The Role Of 14q32 MiRNA Cluster In The Development Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related death and it takes more than 500,000 people’s lives each year.Traditional strategies for treating HCC include resection,liver transplantation and local ablation.In recent years,studies on molecular classification based on the cancer genome,transcriptome,metabolome and proteome have provided effective theoretical basis for clinical diagnosis and individualized treatment with the development of high throughput technology.Diagnosis and treatment of cancer at the molecular level is drawing people’s attention.MicroRNAs(miRNAs)are 20～25 nucleotides non-coding RNAs that modulate various biological processes.They may act as oncogenes or tumor suppressors according to the target genes.Investigation on miRNA expression profiles may provide new biomarkers or therapeutic targets for the diagnosis and treatment of HCC.By high-throughput sequencing technologies,we obtained the miRome of tumor tissues derived from 3 1HBV-related HCC patients.We then clustered the HCC patients into five subclusters by using consensus clustering based on the 210 miRNAs with standard variation larger than 2.We identified 5 clusters and the cluster 5 tumors showed significant up-regulation of 47 miRNAs among which 37 miRNAs belongs to the 14q32 miRNA gene cluster.Previous studies have revealed that this miRNA cluster plays an important role in the process of multiple tumors,including HCC.Some miRNAs in this cluster were reported associated with HCC progression.For exemple,miR-127,miR-433 and miR-431 can promote HCC cell migration while miR-494,miR-664,miR-485-3p and miR-495 enhance HCC cell proliferation ability.However,there are still many other miRNAs in this cluster may function dramatically in the progression of HCC.miR-411-5p is located in the miRNA cluster and previous research showed that enhancing miR-411-5p promote the growth of Hep3B cells.We collected and analysed the clinical data from gene expression omnibus(GEO)and the results showed that high expression of miR-411-5p is accompanied by HCC patients’ low overall survival rate and disease-free survival rate.Our subsequent work revealed that miR-411-5p could promote migration and invasion of HCC cells and L02 cells.These results indicate that miR-411-5p may play a role during cancer progression by promoting migration and invasion of the cancer cell.As is known,miRNAs biologically function through regulating their target genes.To search for the target gene of miR-411-5p,we used the TargetScan software to predict the genes targeted by miR-411-5p and integrated them with the argonaute-crosslinking immunoprecipitation(AGO-CLIP)data set.Three potential genes were found and among them was SIRT7 whose mRNA and protein expression level could be down-regulated by miR-411-5p mimics.Then we sought for the mechanism for the abnormal expression of 14q32 miRNA cluster.Co-expression of clustered miRNAs mainly accounts to two reasons,the first is the genomic variations in this region,and the second is that these miRNAs lies in a polycistronic position,causing the clustered miRNAs regulated by the promoter region methylation or transcription factors.Subsequent research revealed that 14q32 miRNA cluster was located in the DLK1-DI03 imprinted gene status.It is noteworthy that there is a differentially methylated region(DMR)upstream of the imprinted region.These results made us speculate that the upstream DMR may account for the abnormal expression of the 14q32 miRNA cluster.In line with this hypothesis,we found that the CpG islands in the DMR were unmethylated along with significant higher expression levels of miR-127-3p and miR-411-5p(two of the miRNAs within the miRNA cluster)after treating the Bel-7402 cells with 5-Aza-CdR.To sum up,by high-throughput sequencing and consensus clustering analysis for the 31 HBV-related HCC,we identified 14q32 miRNA cluster which can be inactivated by the methylation of upstream CpG islands.As one of the miRNA in this cluster,miR-411-5p plays a role during HCC progression by promoting migration and invasion of the cancer cell.Among the 3 candidate targets,SIRT7is most likely to be the target gene as its mRNA and protein expression could be down-regulated by miR-411-5p mimics.This research provides a new reference for the diagnosis and treatment of HCC.