Establishment Of A Double-stranded RNA Library Of Human Hepatocellular Carcinoma Cells And Preliminary Analysis On The New Anti-tumor Mechanism Of 5-Aza-2’-deoxycytidine

Cancer is a great threat to human life and health with the second mortalityimmediatelyafter cardiovascular disease.5-Aza-2’-deoxycytidine,a kind of DNA methyltransferase inhibitor,can inhibit DNA methylation and induce the expression of genes regulated by DNA methylation.It is found that AZA has a significant anti-tumor effect and has been used in the clinical treatment of leukemia.Recent studies have shown that AZA treatment can activate tumor suppressor genes and double stranded RNA-depended innate immune genes.However,it is not clear whether there exist other anti-tumor mechanisms of AZA.Antisense RNA is an endogenous transcript or a natural antisense transcript complementary to the known genes.Antisense RNA is an important gene expression regulator that can be involved in gene regulation at both transcriptional and post-transcriptional levels.Studies have shown that many genes,including genes associated with tumors,have antisense RN As.However,it is still very lack of studies on the functions of most antisense RNAs and their roles in tumor therapy.In this study,we first constructed a double stranded RNA library of human hepatocellular carcinoma cells,and then selected some representative dsRNAs for further study to reveal whether the anti-tumor effect of AZA was mediated by dsRNAs.The main research methods and results are as follows:1.Construction of double-stranded RNA library.HepG2 cells(from a human carcinoma cell line)treated with or without AZA were used to establish a double stranded RNA library.The HepG2 cells treated with DMSO were used as control group,while those treated with AZA dissolved in DMSO were used as experimental group.After 48 hours of treatment,total RNA was extracted from the cells.The RNA samples were then treated with DNase I and reverse transcribed into cDNA.Subsequently,the cDNA samples were treated with RNase H,followed by hybridization and treatment with nuclease S1.The final double-stranded cDNA samples were subjected to high-throughput sequencing analysis.As a result,a total of 2364 genes possesssense and anti-sense transcripts that could form dsRNA,including 2066 genes from the experimental group and 843 genes from the control group.2.Pathway analysis.In the control group,the genes that could form dsRNAs were mainly enriched in metabolic and proliferative pathways,especially the ’Protein processing endoplasmic reticulum’ and ’Cell cycle’ pathways.In the experimental group,dsRNAs were not only enriched in the pathways shared by the control group,but also enriched in other signaling pathways,such as those related to the occurrence and development of cancer(’Pathway in cancer’,’TGF-beta signaling pathway’,’Pancreatic pathway’),and those related to cell growth(’Hippo signal pathway’).Among them,dsRNAs were most enriched in ’Pathway in cancer’,followed by the’Huntington’s disease’ pathway and ’Protein processing in endoplasmic reticulum’ pathway.3.Verification of double-stranded RNA Library.Four categories of genes were selected from the double-stranded RNA library,including tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten(PTEN),innate immunity-related gene signal transducers and activators of transcription 1(STAT1),oncogenes Ras-like proto-oncogene B(RALB)and MET,as well as cancer-related genes CD44 and heat-shock protein A4(HSPA4).The expressions of sense and antisense RNAs for the selected genes were detected by strand-specific RT-PCR,respectively.The results showed that all the six genes had antisense RNAs,which validated the double-stranded RNA library established abovementioned.4.The anticancer effect of AZA was mediated by antisense RNAs.The expressions of sense and antisense RNAs for the selected genes were detected by quantitative PCR,respectively.AZA induced the expressions of tumor suppressor gene PTEN(P<0.01)and innate immunity-related gene STAT1(P<0.001).In contrast,the expressions of cancer-related genes CD44 and HSPA4were obviously inhibited by AZA treatment(P<0.01)with decreaced antisense RNA expression.In conclusion,the construction of the double-stranded RNA library provides an important resource for study on the role of antisense RNA in hepatic carcinogenesis and related mechanism.Further study on the representative antisense RNAs not only confirmed the known mechanism by which AZA plays its anti-tumor role,through activating the expression of tumor suppressor genes and innate immune genes,but also revealed a new mechanism by which AZA inhibited the expression of oncogenes and cancer-related genes,through activating their antisense RNAs.