ARHGEF10L Promotes Cervical Tumorigenesis via RhoA-Mediated Signaling.

Junyi Tang,Chang Li,Kehua Fang,Xiaotian Chang,Xiaosong Liu

doi:10.1155/2021/6683264

Abstract

Background Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) is a member of the guanine nucleotide exchange factor family, which regulates Rho GTPase activities, thus contributing to tumorigenesis. Our previous study demonstrated a strong association between the ARHGEF10L gene and the risk of cervical carcinoma. This study investigated the pathogenic role and mechanism of ARHGEF10L in cervical tumors. Methods The HeLa cell line, which was derived from cervical carcinoma, was transfected with ARHGEF10L-overexpressing plasmids or anti-ARHGEF10L siRNA. Cell counting kit-8 assays, wound-healing assays, and cell apoptosis assays were performed to investigate the effects of ARHGEF10L on cell activities. A Rho pull-down assay and RNA-sequencing analysis were performed to investigate the pathogenic pathway of ARHGEF10L involvement in cervical tumors. Results ARHGEF10L overexpression promoted cell proliferation and migration, reduced cell apoptosis, and induced epithelial-to-mesenchymal transition (EMT) via downregulation of E-cadherin and upregulation of N-cadherin and Slug in transfected HeLa cells. The overexpression of ARHGEF10L also upregulated GTP-RhoA, ROCK1, and phospho-ezrin/radixin/moesin (ERM) expression in HeLa cells. RNA-sequencing analysis detected altered transcription of 31 genes in HeLa cells with ARHGEF10L overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway analyses identified significant differences in cyclin-dependent protein serine/threonine kinase activity, cell responses to vitamin A, and Toll-like receptor signaling pathways. Both real-time PCR and Western blotting verified the increased expression of heat shock 70 kDa protein 6 (HSPA6) in ARHGEF10L-overexpressing HeLa cells. Since we reported that ARHGEF10L played a role through RhoA-ROCK1-ERM signaling, an important pathway in tumorigenesis, and stimulated EMT and HSPA6 expression in liver tumors and gastric tumor cells, we suggest that ARHGEF10L is a novel oncogene in many tumors.

Highlights

Rho GTPases are a family of approximately 20 small G proteins that are key regulators of diverse cellular functions, such as cell growth, survival, motility, morphogenesis, and differentiation [1]. e most well-characterized members are Cdc42, Rac1, and RhoA. e active GTP-bound state and the inactive GDP-bound state are the two different Rho GTPase conformations
We used the Illumina GoldenGate Assay, Sequenom MassARRAY, and TaqMan polymerase to analyze the possible correlations between tag single nucleotide polymorphism (SNP) in the ARHGEF10L locus and various tumor risks. e genotyping analyses demonstrated a strong association of rs2244444 and rs12732894 in ARHGEF10L with liver cancer. at study detected increased expression of ARHGEF10L in hepatocellular carcinoma tissues and demonstrated that this increased expression stimulates hepatocellular carcinoma cell proliferation and migration by activating the RhoA-ROCK1-phospho-ERM pathway and epithelial-to-mesenchymal transition (EMT) in two liver tumor cell lines and in tumor-bearing mice. is finding suggests that increased expression of ARHGEF10L plays an important role in hepatocellular tumorigenesis [8]
Effect of ARHGEF10L Expression on the Proliferation, Migration, and Apoptosis of HeLa Cells. e ARHGEF10Lexpressing plasmids and the mock expression vectors were transfected into HeLa cells. e expression of exogenous ARHGEF10L was detected by qRT-PCR and Western blotting. e qRT-PCR results showed increased mRNA levels of ARHGEF10L in the transfected HeLa cells compared with the cells transfected with blank expression plasmids (Figure 1(a)). e Western blotting results showed increased protein levels of ARHGEF10L in the transfected HeLa cells compared with the mock-transfected cells (Figure 1(b))

Summary

Introduction

Rho GTPases are a family of approximately 20 small G proteins that are key regulators of diverse cellular functions, such as cell growth, survival, motility, morphogenesis, and differentiation [1]. e most well-characterized members are Cdc, Rac, and RhoA. e active GTP-bound state and the inactive GDP-bound state are the two different Rho GTPase conformations. Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) is a member of the guanine nucleotide exchange factor family, which regulates Rho GTPase activities, contributing to tumorigenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway analyses identified significant differences in cyclin-dependent protein serine/threonine kinase activity, cell responses to vitamin A, and Toll-like receptor signaling pathways. Both real-time PCR and Western blotting verified the increased expression of heat shock 70 kDa protein 6 (HSPA6) in ARHGEF10L-overexpressing HeLa cells. Since we reported that ARHGEF10L played a role through RhoA-ROCK1-ERM signaling, an important pathway in tumorigenesis, and stimulated EMT and HSPA6 expression in liver tumors and gastric tumor cells, we suggest that ARHGEF10L is a novel oncogene in many tumors

Methods

Results

Conclusion