Research On The Mechanism Of ARHGAP11A Promoted The Malignant Biological Behaviors Of Lung Adenocarcinoma

Objective:Lung cancer is responsible for more mortality than any other malignancy in the world.Due to changes in lifestyle and increasing air pollution,lung cancer is rising in incidence every year.In recent years,lung adenocarcinoma(LUAD)has replaced lung squamous cell carcinoma as the most common histological type of lung cancer.With the rapid progress of medical imaging and early screening technology for lung cancer,as well as the continuous improvement of targeted lung cancer therapies,more and more lung cancer patients have benefited and significantly extended their survival.Nevertheless,at present,the 5-year survival rate of patients with non-small cell lung cancer is still less than 15%.Therefore,there is an urgent need to discover new diagnostic markers and therapeutic targets to improve the survival of patients with non-small cell lung cancer.Rho GTPase-activating protein 11A(ARHGAP11A),also known as MP-GAP,is a member of the Rho GTPase-activating protein(Rho GAP)subfamily.Rho GAPs are down-regulated in tumors and are usually associated with malignant progression.Currently,the role of ARHGAP11 A in cancer is still controversial.In order to clarify the expression and clinical significance of ARHGAP11 A in lung adenocarcinoma,bioinformatics and experiments are planned to investigate the effect of ARHGAP11 A on malignant progression of lung adenocarcinoma,to elucidate the potential mechanism of ARHGAP11 A in the development of lung adencarcinoma.Methods:1.The original gene expression profile,survival data,and clinical information of patients with lung adenocarcinoma(LUAD)were downloaded from The Cancer Genome Atlas(TCGA)database.The expression difference of ARHGAP11 A between LUAD tissues and adjacent normal tissues in the TCGA database was analyzed by using R software and verified by the Oncomine database and immunohistochemical(IHC)assay of LUAD sections.Logistic regression was applied to analyze the relationship between the expression of ARHGAP11 A and clinicopathological factors of LUAD.Kaplan-Meier(KM)survival curves and a Cox proportional-hazards model were selected to evaluate the prognostic significance of ARHGAP11 A expression.Gene set enrichment analysis(GSEA)software was applied to screen the tumor signaling pathways associated with the low and high expression group of ARHGAP11 A in LUAD.2.For the study of human LUAD tissue specimens,we collected paraffin specimens including cancer and adjacent tumors.Immunohistochemistry was used to detect the expression of ARHGAP11 A to analysis its correlation with clinicopathological factors,lymph node metastasis and prognosis.For the study of ARHGAP11 A on the biological functions of LUAD,we first used RT-PCR and Western blot to detect the expression of ARHGAP11 A in LUAD cell lines and normal bronchial epithelial BEAS-2B cells,then screened relatively high and low expression cell lines of ARHGAP11 A for further studies in vitro.Expression of ARHGAP11 A in A549 and PC-9 was inhibited by ARHGAP11A-sh RNA or ARHGAP1A-NC(negative control)respectively.The effect of ARHGAP11 A on cell proliferation was analyzed by CCK-8 assay.Transwell and wound healing assay were used to measure the changes in cell invation and migration after transfected with sh RNA or NC.We explored the effect of ARHGAP11 A on cell cycle and apoptosis via Flow cytometry.The effect of ARHGAP11 A on tumor growth was investigated by subcutaneous tumorigenesis in nude mice.3.In the study of ARHGAP11 A promoted LUAD invasion by regulating Hippo-YAP pathway,we first used RT-PCR and Western blot to detect the expression of YAP and p YAP to evaluate the effect of ARHGAP11 A on the Hippo-YAP pathway.Rescue experiment was performed to determine whether ARHGAP11 A regulates cell functions by over-expression YAP.Results:1.The TCGA database showed that the expression of ARHGAP11 A was significantly higher in LUAD tissues than in normal tissues(P<0.001).The upregulation of ARHGAP11 A in LUAD was verified by the Oncomine database(P<0.001)and IHC assay(P<0.001).Logistic regression analysis revealed the high expression of ARHGAP11 A to be closely related to age,sex,advanced pathological stage,advanced T stage,and lymph node metastasis.The KM plots based on the TCGA and KM plotter databases indicated that patients with LUAD highly expressing ARHGAP11 A had a poorer overall survival(OS)than patients with low expression of ARHGAP11 A.Multivariate Cox regression analysis showed that the high expression of ARHGAP11 A could be an important independent predictor of a poor prognosis of LUAD [hazards ratio (HR)=1.385;P<0.001].GSEA indicated that 10 signal pathways were significantly enriched in LUAD samples with the ARHGAP11 A expression phenotype.2.RT-PCR and Western blot confirmed that ARHGAP11A-sh RNA could significantly knockdwon the expression of ARHGAP11 A in A549 and PC-9 cells.3.CCK-8 results displayed that ARHGAP11 A deletion decreased the proliferation ability of two LUAD cell lines significantly.ARHGAP11 A deficiency increased the percentage of cells in S/G2/M phase and decreased the percentage of cells in G0/G1 phase.Curiously,while there was no difference in A549 cell apoptosis level,there was a difference founded in PC-9 cell apoptosis level.While knockdown the ARHGAP11 A could inhibited the invasion capacity of LUAD cells indicated by a Transwell assay test.Cells migration capacity was also confirmed to be restricted following ARHGAP11 A knockdown via a wound healing assay test.4.ARHGAP11 A deletion suppresses inhibits tumour xenografts growth in vivo.5.In the study of ARHGAP11 A promoted LUAD invasion by regulating Hippo-YAP pathway,first the RT-PCR and Western blot showed that knock down ARHGAP11 A in PC-9 was down regulate the expression of total YAP,indicate the positively correlated of ARHGAP11 A and YAP expression.Then subcellular fractionation experiment showed that while knocking down ARHGAP11 A down-regulated the expression of YAP in the nucleus significantly,and there is no differently in the cytoplasm,p YAP and p YAP/YAP were up-regulated in the cytoplasm significantly.These results indicated that ARHGAP11 A enhance the invasion ability of LUAD by inhibiting the Hippo pathway.6.We used in the Rescue experiments showed over expression of YAP significantly reversed the inhibition by ARHGAP11A-sh RNA on PC-9 cell line,such as promote the proliferation;decrease the cell apoptosis level,promote invasion capacity,increased the percentage of cells in S/G2/M phase and decreased the percentage of cells in G0/G1 phase as the same as before.These results indicated that ARHGAP11 A inhibited Hippo pathway activity by down-regulating YAP expression in the nucleus.Conclusion : 1.ARHGAP11 A is frequently upregulated in lung adenocarcinoma,correlated with the tumor size,lymph node metastasis,tumor stage and patient prognosis,ARHGAP11 A may play a carcinogenic role in the malignant progression of LUAD,and it can be considered as a new independent prognostic factor and potential therapeutic target for LUAD.2.ARHGAP11 A promoted the malignant biological behavior in LUAD cell lines,and promoted the growth of subcutaneous transplanted tumor in nude mice.3.ARHGAP11 A up-regulated the YAP expression in nucleus at the m RNA level and promoted the YAP protein turn into the nucleus which in turn promotes the invasion of LUAD.