Application of Oct4-pg4 and Oct4-pg5 interference long-chain non-coding RNA (Ribonucleic Acid) in liver cancer treatment and verification mechanism

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA (Ribonucleic Acid) interference in liver cancer treatment and a verification mechanism. The interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA inhibits proliferation, migration and invasion of liver cancer cells, induces apoptosis of the liver cancer cells, and regulates and controls the cycle of the liver cancer cells and expression of immune escape related genes. Through in-vivo and in-vitro experiments, the expression of Oct4-pg4 and Oct4-pg5 is specifically interfered by using an RNAi technology, and the influence of the Oct4-pg4 and Oct4-pg5 on biological behaviors such as proliferation, migration, invasion and cell cycle of liver cancer cells is observed. Meanwhile, the regulation and control effects of Oct4-pg4 and Oct4-pg5 expression changes on the original gene Oct4 and cell cycle, apoptosis and differentiation related genes are further discussed, and it is proved that interference on Oct4 pseudogenes pg4 and pg5 regulates and controls expression of a plurality of key genes, and a new thought is provided for treatment of liver cancer.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to an application and verification mechanism of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in liver cancer treatment. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is a common cancer type worldwide and a common solid malignant tumor. Although with the development of medical technology, targeted chemotherapy, radiotherapy, immunotherapy and combined therapy have made great breakthroughs in the treatment of HCC, but later still face problems such as tumor drug resistance, recurrence, metastasis, short survival time, etc. Early symptoms are not easy to be detected, and when found, it is too late, so the best treatment time is missed. Therefore, discovering a new treatment target and early diagnosing HCC are important research directions for treating HCC.

[0003] HCC is prone to drug resistance. At present, targeted drugs include angiogenesis inhibitors and kinase inhibitors. Sorafenib, as a targeted drug for advanced liver cancer, has greatly changed the treatment mode of liver cancer, but long-term use will also produce drug resistance. The overall survival of the new drug Lenvatinib is slightly higher than that of Sorafenib, but the treatment effect is not ideal, so there are deficiencies in the targeted treatment of liver cancer. Injection of vaccines or use of immune checkpoint inhibitors for immunotherapy. The main targets of ICIs are programmed cell death protein 1 / programmed cell death ligand 1 (PD-1 / PD-L1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). PD-L1 regulates T cell activation, and CTLA-4 pathway hinders T cell response, which is excessively activated in tumor cells. Liver cancer targeting and immunotherapy have been widely used in clinical practice, but drug resistance occurs in single therapy, so combined therapy is generally used in clinical practice. The application provides an application and verification mechanism of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in liver cancer treatment, which provides a new idea for the treatment of liver cancer. SUMMARY

[0004] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, the abstract and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0005] To solve the above technical problems, according to one aspect of the present application, the present application provides the following technical solutions: Application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in liver cancer treatment.

[0006] As a preferred scheme of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 inhibits the proliferation, migration and invasion of liver cancer cells, induces apoptosis of liver cancer cells, and regulates the expression of liver cancer cell cycle and immune escape related genes.

[0007] The verification mechanism of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer includes in vitro experiments and in vivo experiments, and the in vitro experiments include the following steps: S1: Detect the effect of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 on the growth activity of liver cancer cell lines SMMC-7721, HepG2 and Huh7 after 24h and 48h by MTT method; S2: Further verify the optimal transfection efficiency of the sequence by qPCR and determine the effect of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 on the original gene Oct4: collect cells and lyse; collect and purify RNA; perform RNA reverse transcription and real-time fluorescent quantitative PCR; S3: Observe the morphological changes of the transfected Huh7 cells, and determine the cell growth activity after 0h, 24h, 48h and 72h of transfection of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5; S4: Detect the expression of CCND1, BIRC5, Bax, Bcl-2, Caspase3, PD-1 and c-Myc genes by qPCR to determine the effect of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 on Oct4 and related genes; S5: Explore the effect of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 on the migration and invasion ability of Huh7 cells by cell scratch and Transwell experiments; S6: Detect the apoptosis and cycle of Huh7 cells by flow cytometry after the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5; S7: Detect the expression of Oct4 protein and related apoptosis proteins Bax, Bcl-2, Caspase3, cycle proteins CCND1, BIRC5, and PD-1, c-Myc of Huh7 cells by WB experiment after the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5; The in vivo experiment includes the following steps: Step 1: Mice liver cancer model modeling, drug administration and section preparation; Step 2: H&E staining; Step 3: Immunohistochemical staining; Step 4: qPCR detection of tumor gene expression; Step 5: Western blot detection.

[0008] As a preferred scheme of the verification mechanism of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the specific detection method of S1 is: setting Control, Si-429, Si-920 and Control, Si-457, Si-635, Si-697, NC group, taking SMMC-7721, HepG2, Huh7 cells, washing, digesting, blowing, centrifuging, counting, 0.8x104 cells per hole, 3 duplicate holes per group; when the cell density reaches 60%-70%, transfection is carried out, 10ul complex is added to each hole, and it is mixed uniformly by blowing, and then it is cultured for 24h and 48h for detection; 2 96-well plates after 24h and 48h are detected respectively, 20ul of prepared MTT solution is added to each duplicate well, incubated for 4h, then 150ul of DMSO is added to each hole, and after waiting for 15min, the absorbance at 490nm is measured, the experiment is repeated three times to take the average value and draw the curve.

[0009] As a preferred scheme of the verification mechanism of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the specific method of cell scratch experiment in S5 is: the experiment is divided into blank group, Si-920 group, Si-635 group, Si-920+Si-635 group, NC group, a horizontal line is drawn every 0.6cm on the back of the 6-hole plate with a water pen, 4 lines are drawn on each hole, and the Huh7 cells in vigorous growth are uniformly laid on the 6-hole plate, and the cells are cultured overnight, the next day, when the cells reach 60%-70% of the fusion degree, 10ul of gun head is used to quickly draw a vertical line on the scratch, and then it is washed with culture medium, and then it is washed with PBS, and then cell transfection is carried out, and it is incubated in a 37℃ incubator, and then the 6-hole plate is taken out at 0h, 24h, 48h and 72h, and the photo is taken and saved.

[0010] As a preferred scheme of the verification mechanism of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the specific method of the Transwell experiment in S5 is: replace the serum-free culture solution, starve the cells for 18h, and prepare Matrix-GelTM matrix glue, melt in the ice block, place in the 4℃ refrigerator, configure the diluent of the matrix glue and the serum-free culture medium on the ice at 1:7, uniformly and slowly add it vertically on the Trantswell chamber, 65ul per hole, incubate for 3h, suck out the unbound matrix glue, and then add 100ul of empty culture medium, incubate for half an hour at 37℃, hydrate, if there is no leakage in the lower chamber of the chamber, inoculate cells in the upper chamber, add 500ul of culture solution containing 20% FBS in the lower chamber, incubate for 48h, discard the liquid, clean the matrix glue and cells in the chamber with a cotton ball, add 600ul of fixing solution and stand for 40min, then add 0.5% crystal violet solution for 15min, and take a picture after washing.

[0011] As a preferred scheme of the verification mechanism of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the specific method of the Transwell experiment in S5 is: replace the serum-free culture solution, starve the cells for 18h, and prepare Matrix-GelTM matrix glue, melt in the ice block, place in the 4℃ refrigerator, configure the diluent of the matrix glue and the serum-free culture medium on the ice at 1:7, uniformly and slowly add it vertically on the Trantswell chamber, 65ul per hole, incubate for 3h, suck out the unbound matrix glue, and then add 100ul of empty culture medium, incubate for half an hour at 37℃, hydrate, if there is no leakage in the lower chamber of the chamber, inoculate cells in the upper chamber, add 500ul of culture solution containing 20% FBS in the lower chamber, incubate for 48h, discard the liquid, clean the matrix glue and cells in the chamber with a cotton ball, add 600ul of fixing solution and stand for 40min, then add 0.5% crystal violet solution for 15min, and take a picture after washing.

[0012] As a preferred scheme of the verification mechanism of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the specific method of the Transwell experiment in S5 is: replace the serum-free culture solution, starve the cells for 18h, and prepare Matrix-GelTM matrix glue, melt in the ice block, place in the 4℃ refrigerator, configure the diluent of the matrix glue and the serum-free culture medium on the ice at 1:7, uniformly and slowly add it vertically on the Trantswell chamber, 65ul per hole, incubate for 3h, suck out the unbound matrix glue, and then add 100ul of empty culture medium, incubate for half an hour at 37℃, hydrate, if there is no leakage in the lower chamber of the chamber, inoculate cells in the upper chamber, add 500ul of culture solution containing 20% FBS in the lower chamber, incubate for 48h, discard the liquid, clean the matrix glue and cells in the chamber with a cotton ball, add 600ul of fixing solution and stand for 40min, then add 0.5% crystal violet solution for 15min, and take a picture after washing.

[0013] As a preferred scheme of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the specific method of step 1 is: to establish a subcutaneous liver cancer model of nude mice: after 4-5 weeks of age, male BALB / c-nu mice are adaptively fed for one week, the Huh7 cells in the logarithmic growth phase are counted, the cell pellet is resuspended to 5x106 / 100ul, and the nude mice are subcutaneously inoculated to build an ectopic liver cancer model, and the body weight and tumor body of the nude mice are measured every three days; grouping and administration: after the modeling is completed, the nude mice are randomly divided into groups, and the tumor body is injected with drugs, and the nude mice are randomly divided into 5 groups, Control group, Si-1 group, Si-2 group, Si-1+Si-2 group, and NC group, at least three positions of the tumor body are injected, and the Control group is injected with normal saline; and finally, paraffin sections are prepared.

[0014] As a preferred scheme of the application of the long-chain non-coding RNA interfering Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer, wherein the specific method of step 3 is: to dewax and hydrate: the section is immersed in xylene, and then sequentially immersed in different concentrations of ethanol solution from high to low, and then washed; antigen repair: the section is immersed in citric acid antigen repair buffer, heated, slowly cooled, and then washed on a shaker; remove the activity of endogenous peroxidase and block; primary and secondary antibody incubation: discard the blocking solution, dilute the primary antibody Bcl-2 (1:200) and Bax (1:200) according to the ratio, incubate overnight, discard the primary antibody, wash 3 times with PBS, and add the secondary antibody and incubate for 1h; section color development and restaining; dehydration and mounting; after mounting, observe and collect images.

[0015] Compared with the prior art, the application has the beneficial effects that: through in vivo and in vitro experiments, the application specifically interferes with the expression of Oct4-pg4 and Oct4-pg5 by using RNAi technology, and observes the influence on the biological behaviors of liver cancer cells such as proliferation, migration, invasion and cell cycle. Meanwhile, the application further explores the regulatory effect of the expression changes of Oct4-pg4 and Oct4-pg5 on the original gene Oct4 and the cell cycle, apoptosis and differentiation related genes, proves that the interference of Oct4 pseudogenes pg4 and pg5 inhibits the invasion, proliferation and immune escape of HCC cells by regulating the expression of multiple key genes, and induces cell apoptosis and differentiation, thereby providing a new idea for the treatment of liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the present application will be described in detail below with reference to the drawings and detailed embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor. Among them: Figure 1 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of cell activity after 24, 48h of Oct4-pg4 sequence interference SMMC-7721 cells; Figure 2 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of cell activity after 24, 48h of Oct4-pg5 sequence interference SMMC-7721 cells; Figure 3 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of cell activity after 24, 48h of Oct4-pg4 sequence interference HepG2 cells; Figure 4 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of cell activity after 24, 48h of Oct4-pg5 sequence interference HepG2 cells; Figure 5 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of cell activity after 24, 48h of Oct4-pg4 sequence interference Huh7 cells; Figure 6 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of cell activity after 24, 48h of Oct4-pg5 sequence interference Huh7 cells; Figure 7 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of Oct4-pg4, Oct4-pg5 gene expression in interfering SMMC-7721 cells; Figure 8 Figure for the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNA in the treatment of liver cancer and verifying the mechanism of the effect of Oct4-pg4, Oct4-pg5 gene expression in interfering HepG2 cells; Figure 9 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 10 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 11 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 12 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 13 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 14 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 15 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 16 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 17 Figure of the effect of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs on the expression of Oct4-pg4 and Oct4-pg5 genes of Huh7 cells in the application of interfering Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism; Figure 18Figure 6 is a diagram showing detection of BIRC5 and CCND1 gene expression after transfection of Huh7 cells according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 19 Figure 7 is a diagram showing detection of Bcl-2, Bax and Caspase3 gene expression after transfection of Huh7 cells according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 20 Figure 8 is a diagram showing detection of c-Myc, PD-1 gene expression after transfection of Huh7 cells according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 21 Figure 9 is a diagram showing a scratch of transfection cells of Huh7 cells under a microscope according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 22 Figure 10 is a diagram showing migration rate of Huh7 cells according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 23 Figure 11 is a diagram showing cell number of Huh7 cells after transfection for 48 h under a microscope according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 24 Figure 12 is a diagram showing cell number of Huh7 cells after transfection for 48 h according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 25 Figure 13 is a diagram showing cell apoptosis of Huh7 cells after transfection for 48 h according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 26 Figure 14 is a diagram showing cell cycle of Huh7 cells after transfection for 48 h according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 27 Figure 15 is a diagram showing growth weight of nude mice according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 28 Figure 16 is a diagram showing tumor volume of nude mice according to the application interfering with Oct4-pg4 and Oct4-pg5 long-chain non-coding RNAs in the treatment of liver cancer and verifying the mechanism thereof; Figure 29The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of nude mice tumor volume column chart; Figure 30 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of nude mice heart, liver, spleen, lung, kidney coefficient change chart; Figure 31 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of each group of mice tumor tissue pathology H&E section change chart under microscope; Figure 32 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of each group of mice tumor Bcl-2 protein expression chart under microscope; Figure 33 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of each group of mice tumor Bax protein expression chart under microscope; Figure 34 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of tumor protein Bcl-2 (left), Bax (right) expression chart; Figure 35 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of Oct4-pg4, Oct4-pg5 gene expression chart in nude mice tumor; Figure 36 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of Oct4 gene expression chart in nude mice tumor; Figure 37 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of Bax, Bcl-2, Caspase3 gene expression in nude mice tumor; Figure 38 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of pseudogene CCND1, BIRC5 gene expression chart in nude mice tumor; Figure 39 The application of the long-chain non-coding RNA Oct4-pg4 and Oct4-pg5 interfering with the present application in the treatment of liver cancer and the verification mechanism of pseudogene PD-1, c-Myc gene expression chart in nude mice tumor; Figure 40 Figure 4 is a diagram showing the expression of CCND1, BIRC5 and Oct4 proteins in tumor tissues of nude mice for verifying the mechanism of application of the long-chain non-coding RNAs Oct4-pg4 and Oct4-pg5 in treatment of liver cancer; Figure 41 Figure 5 is a diagram showing the expression of Bax, Bcl-2 and Caspase3 proteins in tumor tissues of nude mice for verifying the mechanism of application of the long-chain non-coding RNAs Oct4-pg4 and Oct4-pg5 in treatment of liver cancer; Figure 42 Figure 6 is a diagram showing the expression of PD-1 and c-Myc proteins in tumor tissues of nude mice for verifying the mechanism of application of the long-chain non-coding RNAs Oct4-pg4 and Oct4-pg5 in treatment of liver cancer. DETAILED DESCRIPTION

[0017] In order to make the above objectives, features and advantages of the present application more apparent, specific embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0018] Secondly, the present application is described in detail in combination with the schematic diagram. In the detailed description of the embodiments of the present application, the cross-sectional view of the device structure will be partially enlarged without the general proportion for the convenience of description, and the schematic diagram is only an example which should not limit the scope of protection of the present application. In addition, the three-dimensional spatial dimensions of length, width and depth should be included in the actual manufacture.

[0019] In order to make the objectives, technical solutions and advantages of the present application more apparent, the embodiments of the present application will be described in further detail below with reference to the accompanying drawings.

[0020] The application of the long-chain non-coding RNAs Oct4-pg4 and Oct4-pg5 in treatment of liver cancer inhibits the proliferation, migration and invasion of liver cancer cells, induces apoptosis of liver cancer cells, and regulates the expression of liver cancer cell cycle and immune escape related genes.

[0021] In order to verify the influence of the expression of the lncRNA Oct4-pg4 and Oct4-pg5 from the pseudogene on human liver cancer cells, the following two experiments were carried out in the present study 1. In vitro experiment S1: The influence of the long-chain non-coding RNAs Oct4-pg4 and Oct4-pg5 on the growth activity of liver cancer cell lines SMMC-7721, HepG2 and Huh7 was detected by MTT method after 24h and 48h.

[0022] 1.1: Set Control, Si-429, Si-920 and Control, Si-457, Si-635, Si-697, NC group, take good growth SMMC-7721, HepG2, Huh7 cells, discard the culture solution, add 2 ml PBS to wash the cells, trypsin digestion, gently blow and collect the cells, centrifuge at 1100 rpm for 3 min, discard the supernatant and resuspend and count, 0.8 x 104 cells per well, set 3 replicate wells per group, and return to the incubator.

[0023] 1.2: When the cell density reaches 60%-70%, transfection is performed. Take out the divided siRNA (20 pmol / μl), according to the recommended amount of transfection reagent instruction and optimization: dilute siRNA and transfection reagent in culture medium without double antibody, incubate at room temperature for 10 min, then mix, incubate for 20 min. Add 10 μl complex to each well, mix gently, continue to culture for 24, 48 h, and then detect without changing the culture medium.

[0024] 1.3: Measure two 96-well plates after 24, 48 h, add 20 μl of freshly prepared MTT solution to each replicate well, incubate in the incubator for 4 h, discard the liquid, add 150 μl of DMSO to each well, wait for 15 min, and measure the absorbance at 490 nm by placing the enzyme marker in the vibration plate for 20 s. Repeat the experiment three times, take the average value and draw the curve.

[0025] The results are as follows Figures 1-6 As shown, it is found that interference of Oct4-pg4, Oct4-pg5 significantly inhibits cell proliferation, and in three cell lines Si-920 and Si-635 sequences are the best, and the effect is better than 24 h at 48 h.

[0026] S2: Further verify the best transfection efficiency of the sequence by qPCR and determine the effect of single and combined interference of the two pseudogenes on the original gene Oct4.

[0027] 2.1: Collect and lyse the cells: centrifuge the adherent cells and discard the supernatant to leave the cell pellet, ensure the cell amount is 1 x 106, place in a 1.5 ml EP tube, add 0.5 ml SevenFast® lysis solution, mix well by blowing, and stand for 60 s for complete lysis. Rinse, remove ethanol, and elute according to the instructions.

[0028] 2.2: Collecting purified RNA: add 30 μl RNase-Free H2O in the middle of the adsorption membrane, stand at room temperature for 60 s, centrifuge at 1100 rpm for 60 s. Further reverse transcription into cDNA. Measure the RNA content with Take3 plate of the enzyme marker, wipe the plate with DEPC water with lens paper and adjust to zero. Take 2 μl of each group of extracted RNA samples, measure three times and take the average, the OD260 / OD280 ratio is about 2.0 to ensure RNA purity.

[0029] 2.3: According to the PrimeScriptTM FASTRT reagent Kit with gDNA Eraser kit instructions, RNA reverse transcription and real-time fluorescent quantitative PCR are carried out.

[0030] The results are shown in Figures 7-13 , wherein Figure 7 the left graph is the expression of interfering Oct4-pg448h; the right graph is the expression of interfering Oct4-pg548h. Figure 8 the left graph is the expression of interfering Oct4-pg448h; the right graph is the expression of interfering Oct4-pg548h. Figure 9 the left graph is the expression of interfering Oct4-pg448h; the right graph is the expression of interfering Oct4-pg548h. Figure 10 the left graph is the expression of interfering Oct4-pg4; the right graph is the expression of interfering Oct4-pg5. Figure 11 the left graph is the expression of interfering Oct4-pg4; the right graph is the expression of interfering Oct4-pg5. Figure 12 the left graph is the expression of interfering Oct4-pg4; the right graph is the expression of interfering Oct4-pg5. Figure 13 the left graph is the expression of interfering Oct4-pg4; the right graph is the expression of interfering Oct4-pg5.

[0031] S3: Observe the morphological changes of Huh7 cells after transfection, and observe the morphological changes of Huh7 cells after transfection, and measure the cell growth activity after transfection for 0h, 24h, 48h, 72h. Observe the morphology of Huh7 cells under a microscope, as shown in Figure 14As shown, the cell morphology after interference was obviously shrunk and irregular, with different sizes, poor integrity, and a large number of dead cells floating, compared with the Control group. The expression of CCND1, BIRC5, Bax, Bcl-2, Caspase3, PD-1 and c-Myc genes was detected by qPCR (experimental method same as S2), to determine the effect of single and combined interference of pseudogenes on Oct4 and related genes. The growth activity curve of Huh7 cells after 0h, 24h, 48h, 72h of single and combined interference was detected by MTT experiment (experimental method same as S1), as shown in Figure Figures 15-17 As shown, the combined interference had better effect than the single interference.

[0032] S4: WB experiment was used to detect the expression of Oct4 protein and related apoptosis proteins Bax, Bcl-2, Caspase3, cyclin CCND1, BIRC5, and PD-1, c-Myc in Huh7 cells after single and combined interference of pseudogenes, to further determine the effect of interfering pseudogenes on hepatoma cells.

[0033] 4.1: Cell total protein extraction: Huh7 cells were prepared into a single cell suspension, and 2x105 per well was uniformly plated in a 60mm dish for overnight transfection. After 48h, the supernatant was discarded, the cells were washed with ice PBS, placed on ice slush, and lysate containing PMSF was added. After 0.5h, the cells were gently scraped and collected in a 1.5ml centrifuge tube with a 200ul gun head. Centrifugation was performed at 12000rpm for 3min at 4°C, and the supernatant was transferred to a new 1.5ml Ep tube.

[0034] 4.2: BCA method for protein content determination and standard curve preparation: 2mg BSA Standard was diluted with 1xPBS to 2mg / ml and aliquoted standard solution was stored at -20°C. The pre-calculated working solution was prepared according to the proportion. If the protein is relatively concentrated, it can be diluted to a certain proportion before measuring the OD value at 562nm.

[0035] 4.3: 5x SDS sample cooking: mix the protein supernatant with 5x protein loading buffer solution, put it in a 95°C pot for 10min, then take it out, cool it down and aliquot to avoid repeated freeze-thawing.

[0036] 4.4: Leaking and making gel: wash the 1mm thick glass plate and drain the water, assemble the gel making device, add distilled water between the two glass plates to the edge of the low plate, and stand for 5min. If there is no leakage, discard the distilled water and absorb the water. First, add 4ml separation gel with distilled water and press it flat for 5min, then absorb the water with filter paper. Then add 2.5ml concentrated gel and insert the comb to avoid air bubbles. After the gel is completely coagulated, it is stored for use.

[0037] 4.5: Loading: Put the gel clip placed in 4℃ refrigerator into the inner tank, fill the tank with the new electrophoresis solution, gently pull out the comb vertically, add the sample into the hole with 20μl per hole, and add the Maker as the reference standard.

[0038] 4.6: Electrophoresis: Run the upper gel at 80V for 30min, and run the lower gel at 120V for 50min. According to the separation of the Maker in the electrophoresis and the molecular weight of the target protein, the electrophoresis can be stopped appropriately.

[0039] 4.7: Transferring: According to the need, cut the gel with the Maker, place it on the pre-prepared "sandwich" filter paper, soak it with the transferring solution, cut the PVDF membrane according to the need, cover the gel after activating it in methanol for 40s, clamp it in the order of transparent plate-sponge pad-filter paper-PVDF membrane-gel-filter paper-sponge pad-blackboard, and get rid of the air bubbles, place the "sandwich" in the "black against black, red against transparent" position, add the transferring solution, set the current to 200mA for 80min, and ensure the low temperature condition during the transferring.

[0040] 4.8: Blocking: After the transferring, disassemble the transferring tank and check whether there is a clear Maker mark on the PVDF membrane, then place the PVDF membrane in the incubation box with the blocking solution for 2h, with the protein facing down.

[0041] 4.9: Washing the membrane: Add the TBST solution to wash the membrane, and place it on the shaker at room temperature for 15min, and wash it three times.

[0042] 4.10: Incubating the primary antibody and washing the membrane: Dilute the primary antibodies Oct4 (1:4000), BIRC5 (1:800), CCND1 (1:800), Bax (1:5000), Bcl-2 (1:5000), Caspase (1:1000), PD-1 (1:800), and c-Myc (1:800) according to the proportion, respectively, and place them in the incubation box for incubation on the shaker and overnight in the 4℃ refrigerator. Recover the primary antibody, add the TBST solution to wash the membrane, and wash it three times for 15min each time.

[0043] 4.11: Incubating the secondary antibody and washing the membrane: Dilute the secondary antibody according to the proportion (1:5000) and place it in the incubation box for incubation at room temperature for 2h. Recover the secondary antibody, add the TBST solution to wash the membrane, and wash it three times for 15min each time.

[0044] S5: Use qPCR to detect the expression of CCND1, BIRC5, Bax, Bcl-2, Caspase3, PD-1, and c-Myc genes, and clarify the effect of single and combined interference of the pseudogenes on Oct4 and related genes (the experimental method is the same as S2). For example, Figures 18-20As shown, after interfering with Huh7 cells, the expression of CCND1, BIRC5 cell cycle genes was down-regulated, the expression of Bax, Caspase3 apoptosis genes was up-regulated, the expression of Bcl-2 gene was down-regulated, the expression of immune gene PD-1 and low differentiation c-Myc gene was down-regulated, and the combined inhibition effect was better.

[0045] S6: The effects of single and combined interference on the migration and invasion ability of Huh7 cells were explored by cell scratch and Transwell experiments.

[0046] Cell scratch experiment: the experiment was divided into blank group, Si-920 group, Si-635 group, Si-920+Si-635 group, NC group, a thin pen was used to draw a horizontal line every 0.6 cm on the back of the 6-hole plate, 4 lines were drawn on each hole, and the Huh7 cells were evenly spread on the 6-hole plate and cultured overnight. The next day, when the cells reached 60%-70% confluence, 10 μl of gun head was used to quickly draw a vertical line on the scratch, ensuring that the drawn trace was a straight line and the width was consistent. After washing with culture medium and PBS, cell transfection was performed, and the 6-hole plate was placed in a 37°C incubator. At 0h, 24h, 48h, and 72h, the 6-hole plate was taken out, photographed, and the photos were saved.

[0047] Transwell experiment: replace the serum-free culture medium, starve the cells for 18h, and prepare Matrix-GelTM matrix glue, melt in the ice block at 4°C. On ice, prepare the diluent of matrix glue and serum-free culture medium at 1:7, add it evenly and slowly vertically on the Transwell chamber, avoid air bubbles, 65 μl per hole, incubate for 3h, absorb the unbound matrix glue, add 100 μl of empty culture medium, incubate for half an hour at 37°C, and hydrate. Check if the lower chamber of the chamber leaks, if not, inoculate cells in the upper chamber, add 500 μl of culture medium containing 20% FBS to the lower chamber, and incubate for 48h. Discard the liquid, use cotton balls to remove the matrix glue and cells in the chamber, add 600 μl of fixing solution and stand for 40 min, discard the liquid, wash with PBS, and then add 0.5% crystal violet solution for 15 min, wash and take a photo. As shown, the scratch experiment showed that the cell migration was inhibited after transfection, especially after combined transfection, a large number of cells floated and died, and the cell migration was more obviously inhibited. Transwell invasion experiment showed that compared with the Control group, the number of cells penetrating the membrane in each transfection group was reduced, and the number of cells penetrating the membrane in the combined transfection group was more significantly reduced. Figures 21-24

[0048] S7: The effects of single and combined interference on the apoptosis and cycle of Huh7 cells were detected by flow cytometry.

[0049] ​Apoptosis experiment: well-grown Huh7 cells were seeded at 2.5x105 per well in 6-well plates, transfected overnight, and collected 48h later. The supernatant was collected together. The cells were centrifuged with pre-cooled PBS and 1.5x105 cells were collected. In a 1.5ml Ep tube, 500μl of 1x Annexin V Binding Buffer was added to resuspend the cells, and 2.5μl of Annexin V-FITC Reagent and 2.5μl of PI were added to the suspension. After mixing, the mixture was incubated at 25°C for 20min in the dark, and then immediately detected by flow cytometry. If the detection cannot be completed in time, the mixture can be stored on ice in the dark and detected within 1h.

[0050] Cell cycle experiment: anhydrous ethanol was prepared in a -20°C refrigerator overnight, and RNase A Reagent was dissolved and mixed. The transfection process was the same as apoptosis, and about 1.5x105 cells were collected after 48h of treatment. Centrifugation at 1100rpm for 5min, discard the supernatant. Add 1ml of PBS and centrifuge at 1100rpm for 3min, discard the supernatant. Add 0.3ml of PBS and 1.2ml of -20°C anhydrous ethanol to resuspend the cells, mix well by blowing, and then store in a -20°C refrigerator overnight. Centrifuge at 12000rpm for 3min, discard the supernatant, resuspend the cells with 1ml of PBS, and place at room temperature for 15min. Discard the supernatant, add 100μl of RNase A Reagent, and mix the cells well by blowing. After 0.5h of 37°C water bath, add 400μl of PI Reagent and mix well. Incubate in a 4°C refrigerator in the dark for 0.5h, and immediately detect the red fluorescence at an excitation wavelength of 488nm. The apoptosis results are shown in Figure 2. Figure 25 The results of the cell cycle are shown in Figure 3. Figure 26 As shown in Figure 3, after 48h of transfection, the G0 / G1 phase of the combined group was blocked compared with the Control group, and the S phase cells were significantly reduced, affecting the cell cycle.

[0051] PD-1 gene plays an important role in tumor immunity and chronic infection by encoding PD-1 protein, and is also related to immune escape of tumor cells. Long-term chronic inflammation can stimulate up-regulation of PD-1 receptor, leading to the occurrence of liver cancer. c-Myc affects cell proliferation, invasion, etc., promotes cell division, causes cell abnormalities, and regulates HCC progression. The qPCR and WB experiments proved that the expression of PD-1 and c-Myc genes and proteins was down-regulated after interference with the pseudo-genes Oct4-pg4 and Oct4-pg5, and the function of tumor cells was regulated.

[0052] 2. In vivo experiment Step one: mouse liver cancer model modeling, drug administration and section preparation (1) Establishing the subcutaneous hepatoma model in nude mice: 4-5 weeks old male BALB / c-nu mice were adaptively fed for one week, then the Huh7 cells in logarithmic growth phase were counted, and the precipitate was resuspended in PBS three times, centrifuged, and placed on ice. Finally, the cell precipitate was resuspended to 5x106 / 100 μl with a 1 ml syringe, and each nude mouse was subcutaneously inoculated with 100 μl. The ectopic hepatoma model was established, and the state of the nude mice was observed every day after inoculation. The body weight of the nude mice was measured every three days, and the tumor volume was measured according to the formula: tumor volume = (A x B2) / 2 (A: tumor long diameter; B: tumor short diameter).

[0053] (2) Grouping and dosing regimen: After the modeling was completed, the nude mice were randomly divided into groups, and the tumor injection was started when the tumor grew to 50 mm3. The injection was given twice a week, and the entire dosing process lasted for two weeks from the start of dosing to the end of dosing. The nude mice were randomly divided into 5 groups: Control group, Si-1 group, Si-2 group, Si-1+Si-2 group, and NC group (wherein Si-1 represents interfering Oct4-pg4, and Si-2 represents interfering Oct4-pg5). At least three positions of the tumor were selected for injection, and the Control group was injected with normal saline. Each mouse in the dosing group was injected with a total of 1.25 nmol siRNA. After the experiment, the nude mice were sacrificed by decapitation, and the tumor was photographed. The tumor was divided and packaged according to the experimental requirements. The mouse was dissected to remove the heart, liver, spleen, lung, and kidney organs, which were placed in normal saline and dried with filter paper. The organs were weighed and recorded, and the organ index (%) was calculated according to the formula: organ index (%) = (total organ weight / nude mouse weight) x 100%.

[0054] (3) Preparation of paraffin sections: The tumor mass packaged in the tissue fixative was washed with running water and then dehydrated with alcohol to remove water from the tumor mass. The tumor mass was dehydrated with 70%, 85%, 95%, and then absolute ethanol, with each dehydration step lasting 1 hour. If the dehydration steps could not be performed sequentially, the tumor mass could be stored in 70% alcohol. The tumor mass was then infiltrated with a mixture of absolute ethanol and xylene in a ratio of 1:1 for 2 hours, followed by immersion in xylene until the tumor mass became transparent. The tumor mass was then embedded in a mixture of melted paraffin and xylene in a ratio of 1:1 for 2 hours, with the largest surface of the tumor mass facing upwards. The embedded tumor mass was then cut into four-sided slices with a thickness of 6 μm. The slices were then adhered to glass slides using glycerol, and the glass slides were then dried in a 50°C oven.

[0055] As shown in Figures 27-30 , no significant abnormal behavior changes were observed in the nude mice during the dosing period. The body weight of the nude mice increased slightly, but there was no significant effect on the normal life of the nude mice, and there was no significant difference. Through observation of the subcutaneous ectopic hepatoma model in nude mice, it was found that the body weight of the nude mice and the organs of the heart, liver, spleen, lung, and kidney were not significantly abnormal during the dosing period. The injection of siRNA into the tumor had no significant toxicity, and the tumor volume was significantly reduced, indicating that siRNA had a therapeutic effect and a better combined effect.

[0056] Step two: H&E staining (1) Dewaxing and hydration: The paraffin slices were soaked in xylene for 10 minutes, then soaked in alcohol of high concentration to low concentration (anhydrous ethanol, 90% alcohol, 80% alcohol, 70% alcohol), and finally washed with distilled water.

[0057] (2) Staining: The sections were stained in hematoxylin solution, washed with tap water for 1 hour, then soaked in distilled water, dehydrated in 70% and 90% alcohol for 10 minutes each, and then stained with eosin staining solution for 3 minutes.

[0058] (3) Dehydration and transparency: The stained sections are dehydrated by 70% alcohol, 80% alcohol, 90% alcohol and anhydrous ethanol, and then soaked in xylene to make the sections transparent.

[0059] (4) Sealing and observation: Apply neutral resin to the transparent section and seal it with a coverslip. After the resin dries, attach a label. Observe and photograph under a microscope, and finally save the image.

[0060] like Figure 31 As shown, H&E staining of tumor cells revealed that, compared with the saline group, the combined treatment group exhibited varying cell sizes and morphologies and lower cell density. Immunohistochemistry showed that the combined treatment group had a significantly increased Bax gene positivity rate and a significantly decreased Bcl-2 gene positivity rate, indicating that interference with the pseudogenes Oct4-pg4 and Oct4-pg5 induced apoptosis.

[0061] Step 3: Immunohistochemical staining (1) Dewaxing and hydration: Soak the sections in xylene for 10 min, and then soak them in anhydrous ethanol, 95% ethanol, 70% ethanol and 50% ethanol solutions for 5 min each, in descending order of concentration. Finally, soak them in distilled water for 5 min and then wash them with PBS.

[0062] (2) Antigen retrieval: Immerse the slide in citrate antigen retrieval buffer, heat in a pressure cooker for 10 minutes, cool slowly, and then wash thoroughly on a shaker.

[0063] (3) Remove endogenous peroxidase activity and block: Add 3% H2O2 solution to the surface of the slide and block for 10 min to remove endogenous peroxidase in the tissue. Immerse the slide completely in BSA and block at 25℃ for 30 min.

[0064] (4) Incubation with primary and secondary antibodies: Discard the blocking solution, dilute the primary antibody Bcl-2 (1:200) and Bax (1:200) at a ratio of 1:200, and incubate overnight at 4°C. Discard the primary antibody, wash 3 times with PBS, and incubate with secondary antibody for 1 hour.

[0065] (6) Slice color and restain: DAB color developing liquid was used for staining, and uniform light brown color was observed on the surface of the slice under the microscope, and the color developing effect was fixed. Hematoxylin was used for restaining. After obvious staining effect was observed, the slice was washed with tap water, and then returned to blue treatment with PBS buffer for 5 min.

[0066] (8) Dehydration and mounting: the slice was sequentially soaked in 50%, 70%, 95% ethanol, anhydrous ethanol for 10 min, and then soaked in dimethylbenzene for 10 min. Neutral balsam was added for mounting.

[0067] (9) Observation and image collection after mounting: the slice was placed under the microscope for observation and photography.

[0068] Through immunohistochemical staining, the expression of antibodies in tumor tissues was detected by using known antibodies Bcl-2 and Bax in proportion, as shown in Table 1. Figures 32-34 The interference of the pseudo-genes Oct4-pg4 and Oct4-pg5 reduced the proportion of Bcl-2 positive in tumor tissues, reduced the expression of Bcl-2 protein, increased the proportion of Bax positive, and increased the expression of Bax protein. The combined interference effect was more significant.

[0069] Step four: qPCR was used to detect the expression of tumor genes (the experimental process was the same as step two).

[0070] Step five: Western blot was used to detect protein expression (the experimental process was the same as S7).

[0071] The expression of Oct4-pg4, Oct4-pg5, BIRC5 and CCND1, Bax, Bcl-2 and Caspase3, c-Myc and PD-1 in the tumor of nude mice was detected by qPCR, and the effects of the interference on BIRC5, CCND1, Oct4, Bax, Bcl-2, Caspase3, PD-1 and c-Myc were detected by Western blot. The results showed that the injection of modified siRNA into the tumor could knock down the expression of Oct4-pg4, Oct4-pg5 and the expression of the original gene Oct4 was also inhibited, and the effect of the combined interference group was more obvious. The expression of the apoptosis gene Bax was increased, the expression of the Bcl-2 gene was decreased, which was consistent with the results of the cell experiment, the expression of Caspase3 gene had an upward trend, which confirmed that the interference of the pseudo gene could cause tumor cell apoptosis. The expression of CCND1 and BIRC5 genes was decreased, which indicated that the interference of the pseudo gene could affect the progress of the cell cycle. The expression of PD-1 and c-Myc genes was decreased, and the inhibition of the pseudo gene also affected cell differentiation. The detection of protein level also confirmed that the expression of Oct4, CCND1 and BIRC5 and Bcl-2 protein was decreased, the expression of Bax and Caspase3 protein was increased, and the expression of PD-1 and c-Myc protein was also decreased (as shown in Figures 35-42 ).

[0072] Although the present application has been described with reference to the embodiments above, it is possible for various modifications and / or alterations to be made to the application without departing from the scope of the application. In particular, various features of the disclosed embodiments can be combined together in any manner, and the combinations of these features are not exhaustively described in the specification only for the sake of brevity and conciseness. Therefore, the present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Application of interference with Oct4-pg4 and Oct4-pg5 long non-coding RNAs in the treatment of liver cancer.

2. The application of the long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 according to claim 1 in the treatment of liver cancer, characterized in that, The interference of Oct4-pg4 and Oct4-pg5 long non-coding RNAs inhibits the proliferation, migration, and invasion of liver cancer cells, induces apoptosis in liver cancer cells, and regulates the expression of genes related to liver cancer cell cycle and immune escape.

3. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 1, characterized in that, This includes in vitro experiments and in vivo experiments, wherein the in vitro experiments include the following steps: S1: The effects of interfering pseudogenes Oct4-pg4 and Oct4-pg5 on the growth activity of hepatocellular carcinoma cell lines SMMC-7721, HepG2, and Huh7 after 24 h and 48 h were detected by MTT assay. S2: Further validate the optimal transfection efficiency of the sequence and clarify the effects of single and combined interference with the two pseudogenes on the original gene Oct4 by qPCR: collect cells and lyse them; collect and purify RNA; perform reverse transcription of RNA and real-time quantitative PCR. S3: Observe the morphological changes of Huh7 cells after transfection, and interfere with the two pseudogenes alone and in combination. Measure cell growth activity at 0h, 24h, 48h and 72h after transfection. S4: The expression of CCND1, BIRC5, Bax, Bcl-2, Caspase3, PD-1 and c-Myc genes was detected by qPCR to clarify the effects of individual and combined interfering pseudogenes on Oct4 and related genes. S5: Investigate the effects of single and combined interference on the migration and invasion abilities of Huh7 cells using cell scratch and Transwell assays; S6: Flow cytometry was used to detect the effects of single and combined interfering pseudogenes on apoptosis and cell cycle in Huh7 cells; S7: Western blot analysis was used to detect the expression of Oct4 protein and related apoptosis proteins Bax, Bcl-2, Caspase3, cyclin CCND1, BIRC5, PD-1, and c-Myc in Huh7 cells by single and combined interference pseudogenes. The in vivo experiment includes the following steps: Step 1: Mouse liver cancer model establishment, drug administration, and section preparation; Step 2: H&E staining; Step 3: Immunohistochemical staining; Step 4: qPCR detection of tumor gene expression; Step 5: Western blot analysis.

4. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 3, characterized in that, The specific detection method for S1 is as follows: Control, Si-429, Si-920 and Control, Si-457, Si-635, Si-697, NC groups were set up. SMMC-7721, HepG2, and Huh7 cells were taken, washed, digested, pipetted, centrifuged, and counted, with 0.8 × 10⁴ cells per well and 3 replicates per group. When the cell density reached 60%-70%, transfection was performed. 10 μl of the complex was added to each well, gently pipetted to mix, and cultured for 24 and 48 hours before detection. The absorbance of two 96-well plates was measured after 24 and 48 hours. 20 μl of freshly prepared MTT solution was added to each replicate well, and after incubation for 4 hours, 150 μl of DMSO was added to each well. After waiting 15 minutes, the absorbance at 490 nm was measured. The experiment was repeated three times, and the average value was plotted as a curve.

5. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 3, characterized in that, The specific method of the cell scratch assay in S5 is as follows: The experiment is divided into blank group, Si-920 group, Si-635 group, Si-920+Si-635 group, and NC group. Draw a horizontal line every 0.6 cm on the back of the 6-well plate with a water pen, and draw 4 lines on each well. Spread the vigorously growing Huh7 cells evenly on the 6-well plate and culture the cells overnight. On the second day, when the cells reach 60%-70% confluence, quickly draw vertical horizontal lines with a 10 μl pipette tip. Wash once with culture medium, then wash with PBS, and then perform cell transfection. Incubate in a 37℃ incubator. Take out the 6-well plate at 0h, 24h, 48h, and 72h, take pictures and save the photos.

6. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 3, characterized in that, The specific method for the Transwell experiment in S5 is as follows: Replace with serum-free culture medium, starve cells for 18 hours, and prepare Matrix-Gel™ matrix gel. Thaw the gel in ice at 4°C. Prepare a 1:7 dilution of matrix gel and serum-free culture medium on ice, and add it evenly and slowly vertically to the Transwell chambers, 65 μl per well. Incubate for 3 hours, aspirate unbound matrix gel, add 100 μl of empty culture medium, and incubate at 37°C for half an hour to hydrate. If there is no leakage in the lower chamber, seed cells in the upper chamber. Add 500 μl of culture medium containing 20% ​​FBS to the lower chamber and incubate for 48 hours. Discard the liquid, remove matrix gel and cells from the chamber with cotton balls, add 600 μl of fixative and let stand for 40 minutes, then add 0.5% crystal violet solution for staining for 15 minutes. Wash and photograph.

7. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 1, characterized in that, The apoptosis assay in S6 was performed as follows: 2.5 × 10⁵ Huh7 cells in good growth condition were seeded in each well of a 6-well plate and transfected overnight. After 48 hours, the cells were digested and the supernatant was collected. Pre-cooled PBS was added, and the cells were centrifuged, collecting 1.5 × 10⁵ cells. 500 μl of 1× Annexin VBinding Buffer was added to a 1.5 ml Eppendorf tube to resuspend the cells. Then, 2.5 μl of Annexin V-FITC Reagent and 2.5 μl of PI were added to the suspension, mixed, and incubated at 25°C in the dark for 20 min. The cells were then immediately analyzed by flow cytometry.

8. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 3, characterized in that, The cell cycle experiment method in S6 is as follows: Prepare anhydrous ethanol and incubate overnight at -20°C. Dissolve RNase A Reagent, mix well, and keep on ice for later use. The transfection process is the same as for cell apoptosis. After 48 h, process and collect 1.5 × 10⁵ cells, centrifuge, discard the supernatant, wash, centrifuge, discard the supernatant, add 0.3 ml PBS and 1.2 ml -20°C anhydrous ethanol to resuspend the cells, mix well by pipetting, incubate overnight at -20°C, centrifuge, discard the supernatant, add 1 ml PBS to resuspend the cells, place at room temperature, centrifuge and discard the supernatant, add 100 μl RNase A Reagent and pipette the cells thoroughly, incubate in water, add 400 μl PI Reagent, mix well, incubate in the dark, and record the red fluorescence at the excitation wavelength of 488 nm.

9. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 1, characterized in that, The specific method of step 1 is as follows: Establish a subcutaneous liver cancer model in nude mice: After one week of acclimatization feeding, 4-5 week old male BALB / c-nu mice were digested and counted in the logarithmic growth phase. The cell pellet was resuspended at 5 × 10⁶ cells / 100 μl and subcutaneously inoculated into nude mice to establish an ectopic liver cancer model. The weight and tumor size of the nude mice were measured every three days. Grouping and drug administration: After modeling, the nude mice were randomly grouped, and drugs were injected into the tumor. The nude mice were randomly divided into 5 groups: Control group, Si-1 group, Si-2 group, Si-1+Si-2 group, and NC group. At least three sites on the tumor were selected for injection. The Control group was injected with physiological saline. Finally, paraffin sections were prepared.

10. The verification mechanism for the application of long non-coding RNAs interfering with Oct4-pg4 and Oct4-pg5 in the treatment of liver cancer according to claim 1, characterized in that, The specific method of step 3 is as follows: Dewaxing and hydration: Immerse the slides in xylene, and wash with ethanol solutions of different concentrations from high to low; Antigen retrieval: Immerse the slides in citrate antigen retrieval buffer, heat, slowly cool, and then wash on a shaker; Remove endogenous peroxidase activity and block; Primary and secondary antibody incubation: Discard the blocking solution, dilute the primary antibody Bcl-2 (1:200) and Bax (1:200) at the ratio, incubate overnight, discard the primary antibody, wash 3 times with PBS, add secondary antibody and incubate for 1 hour; Slide staining and counterstaining; Dehydration and mounting; Observe and acquire images after mounting.

Images