PUB131 MIR155 Mediated Down-Regulation of Ubiquilin 1 Induces EMT in Lung Cancer Cells

Abstract

Ubiquilin 1 (UBQLN1) functions as an adaptor protein that links the ubiquitination machinery to the proteasome for protein degradation. However, recently our lab has shown that despite the role in protein degradation, UBQLN1 also plays a role in cancer biology. In the present study, we have examined the post-transcriptional regulation of UBQLN1 and identified a miRNA responsible for regulation of UBQLN1 in A549 and H358 cells. In silico approaches involve the use of TargetScan and miRDB web portals for identification of miRNA sites in 3'-UTR of UBQLNs. Transfections of miRNA mimics and inhibitors were performed using Dhafrmafect reagent. Epithelial to mesenchymal transformation (EMT) was assayed by colony formation, scratch assay and cell migration and cell invasion assays. Dual luciferase assay was carried out to validate the binding of MIR155 on 3'-UTR of UBQLN1. In silico approaches suggested miR-200, miR-425 and miR-155 as probable miRNAs capable of regulating levels of UBQLN1. However, over-expression of only miR-155 significantly down-regulated levels of UBQLN1 protein in A549 and H358, two different lung adenocarcinoma cell lines. Interestingly, introduction of miR-155 significantly increased the proliferation of A549 and H358 cells. Flow cytometry based cell cycle analysis of miR-155 over-expressing cells showed a significant decrease in the G1 phase of the cell cycle. Over-expression of miR-155 induced colony formation, cell invasion and migration of A549 and H358 cells. Further, dual luciferase assays carried out with 3’-UTR of UBQLN1 and miR-155 mimic confirmed in vivo binding capability of miR-155 on 3’-UTR of UBQLN1. Moreover, when MIR155 transfected A549 cells are injected in immunodeficient mice they colonize and grow faster in lungs of mice than non-targeting control transfected A549 cells. In conclusion, for the first time our studies have identified a miRNA that regulates UBQLN1 in lung carcinoma cells.