Study On The MiRNAs Expression Profile Of The HepG2 Stimulated By UC-MSCs Conditioned Medium And Biologicol Function Of MIR-3065-5p In HepG2

Mesenchymal stem cells (MSCs) are multipotent adult stem cells. MSCs widely exist in animal feral and muture tissues, such as adipose tissue, cartilage, bone marrow, amniotic fluid, umbilical cord and placenta[M] MSCs have an ability of home-backing to tumor site and are criticial for tumorigenesis. They can affect the proliferation, migration and angiogenesis of tumor by secreting cytokines. Moreover, MSCs can alter the expression level of tumor miRNAs, and then affect the biological behaviour of cancer cells[6].Based on our previous study, the conditioned medium of UC-MSCs in a certain concentration could inhibit the proliferation of HepG2. In order to explore the molecular mechanism, we first stimulated HepG2 with conditioned medium of UC-MSCs in different stages (Oh, 1h,6h,12h,24h) and obtained the miRNAs expression profiles by small RNAs sequencing. As a result,10.91,10.79, 11.64,10.86 and 11.93 million clean reads were obtained respectively; Clean reads were mapped to a reference sequences, the genome coverage of the samples were over 67%; Mapping the miRBase, 942,995,1031,976 and 958 types of mature miRNAs were detected respectively. Moreover, comparing the number of differential expressed miRNAs between treatment and control samples, we found that the number of differential expressed miRNAs was increased and expression of miRNAs altered constantly with the extended time in conditioned medium stimulating. In order to verify reliability of the results of high-throughput sequencing, we used qRT-PCR detecting 10 differential expressed miRNAs and indicated that the tendency of miRNAs expression were highly consistent with the results of sequencing.Then we predicted the alternative target genes of differential expressed miRNAs by TargetScan software, and analysized GO and KEGG of the identified target genes. We found that the alternative target genes of these miRNAs were involved in transcriptional regulation, cell proliferation, apoptosis and signal transduction, etc. However, there were significantly enriched in multiple pathway, such as MAPK pathway、wnt pathway、calcium pathway、herpes simplex infection and amino sugar metabolism, etc. The result indicated that the conditioned medium of UC-MSCs effect the signal pathways related to physiology and biochemistry.Furthermore, in order to illuminate the molecular mechanism that the supernate of UC-MSCs had an inhibitory effect on the proliferation of HepG2, we made correlation analysis of HepG2 small RNAs and mRNAs data. As a result, there were 12 items of interactive patterms between up-regulated miRNAs and down-regulated mRNAs; 5 items of interactive patterms between down-regulated miRNAs and up-regulated mRNAs. For instance, up-regulated miR-375 negtively regulated the expression of ARHGAP21 and JUND, high expressed let-7f-3p and miR-34a-3p synergistically inhibited the expression of IL6.In the end, we detected miR-3065-5p function. It was demonstrated that miR-3065-5p could inhibit the proliferation and invasion of HepG2 in vitro, which suggested miR-3065-5p was a potential tumor suppressor miRNA. Moreover, overexpression of miR-3065-5p down regulated the expression of SATB1 in HepG2, suggesting STAB1 may be a target gene of miR-3065-5p.