The Role Of MiR-132/212 Family In Lung Cancer And The Mechanism Of Regulation Of Hedgehog Signaling Pathway

Lung cancer is the leading cause of cancer-related deaths in both men and women. According to histological type, there are two most prevalent histological types of lung carcinoma:non-small-cell lung cancer (NSCLC) and small-cell lung carcinoma, and the most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. MicroRNAs are a class of22-nucleotide, noncoding RNAs that are evolutionarily conserved and function as negative regulators of gene expression. MiRNAs are aberrantly expressed or mutated in human cancer, indicating that they may function as a novel class of oncogenes or tumor suppressor genes. MicroRNAs are always clustered on chromosome, with the exception of a few individual cases, so far little is known about the functional consequence of this conserved clustering of miRNA loci.In this article, we investigated the role of miR-132/212cluster in NSCLC and their regulatory mechanism. Utilizing microRNA array, we explored the change expression of miRNAs in NSCLC cell lines A549treated with TPA or not, and we found the expression of several miRNAs was changed including two members of miR-132/212cluster, miR-132and miR-212. To exclude the false positive results of miRNA array, we detected the expression of miR-132and miR-212in A549, H1299and BEAs-2B cells treated with TPA, and found miR-132and miR-212were up-regulated by TPA, which was in consistent with miRNA array. We then investigated the role of miR-132and miR-212in NSCLC. Transient transfection and stably overexpressing miR-132or miR-212in NSCLC cells promoted cell proliferation, altered cell cycle progress, increased the ablility of cell anchorage-independent growth, and promoted cell migration and invasion, and blocking the overexpression of miR-132or miR-212by anti-miR-132or anti-miR-212partially reversed the promoting effect, which indicated miR-132and miR-212displayed oncogenic properties in NSCLC. MiR-132and miR-212belongs miR-132/212cluster which located at chromosome17p13.3, they are separated by263bp. To explore the relationship between miR-132and miR-212, utilizing reverse transcription PCR, we showed miR-132and miR-212was derived from the same primary transcript. We screened bulk-selected H1299cell which stably overexpressed both miR-132and miR-212at the same time, and designated as "H1299-pEGP-miR-T", the control cells overexpressing GFP but no miRNA were designated as "H1299-pEGP-miR-null". We examined the influence of miR-T on cell functions. We showed compared with control cells, overexpressing both miR-132and miR-212(miR-T) promoted cell proliferation, but suppressed cell anchorage-independent growth, inhibited the capacity of cell to migrate and invade. Interesting, in H1299-pEGP-miR-T cells, knock down the overexpression of either miR-132or miR-212by anti-miR-132or anti-miR-212reversed the inhibiting effect of miR-T on cell migration and invasion, suggesting the different expression strength of miR-132or miR-212may cause different influence on cell migration and invasion.To investigate the regulatory mechanism of miR-132/212cluster in NSCLC, using bioinformatics, we predicted the potential targets of miR-132/212, and found hedgehog pathway receptor PTCH1may be regulated by miR-132/212. Hedgehog pathway is one of major cell-cell signaling pathways which control the vast majority of cell fate decisions during the development of bilaterian animals. Secreted Hh molecules (Shh, Dhh, and Ihh) bind to the receptor (Hipl, PTCH1, and PTCH2), thereby activating a putative transmembrane protein smoothened (Smo). Smo initiates a cascade of events resulting in Gli entering the nucleus and acting as a transcription activator. Utilizing dual luciferase assay, real time RT-PCR and western blot, we showed PTCH1was a target of miR-132/212. We synthesis PTCH1siRNA to block the expression of PTCH1, and we showed in A549, H1299and BEAs-2B cells, knocking down PTCH1by PTCH1siRNA promoted cell proliferation, which was in consistent with the effect of PTCH1was down-regulated by miR-132and miR-212, this indicated the promoting role of miR-132/212cluster on cell proliferation was dependent on their regulation to PTCH1, in these three cell lines, blocking PTCH1by PTCH1siRNA suppressed cell migration and invasion, which was opposite with the effect of miR-132or miR-212, but in consistent with the effect of miR-T, this result suggested PTCH1was not the effector which mediates the role of miR-132/212cluster on cell migarion and invasion. We also proved that another hedgehog pathway memebers, HHIP1and SUFU, may be targeted by miR-132/212. PTCH1, HHTP1and SUFU are inhibitor of hedgehog pathway, we examined the influence of miR-132/212cluster on the acitivty of hedgehog pathway, and showed blocking miR-132/212cluster decreased the expression of GLI1, which is a faithful marker of hedgehog pathway activity.The present study, we showed in NSLCL cells, overexpressing miR-132or miR-212promoted cell proliferation, anchorage-independent growth, cell migration and invasion, suggesting miR-132or miR-212displayed oncogenic properties. PTCH1was a shared target of miR-132and miR-212, the role of miR-132/212cluster was partially dependent on their regulation to PTCHl. MiR-132/212cluster may influence the activity of hedgehog pathway.