3. Protein extraction from fresh liver cancer tissue

[0040] a. Cut 14 pairs of liver cancer tissue samples, each about the size of a soybean, on ice. Transfer the tissue to round-bottom centrifuge tubes, peel, and weigh. Add 200 μL of ice-cold RIPA for every 1 mg of tissue and homogenize using an electric homogenizer.

[0041] b. Stir the contents at 4°C for 2 hours.

[0042] c. Centrifuge the centrifuge tube at 16,000 g for 20 minutes at 4°C. Collect the supernatant in a new tube and place it on ice. Discard the precipitate.

[0043] 4. Western blotting and RT-qPCR

[0044] To achieve the goal of knocking down the PTBP2 gene, we purchased short hairpin RNA (sh-RNA) targeting the PTBP2 gene and control sh-RNA (sh-NC) from Shanghai Qingke Company. To achieve the goal of overexpressing the PTBP2 gene, we purchased a PTBP2 plasmid with a Flag tag (http: / / www.miaolingbio.com / plasmid / P1474.html) and an empty vector (EV) for the overexpression control group from Miaoling Company. We designed experimental groups: sh-NC+EV (control group), sh-PTBP2+EV (PTBP2 knockout), and sh-NC+Flag-PTBP2 (PTBP2 overexpression), along with primers and shRNA sequences.

[0045] Table 1 Primer and shRNA sequences

[0046]

[0047] a. Western blotting technique

[0048] (1) Transfect sh-NC+EV (control group), sh-PTBP2+EV (PTBP2 knockout), and sh-NC+Flag-PTBP2 (PTBP2 overexpression) into 12-well plates respectively. After 48 h, well-grown HepG2 and Huh7 liver cancer cells were aspirated, and 100 μL of cell lysis buffer RIPA was added to each well. After shaking at 4°C for 20 minutes, the cells were transferred to -80°C and frozen overnight.

[0049] (2) Take the sample out of -80℃, melt it at 4℃ and transfer it into a 1.5ml Ep tube. After centrifuging at 4℃ for 12000r for 20min, take the supernatant, add 4XSDS and boil it in a metal bath at 95℃ for 5min.

[0050] (3) Prepare 10% WB gel in advance for use. Run electrophoresis at constant voltage of 80V, transfer membrane at constant current of 220mA for 120min, wash membrane with TBST for 5min, block with 5% milk powder prepared with TBST for 40min, wash membrane, and apply corresponding antibody overnight.

[0051] (4) Prepare developer, water, and fixer in the darkroom in advance. After the film has been dripped with ECL luminescent solution, spin it dry and lay it flat on plastic wrap. Then fix it in the darkroom and press and expose it in the darkroom. After exposure, open the darkroom, take out the film, immerse it in developer, and observe it under a red light until the bands appear. After rinsing it in water, put it in fixer and then turn on the light to observe the exposure results. Finally, scan the film to analyze the results.

[0052] b. RT-qPCR technology

[0053] (1) Take HepG2 and Huh7 liver cancer cells that have been transiently transfected with sh-NC+EV, sh-PTBP2+EV, and sh-NC+Flag-PTBP2 respectively and have grown well after 48 h. Aspirate the culture medium, add 1 ml of Trizol to each well, pipette several times to ensure complete cell lysis, and then transfer to centrifuge tubes.

[0054] (2) The lysate was left at room temperature for 5 min to allow the nucleic acid-protein complex to be completely separated;

[0055] (3) Add 0.2 ml of chloroform to each 1 ml of Trizol, tighten the cap, shake vigorously for 15 s, and let stand at room temperature for 2-3 min;

[0056] (4) Centrifuge at 12000g at 4℃ for 15 min. The sample will be separated into three layers: an orange-yellow lower organic phase, a middle layer, and a colorless upper aqueous phase.

[0057] (5) Transfer the upper aqueous phase containing total RNA to a new centrifuge tube, and transfer the volume of the aqueous phase to 60% of the Trizol reagent used;

[0058] (6) Add 0.5 ml of isopropanol for each 1 ml of Trizol used initially, invert several times to mix, and let stand at room temperature for 10 min;

[0059] (7) Centrifuge at 12000g at 4℃ for 10 min, discard the supernatant, and a gel-like RNA precipitate will be visible;

[0060] (8) Add 1 ml of 75% ethanol for each 1 ml of Trizol used initially, invert several times to mix, and wash the precipitate.

[0061] (9) Centrifuge at 12000g for 5 min at 4℃, and discard the supernatant;

[0062] (10) Invert at room temperature for 5-10 minutes to air dry;

[0063] (11) Add 25 μl DEPC-ddH2O and pipette several times to dissolve the RNA;

[0064] (12) The concentration, purity and integrity of RNA were detected by RNA electrophoresis and ultraviolet spectrophotometry;

[0065] (13) The obtained RNA should be used immediately or aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles;

[0066] (14) Reverse transcription reaction system:

[0067] 1 μL Enzyme Mix, 4 μL 5 × All-in-one qRT SuperMix, 1 pg - 1 μg template RNA, add free water to a volume of 20 μL; reaction program: 50℃, 15 min; 85℃, sec 5;

[0068] (15) RT-qPCR detection:

[0069] Prepare the following mixture in a qPCR tube: 10.0 µL 2 × Taq Pro Universal SYBR qPCRMaster Mix, 0.4 µL Primer1 (10 µM), 0.4 µL Primer2 (10 µM), x µL Template DNA, and add free water to a volume of 20 µL; reaction steps: pre-denaturation 95 °C: 30 sec; 40 cycles, 95 °C: 3 - 10 sec, 60 °C: 10 - 30 sec; melting curve 95 °C: 15 sec, 60 °C: 60 sec, 95 °C: 15 sec.

[0070] 5. CCK8 cell proliferation assay

[0071] (1) Take HepG2 and Huh7 liver cancer cells that have grown well after transient transfection with sh-NC+EV, sh-PTBP2+EV, and sh-NC+Flag-PTBP2 for 24 h respectively, and digest the adherent cells into a uniform cell suspension.

[0072] (2) Take 20 µL of the above homogeneous cell suspension and add it to a cell counting chamber. Measure its concentration using a cell counter. Dilute the cell suspension according to the corresponding ratio and pipette to mix it evenly.

[0073] (3) Take 6 96-well cell plates and add 2×10 to each well. 3 0.1 mL of cells were used for quantitative analysis. Five replicates were set up for each treatment group. The cells were shaken well using the figure-eight method and placed in a constant temperature incubator.

[0074] (4) When the cells in the six 96-well cell culture plates were cultured for 0, 1, 2, 3, 4, 5, 6, and 7 days respectively, 10 µL of CCK8 reagent was added to each well under dark conditions and the plates were incubated in a constant temperature incubator for 2 hours until the plates turned orange-yellow. The absorbance of each well was measured by setting the wavelength of the microplate reader to 450 nm, and the readings were repeated 3 times. The data were recorded and organized.

[0075] 6. Transwell migration experiment

[0076] (1) Take HepG2 and Huh7 liver cancer cells that have grown well after transient transfection with sh-NC+EV, sh-PTBP2+EV, and sh-NC+Flag-PTBP2 for 24 h respectively, and digest the adherent cells into a uniform cell suspension.

[0077] (2) Collect the cell suspension into a 1.5 mL centrifuge tube and centrifuge at 1,000 rpm for 4 min;

[0078] (3) Discard the supernatant, resuspend the cell pellet in 1 mL PBS solution, centrifuge again as described above, resuspend the cells in 1 mL DMEM, count the cells using a cell counting chamber, and dilute with DMEM as needed.

[0079] (4) Using sterile forceps, pick up 3 Transwell chambers and place them into a new 24-well cell plate. Add 150 µL of the above cell suspension to each chamber and quantify 5 × 10⁻⁶ cells / mL. 4 For each cell, add 500 µL of complete culture medium to the lower chamber, let stand for 30 min, and then place the well plate into a constant temperature incubator.

[0080] (5) After 48 h, discard the culture medium inside and outside the chamber, and rinse the chamber and the bottom of the well plate twice with PBS buffer;

[0081] (6) Add 500 µL of paraformaldehyde fixative to the small chamber and let it stand for 30 min;

[0082] (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 for staining;

[0083] (8) Recover the staining agent, rinse three times with PBS solution, air dry the chamber naturally, take photos and collect data.

[0084] 7. Cloning

[0085] (1) Take HepG2 and Huh7 liver cancer cells that have grown well after transient transfection with sh-NC+EV, sh-PTBP2+EV, and sh-NC+Flag-PTBP2 for 24 h respectively, and digest the adherent cells into a uniform cell suspension.

[0086] (2) Take 20 µL of the above homogeneous cell suspension and add it to a cell counting chamber. Measure its concentration using a cell counter. Dilute the cell suspension according to the corresponding ratio and pipette to mix it evenly.

[0087] (3) Take several 6-well cell plates and add 1×10 to each well. 3 10 cells, 2 mL of quantified solution, 3 replicates per treatment group, shake well using the figure-eight method, place in a constant temperature incubator, change the medium every 5 days, and culture for a total of 10–15 days;

[0088] (4) When cell colonies are visible to the naked eye in the six-well plate, discard the culture medium in the well, rinse twice with PBS buffer, add an appropriate amount of 4% paraformaldehyde to each well to fix the cells, and let stand for 30 min.

[0089] (5) Discard the fixative, rinse twice with PBS buffer, add 800 µL of 0.1% crystal violet dye to each well, stain on a shaker for 10 min, recover the dye, rinse three times with PBS buffer, place in an oven at 37℃ overnight, and take pictures and collect data after the six-well plate has dried.

[0090] 8. In vitro mouse tumor formation experiment

[0091] With the approval of the Animal Ethics Committee of Ningbo University, tumor size was examined in immunodeficient mice in different groups using human liver cancer cells to induce tumor formation. In the in vivo xenotransplantation experiment, NOD-SCID mice (T001492), aged 4-6 weeks and weighing 15-25 g, were used. Four sh-PTBP2 Huh7 cell lines and eight sh-NC Huh7 cell lines (3 × 10⁶ cells per line) were prepared in advance. 6 Two days before the in vitro experiment, eight sh-NC Huh7 cells (10 μg / 3 × 10⁶ cells) were transiently transfected with Flag-PTBP2 plasmid and EV (empty vector) plasmid, respectively. 6 Transfect 4 sh-PTBP2 Huh7 cells (10 μg / 3 × 10⁻⁶ cells) with EV plasmid. 6 (Cells). After digestion and centrifugation, each group of cells was resuspended in 400 μL PBS and 400 μL of matrix gel, and then evenly distributed. The cultured Huh7 cells were divided into three groups: sh-NC+EV, sh-NC+Flag-PTBP2, and sh-PTBP2+EV. These cell groups were then subcutaneously transplanted into the right side of 12 mice. The mice were randomly assigned to different groups. During transplantation, each mouse received 200 μL of cell suspension. Tumor volume was monitored and measured every 4 days for 20 days, starting from day 4 post-transplantation. The tumor volume was calculated using the formula: Tumor volume = (length × width^2) × 1 / 2. At the end of the experiment, the mice were euthanized, the tumors were carefully removed, and the mice were weighed to determine their body weight. Four mice were placed in each group, and the experiment was not blinded. All animal experiments were conducted according to protocols approved by the Ningbo University Animal Protection Committee. These protocols ensured that the animals involved in the research were treated ethically and responsibly.

[0092] 9. Construction of stable hepatocellular carcinoma cell lines

[0093] To construct sh-PTBP2 and sh-NC hepatocellular carcinoma cells (Huh7), we purchased shRNA targeting PTBP2 (gene ID: 58155, Ref Seq: NM_021190) and sh-NC from Shanghai Qingke Biotechnology Co., Ltd. Day 1: 293T seeding: 293T cells were pre-seeded in 10 cm culture dishes, achieving a confluence rate of 70-80% on the day of transfection. Day 2: Before transfection, the old cell culture medium was discarded, and the cells were washed with 3-5 mL of PBS, then serum-free DMEM was added and equilibrated in an incubator. Transfection reagents were prepared as follows: Mixture 1 (25 μg sh-PTBP2 or sh-NC, 7.5 μg PMD2G, 15 μg PSPAX2 dissolved in 1000 μL serum-free DMEM). Mixture 2 (100 μL Lipo6000™ transfection reagent dissolved in 1000 μL serum-free DMEM). Mixture 1 and Mixture 2 separately and incubate at room temperature for 5-10 min. Then, mix Mixture 1 and Mixture 2 and incubate at room temperature for 30 min, then transfer to 10 cm culture dishes. Day 3: Replace with fresh culture medium (containing 10% FBS) within 6-24 hours, observe transfection efficiency and take photos. Day 5: Observe cell status and take photos. Collect viral supernatant and filter through a 0.45 μm filter membrane. Add the collected viral supernatant to centrifuge tubes and centrifuge at 25000 rpm for 1.5 h at 4 °C. Collect the supernatant, redissolve it in an appropriate viral preservation solution, mix and dissolve overnight. Day 6: Collect and package the virus for viral titer determination. The collected viral supernatant was used to infect liver cancer cells (mixed with complete culture medium at a 1:1 ratio). To screen for stable lines, we selected puromycin to obtain stable sh-PTBP2 and sh-NC liver cancer cell lines. Compared with stable sh-NC hepatocellular carcinoma cells, the low expression of PTBP2 in established sh-PTBP2 hepatocellular carcinoma cells has been confirmed at both protein and RNA levels.

[0094] 10. Results Analysis

[0095] This experiment ensured that all experimental data and collected raw clinical data underwent comprehensive review, verification, and organization to guarantee the data's completeness, accuracy, and error-free nature. Then, Excel software was used to establish separate tissue and cell databases, and the relevant raw clinical data were grouped, summarized, and entered into spreadsheets. Finally, GraphPad Prism 9.0 statistical software was used to organize and analyze the final experimental data. All statistical tests were performed using two-tailed probabilities at a significance level of α=0.05. pA value <0.05 was considered statistically significant between the two groups; otherwise, the value >0.05 was considered not statistically significant. The plotting of experimental results, including bar charts and line graphs, was done using GraphPad Prism 9.0 software.

[0096] II. Experimental Results

[0097] To preliminarily detect the expression of PTBP2 in hepatocellular carcinoma (HCC) tissues, we visualized the PTBP2 expression of 165 HCC tissue samples and corresponding normal liver tissues using the online database UALCAN. The results showed that the PTBP2 protein expression level in HCC tissues was significantly higher than that in normal liver tissues. p <0.001) Figure 1 A). To further examine the expression of PTBP2 in hepatocellular carcinoma (HCC) tissues, we collected 50 pairs of paraffin-embedded human HCC tissues and 14 pairs of fresh human HCC tissues. Western blotting and immunohistochemical staining were used to detect the expression level of PTBP2 in clinical samples. The results of Western blotting showed that the expression level of PTBP2 protein in HCC tissues was significantly higher than that in normal adjacent normal tissues. p <0.05) Figure 1 BC). Furthermore, by detecting and analyzing the expression levels of PTBP2 protein in two hepatocellular carcinoma cell lines and one normal hepatocyte cell line, the results showed that the protein expression level of PTBP2 in HepG2 cells was higher than that in normal hepatocyte LO2 (BC). Figure 1 DE).

[0098] Immunohistochemical staining results showed that PTBP2 stained as varying shades of brownish-yellow and was mainly located within the cell nucleus. Compared with normal tissue near hepatocellular carcinoma (HCC), PTBP2 expression was stronger in HCC tissue. Analysis of the immunohistochemical staining images using ImageJ software showed that the PTBP2 protein expression levels in HCC tissue and surrounding normal tissue could be classified into three grades: negative, low positive, and high positive. In HCC tissue, the positive expression rate of PTBP2 was as high as 78% (39 / 50), while in surrounding normal tissue it was 40% (20 / 50), a statistically significant difference (X). 2 =14.93, p <0.001. The expression level of PTBP2 in hepatocellular carcinoma tissues was significantly higher than that in the corresponding adjacent normal tissues ( p <0.001)( Figure 2 AB).

[0099] To further validate the PTBP2 protein function in subsequent cellular functional experiments, we transiently transfected two hepatocellular carcinoma cell lines according to the sh-NC+EV, sh-PTBP2+EV, and sh-NC+Flag-PTBP2 protocols. We then performed Western blotting and RT-qPCR to verify the transfection efficiency. Figure 3 AB). In further experiments to verify the effects of PTBP2 on the proliferation and migration of hepatocellular carcinoma cells, we used the CCK8 assay, Transwell migration assay, and colony formation assay. The CCK8 assay showed that, compared with the control group, the PTBP2 high-expression group showed significantly faster cell proliferation, while the PTBP2 low-expression group showed slower proliferation than the control group. Figure 4 (AB). This indicates that PTBP2 has the ability to promote the proliferation of liver cancer cells.

[0100] Colony formation assays showed that, compared with the control group of liver cancer cells, the PTBP2 high-expression group had a significantly increased number of cell colonies, while the PTBP2 low-expression group showed a significantly reduced number of cell colonies compared with the control group. Figure 5 (AB). This indicates that PTBP2 has the ability to promote the proliferation and colony formation of hepatocellular carcinoma cells.

[0101] Transwell migration assays showed that in liver cancer cells, the number of cells that crossed the chamber was significantly increased in the PTBP2 high-expression group compared to the control group, while the number of cells that crossed the chamber was significantly reduced in the PTBP2 low-expression group. Figure 6 A and B) indicate that PTBP2 has the ability to promote the migration of hepatocellular carcinoma cells.

[0102] Subcutaneous tumorigenesis experiments in mice showed that, in liver cancer cells, the tumor tissue weight was larger in the PTBP2 high-expression group than in the control group, while the tumor weight was smaller in the PTBP2 low-expression group than in the control group. Figure 7 AB). Furthermore, measurements of subcutaneous tumor volume in mice revealed that the growth rate of the PTBP2-low expression group was significantly lower than that of the control group (AB). Figure 7 C).

[0103] These results suggest that detecting PTBP2 protein has relatively high diagnostic value, especially immunohistochemistry. The expression level of PTBP2 in hepatocellular carcinoma tissues is significantly lower than that in the corresponding adjacent normal tissues (p<0.001). Therefore, detecting the expression of PTBP2 protein in hepatocellular carcinoma tissues can achieve a diagnosis of hepatocellular carcinoma with high sensitivity and specificity.

[0104] This invention provides a highly sensitive, specific, short-cycle, and stable detection method by detecting the expression of PTBP2 in hepatocellular carcinoma tissue and the effects of high and low PTBP2 expression on the proliferation, migration, and development of hepatocellular carcinoma cells. Combining statistical principles and modern biological techniques, it offers a scientific basis for the diagnosis and treatment of hepatocellular carcinoma patients and provides possibilities for molecularly targeted liver cancer therapy.

[0105] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. The application of a non-therapeutic PTBP2 protein in regulating the proliferation and / or migration of liver cancer cells, characterized in that: Upregulation of PTBP2 expression promotes the proliferation and / or migration of liver cancer cells. The forward amplification primer sequence used for upregulating PTBP2 expression is 5′-AGGACTGTATTGCCACCAGC-3′, and the reverse amplification primer sequence is 5′-GCTTCCTGTTGCAGCTGTTC-3′.

Images