Study On The Role Of MiR-486-5p On The Expression Of Pim-1Gene In Non-small-cell Lung Cancer

Objective: MicroRNAs （miRNAs） are endogenous²3nt RNAs thatplay important gene-regulatory roles by pairing to the3’-UTR of specifictarget mRNAs, resulting in the disruption of mRNA stability and/or translation.Our previous studies have identified that miR-486-5p is one of the mostdownregulated miRNAs in Non-small-cell lung cancer tissues （NSCLCs）, aswell as a valuable diagnostic biomarker for early detection of NSCLC insputum and plasma. Furthermore, we found that miR-486-5p could act as atumor-suppressor in the development and progression of NSCLC. However,the role of miR-486-5p in the development and progression of NSCLC is stilllargely unknown. Therefore, in the present study, we aimed to identify thepossible targeted genes of miR-486-5p to further elucidate the mechanismsresponsible for the tumor-suppressive abilities of miR-486-5p in NSCLC.Methods: Bioinformatics analysis was used to predicate candidate targetsof miR-486-5p firstly. The3’-UTR luciferase reporter construct of Pim-1wascreated,and Luciferase reporter assay was performed to determine whetherPim-1could be regulated by miR-486-5p. In addition, the expressions ofPim-1both at mRNA and protein level were detected in NSCLC cells stablyoverexpressing miR-486-5p using Western Blot and Real-time PCR assay,respectively. Furthermore, miR-486-5p and Pim-1expression was examined intumor tissues and the matched normal tissues from24cases of human primaryNSCLC.Results:1Bioinformatics analysis to predict the target genes of miR-486-5pWe used the miRecords software programs and miRsearch tool to identifythe candidate targets of miR-486-5p. Pim-1was the top25high-scoring candidates among the32853genes predicted by miRecords（http://mirecords.biolead.org/）. Conversely, if predicting the miRNAs that bindto the3’-UTR region of Pim-1gene, miR-486-5p was the top ten miRNAswith high-scoring. The above results suggested that Pim-1might be a targetedgene of miR-486-5p.2Construction of Pim-1-3’ untranslated region （UTR） luciferase reporterTo create3’-UTR luciferase reporter construct of Pim-1,1200-bpsequences from putative miR-486-5p binding sites were synthesized andcloned into the pGL3-REPORT vector according to the prediction results bymiRecords software programs. The positive plamids were extracted andunderwent identification by restriction enzymes test. After that, the possiblepositive plasmid was further sequenced and blasted. The results showed that itis fidelity to that in Genebank （NM₀02648.3）, which means that thepGL3-Pim-1report vector was constructed successfully.3. Using Luciferase reporter assay to examine the regulation of miR-486-5pon Pim-1expressionTo determine whether Pim-1could be regulated by miR-486-5p, weperformed luciferase reporter assay. The luciferase activity of Pim-1-3’-UTRwas reduced by⁷0%in cells expressing miR-486-5p compared with thoseexpressing the control miRNA. It is likely that miR-486-5p may bind to the3’-UTR sequences of Pim-1mRNA to regulate its expression.4Generating NSCLC cells stably overexpressing miR-486-5pTo force expression of miR-486-5p in NSCLC cells, cells weretransfected with miR-486-5p expressing vector or the empty vector, andselected with2μg/ml puromycin to obtain the miR-486-5p expressing clones.Compared with the empty vector cells, the level of miR-486-5p wassignificantly overexpressed in H1299, H157and A549cells expressingmiR-486-5p by Taqman microRNA assays, which confirmed that the NSCLCcells stably overexpressing miR-486-5p were generated.5Pim-1expression in cells stably overexpressing miR-486-5pPim-1protein expression was detected in the cell overexpressing miR-486-5p using Western blot assay. It showed that forced expression ofmiR-486-5p in the cells resulted in an obvious decrease of Pim-1proteinexpression compared with control cells. However, this was not accompaniedby a significant reduction in mRNA expression in cells overexpressingmiR-486-5p relative to their control cells, respectively. Together, miR-486-5pmight directly bind to the3’-UTR sequences of Pim-1, as well as inhibiting itsexpression mainly through posttranscriptional regulation.6MiR-486-5p and Pim-1expression in human primary NSCLCsTo confirm the correlation between miR-486-5p and its target in clinicalspecimens, we analyzed miR-486-5p expression and Pim-1protein expressionin24cases of frozen NSCLC tissues. MiR-486-5p is downregulated in87.5%（21of24） tumor tissues compare with the matched normal lung tissues,whereas Pim-1protein expression is upregulated in87.5%（21of24） tumortissues by Western bolt analysis. Analysis showed that the level of miR-486-5pin tumors is significantly lower than that in normal tissues using Wilcoxonmatched pair rank test （Z=-3.971, p=.000）, while Pim-1protein expression intumors was statistically higher than that in normal tissues （Z=-4.257, p=.000）.Moreover, in five NSCLC cell lines （A549, H1299, H157, SK-MES-1andH358） which all showed markedly decreased miR-486-5p expression relativeto that of human normal lung tissue, Pim-1protein expressions were allindicated at relatively higher level.Conclusion:1MiR-486-5p expression is frequently downregulated in tumor tissuescompared with the normal lung tissues, and reduced miR-486-5p expression isa frequent molecular event in NSCLC.2Pim-1is one of the target genes of miR-486-5p, and miR-486-5p maynegatively regulate Pim-1expression at posttranscriptional level.3Downregulation of miR-486-5p in NSCLC confer to Pim-1upregulation byrelieving the inhibitory effect of the3’-UTR, which is also a pathway ofmiR-486-5p involved in the development and progression of NSCLC.