Study On The Mechanism Of ARHGAP9 Regulating The Invasion And Metastasis Of Lung Adenocarcinoma Via Wnt/β-catenin Signaling Pathway

Background and ObjectiveInvasion and metastasis are important causes of cancer-related deaths in patients with lung adenocarcinoma,but its molecular mechanism is not yet clear.As a negative regulator of Rho-GTPase,ARHGAP9 plays a key role in cell proliferation,metastasis,invasion and other processes,and is related to the occurrence and development of many tumors.This study aims to clarify the role of ARHGAP9 in the invasion and metastasis of lung adenocarcinoma,and to preliminary explore its molecular mechanism.Materials and methodsTo preliminarily analysis the role of ARHGAP9 gene in lung adenocarcinoma in the TCGA database,and compare the different expressions of ARHGAP9 between lung adenocarcinoma patients and normal lung tissues;To construct a cell line in which ARHGAP9 is silenced or overexpressed,and perform further detection of apoptosis,cell cycle,western blot,etc.;The effects of ARHGAP9 on cell growth and migration were studied through colony formation and migration experiments.Statistical AnalysisThe SPSS standard version 21.0 was used for statistical analysis.The Chi-Square test was used to evaluate the relationship between ARHGAP9 expression and clinicopathological characteristics.The survival curve was generated by the Kaplan-Meier(KM)method,and the Log-Rank test was used for statistical analysis.P-value<0.05 is considered statistically significant.Results1.The low expression of ARHGAP9 is significantly related to the clinical grade and poor prognosis of lung adenocarcinomaWe conducted a preliminary analysis of the role of ARHGAP9 gene in The Cancer Genome Atlas(TCGA)database,and the results showed that the expression of ARHGAP9 in lung adenocarcinoma tissues was significantly lower than that in adjacent normal tissues.The low expression of ARHGAP9 has a significant correlation with the clinical stage of tumor.The higher of the clinical stage was,the lower of the expression of ARHGAP9 was.The survival time of patients with low ARHGAP9 expression is significantly shorter than that of patients with high expression.2.Down-regulating the expression of ARHGAP9 in lung adenocarcinoma cells can promote cell proliferation,metastasis and invasionRT-q PCR and western blot were used to detect the m RNA and protein levels of the ARHGAP9 gene in four lung adenocarcinoma cell lines H1299,H1650,HCC827 and A549.HCC827 with relatively low ARHGAP9 expression and H1299 with relatively high ARHGAP9 expression were selected to study the mechanism at the cellular level.The results of CCK-8 cell proliferation experiments and clone formation experiments showed that transient down-regulation of ARHGAP9 can promote the proliferation of H1299 cells,while transient overexpression of ARHGAP9 can inhibit the proliferation of HCC827 cells.Scratch and Transwell showed that transient down-regulation of ARHGAP9 can promote the metastasis and invasion of H1299 cells,while transient overexpression of ARHGAP9 can inhibit the metastasis and invasion of HCC827 cells.3.Down-regulating the expression of ARHGAP9 in lung adenocarcinoma cells can inhibit cell apoptosis and G0/G1 cell cycle arrestFlow cytometry was used to detect the apoptosis and cell cycle distribution of lung adenocarcinoma cells after down-regulation and over-expression of ARHGAP9,respectively.The results showed that transiently down-regulated ARHGAP9 in H1299 cells can significantly inhibit cell apoptosis and reduce G0/G1 cycle arrest.The transient over-expression of ARHGAP9 in HCC827 cells can significantly increase cell apoptosis and G0/G1 cell cycle arrest.4.Down-regulating ARHGAP9 activates the Wnt/β-catenin signaling pathway by reducing the expression of DKK2,thereby promoting the invasion and metastasis of lung adenocarcinomaTo further explore its molecular mechanism,we sequenced the transcriptome of H1299 cells that down-regulated ARHGAP9 and found that down-regulation of ARHGAP9 can cause the transcription and protein levels of DKK2,an antagonist of the Wnt/β-catenin signaling pathway,to decrease.Western blot results showed that after down-regulation of ARHGAP9,the expression of β-catenin significantly increased and the expressions of the proteins Bcl-2,Cyclin D1 and c-Myc regulated by the nucleus as transcription factors were significantly up-regulated.The results of cytoplasmic nuclear isolation experiments showed that after down-regulating ARHGAP9,the amount of β-catenin into the nucleus significantly increased.ConclusionIn summary,this study found that down-regulation of ARHGAP9 can significantly reduce the expression of DKK2 and relieve its antagonistic effect on the Wnt/β-catenin signaling pathway to increase the expression of β-catenin,and nucleus,which make its downstream genes(Bcl-2,Cyclin D1 and c-Myc)overexpressed,thereby inhibiting cell adhesion,apoptosis and cycle arrest,and promoting cell proliferation,metastasis and invasion.It also provides a theoretical basis that ARHGAP9 may become a potential new target for the treatment of lung adenocarcinoma.