Study On Novel Anti-cancer Mechanism And Identification Of Antisense RNAs Of Three HCC-associated Genes

Aim:Hepatocellular carcinoma(HCC)has become the most common malignant tumor in China.In recent years,in-depth studies on hepatocellular carcinoma have yielded significant achievements,particularly at the genetic level.However,due to the complexity of the pathogenesis of HCC,the research progress is still very limited.5-Aza-2'-deoxycytidine(AZA)is a DNA methylation inhibitor that is currently used not only for the treatment of blood malignancies,such as acute leukemia,myelodysplastic syndrome MDS),etc.,are also used in the treatment of other solid tumors.Antisense RNA is a ubiquitous endogenous regulatory RNA.It is the first RNA molecule to be found to have inhibitory function.The study found that its function is not only related to post-transcriptional regulation,but also related to transcriptional regulation,including epigenetic regulation,such as DNA methylation and histone modification.Transcriptional regulation is mainly caused by transcriptional repression and chromatin remodeling,and post-transcriptional regulation is mainly related to the interaction of RNA.Antisense RNA also plays a major regulatory role in various physiological and pathological processes,such as organogenesis,cell differentiation,and disease progression.Most of genes can transcribe antisense RNA transcripts.Some studies have confirmed that mice and humans have a large number of antisense transcripts.Several laboratories have established methods for screening antisense RNA,such as Quere et al.Combined high throughput SAGE with bioinformatics to identify antisense RNA.Bioinformatics analysis for Genomic transcripts found that abnormal antisense RNA can affect the normal expression of normal genes.For example,P15 antisense RNA can inhibit the expression of P15 gene by affecting DNA methylation and histone modification The purpose of this study is to try to answer this question.Method:In this study,we first constructed double-stranded RNA library,and then screened antisense RNAs related to hepatocellular carcinoma from established double-stranded RNA library.Secondly,three genes,MEF2D,CDK16 and SMARCA4 were verified to have antisense RNAs in HepG2 cells..Finally,the full-length sequences were identified by cDNA rapid amplification technique.Results:in the case of established double-stranded cDNA select three genes for validation,after a series of experiments,the results are as follows:(1)by using the strand-specific reverse transcription primers and ordinary PCR,antisense RNAs of MEF2D,CDK16 and SMARCA4 were confirmed;(2)the expression of MEF2D,CDK16 and SMARCA4 and their antisense RNA levels were analyzed using RT-qPCR before and after treatment by the 5-Aza-dC in HepG2 cells.Data showed that antisense RNAs of these genes were increased significantly by DNA methyltransferase inhibitor treatment,(P<0.01),while the expression of these genes were down regulated(P<0.05);(3)Antisesne Genes of these genes were identified by using the rapid amplification of cDNA end(RACE)technique;(4)the open reading frame of the these antisense RNAs were predicted with the help of ORF finder software.We found that these antisense RNAs may be long non-coding RNAs(lncRNAs).Conclusion:(1)the MEF2D,CDK16 and SMARCA4 genes do exist double stranded RNA and there are an inverse correlation between.the sense and antisesne RNAs of the three genes.(2)DNA methylation inhibitor AZA downregulated the expression of cancer associated genes by activating the expression of their antisense RNAs.It may be a new mechanism of AZA anticancer.The results of this study are of great significance for revealing the mechanism of HCC and cancer therapy.