The Screen Of The Diverse MicroRNA Within The EMT Transcription Factor Twist1Induced Vasculogenic Mimicry In HCC

Backgroud&Object i ve:MicroRNA (miRNA) contain a class of small noncoding RNAs that regulate expression of target genes either by decreasing the stability of the target mRNA or by translational inhibition. They are involved in diverse processes including cellular differentiation proliferation and apoptosis. Recently there is evidence suggests their importance for cancer genesis. Research on vasculogenic mimicry-related miRNA is becoming a hot point."We used the miRNA gene chip (Capitalbio Corporation) to screen the differentially expressed miRNA in Twistl-overexperssing hepatocellular carcinoma cell lines, and confirmed the results using real time-PCR; then we predicted the target genes of the diverse miRNAs by bioinformatics methods and got the VM related miRNA, then we discussed the conceivable mechanism of the miRNAs in the VM formation. The result of this study can provide theoretical support and experimental basis for further research on the function of miRNA in the signal pathway of molecular Twistl regulating the formation of VM by tumor cell via EMT.Methods:We used the human hepatocellular carcinoma cell line HepG2as research object. Twistl expression plasmid was constructed and Twistl overexpressing HCC models were established by the transfection. The nontransfected HepG2cells were used as control, the total RNA was extracted respectively. MiRNA gene chip(Capitalbio Corporation) was used to analyze the alteration of the miRNA profile caused by the overexpression of twistl, and find out the genes that are up-regulated or down-regulated obviously.Four of the identified differentially expressed miRNA were selected, hsa-miR-128was up-regulated notably, and has-miR-27a, has-miR-26b, has-miR-17were down-regulated obviously. At last we enquiry the target genes of miRNA in the Internet with the TargetScan, and find out the miRNA which likely control VM through screening the target genes which are related with VM.Results: 1. In Twist l-overexpressing HepG2cells there are18differentially expressed miRNA.5up-regulated miRNA genes:has-miR-128, has-miR-132, has-miR-151-5p, has-miR-26a and has-miR-319, and13down-regulated miRNA genes: has-miR-25, has-miR-1246, has-miR-19a, has-miR-378, has-miR-486-3p, has-miR-140-3p, has-miR-26b, has-miR-27a, has-miR-19b, has-miR-1260b, has-miR-23b, has-miR-17and has-miR-9.2. It was confirmed that results of real time-PCR were consistent with the gene chips has-miR-27a, has-miR-26b, has-miR-17were down-regulated relatively in Twist1-overexpressing HepG2cells and has-miR-128was up-regulated relatively.3. The bioinformatics study of the target genes:There were different amount of target genes for miRNAs; Some of them were related with the vasculogenic mimicry: VE-cadherin, EphA2, MMP2, VEGF-A, and relevant miRNA:has-miR-27a, has-miR-26b, has-miR-17.Conclusions:1. MiRNA expression profile of Twist1-overexpressing HepG2cells was established compared to the nontransfected HepG2cells.There were5up-regulated miRNAs:has-miR-128, has-miR-132, has-miR-151-5p, has-miR-26a, has-miR-3195, and13down-regulated miRNAs genes:has-miR-25, has-miR-1246, has-miR-19a, has-miR-378, has-miR-486-3p, has-miR-140-3p, has-miR-26b, has-miR-27a, has-miR-19b, has-miR-1260b, has-miR-23b, has-miR-17, has-miR-9.2. Has-miR-27a,has-miR-26b,has-miR-17maybe have suppression effect by regulate the expression of VE-cadherin,EphA2,MMP2,VEGF-A in the formation of tumor vaculogenic mimicry.3. After the happening of EMT urged by Twistl, the expression of has-miR-27a,has-miR-26b,has-miR-17in tumor cell was changed, then regulated the expression of VE-cadherin,EphA2,MMP2,VEGF-A, final affected VM.