Dual-strand tumor suppressor miR-193b-3p and -5p inhibit malignant phenotypes of lung cancer by suppressing their common targets

Kyung Hee Choi,Haein Ji,Chang Hoon Shin,Hyeon Ho Kim,Woo Joo Lee

doi:10.1042/bsr20190634

Abstract

Emerging studies suggest that microRNAs (miRNAs) play multiple roles in cancer malignancy, including proliferation and acquisition of metastatic potential. Differentially expressed miRNAs responsible for the malignancy of lung cancer were searched by miRNA microarray using a previously established brain metastatic lung cancer model. Twenty-five miRNAs were down-regulated in brain metastatic lung cancer cells. Among those, miR-193b-3p and -5p were chosen for further studies. Their function in metastatic potential and proliferation was examined using Transwell invasion, wound healing, and colony forming assays. The underlying mechanism of tumor-suppressor miR-193b-3p and -5p was explored using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Western blot, Argonaute 2-RNA immunoprecipitation (Ago2-RIP), and reporter assays. Both strands of miR-193b were down-regulated in brain metastatic lung cancer cells and in tissues from lung cancer patients. Overexpression of miR-193b-3p and -5p inhibited invasive and migratory activities and diminished clonogenic ability. Conversely, inhibition of miR-193b-3p or -5p increased the metastatic potential and colony forming ability. Cyclin D1 (CCND1), Ajuba LIM Protein (AJUBA), and heart development protein with EGF like domains 1 (HEG1) were identified as common target genes of miR-193b-3p and -5p. A reporter assay and an Ago2-RIP experiment showed that both miRNAs directly bind to the 3′ untranslated region (3′UTR) of the target mRNA. Knockdown of target gene reduced the proliferative and metastatic potential of primary and metastatic lung cancer cells. Our results demonstrate miR-193b is a dual-strand tumor suppressor and a novel therapeutic target for lung cancer.

Highlights

Despite the remarkable development of lung cancer therapies, lung cancer has been classified as a major cause of death [1]
reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analyses using tissues from patients with lung cancer and brain metastases revealed that the expression level of primary miR-193b further decreased as the cancer developed into brain metastasis (Figure 1C)
Suppression of target genes by miR-193b-3p and -5p was abolished by the disruption of the direct binding between miR-193b and the 3 3 -untranslated region (UTR) of the target mRNAs by point mutations in the seed sequence. These results demonstrate that miR-193b-3p and -5p inhibit the expression of common target genes (e.g., Cyclin D1 (CCND1), Ajuba LIM Protein (AJUBA), and HEG1) through direct interaction with the 3 untranslated region (3 UTR) of their mRNA

Summary

Introduction

Despite the remarkable development of lung cancer therapies, lung cancer has been classified as a major cause of death [1]. Within a few months of diagnosis, distant metastasis of lung cancer occurs mainly to the brain and bone [4]. The limited efficacy of lung cancer therapy can be largely attributed to two main difficulties. Since conventional anticancer drugs are targeted for lung cancer, effective treatment for metastatic lung cancer is limited [6]. For these reasons, the regulatory molecules that control the metastatic process need to be discovered to build the basis for the development of new therapies

Methods

Results

Conclusion