Application of long non-coding RNA OIP5-AS1 in inhibiting HBV-positive liver cancer

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to the use of long non-coding RNA OIP5-AS1 as a molecular target for inhibiting the growth of HBV-positive liver cancer cells. The present invention discloses the application of lncRNA OIP5-AS1 as a target molecule in the preparation of drugs for anti-HBV-related hepatocellular carcinoma. By overexpressing lncRNA OIP5-AS1, the proliferation, migration and invasion abilities of HCC cells are inhibited. After the overexpression of lncRNA OIP5-AS1, the proliferation, invasion and migration abilities of HBV-related liver cancer cells are significantly inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the use of long non-coding RNA OIP5-AS1 as a molecular target for inhibiting the growth of HBV-positive liver cancer cells. Background Art

[0002] Primary liver cancer is an important cause of cancer-related deaths globally, mainly including three histopathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, and combined carcinoma, among which HCC is the most common. The clinical registration survey statistics results of primary liver cancer in a certain region in 2022 showed that HCC accounted for 91.6% of liver cancer, and 84% of them were caused by hepatitis B virus (HBV) infection. The complete HBV consists of a double envelope and a core particle. The genome of HBV is a partially circular double-stranded DNA with a genome length of 3.2 kb, containing four open reading frames (ORFs), namely ORF-P, S, C, and X. HBV infection can induce host gene mutations, activate cancer-related signaling pathways, and thus cause the malignant transformation of normal hepatocytes. Currently, most HCC cases have insidious onset, high recurrence and metastasis rates, and high mortality rates, but there is still a lack of effective early diagnosis and treatment methods for HCC clinically.

[0003] Early HCC-related research mainly focused on protein-coding genes. However, with the continuous progress of sequencing technology, more and more non-coding RNAs have been detected, which play important roles in various physiological and pathological processes. Long non-coding RNA (lncRNA) refers to RNA with a length exceeding 200 nucleotides and does not encode proteins. A large number of studies have shown that lncRNAs are abnormally expressed in cancers and are closely related to tumor growth and invasion. As a conserved lncRNA, lncRNA OIP5-AS1 has recently been reported to be involved in a variety of biological and pathological processes, including tumorigenesis and development. OIP5-AS1 plays its carcinogenic or anti-tumor role in human cancers by regulating various miRNAs or mRNAs. However, the function and mechanism of OIP5-AS1 in HBV-related HCC are still unclear. And a variety of lncRNAs have been reported in the published academic papers or invention patents to play important roles in the occurrence, development, diagnosis, and treatment of liver cancer. For example, Wang Yunqiu et al. disclosed LncRNA AC026412.3 and found that the expression level of LncRNA AC026412.3 derived from serum Exosomes of liver cancer patients was up-regulated, which can be used as a screening, early diagnosis, or prognostic judgment marker for high-risk populations of liver cancer.

[0004] There are reports indicating that long non-coding RNA OIP5-AS1 and miR-378a-3p regulate the occurrence and development of non-small cell lung cancer.

[0005] "Downregulation of lncRNA OIP5-AS1 inhibits EMT, invasion and migration of hepatocellular carcinoma cells through the miR-217 / USP7 axis"

[0006] An article informed that: As a result, lncRNA OIP5-AS1 was significantly highly expressed in hepatocellular carcinoma cell lines (P < 0.05 or P < 0.01). Downregulating the expression of lncRNA OIP5-AS1 in hepatocellular carcinoma cells could significantly inhibit EMT, invasion and migration of hepatocellular carcinoma cells (P < 0.05); lncRNA OIP5-AS1 targeted and bound to miR-217, and USP7 was a target gene of miR-217 (P < 0.05); further research confirmed that IncRNA OIP5-AS1 upregulated the expression level of USP7 through miR-217, thereby promoting EMT and invasion and migration of hepatocellular carcinoma cells (P < 0.05 or P < 0.01). Conclusion: IncRNA OIP5-AS1 promotes EMT and invasion and migration of hepatocellular carcinoma cells through the miR-217 / USP7 molecular axis. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a new use of a target, that is, to effectively inhibit the disease progression of HBV-related hepatocellular carcinoma.

[0008] To solve the above technical problem, the present invention provides the application of lncRNA OIP5-AS1 as a target molecule in the preparation of drugs against HBV-related hepatocellular carcinoma.

[0009] As an improvement of the application of the present invention: By overexpressing lncRNA OIP5-AS1, the proliferation, migration and invasion abilities of HCC cells are inhibited. The method of overexpressing lncRNA OIP5-AS1 is: by transfecting a specific plasmid of lncRNA OIP5-AS1.

[0010] As a further improvement of the application of the present invention: After overexpression of lncRNA OIP5-AS1, the proliferation, invasion and migration abilities of HBV-related hepatocellular carcinoma cells are significantly inhibited.

[0011] As a further improvement of the application of the present invention: lncRNA OIP5-AS1 is significantly associated with the glycolysis / gluconeogenesis pathway; lncRNA OIP5-AS1 can play an anti-cancer role through the glycolysis / gluconeogenesis pathway.

[0012] The present invention first provides that the expression of lncRNA OIP5-AS1 is significantly correlated with poor prognosis of HCC patients. According to the embodiments of the present invention, HCC patients in the low-expression level group of lncRNA OIP5-AS1 have a poor prognosis.

[0013] Furthermore, lncRNA OIP5-AS1 has an anti-cancer effect. According to the embodiments of the present invention, after the overexpression of lncRNA OIP5-AS1, the proliferation, invasion and migration abilities of liver cancer cells are significantly inhibited.

[0014] Moreover, lncRNA OIP5-AS1 is significantly correlated with the glycolysis / gluconeogenesis pathway. According to the embodiments of the present invention, lncRNA OIP5-AS1 may exert its anti-cancer effect through the glycolysis / gluconeogenesis pathway.

[0015] The present invention has the following technical advantages:

[0016] The present invention confirms that lncRNA OIP5-AS1 is significantly correlated with HBV-related hepatocellular carcinoma. LncRNA OIP5-AS1 is significantly down-regulated in HBV-positive HCC tissues, and its low expression is significantly correlated with poor prognosis of HBV-positive HCC patients. When lncRNA OIP5-AS1 is overexpressed, the proliferation, invasion and metastasis of liver cancer cells are inhibited. The present invention confirms that lncRNA OIP5-AS1 can be used as an important target for effectively inhibiting the proliferation of HBV-related liver cancer cells.

[0017] It is of great significance to explore the function of lncRNA OIP5-AS1 in HBV-related hepatocellular carcinoma and whether it can be used as a molecular target for anti-HBV-related hepatocellular carcinoma.

[0018] It should be noted that: The EMT of liver cancer cells is different from the etiology of the hepatitis B virus (HBV)-positive liver cancer described in the present invention. Liver cancer is one of the high-incidence tumors globally, and its etiology can be developed from HBV infection, alcoholic fatty liver, etc. Hepatitis B liver cancer is caused by HBV infection. The different etiologies also determine the differences in diagnosis and treatment. The oncogenes are also different between hepatitis B liver cancer and non-hepatitis B liver cancer, and the pathogenic mechanisms are also different. There are few reports on the treatment methods for liver cancer cell EMT. It has been reported that dihydroartemisinin can be used to inhibit liver cancer cell EMT-mediated liver cancer metastasis; while for the treatment of hepatitis B liver cancer, antiviral treatment is the first, and it blocks a series of changes in intracellular signal transduction caused by the hepatitis B virus (HBV) and its components.

[0019] In addition, the statement in the background art that "downregulating the expression of lncRNA OIP5-AS1 in liver cancer cells can significantly inhibit EMT and invasion and migration of liver cancer cells" is diametrically opposed to the conclusion of the present invention that "by overexpressing lncRNA OIP5-AS1, the proliferation, migration and invasion abilities of HCC cells are inhibited". Therefore, the above prior art cannot provide the present invention with a technical inspiration. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following further elaborates on the specific embodiments of the present invention with reference to the accompanying drawings.

[0021] Figure 1 For analyzing the correlation between the expression level of lncRNA OIP5-AS1 and the overall survival of HCC patients in the TCGA database. Among them, "group = high" represents the group with a higher expression level of lncRNA OIP5-AS1, and "group = low" represents the group with a lower expression level of lncRNA OIP5-AS1.

[0022] Figure 2 For the effect of overexpressing lncRNA OIP5-AS1 on the proliferation and metastasis abilities of liver cancer cells; among them, "NC" is Hep3B cells transfected with a blank control plasmid, and "OIP5-AS1" is Hep3B cells transfected with an lncRNA OIP5-AS1 plasmid;

[0023] Figure 2 In:

[0024] A shows the proliferation ability of Hep3B cells detected by a real-time cell proliferation experiment after transfection with an lncRNA OIP5-AS1 overexpression plasmid;

[0025] B shows the invasion ability of Hep3B cells detected by a Transwell experiment after overexpressing lncRNA OIP5-AS1;

[0026] C shows the migration ability of Hep3B cells detected by a cell scratch experiment (0h, 24h, 48h) after overexpressing lncRNA OIP5-AS1.

[0027] Figure 3 For the effect of knocking down lncRNA OIP5-AS1 on the proliferation and metastasis abilities of liver cancer cells; among them, "NC" is Huh7 cells transfected with a blank control plasmid, and "Si-OIP5-AS1" is Huh7 cells transfected with an lncRNA OIP5-AS1 si sequence;

[0028] Figure 3 In:

[0029] A was to observe the real-time cell proliferation of Huh7 cells and Huh7 cells transfected with the lncRNA OIP5-AS1 si sequence; B was to observe the migration of Huh7 cells and Huh7 cells transfected with the lncRNA OIP5-AS1 si sequence;

[0030] C was to observe the migration of Huh7 cells and Huh7 cells transfected with the lncRNA OIP5-AS1 si sequence after scratching.

[0031] Figure 4 It was the result of the tumorigenesis experiment in nude mice after knocking down lncRNA OIP5-AS1;

[0032] Figure 4 Among them:

[0033] A was to detect the luciferase signal of subcutaneous tumors in nude mice through an in vivo imaging system. Right side: lncRNA OIP5-AS1 knockdown group; Left side: NC group (n = 6);

[0034] B-D were that knocking down lncRNA OIP5-AS1 promoted the growth (size, volume and weight) of liver cancer in nude mice.

[0035] Figure 5 It was the result of bioinformatics analysis of proteomic data;

[0036] Figure 5 Among them:

[0037] A was to show the genes with differential expression in lncRNA OIP5-AS1 knockdown group and control group of liver cancer cells through a volcano plot; The points on the right represented up-regulated genes; The points on the left represented down-regulated genes;

[0038] B was to perform KEGG functional analysis on the differentially expressed genes.

[0039] Figure 6 It was the result graph of lncRNA OIP5-AS1 regulating the glycolytic metabolism of HCC; Among them, "NC" were Huh7 cells transfected with a blank control plasmid, and "Si-OIP5-AS1" were Huh7 cells transfected with the lncRNA OIP5-AS1 si sequence;

[0040] Figure 6 Among them:

[0041] A was the glucose consumption of Huh7 cells transfected with blank control and lncRNA OIP5-AS1 siRNA respectively;

[0042] B was the lactic acid production level of Huh7 cells transfected with blank control and lncRNA OIP5-AS1 siRNA respectively;

[0043] C represents the ATP concentration in Huh7 cells transfected with blank control and lncRNA OIP5-AS1 siRNA respectively. Specific implementation manners

[0044] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0045] The experimental methods and procedures involved in the specific embodiments of the present invention have all obtained the approval of the Ethics Committee of Zhejiang University.

[0046] Example 1: The expression of lncRNA OIP5-AS1 is significantly correlated with poor prognosis of HCC patients

[0047] In this example, the present invention performed a prognostic survival analysis of lncRNA OIP5-AS1 using the sample data of 271 HBV-related HCC patients in the TCGA database, and found that HCC patients in the low-expression level group of OIP5-AS1 had a poor prognosis. According to the median expression level of OIP5-AS1 in HBV-related HCC samples in the TCGA database, the high-expression group and the low-expression group were determined, and the Log-rank test was performed using the Kaplan-Meier (KM) method. The obtained results are as Figure 1 , according to Figure 1 , it can be known that the expression of lncRNA OIP5-AS1 is significantly correlated with poor prognosis of HCC patients, and the lower the expression of OIP5-AS1, the worse the prognosis of the patients.

[0048] Example 2: Overexpression of lncRNA OIP5-AS1 inhibits the proliferation and metastasis ability of liver cancer cells

[0049] In this example, a plasmid overexpressing lncRNA OIP5-AS1 was synthesized and transfected into Hep3B cells to obtain Hep3B cells after transfection; first, 50 μL of DMEM medium was added to a special ACEA well plate, the ACEA software was connected, the observation time was set to 120 h, and baseline calibration was performed, and then 1×10 4 cells / well of Hep3B cells after transfection were seeded and placed in an ACEA analyzer for real-time observation of cell growth. The obtained results are as Figure 2 shown in A of, therefrom, it can be known that overexpression of lncRNA OIP5-AS1 can inhibit the proliferation ability of liver cancer cells.

[0050] Note: The plasmid overexpressing lncRNA OIP5-AS1 can be synthesized and provided by Vigen Company, for example. The sequence of the plasmid overexpressing lncRNA OIP5-AS1 is as shown in SEQ ID NO:1.

[0051] Seed 2.5x10 5 Hep3B cells per well in a 12-well plate. After culturing overnight at 37°C in a cell incubator, transfect the plasmid overexpressing lncRNA OIP5-AS1 into Hep3B cells according to the above method. Place the cells in a 37°C cell incubator until the cells grow to about 90%. Use a sterile 200 μL pipette tip to draw a straight line in the middle of the well plate to completely scrape up the cells. Aspirate the culture medium, add 1 mL of sterile 1x PBS for washing, and then observe and photograph using an inverted microscope. The obtained results are as shown in Figure 2 Panel B of . From this, it can be known that overexpression of lncRNA OIP5-AS1 can inhibit the migration ability of liver cancer cells.

[0052] In a 24-well plate, add 600 μL of cell culture medium containing 10% FBS to each well, and then gently place a Transwell chamber (pore size 8 μm); Dilute the Hep3B cells transfected with the plasmid overexpressing lncRNA OIP5-AS1 with serum-free medium and seed 200 μL per well at the corresponding density on the upper layer of the Transwell chamber. Place the cells in a 37°C cell incubator for culture; After culturing the Hep3B cells for 6 hours, take out the Transwell chamber, place it in a new 24-well plate, aspirate the upper layer of the culture medium, add 800 μL of 1x PBS and wash 3 times; Add 800 μL of 4% paraformaldehyde at room temperature and fix for 15 minutes. Aspirate the paraformaldehyde and wash 3 times with 800 μL of 1x PBS; Add 500 μL of 0.1% crystal violet staining solution and stain for 10 minutes. Aspirate the crystal violet and wash 3 times with 800 μL of 1x PBS; Take out the Transwell chamber, gently wipe off the cells on the upper layer of the Transwell chamber with a cotton swab, and then observe and count using an inverted microscope. The obtained results are as shown in Figure 2 Panel C of . From this, it can be known that overexpression of lncRNA OIP5-AS1 can inhibit the invasion ability of liver cancer cells.

[0053] The above results show that after transfecting the plasmid overexpressing lncRNA OIP5-AS1 into Hep3B cells, the proliferation rate of liver cancer cells is significantly slower than that of the control group cells. At the same time, the results of the Transwell experiment and the cell scratch experiment show that after transfecting the plasmid overexpressing lncRNA OIP5-AS1, the invasion and migration abilities of Hep3B cells are significantly inhibited.

[0054] Example 3: Effects of knocking down lncRNA OIP5-AS1 on the proliferation and metastasis abilities of liver cancer cells

[0055] In this example, small interfering RNA (siRNA) was synthesized, and after transfection of lncRNA OIP5-AS1 siRNA into HBV-negative hepatocellular carcinoma cell line Huh7 cells (the resulting cells were named Huh7 cells with knockdown of lncRNA OIP5-AS1), functional phenotype experiments of tumor cells (proliferation, invasion, migration) were carried out to illustrate the effect of OIP5-AS1 on the biological functions of HCC cells from another aspect.

[0056] The above small interfering RNA (siRNA) can be synthesized and provided by GenePharma Co., Ltd., and the sequence is 5'-GCAGCAGUAAAUUCCAAAUTT-3′.

[0057] First, add 50 μL of DMEM medium to a dedicated ACEA well plate, connect the ACEA software, set the observation time to 120 h, perform baseline calibration, and then seed 1x10 4 Huh7 cells with knockdown of lncRNA OIP5-AS1 per well, and place them in an ACEA analyzer for real-time observation of cell growth. The obtained results are as Figure 3 shown in A. Accordingly, it can be known that after knockdown of lncRNA OIP5-AS1, the results of the real-time cell proliferation experiment show that the proliferation ability of Huh7 cells is significantly enhanced.

[0058] Seed 2.5x10 5 Huh7 cells with knockdown of lncRNA OIP5-AS1 per well in a 12-well plate, place them in a 37 °C cell culture incubator until the cells grow to about 90%, use a sterile 200 μL pipette tip to draw a straight line in the middle of the well plate to completely scrape up the cells, aspirate the medium, add 1 mL of sterile 1x PBS for washing, and then observe and photograph using an inverted microscope. The obtained results are as Figure 3 shown in B. Accordingly, it can be known that after knockdown of lncRNA OIP5-AS1, the results of the real-time cell proliferation experiment show that the migration ability of Huh7 cells is significantly enhanced.

[0059] In a 24-well plate, add 600 μL of cell culture medium containing 10% FBS to each well, and then gently place a Transwell insert (pore size: 8 μm). Huh7 cells with knocked-down lncRNA OIP5-AS1 were diluted with serum-free medium and seeded into the upper layer of the Transwell insert at a corresponding density of 200 μL per well, and then cultured in a 37 °C cell incubator. After 6 hours, take out the Transwell insert, place it in a new 24-well plate, aspirate the upper layer of the medium, add 800 μL of 1x PBS and wash 3 times; add 800 μL of 4% paraformaldehyde at room temperature and fix for 15 minutes, aspirate the paraformaldehyde, and wash 3 times with 800 μL of 1x PBS; add 500 μL of 0.1% crystal violet staining solution and stain for 10 minutes, aspirate the crystal violet, and wash 3 times with 800 μL of 1x PBS; take out the Transwell insert, gently wipe off the cells on the upper layer of the Transwell insert with a cotton swab, and then use an inverted microscope to take pictures and count. The results are as shown in Figure 3 shown in C of Figure 3 . Accordingly, it can be known that after knocking down lncRNA OIP5-AS1, the results of the real-time cell proliferation experiment show that the invasion ability of Huh7 cells is significantly enhanced.

[0060] Example 4: Results of the xenograft tumor formation experiment in nude mice after knocking down lncRNA OIP5-AS1

[0061] In this example, small interfering RNA (siRNA) was synthesized. After transfection of lncRNA OIP5-AS1 siRNA into the HBV-negative hepatocellular carcinoma cell line Huh7 cells (the resulting cells were named Huh7 cells with knocked-down lncRNA OIP5-AS1), sufficient amounts of Huh7 cells transfected with si lncRNA OIP5-AS1 and untransfected si lncRNA OIP5-AS1 were collected, resuspended with sterile 1x PBS, and 2×10 6 cells of the stable lncRNA OIP5-AS1 knockdown group and Huh7 cells as the control group were respectively inoculated on the left and right sides of nude mice. After waiting for one week, the subcutaneous stable transfected cells successfully proliferated. Use an electronic caliper to measure the length and width of the tumor, and measure it every two days and record it. Until the subcutaneous tumor grows to 1000 mm 3 , stop the measurement and take pictures of the nude mouse tumor imaging. Then sacrifice the nude mouse, dissect the tumor subcutaneously, take pictures and weigh it. The results are as shown in Figure 4 shown in Figure 4 . The results of in vivo imaging of nude mice show that the tumor in the left lncRNA OIP5-AS1 knockdown group is larger in volume and has richer blood flow compared with the control group; subsequently, after dissecting the tumor subcutaneously, compared with the control group, the tumor volume and weight of the lncRNA OIP5-AS1 knockdown group are both significantly increased.

[0062] Example 5: KEGG pathway analysis results showed that the differentially expressed genes were mainly enriched in the glycolysis / gluconeogenesis pathway

[0063] In this example, proteomic detection was performed on Huh7 cells with knocked-down lncRNA OIP5-AS1. The mass spectrometry results showed that compared with the negative control group, a total of 558 differentially expressed genes were found, including 107 up-regulated genes and 451 down-regulated genes (fold-change≥2.0, P≤0.05). To further study the biological functions of the differentially expressed genes, these differentially expressed genes were also subjected to KEGG pathway analysis. The results are as follows Figure 5 , and it was found that the differentially expressed genes were mainly enriched in the glycolysis / gluconeogenesis pathway, suggesting that lncRNA OIP5-AS1 may play an anti-cancer role through the glycolysis / gluconeogenesis pathway.

[0064] Example 6: lncRNA OIP5-AS1 regulates glycolytic metabolism in HCC

[0065] In this example, the glucose consumption and lactate production levels of Huh7 cells transfected with the blank control si sequence and Huh7 cells transfected with the lncRNA OIP5-AS1 si sequence were detected respectively to reflect the glycolysis process. Take 3x10 6 cells for lysis, and use a glucose uptake kit to detect the OD value at a wavelength of 570 nm. Calculate the standard curve based on the OD values of the standard products, and then calculate the glucose consumption of the experimental wells. Take 1x10 6 cells for lysis, and use a lactate production kit to detect the OD value at a wavelength of 450 nm. Calculate the standard curve based on the OD values of the standard products, and then calculate the lactate production of the experimental wells. Lyse the cells with lysis buffer, add the ATP detection working solution to each tube, measure with a chemiluminescence instrument, calculate the standard curve, and then calculate the ATP concentration of the experimental group. The results are as follows Figure 6 , after knocking down lncRNA OIP5-AS1, the glucose consumption and lactate production levels in Huh7 cells were significantly reduced, indicating that OIP5-AS1 can regulate the glycolysis process of hepatoma cells. In addition, the results of the ATP concentration detection experiment showed that compared with the control group cells, the intracellular ATP generation ability of hepatoma cells transfected with OIP5-AS1 siRNA was significantly enhanced.

[0066] It should be emphasized that: IncRNA OIP5-AS1 increases the expression level of USP7 through miR-217, thereby promoting the EMT and invasion and migration of hepatoma cells. That is, IncRNA OIP5-AS1 promotes the EMT and invasion and migration of hepatoma cells through the miR-217 / USP7 molecular axis.

[0067] However, the lncRNA OIP5-AS1 of the present invention can exert an anti-cancer effect through the glycolysis / gluconeogenesis pathway. Its actual effect is that lncRNA OIP5-AS1 reduces glycolysis / gluconeogenesis, reduces the energy supply of cancer cells, and thus inhibits the growth of tumor cells.

[0068] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of lncRNA OIP5-AS1 in the preparation of drugs against HBV-related hepatocellular carcinoma.

2. The application according to claim 1, characterized in that: Overexpression of lncRNA OIP5-AS1 inhibits the proliferation, migration and invasion of HCC cells.

3. The application according to claim 2, wherein The method of overexpressing lncRNA OIP5-AS1 is: transfecting lncRNA OIP5-AS1 specific plasmid.

4. The application according to any one of claims 1 to 3, characterized in that: After overexpression of lncRNA OIP5-AS1, the proliferation, invasion and migration of HBV-related hepatoma cells are significantly inhibited.

5. The application according to any one of claims 1 to 3, characterized in that: lncRNA OIP5-AS1 is significantly associated with the glycolysis / gluconeogenesis pathway; lncRNA OIP5-AS1 can play an anti-cancer role through the glycolysis / gluconeogenesis pathway.

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