| Objective: Lung cancer is by far the leading cause of cancer death.This study aims to find the differentially expressed molecules in lung adenocarcinoma(LUAD)and investigate the molecular mechanisms of oncogenesis to provide a basis for tumor detection and targeted therapy.ARHGEF16 is a Rho guanine nucleotide exchange factor(GEF)that mainly regulates the GDP/GTP exchange to activate Rho GTPases,affecting important biological processes.The earliest studies have shown that ARHGEF16 promotes malignant transformation of untransformed NIH 3T3 mouse fibroblasts.But this transformation can be inhibited when the ROCK inhibitor Y-27632 is used.ARHGEF16 can also mediate the activation of CDC42 through the viral protein HPV16E6.Anti-anoikis is thought to be strongly associated with tumor invasion and metastasis,and studies have found that knockdown of ARHGEF16 in Hela cells and MCF7 cells increased the number of apoptotic cells and significantly decreased levels of PI3 K and AKT related to proliferation.ARHGEF16 has been reported to interact with other proteins as an oncogene to promote malignant progression in breast,glioma and colon cancers.In breast cancer,ARHGEF16 interacts with the receptor Eph A2 to activate Rho G,to form a complex on the cell membrane to mediate the activation of Rac and promote the metastasis and invasion of breast cancer cells.In glioma,GLI2 is found to upregulate the transcriptional level of ARHGEF16,and the overexpression of ARHGEF16 promotes the malignant phenotype of glioma cells.In colon cancer tissues,ARHGEF16 expression is upregulated and ARHGEF16 promotes the malignant progression of colon cancer cells with the non-receptor tyrosine kinase FYN.In contrast,transcriptomic data from melanoma cells and quantitative real-time PCR experiments confirm that ARHGEF16 may be a candidate tumor suppressor gene and may be associated with transcriptional inactivation due to ARHGEF16 promoter methylation.The role of ARHGEF16 in LUAD and the oncogenic mechanisms in tumor progression have not been reported so far.It has been found that the expression level of Wnt5 b m RNA is elevated and the expression levels of Wnt10 a m RNA and ARHGEF16 m RNA are decreased in the zebrafish retina after optic nerve injury.The changes in expression levels of these molecules are associated with the downregulation of β-catenin in the nucleus.Based on this,we speculated that ARHGEF16 might function in LUAD through the oncogenic mechanism of Wnt signaling pathway.Immunohistochemical staining was performed to assess the expression of ARHGEF16 in LUAD sections,and protein immunoblotting was carried out to detect the expression of GEF16 protein in LUAD and paracancerous tissues.Cellular functional changes were observed by transfection of plasmid ARHGEF16(p-ARHGEF16)and si RNA ARHGEF16(si ARHGEF16)and Western blot was used to detect changes in key proteins of the Wnt signaling pathway.The inhibitor XAV-939 was used to validate that the activation of Wnt signaling pathway was mediated by ARHGEF16 overexpression.Methods: 1.123 non-small cell lung cancer(NSCLC)paraffin-embedded tissue specimens were obtained,including 75 LUAD cases from the database of the pathology department,the Affiliated Hospital of Chengde Medical University.Besides,paraffin-embedded(FFPE)blocks of paired 42 cases of paracancerous lung tissues were collected.And six pairs of fresh lung adenocarcinoma tissues and paracancerous tissues were also collected,and the difference of ARHGEF16 protein expression between the two group was detected by Western blot.2.Immunohistochemistry(IHC): Paraffin-embedded blocks NSCLC tissues and paired paracancerous tissues fixed by formalin were stained according to the IHC kit instructions to analyze the correlation between ARHGEF16 expression and clinicopathological features.3.Database analysis: The transcript levels of ARHGEF16 in NSCLC tissues and normal lung tissues was compared by the databases GEPIA and Oncomine.The c Bio Portal database was used to investigate the type and frequency of ARHGEF16 mutations in LUAD.The Kaplan-Meier database was performed to analyzed the expression and prognostic value of ARHGEF16 in lung adenocarcinoma.4.Cell culture and transfection: Five NSCLC cell lines(H1975,A549,H1299,SK-MES-1 and H460)and normal bronchial epithelial cell lines(HBE)were used to compare the protein levels of endogenous ARHGEF16.Finally,p-ARHGEF16 was transfected into H1299 and A549 cell lines and si ARHGEF16 was transfected into H1975 and A549 cells using Lipo3000,followed by subsequent cell function experiments and protein detection.5.Cellular immunofluorescence: H1299 and A549 cells seeded in 24-well plates were incubated with ARHGEF16 antibody to observe the subellular localization of ARHGEF16 in the cell lines.H1299 cells transfected with ARHGEF16 plasmid and A549 cells transfected with si ARHGEF16 in 24-well plate were incubated with β-catenin antibody overnight.The second antibody was incubated for 2 hours on the second day,and then the nucleus was stained with DAPI.The entry of β-catenin into the nucleus regulated by ARHGEF16 was observed.6.CCK-8 and clone formation experiment: The regulatory effect of ARHGEF16 on the proliferation of H1299 and A549 cells was observed after transfected with p-ARHGEF16.The effect of ARHGEF16 on the proliferation of H1975 and A549 cells was detected after transfected with si ARHGEF16.7.Cell wound healing: The wound healing ability of A549 cells was determined after transfected with p-ARHGEF16 for 24 h or si ARHGEF16 for 36 h.Transwell migration assay: 24 hours after H1299 and A549 cells transfected with p-ARHGEF16,or 36 hours after H1975 and A549 cells transfected with si ARHGEF16,the number of migratory cells was statistically analyzed.The invasive ability of cells were detected after overexpression of ARHGEF16 in H1299 cells and knockdown of ARHGEF16 in A549 cells.8.Flow cytometry: The changes of cell cycle progression were detected after overexpression of ARHGEF16 in H1299 cells or knockdown of ARHGEF16 in A549 cells.Apoptosis: after overexpression of ARHGEF16 in H1299 cells or knockdown of ARHGEF16 in A549 cells,the change of apoptosis rate was observed.9.Western blot: The protein level of ARHGEF16 in fresh LUAD and normal paracancerous lung tissues was compared by Western blot.After H1299 and A549 cells transfected with p-ARHGEF16 or H1975 and A549 cells transfected with si ARHGEF16,the changes of key proteins of Wnt signaling pathway were detected by Western blot.The changes of EMT phenotypic proteins,cell cycle and apoptosis related proteins and migration related proteins were detected by Western blot.10.Nuclear and cytoplasmaic fractionation experiment: After overexpression of ARHGEF16 in H1299 cells or knockdown of ARHGEF16 in A549 cells,nuclear and cytoplasmic proteins were extracted,and the changes in β-catenin protein levels in cell nuclear and cytoplasm were detected by Western blot,respectively.11.XAV-939 inhibitor experiment: XAV-939 was used to inhibit the activity of Wnt signaling pathway.After transfected with p-ARHGEF16 for 36 hours,the cells were incubated with XAV-939 for 12 hours.The changes of cell proliferation and migration were found by cell function assays,and the changes of phosphorylated GSK-3β(ser9),active-β-catenin and target proteins c-Myc,Cyclin D1 and MMP-7 were detected by Western blot.12.q RT-PCR experiment: 24 h after H1299 cells transfected with p-ARHGEF16 or H1975 cells transfected with si ARHGEF16,total RNA was extracted using Trizol.Then c DNA is made by reverse transcription using RNA as a template.The m RNA levels of GSK-3β,β-catenin and Snail were determined after ARHGEF16 regulation.Results: 1.The protein level of ARHGEF16 in LUAD was higher than that of ARHGEF16 in paracancerous tissues by Western blot results.The positive expression of ARHGEF16 was positively correlated with tumor size,lymph node metastasis and p-TNM stage of LUAD by immunohistochemistry staining.It is showed that patients with high ARHGEF16 expression in LUAD had a poor prognosis by Kaplan-Meier database.It was found that ARHGEF16 mutations in LUAD were mainly missense mutations via c Bio Portal database.2.Through CCK-8 and clone formation experiment,we found that overexpression of ARHGEF16 enhanced the viability and colony-formation ability of H1299 and A549 cells,whereas knockdown of ARHGEF16 weakened the viability and colony-formation ability of H1975 and A549 cells.3.After transfection of plasmid ARHGEF16,the migratory ability of H1299 and A549 cells and the invasive ability of H1299 cells were enhanced in cell function assays,while the migratory ability of H1975 and A549 cells and invasive ability of A549 cells were weakened after transfection of si ARHGEF16.The levels of migration-related proteins Rho B,Rho C,Rac1 and CDC42 increased after overexpression of ARHGEF16 in transfected cells by Western blot analysis and the expression of Rho A was not statistically significant.In contrast,the knockdown of ARHGEF16 had the opposite result.4.After ARHGEF16 overexpression in H1299 cells,the percentage of S-phase cells increased and that of G1-phase decrease by cell cycle analysis,meanwhile,the expression of CDK2,CDK4 and cyclin D1 of cycle related proteins increased by Western blot analysis.On the contrary,after ARHGEF16 knockdown in transfected A549 cells,the cycle progression and related peoteins expression showed opposite trends.We also found that the apoptosis rate after in transfected H1299 and A549 cells was not statistically significant compared with the control group.5.The levels of phosphorylated GSK-3β(ser9)and active-β-catenin protein in Wnt signaling pathway increased after overexpression of ARHGEF16 in H1299 and A549,and similarly the levels of c-Myc,Cyclin D1 and MMP-7 showed a consistent trend.On the contrary,after knocdown of ARHGEF16 in H1975 and A549 cells,the expression level of these proteins showed the opposite result.In the process of EMT,when ARHGEF16 was upregulated,the protein levels of N-cadherin,Snail and Slug which are related to mesenchymal phenotypic increased,while the expression of E-cadherin,which represents epithelial adhesion,decreased.When knockdown of ARHGEF16 in H1975 and A549 cells,the levels of N-cadherin,Snail and Slug decreased,while the level of E-cadherin increased.These results suggest that ARHGEF16,as a potential oncogenic marker,activates the Wnt pathway and changes in EMT.6.The level of nuclear β-catenin increased after overexpression of ARHGEF16 in H1299 cells compared with the control group,on the contrary,after exogenous interference with ARHGEF16 by si RNA,the level of nuclear β-catenin in A549 cells decreased.7.The incubation of H1299 and A549 cells overexpressing ARHGEF16 with XAV-939,compared with DMSO group,induced a clear reduction in the protein levels of active-β-catenin,phosphorylated GSK3β(ser9),as well as downstream molecules of Wnt pathway: c-Myc,Cyclin D1,and MMP-7.Additionally,we could also observe that,in cell function experiments,XAV-939 significantly inhibited the proliferation and migration of A549 and H1299 cells,reversing the effect caused by ARHGEF16 overexpression.These results suggest that XAV-939 can reverse the increase of key proteins in Wnt signaling pathway caused by overexpression of ARHGEF16.8.The m RNA levels of GSK-3β,β-catenin and Snail were all up-regulated after overexpression of ARHGEF16 in H1299 cells,and after knockdown of ARHGEF16 in H1975 cells,they showed an opposite trend.Therefore,we speculate that overexpression or knockdown of ARHGEF16 will affect the transcriptional activity of GSK-3β,β-catenin and Snail,and then affect the activity of Wnt signaling pathway.Conclusion: 1.ARHGEF16 showed high expression in LUAD tissues and cells,which is related to the poor prognosis and pathological features of LUAD;2.After transfection with ARHGEF16 plasmid and si ARHGEF16 respectively,it was found in cell function and protein levels that ARHGEF16 could promote cells proliferation,migration and invasion in LUAD cells;3.The results of cell cycle flow assays and Western blot showed that ARHGEF16 could promote the G1/S phase transformation in LUAD cells;4.Western blot and q RT-PCR results showed that ARHGEF16 could positively regulate the activity of Wnt signaling pathway and EMT,and promote the malignant progression of LUAD. |
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