Functions And Regulatory Mechanism Of MicroRNA-23a-27a-24-2 Cluster In Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death worldwide due to its high recurrence rate and frequent metastasis.There are lots of studies about the tumorigenesis,developing and metastasis mechanisms of HCC theses years,illustrating the importance of basic research for HCC targeted therapy.Mi R?23a?27a?24?2 is located in the human chromosome 19p13.12,forming three mature mi RNAs: mi R?23a,mi R27 a,and mi R?24,which are expressed abnormally in many malignant tumors.Most studies focus on the targets of some individual mi RNAs;the interactive and global functions of diverse mi RNAs are still unclear and the phenomenon of the gathering of mi RNAs in clusters has always been ignored.This study will concentrate on the interactive functions and regulatory mechanism of mi RNAs in the mi R?23a?27a?24?2 cluster in HCC with mi RNA knock out(CRISPR Cas9)and endogenous regulatory mi R-23a-27a-24-2 cluster overexpress/knock down(CRISPR d Cas9)cell models.Methods(1)The expression level and survival analysis of mi R-23a/mi R-27a/mi R-24 in normal and HCC patients(from The Cancer Genome Atlas,TCGA).(2)Establishment of mi R-23a/mi R-27 a knock out stable cell lines with CRISPR Cas9 technology;Establishment of endogenous regulatory mi R-23a-27a-24-2 cluster overexpress/knock down cell lines with CRISPR d Cas9 technology.(3)Proliferation and regulation mechanism analysis of mi R-23a/mi R-27 a in Hep G2 cells and Xenograft tumor(Cell growth curve/MTT/Clone forming/Soft agar assay/Flow of cell cycle/Brd U staining;RNA-seq/ RT-q PCR/Western Blot/IHC/Dual luciferase/RNA immunoprecipitation/si RNA).(4)Apoptosis analysis of mi R-23a/mi R-27 a in HCC.(5)Role of invasion/metastasis of mi R-23a/mi R-27 a in Hep G2 cell line(Transwell/Scratch assay)and regulation mechanism analysis(Western Blot/IHC/RNA immunoprecipitation).Results(1)The expression level of mi R-23a/mi R-27a/mi R-24 between normal tissue and HCC showed no significant difference.Survival curve of mi R-23a/mi R-27a/mi R-24 showed there was a relationship with high risk and poor prognosis.(2)The expression of mi R-23a/mi R-27a/mi R-24 in normal cell lines(HEK293T)is significantly lower than that in tumor cell lines;In tumor cell lines,a high expression of mi RNAs was exhibited in Hep G2,MDA-MB-231,DU145 and PC3 cells,and there was a lower expression in Huh7 cells.(3)Both in vitro and in vivo experiments confirmed that mi R-23 a and mi R-27 a could promote HCC cell proliferation.(4)Mi R-23 a and mi R-27 a promote cell proliferation through the regulation of Cyclin A/Cyclin B and phosphor-CDK1(Tyr15).Ki67 was downregulated in the Xenograft tumor injected with mi R-23 a ko/mi R-27 a ko Hep G2 cells.(5)PURA was the candidate target gene of both mi R-23 a and mi R-27 a selected by RNA-seq and online prediction website.E2 F targets/G2-M checkpoint/MYC targets/ Mitotic targets/DNA repair were the gene set enriched by GSEA.PURA was the direct target of mi R-23 a and mi R-27 a confirmed by RT-q PCR/Western Blot/Dual luciferase/ RNA immunoprecipitation.PURA could partially reverse the phenotype of mi R-23 a and mi R-27 a in cell proliferation.(6)Mi R-23a-27a-24-2 could regulate the proteins of G2/M phase of cell cycle.There was a regulation network of mi R-23a-27a-24-2 cluster,PURA and STAG1/TTK.(7)Mi R-23a-27a-24-2 cluster has no significant effect on the apoptosis of HCC.(8)Mi R-23 a suppressed cell invasion/metastasis while mi R-27 a facilitated cell invasion/metastasis.Mi R-23 a inhibited the phosphorylation of Smad1/5/9 by targeting BMPR2,downregulated the transcription factor Snail,affected the expression of epithelial and mesenchymal markers,and inhibited the invasion and metastasis of HCC.Conclusions(1)Mi R-23a-27a-24-2 cluster mainly affects the proliferation,invasion/metastasis of HCC.(2)Both mi R-23 a and mi R-27 a can promote cell proliferation in HCC,and mi R-27 a play a more significant role.Mi R-23a-27a-24-2 cluster promotes cell proliferation through the regulation of signaling network of G2/M phase of cell cycle.(3)Functions of mi R-23 a and mi R-27 a on invasion/metastasis of HCC are different.Mi R-27 a promotes invasion and metastasis of HCC,while mi R-23 a inhibits the phosphorylation of Smad1/5/9 by targeting BMPR2 and inhibits invasion/metastasis of HCC.