P53-Regulated MicroRNAs Expression Profiling And Regulatory Network Analysis In Hepatocellular Carcinoma Cell HepG2

Acting as a sequence-specific transcription factor, p53 tumor suppressor involves in a variety of biological processes including cell cycle arrest, cell proliferation, apoptosis, cell invasion and migration after being activated by cellular stresses such as DNA damage. As a crucial tumor suppressor, p53 participates in the regulation of cancer-related cell processes and signaling pathway via activating or repressing target gene expression involved in cancers. MicroRNAs (miRNAs) are a subset of endogenous non-coding RNAs (-22 nucleotides long) which play vial roles in regulating genes expression via targeting the specific sites in 3’untranslated region (3’UTR) of miRNAs. In recent years, miRNAs have been confirmed to be regulated by p53 in several cancer types. However, it is still unclear how miRNAs orchestrate their regulation and function in p53 network after p53 activation in hepatocellular carcinoma (HCC).In this study, we used small RNA sequencing and systematic bioinformatic analysis to characterize the regulatory networks of differentially expressed miRNAs after the p53 activation in HepG2. Here, doxorubicin (Dox) was used as the p53 activating agent to induce the expression of p53 protein in hepatocellular carcinoma cell HepG2. After the Dox treatment, p53 expression was up-regulated in response to DNA damage. Later, small RNA-seq was used to identify the differential expression of p53-regulated miRNAs. Targetscan, miRanda, Tarbase were applied to predict the target of p53-related miRNAs. We then analysed five p53 ChlP-seq database and miRNA TSSs data to identify the potential p53 binding site around the 10 kb upstream and lkb downstream of TSSs of p53-related miRNAs. Moreover, hsa-miR-3661 and hsa-miR-4521 were selected for qRT-PCR validation, CCK-8 array was applied to study the effect of miR-3661 on the proliferation of HepG2.Here, p53 expression was significantly up-regulated in response to DNA damage in HepG2 cells.33 miRNAs significantly regulated by p53 (12 up-regulated and 21 down-regulated) were detected between the doxorubicin-treated and untreated HepG2 cells in two biological replicates for small RNA sequencing and 8 miRNAs have been reported previously to be associated with HCC. Gene ontology (GO) and KEGG pathway enrichment analysis showed that 87.9%(29 out of 33) and 90.9%(30 out of 33) p53-regulated miRNAs were involved in p53-related biological processes and pathways with significantly low P-value, respectively. Remarkably,18 out of 33 p53-regulated miRNAs were identified to contain p53 binding sites around their transcription start sites (TSSs). The qRT-PCR result showed that hsa-miR-3661 was 10.17-fold increased and hsa-miR-4521 was down-regulated 6.25-fold which was consistent with sequencing results. Result of CCK-8 array showed that miR-3661 was able to inhibit the proliferation of HepG2 cell. Finally, comprehensive p53-miRNA regulatory networks were constructed and 64 p53-miRNA-mRNA feed forward loop analyzed. These observations provide a new insight into p53-miRNA co-regulatory network in the context of HCC.