Study On The Role Of LncRNA Gas5 In Papillary Thyroid Carcinoma

Objective Thyroid cancer has become the most common malignant tumor in endocrine system,but its specific molecular mechanism is not quite clear.Accumulating evidence demonstrates that the lncRNA(Gas5)has practical significance in cancer progression and metastasis.However,its role and function in papillary thyroid carcinoma(PTC)remains unknown.In this study,we aimed to explore the potential involvement of Gas5 in papillary thyroid carcinogenesis and to highlight the emerging roles of ceRNAs in the biological regulation of PTC cells.Method We conducted a review of previous microarray data for the expression of Gas5 in human PTC in Oncomine database(www.oncomine.com)(Rhodes et al.,2004).Furthermore,to confirm the reduced expression of Gas5 in PTC cell lines,we performed qRT-PCR analysis to detect the expression of Gas5 in 4 PTC cell lines(BHP5-16,TPC,K1,BHP2-7)and a normal human thyroid cell line(Nthy-ori 3-1).After transient transfection,the proliferation of PTC cells was detected by MTT and colony formation assay in vitro and by Tumor formation assay in vivo.Starbase 2.0 and RNA22 predictions revealed that the 3 ′-UTR of Gas5 contained two miR-222-3p binding sites.And then,we used the dual-luciferase assay to confirm that Gas5 was a target of miR-222-3p.Meanwhile,we detected the expression level of endogeneous miR-222-3p in BHP5-16 and K1 cells by qRT-PCR.To investigate the role of miR-222-3p on PTC carcinogenesis,miR-222-3p mimics or inhibitor were transfected into both BHP5-16 and K1 cells and the proliferation capacitiy was observied by using MTT and colony formation assays.Among the many predicted targets of miR-222-3p by bioinformatics software(TargetScan,MiRanda),we focused on PTEN.And then,to experimentally confirm that PTEN was a target of miR-222-3p,we did qRT-PCR、western blotting and dual-luciferase assay.Western blotting analysis showed the level of PTEN and the phosphorylation level of Akt(P-Akt)in cells co-transfected with Gas5 and miR-222-3p.In order to demonstrate that Gas5 modulates the expression of PTEN partly by competitively binding to miR-222-3p in BHP5-16 and K1 cells,we carried out the dual-luciferase assay.Results Gas5 was down-regulated in papillary thyroid carcinoma(PTC)tissue specimens and PTC cell lines.Gas5 represses the proliferation of PTC cells in vitro and in vivo.Co-transfection of psiCHECK-2-Gas5-WT and miR-222-3p mimics was conducted in both BHP5-16 and K1 cells.Co-transfection with psiCHECK-2-Gas5-WT and miR-nc was served as a control.The dual-luciferase assay demonstrated that Gas5 was a potential target gene of miR-222-3p.Our data showed that the cell viabilities of PTC cells were time-dependently increased by miR-222-3p mimics,and,time-dependently decreased by miR-222-3p inhibitor.QRT-PCR showed that no significant difference of PTEN mRNA level was found in the miR-222-3p mimics transfected cells as compared with cells transfected with miR-nc.By western blotting analysis,we found that up-regulation of miR-222-3p significantly decreased the expression of PTEN while down-regulation of miR-222-3p significantly increased the expression of PTEN.And then,the dualluciferase assay confirmed that PTEN was a target of miR-222-3p.Western blotting analysis showed that forced expression of Gas5 in BHP5-16 and K1 cells exerted a promoting role on endogenous PTEN protein expression and repressing effect on P-Akt protein expression.In addition,we further observed the PTEN level of the group co-transfected with Gas5 and miR-222-3p was much higher while the P-Akt level of that was much lower than those of the group transfected with miR-222-3p.In the end,we carried out the dual-luciferase assay and the result showed that the luciferase activity of the group of pcDNA3.1/Gas5 co-transfected with miR-222-3p mimics and psiCHECK-2-PTEN-3′-UTR-WT was much higher than the control group of co-transfection of psiCHECK-2-PTEN-3′-UTR-WT、miR-222-3p mimics and pcDNA3.1.Conclusion Gas5 can suppress the proliferation of papillary thyroid carcinoma cells.MiR-222-3p can mediate the tumor proliferation possibly through PTEN/Akt signaling pathway in PTC cells.Gas5 and PTEN are the potential target genes of miR-222-3p.Aberrant expression of Gas5,a ceRNA of miR-222-3p,may be involved in the progression of PTC by abolishing the miR-222-3p induced repressing activity on PTEN.