LncRNA AFAP1-AS1 Induces Renal Clear Cell Carcinoma Growth And Metastasis Via Inhibiting PTEN-AKT Signaling

Objective: Clear cell renal cell carcinoma(cc RCC)accounts for 60~85 percent of renal cell carcinoma.Cc RCC is insensible to chemotherapy and radiotherapy.Targeting therapy and early diagnosis can improve cc RCC survival significantly.Actin filament-associated protein 1-antisense RNA1(AFAP1-AS1)is overexpressed in human digestive tumors,and is associated with unfavorable prognosis.This study evaluated the role of AFAP1-AS1 in cc RCC growth and metastasis,and explored the underlying mechanisms,to find a novel target for cc RCC prevention and treatment.Methods: 60 human cc RCC speciments,20 cancer adjacent specimens,human renal tubule cell HK-2 and cc RCC cell 786-O,Caki-1,ACHN and A498 were studies in this study.Real-time PCR was used to detect AFAP1-AS1 and PTEN expression in human cc RCC specimens,and examine AFAP1-AS1 expression in HK-2 cell,cc RCC cells and mouse tumor tissues.AFAP1-AS1 sh RNA(sh AF)was used to transfect cc RCC cells to downregulate AFAP1-AS1.CCK-8 kit was used to analyze cell viability.Western blot was used to assess proliferating cell nuclear antigen(PCNA),Vimentin,E-cadherin,PTEN,phospho-AKT(p AKT)and AKT expression in cc RCC cells and mouse tumor tissues.Transwell was used to analyze cell migration and invasion.PTEN si RNA was used to transfect cc RCC cells to downregulate PTEN expression.Xenograft tumor model was established to observe tumor growth and metastasis.SPSS 13.0 was used to perform statistical analysis.Values are expressed as mean±SD.Student's t test was used to compare between two groups.Survival rate was analyzed by Kaplan-Meier method.P value less that 0.05 was considered as significant.Results: 1.AFAP1-AS1 expression level in human cc RCC tissues was higher than that in cancer adjacent tissues.AFAP1-AS1 expression level was correlated with tumor TNM stage,lymph node metastasis and prognosis.2.AFAP1-AS1 expression level in Caki-1,ACHN and A498 cells was higher than that in HK-2 cell.Sh AF stable transfection downregulated AFAP1-AS1 expression in cc RCC cells.3.AFAP1-AS1 promoted cc RCC cell proliferation.AFAP1-AS1 downregulation reduced cc RCC cell viability and PCNA expression.4.AFAP1-AS1 induced cc RCC cell epithelial-mesenchymal transition(EMT),and migration and invasion.AFAP1-AS1 downregulation inhibited cc RCC cell migration and invasion,and vimentin expression,but increased ccRCC cell E-cadherin expression.5.AFAP1-AS1 suppressed PTEN-AKT signaling.PTEN m RNA level in human cc RCC tissues was higher than that in cancer adjacent tissues,and correlated inversely with AFAP1-AS1 expression.AFAP1-AS1 downregulation increased PTEN expression whereas reduced p AKT expression in cc RCC cells.6.PTEN mediated AFAP1-AS1-induced cc RCC cell proliferation,migration and invasion.PTEN downregulation restored cc RCC cell viability,migration and invasion impaired by AFAP1-AS1 downregulation.7.PTEN mediated AFAP1-AS1-induced cc RCC cell proliferation,migration and invasion through regulating PCNA expression and EMT.PTEN downregulation restored cc RCC cell PCNA expression and EMT impaired by AFAP1-AS1 downregulation.8.AFAP1-AS1 induced xenograft tumor growth and metastasis.Sh AF transfection reduced AFAP1-AS1 expression in mouse tumor tissues.AFAP1-AS1 downregulation suppressed xenograft tumor growth and metastasis.AFAP1-AS1 downregulation increased E-cadherin and PTEN expression but reduced Vimentin,PCNA and p AKT expression in mouse tumor tissues.Conclusion: 1.AFAP1-AS1 is overexpressed in human cc RCC tissues,and its expression level is correlated with tumor TNM stage,lymph node metastasis and prognosis,suggesting AFAP1-AS1 as a marker for prognosis.2.AFAP1-AS1 downregulation impaires cc RCC cell proliferation,migration and invasion in vitro,and suppresses cc RCC growth and metastasis in vivo,indicating that AFAP1-AS1 functions as tumor promoter and may be as s novel target for cc RCC prevention and treatment.3.AFAP1-AS1 inhibites PTEN-AKT signaling,and PTEN downregulation restores the protumor effect impaired by AFAP1-AS1 downregulation,demonstrating that PTEN-AKT signaling mediates AFAP1-AS1-induced cc RCC growth and metastasis.