| IntroductionHepatocellular carcinoma(HCC)is among the most prevalent and lethal cancers in the human population.We ever reported that co-overexpression of c-Myc and EZH2 was associated with poorer prognosis of HCC patients.Each microRNA could target multiple downstream genes,so it is a worthwhile thing to do research on c-Myc and EZH2 double positive HCC therapy by using microRNA.Part?Identification of miRNAs negatively regulated both c-Myc and EZH2 in cancer cells [Background]It was reported that co-overexpression of c-Myc and EZH2 is associated with poorer prognosis of multi-Tumors patients.MicroRNAs(miRNAs),a class of endogenous 21-to 25-nucleotide highly conserved non-codingRNAs,negatively regulate gene expression in a posttranscriptional manner through directly degrading multiple messengerRNA(mRNA)or indirectly repressing their translation.[Aims] Identification of miRNAs negatively regulated both c-Myc and EZH2 in cancer cells.[Methods]We screen miRNAs that were inverse correlative with both c-Myc and EZH2 by an online dataset.By over-expression of these candidates in hepatocellular carcinoma(HCC)cell line,we identified their effect in inhibiting c-Myc and EZH2.[Results]We found 15 candidate miRNAs which were inverse correlative with c-Myc and EZH2 using the dataset mentioned above.Nine of them were tested by Western Blot.It was miR-26 a that could negative regulate c-Myc and EZH2.[Conclusions] We successfully found miR-26 a,which could negative regulate c-Myc and EZH2 in HCC.Part? The mechanism that miR-26 a negatively regulated c-Myc and EZH2 in HCC [Background]In previous part,we have found that miR-26 a negatively regulate c-Myc and EZH2 in HCC,but the mechanism of this regulation is still unknown.[Aims]Put light on the mechanism that miR-26 a negatively regulated c-Myc and EZH2 in HCC.[Methods]Firstly,we predicted the binding sites of miR-26 a in c-Myc's mRNA by an online tool.We also identified this interaction between miR-26 a and c-Myc by dual-luciferase report assay and Western Blot assay.Secondly,we analyzed associated genes in wnt pathway,which can stimulate c-Myc,in order to clarify whether there was target gene of miR-26 a in this pathway.Then,in one hand,we tested whether this candidate was regulated by miR-26 a by dual-luciferase report assay and Western Blot assay.In another hand,we tested whether this candidate regulate c-Myc by knockdown and over-express this candidate gene.Finally,we tested whether miR-26 a regulate EZH2 by dual-luciferase report assay.[Results] Firstly,we found the binding sites of miR-26 a in c-Myc's mRNA byRNA22 V2.We also confirmed that miR-26 a could bind to CDS region of c-Myc's mRNA by dual-luciferase report assay and miR-26 a could negatively regulate expression of c-Myc by Western Blot assay.Secondly,we screen out CDK8 which is not only a key molecule in wnt pathway but a candidate target of miR-26 a.In one hand,we confirmed that miR-26 a could bind to 3'UTR region of CDK8's mRNA by dual-luciferase report assay and miR-26 a could negatively regulate expression of CDK8 by Western Blot assay.In another hand,when we changed the expression level of CDK8,the level of c-Myc was also changed correspondingly.Finally,the result of dual-luciferase report assay showed that miR-26 a could directly bind to 3'UTR of EZH2.[Conclusions]We clarified the mechanism that miR-26 a regulate c-Myc and EZH2.Firstly,miR-26 a directly suppressed c-Myc by binding to its CDS region.Secondly,miR-26 a negatively regulated c-Myc by targeting CDK8.Intriguingly,the indirect effect was much stronger than the direct effect.Mi R-26 a could negatively regulated EZH2 by directly binding to its 3'UTR.Part ? The mechanism that c-Myc and EZH2 negatively regulated miR-26 a in HCC [Background] It was reported that c-Myc could inhibit miR-26 a in HCC.Besides,it was also reported that c-Myc could recruit EZH2 to repress miR-26 a in other cancer cells.But whether this mechanism is appropriate for HCC is still unknown.In the previous part,we have found that miR-26 a ne gatively regulate.[Aims] Clarify the mechanism that c-Myc and EZH2 negatively regulate miR-26 a in HCC.[Methods] Firstly,in order to identify which precursor of miR-26 a contribute more to mature miR-26 a,we not only analyzed normal liver data in TCGA dataset,but also tested their expression level in paired HCC clinical samples.Secondly,we tested the effect of JQ1(c-Myc inhibitor)and DZNep(EZH2 inhibitor)on CTDSP2(pre-miR-26a-2)promoter by dual-luciferase report assay in BEL-7404 cell line.Besides,we also tested the effect of JQ1 and DZNep on mature miR-26 a and pre-miR-26a-2 expression level by q RT-PCR assay in two HCC cell lines.Finally,we identified the interaction between c-Myc/EZH2 and CTDSP2 promoter by Ch IP-qPCR.In another hand,we detect the role of c-Myc in this interaction by using siRNA.[Results] Firstly,we found that the expression level of pre-miR-26a-2 was thousands of times more than pre-miR-26a-1 by analyzing TCGA normal liver data.Besides,we tested the expression level of mature miR-26 a,pre-miR-26a-1 and pre-miR-26a-2 in 8 paired HCC samples.The result showed that mature miR-26 a was down-regulated in 6 tumor samples compared with adjacent normal liver tissue.For pre-miR-26a-2,the number was 6,but for pre-miR-26a-1,the number was only 1.Secondly,the activity of CTDSP2 promoter could be stimulated by JQ1 and/or DZNep.The expression level of mature miR-26 a and pre-miR-26a-2 could also be stimulated by JQ1 and/or DZNep.Finally,we detected that c-Myc and EZH2 both could bind to the promoter region of CTDSP2.If without c-Myc,the binding of EZH2 and the promoter was lost.[Conlusions] Almost all of mature miR-26 a was generated from pre-miR-26a-2 at least in liver.c-Myc recruit EZH2 to silence pre-miR-26a-2 in HCC,and c-Myc is the bridge molecule in this interaction.Part ? Identification of c-Myc/EZH2-miR-26 a feedback loop in HCC[Background] Summing up previous three parts,we hypothesized a c-Myc/EZH2-miR-26 a feedback loop in HCC.[Aims]Identify the feedback loop in cell lines,animal models and clinical samples.[Methods] Firstly,we transfected miR-26 a mimics in HCC cell lines,and tested the expression level of pre-miR-26a-2.Secondly,we build a DEN-induced liver cancer model in mouse,and tested expression level of associated genes in feedback loop by q RT-PCR.Finally,we detected the expression level of miR-26 a in HCC samples by q RT-PCR,and level of c-Myc,EZH2,CDK8 in HCC samples by IHC staining.Besides,we also tested the relation between associated genes in this loop in liver cancer GEO datasets.[Results] Firstly,we found up-regulation of pre-miR-26a-2 in both BEL-7404 and SMMC-7721 cell lines,when they were transfected by mature miR-26 a mimics.Secondly,we successfully built DEN-induced liver cancer model in mouse,and found that,in one hand,c-Myc,EZH2 and CDK8 were all up-regulated in tumor tissues when compared with normal liver tissues;in another hand,c-Myc,EZH2 and CDK8 were all up-regulated in lung metastatic clonies when compared with primary liver tumors.Intriguingly,miR-26 a was found down-regulated in tumor tissues,and even much less in lung metastatic clonies.Finally,the result from clinical sample showed that the expression level of miR-26 a in normal liver tissue was 32 times higher than that in tumor tissues.However,the expression of c-Myc,EZH2 and CDK8 was hard to detect in normal liver tissue by IHC staining.Besides,it was shown that miR-26 a was inverse correlative with c-Myc,EZH2 and CDK8 by analyzing of GEO datasets.[Conclusions] We successfully identify the feedback loop in cell lines,animal models and clinical samples. |
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