Cell specific tumor suppressor effect of Hsa-miR-1226-3p through downregulation of HER2, PIK3R2, and AKT1 genes

https://doi.org/10.1016/j.biocel.2021.105965Get rights and content

Abstract

PI3K/AKT signaling has a crucial role in breast cancer incidence and finding the miRNAs that regulate this pathway enhances our understanding of breast cancer regulation. Firstly, our bioinformatics analysis suggested miR-1226-3p as a bona fide regulator of HER2, PIK3R2, and AKT1 putative target genes. Secondly, RT-qPCR, ELISA test and western blotting showed that overexpression of miR-1226 was followed by reduced expression of HER2, PIK3R2, and AKT1 putative targets genes in SKBR3 cells. Third, dual luciferase assay verified direct interaction of miR-1226-3p with 3′UTR sequences of these target genes. Then, overexpression of miR-1226 in SKBR3 cells brought about increased population of sub-G1 and decreased populations of G1 cells, measured by flow cytometry. This was consistent to the reduction of p-AKT protein and increased BAX protein levels, detected by western analysis and consistent to decreased CCND1 genes expression, detected by RT-qPCR. The reduced survival and increased apoptosis rate of these cells was also verified through MTT, Annexin V-FITC and Live-Dead cell staining assays. Our results suggest that miR-1226-3p is a tumor suppressor in SKBR3 cells. However, following the overexpression of miR-1226 in MDA-MB-231 cells, Bax/Bcl2 ratio and CCND1 genes expression levels were not significantly changed, sub-G1 and G1 cell cycle population were reduced while, S and G2/M cell populations were increased, consistent to the results acquired from the apoptosis and staining assays. Finally, TCGA data analysis and RT-qPCR against 20 pairs of Normal/Tumor breast tissues indicated that miR-1226-3p has been downregulated in breast cancer. Overall, the present study gathered shreds of evidence that suggest miR-1226-3p as a tumor suppressor that exerts its inhibitory effect on SKBR3 cells through targeting of HER2, PIK3R2, and AKT1 genes and downregulates PI3K/AKT pathway.

Introduction

Regardless of the great progress in therapy, breast cancer is still one of the main causes of death in women across the globe with 25 % of all cancer cases and 15 % of all cancer-associated mortality (Sun et al., 2017). Four subtypes for breast cancer are known as HER2-positive (HER2-like), ER-negative/HER2-negative (basal-like), and low- and high-proliferative ER-positive/HER2-negative (luminal A and B, respectively). Recent studies reported that HER2 positive breast cancer has a higher incidence in Asian women compared with European women (Pathmanathan et al., 2016).

Her2 is a transmembrane protein that is encoded by HER2 oncogene and is located at 17q21-22 chromosome (Kwa et al., 2017). Her1, Her2, Her3, and, Her4 are also known as (EGFR/ErbB1, HER2/Neu, ErbB3, ErbB4, respectively) and are four members of ERBB class of receptor tyrosine kinases. These cell surface proteins are normally involved in epithelial cell survival and growth (Wieduwilt and Moasser, 2008). Furthermore, HER2 gene amplification and overexpression have been shown to be responsible for aggressive metastatic breast cancer (Yarden, 2001). Through cell surface interaction of Her2 receptor with hormones, cytokines, growth factors, and other extracellular signaling molecules, Her2 signaling pathway is activated which in downstream, leads to proliferation, differentiation, survival and cell migration (De Luca et al., 2012; Lemmon and Schlessinger, 2010).

Her family monomers have several domains including extracellular (N-terminal), transmembrane and cytoplasmic kinase domains. Autophosphorylation of specific tyrosine residues within the catalytic kinase domain of Her2 occurs upon the binding of ligand which induces homodimerization or heterodimerization of it (Hynes, 2016). Cell signaling such as MAPK, PI3K/AKT, and phospholipase Cγ pathways are activated following Her2 dimer complex activation (Liu et al., 2008). PI3K kinas phosphorylates 4, 5-diphosphate (PIP2) to phosphatidylinositol 3, 4, 5-triphosphate (PIP3) which in turn activates its downstream Akt (P. Liu et al., 2009). Phosphorylated Akt targets mTOR which eventually ends up with inhibition of apoptosis and stimulation of cell proliferation (Oh and Jacinto, 2011). To date, no specific ligand has been discovered for Her2 receptor and that’s the reason why Her2 tends to form potent heterodimers with EGFR and Her3 (Butti et al., 2018).

MicroRNAs are a category of short noncoding RNAs which regulate gene expression at the transcriptional or post-transcriptinal levels (Tani et al., 2012). Direct interaction between miRNA and target mRNAs occurs through base pairing of their complementary sites presented in 3′UTR, CDS and 5′UTR (Guo et al., 2010). If miRNA targets 3′UTR, it usually recruits RISC to the sequence and downregulates the expression (Seol et al., 2012). However, interaction of miRNA with coding sequences of mRNA might result in overexpression of the target gene (Wongfieng et al., 2017). Aberrant expression of miRNAs has been reported in several malignancies such as breast cancer (Mishra et al., 2015). In the present study we evaluated the effect of miR-1226-3p on PI3K/AKT pathway in breast cancer.

Section snippets

Bioinformatics study

In order to find the putative target genes that miR-1226-3p may regulates, a bioinformatics search was conducted. The miRNA expression analysis of breast cancer cell lines and tissues was adopted from GEO (GSE26659/26666/40525/81000/40059) (https://www.ncbi.nlm.nih.gov/geo/) database. The miRNA targets were predicted using TargetScan (http://www.targetscan.org/vert_71/), DIANA-microT (http://diana.cslab.ece.ntua.gr/pathways/), miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) and

Hsa-miR-1226 as a candidate regulator of PI3K/AKT signaling pathway in breast cancer

In order to investigate about the miRNAs that are capable of regulating PI3K/AKT signaling pathway, Targetscan, DIANA and miRWalk bioinformatics tools were used. Several miRNA recognition sites (MREs) specific for miR-1226-3p were predicted within the 3′UTR sequences of HER2, PIK3R2 and AKT1 genes (Fig. 1A). Six, Six, and three MREs were predicted within the 3′UTR sequences of HER2, PIK3R2, and AKT1 genes, respectively. Compared to the adjacent sequences within 3′UTRs, some of these MREs were

Discussion

Her2 is an important tyrosine kinase receptor that is involved in development of breast cancer and is considered as a switch to turn on PI3K/AKT signaling pathway. This pathway serves as a key regulator of cell survival, proliferation, migration and apoptosis (Paplomata and O’Regan, 2014; Ruchi Sharma et al., 2017). PI3K/AKTsignaling pathway is known to be affected by dysregulated miRNA expression levels (Josse et al., 2014). Here we intended to find miRNA candidate(s) that are involved in the

Author contributions

Z.M., B.M.S., conceived and designed the study and Z.M., ZG and B.M.S. wrote the paper. Z.M., ZG and B.M.S. performed experiments and analyzed the results. P.H provided cancer tissue samples with preparation of pathological features. All authors reviewed the results and approved the final version of the manuscript.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This study was supported by Tarbiat Modares University and Iran National Science Foundation (INSF) financial aids (Grant Number: 96012144).

References (33)

  • N.E. Hynes

    ErbB2: from an EGFR Relative to a central target for cancer therapy

    Cancer Res.

    (2016)
  • S. Jafarinejad-Farsangi et al.

    MicroRNA-29a induces apoptosis via increasing the Bax: Bcl-2 ratio in dermal fibroblasts of patients with systemic sclerosis

    Autoimmunity

    (2015)
  • C. Jin et al.

    miR-1226 targets expression of the mucin 1 oncoprotein and induces cell death

    Int. J. Oncol.

    (2010)
  • C. Josse et al.

    Identification of a microRNA landscape targeting the PI3K/Akt signaling pathway in inflammation-induced colorectal carcinogenesis

    Am. J. Physiol.-Gastrointest. Liver Physiol.

    (2014)
  • T. Khanzadeh et al.

    Investigation of BAX and BCL2 expression and apoptosis in a resveratrol-and prednisolone-treated human T-ALL cell line, CCRF-CEM

    Blood Res.

    (2018)
  • M. Kwa et al.

    Clinical utility of gene-expression signatures in early stage breast cancer

    Nat. Rev. Clin. Oncol.

    (2017)
  • View full text