MiR-30a-5p Antisense Oligonucleotide Suppresses Glioma Cell Growth by Targeting SEPT7

Zhifan Jia,Kun Wang,Guangxiu Wang,Peiyu Pu,Anling Zhang

doi:10.1371/journal.pone.0055008

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting the mRNAs of hundreds of human genes. Variations in miRNA expression levels were shown to be associated with glioma. We have previously found miR-30a-5p overexpression in glioma cell lines and specimens. Bioinformatics analyses predict that several miRNAs, including miR-30a-5p, are involved in the post-transcriptional regulation of SEPT7. SEPT7 is a member of the septin family, which is a highly conserved subfamily of GTPases implicated in exocytosis, apoptosis, synaptogenesis, neurodegeneration and tumorigenesis. Our previous study has also demonstrated that SEPT7 expression is decreased in astrocytic gliomas with different grades and plays a tumor suppressor role. In the present study, we knocked down miR-30a-5p with antisense oligonucleotide (miR-30a-5p AS) in LN229 and SNB19 glioblastoma(GBM) cells, and found that cell growth and invasion were inhibited, while apoptosis was induced. miR-30a-5p AS treated cells showed upregulation of SEPT7 and downregulation of PCNA, cyclin D1, Bcl2, MMP2 and MMP9. In contrast, when miR-30a-5p mimics were transfected into LN229 and SNB19 GBM cells, cell growth and invasion were promoted and the expression of relevant proteins increased. Meanwhile, the effect of miR-30a-5p mimics on glioma cells can be reversed by transfection of SEPT7 construct. Additionaly, miR-30a-5p directly targeting SEPT7 was identified by the reporter gene assay. Our study demonstrates,for the first time, that miR-30a-5p is a bona fide negative regulator of SEPT7 and the oncogenic activity of miR-30a-5p in human gliomas is at least in part through the repression of SEPT7.

Highlights

Malignant gliomas, such as glioblastoma multiforme (GBM), are the most common and aggressive malignant primary brain tumors
When miR-30a-5p was knocked down, the expression of SEPT7 was upregulated while the expression of PCNA, Cyclin D1, Bcl2, MMP2 and MMP9 downregulated in SNB19 cells and in LN229 cells (Figure 3A, 3B), which coincided with the results determined by MTT, cell cycle kinetics, apoptosis and transwell assay
Recent studies indicate that various miRNAs play important roles in the initiation and progression of cancer. miRNAs may function as tumor suppressors by down-regulating the expression of tumor-promoting genes, or may have oncogenic role by inhibiting the expression of tumor suppressor genes

Summary

Introduction

Malignant gliomas, such as glioblastoma multiforme (GBM), are the most common and aggressive malignant primary brain tumors. We showed that SEPT7 overexpression suppressed the invasion and migration ability of human gliomas, reversed the imbalanced state of MMPs/TIMPs, downregulated the expression of integrin alpha(v)beta(3) and altered the structure of tubulinalpha [15]. These data strongly supported that SEPT7 is an important molecule in gliomagenesis, and should be considered as a tumor suppressor. The effect of miR-30a-5p mimics promoting cell proliferation and inhibition of apoptosis can be reversed by enforced overexpression of SEPT7 in glioma cell lines

Materials and Methods

Results

Discussion