ma.uni-heidelberg.de/apps/zmf/mirwalk). This search strategy identified miR-183 and miR-186 as potential regulators of AKAP12 in hepatocarcinogenesis. We then performed an expression analysis of miR-183 and miR-186 in FFPE and fresh-frozen tissue samples. Expression analysis in FFPE samples showed a significant up-regulation of miR-183 (P < 0.01, all groups versus NL) and a trend toward miR-186 up-regulation in DN and CL tissues (Fig. 7A). Further analysis of miR-186 expression from fresh-frozen tissues showed an up-regulation of miR-186 in CL (see Supporting Fig. 3; P < 0.05). We next determined if miR-183 and miR-186
can directly regulate AKAP12 levels. The 3′UTR of AKAP12α/β harbors three and four putative target Alvelestat sites for miR-183 and miR-186, respectively, as predicted by TargetScan (Fig. 7B).19 The highly conserved 3′ end of the AKAP12 3′UTR contains two putative binding sites for each miRNA and was cloned into the 3′ end of Firefly luciferase. Nucleotide sequences were mutated to ablate either both miR-183 or both miR-186 sites. MiR-183 and miR-186 were cloned into expression plasmids and transfected in HEK293T cells
(see Supporting Fig. 4). Expression of increasing amounts of both miRNAs caused a dose-dependent increase in the mature miRNAs above endogenous levels (range 9 to 27 and 1.8 to buy NVP-AUY922 20-fold for miR-183 and miR-186, respectively). Coexpression of miR-183 上海皓元医药股份有限公司 with the wild-type (WT) and 186-mutated UTR resulted in a measurable (1.3-fold) decrease in luciferase expression as compared to the 183-mutated UTR (Fig. 7C). Coexpression of miR-186 with the WT UTR resulted in a distinct decrease (two-fold) in luciferase expression compared to the 186-mutated UTR.
Expression of miR-186 in HEK293T cells resulted in a 2-fold reduction of endogenous AKAP12 mRNA levels (Fig. 7D). Expression of miR-183 did not reduce endogenous AKAP12 levels. Both α and β isoforms were reduced to similar levels following miRNA-dependent mRNA knockdown (data not shown). Down-regulation and tumor suppressor activity of the scaffold protein AKAP12 has been shown in several malignancies,7, 8, 20 but, so far the role of AKAP12 in hepatocarcinogenesis is almost completely unknown. Here, we present protein expression data of AKAP12 in a large number of human liver tissue specimens (n = 388), revealing a significant down-regulation of AKAP12 in HCC compared to NL. Remarkably, CL and DN already showed reduced AKAP12 expression, suggesting that down-regulation of AKAP12 is an early event in hepatocarcinogenesis. Indeed, recent findings in prostate cancer have led to the theory that AKAP12 may act as a key player in early stages of carcinogenesis.6 TMA data demonstrate that AKAP12 expression is progressively down-regulated during HCC progression.