To study the biological effect of miR-216a elevation further in early hepatocarcinogenesis, we tried to identify its target
gene(s) in hepatocytes. By comparing the gene expression profile between HepG2 cells infected with lenti-miR-216a and with lenti-si-GFP control viruses, the results from our microarray analysis indicated the increase in proliferation and migration activities as putative biological functions affected by the elevation of miR-216a (Supporting Table 2S). As predicted by the miRanda algorithm (MicroRNA.org, http://www.microrna.org, September 2008 release), the tumor suppressor gene TSLC1/IGSF4/CADM1 (Tumor suppressor in lung cancer 1/Immunoglobulin superfamily 4/Cell adhesion molecule 1) was pointed out as one putative B-Raf inhibitor drug target for miR-216a (ranked
second on the list), with major functions to control the cell proliferation and migration activities. Three putative miR-216a target sites were predicted in the 3′ untranslated region (UTR) of the TSLC1 gene, targeting to nucleotide 400–421 (target site 1), 736–759 (target site 2), and 1155–1177 (target site Depsipeptide datasheet 3), respectively (Fig. 5A). Two reporter constructs were established to evaluate the regulation of TSLC1 by miR-216a through these putative target sites. One is pGL3-TSLC1-3′ UTR(WT), which contains the wildtype target sites; the other is pGL3-TSLC1-3′ UTR(Mut), which contains the mutated target sites (Fig. 5A). HepG2 cells expressing either reporter constructs Carnitine palmitoyltransferase II or the pGL3-vector (as a control) were infected with lenti-si-GFP or lenti-miR-216a, with the aim of evaluating the effect of miR-216a on the reporter activity. As a control, the cells transfected with pGL3-vector were not affected by either lenti-si-GFP or lenti-miR-216a (Fig. 5B, lanes 1-3). In contrast, infection with lenti-miR-216a (≈5-fold increase of miR-216a expression, revealed by RT-qPCR) led to a decrease of luciferase activity in cells transfected with TSLC1-3′ UTR(WT) compared with that caused by lenti-si-GFP (Fig. 5B, lane 6 versus lane 5). This effect was diminished when the three putative
target sites were mutated in cells transfected with the TSLC1-3′ UTR-mut reporter construct (Fig. 5B, lane 9 versus lane 6). The results suggested that through these three putative target sites within 3′ UTR, miR-216a can regulate the expression of TSLC1. Moreover, we evaluated the effect of elevated miR-216a on the endogenous TSLC1 protein. Relative to the cells infected with lenti-si-Luc or lenti-si-GFP control viruses, TSLC1 protein was decreased ≈50% in cells infected with lenti-miR-216a (Fig. 5C), suggesting the targeting of TSLC1 by miR-216a. To examine any biological functions for the elevation of miR-216a in hepatocytes, we focused on the proliferation and migration activities due to the well-characterized function of its target gene, TSLC1.
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