Mechanism And Effect Of POSTN On Biological Behavior Of Renal Cell Carcinoma

As a crucial type of renal cancer,renal cell carcinoma(RCC)seriously threatens the health of human beings.RCC is characterized by occult onset and diagnostic metastasis,and exhibits poor prognosis as it cannot be treated with radiotherapy or chemotherapy.Based on previous studies,POSTN presents overexpression in various malignant tumors,inducing tumor angiogenesis,invasion and metastasis.High POSTN level can lead to tumor differentiation,microvascular invasion as well as lymph node metastasis.Epithelial phenotype cells adopt the epithelial mesenchymal transition to physiologically transform into mesenchymal phenotype(EMT),and EMT significantly affects RCC cell metastasis and invasion.POSTN has the function of promoting the migration and transformation of cells via EMT in the process of embryonic development,thus may induce EMT.However,the regulatory mechanism of POSTN is not well understood.POSTN is capable of enhancing ILK expression in tumor cells,and as the downstream signaling pathway of ILK/Akt/m TOR significantly affects the EMT process.ILK together with its downstream effectors associated with EMT,show high expression in malignant tumors.They regulate the phosphorylation regarding crucial signaling protein intermediates such as Akt,and maintain the migration and invasion of tumor cells.According to evidence,Akt signaling pathway and m TOR,the downstream protein complex of Akt,significantly induce EMT,metastasis as well as invasion of tumor cells.According to the present research background,four questions are proposed.If POSTN expression is different in RCC tissues and adjacent tissues? How does POSTN biologically affect RCC? How does POSTN affect the RCC development and progression? Is it associated with the activation of the ILK/Akt/m TOR signaling pathway? For figuring out the answers to the four questions,we adopt RCC and POSTN as the basic model and the core,respectively,exploring the biological and clinical impact of POSTN on RCC in terms of the clinical specimens and the in vivo and in vitro cell level.Methods:The study adopted qRT-PCR and Western blot for detecting the expression of POSTN m RNA and protein in RCC tissues;si RNA and lentivirus overexpression plasmids assisted in constructing RCC cell model which involved POSTN interference and POSTN overexpression;cellular immunofluorescence served for observing POSTN expression and distribution in RCC cells;how POSTN affects the biological activity of RCC cells was explored by using colony formation,wound healing assay,Transwell as well as flow cytometry.Western blot served for detecting the expression of EMT marker proteins,namely E-cadherin,N-cadherin and vimentin,meanwhile detecting the expression of protein related ILK/Akt/m TOR signaling pathway;we conducted in vivo study by using the subcutaneous tumorigenesis model of nude mice,daily recording the tumor tissue weight and volume for monitoring RCC cell proliferation in vivo.When the experiment ended,we separated the tumor tissues for performing immunohistochemistry and Western blot,aiming at further examining how POSTN regulates RCC proliferation as well as its molecular mechanism.Results:1.According to qRT-PCR,POSTN m RNA presented an obviously upregulated expression in RCC tissues compared with adjacent tissues.The Western blot analysis verified the significant increase in POSTN expression in RCC tissues(P<0.01).The high POSTN expression exhibited a statistical difference from tumor TNM staging and from whether it invaded the adjacent lymph nodes,nerves and blood vessels(P<0.05).Immunohistochemical results showed that postn was highly expressed in RCC,most of which were located in the cytoplasm and stroma of tumor cells;The expression of postn was correlated with TNM stage,peripheral lymph node and vascular invasion(P < 0.05).2.Based on the transfection efficiency assay,following the si RNA-POSTN transfection,POSTN expression presented an obvious decrease in A498 cells and ACHN cells in terms of its m RNA and protein,showing a significant difference relative to negative control group(P<0.001).By contrast,the expression of POSTN m RNA and protein presented an obvious increase due to LV-POSTN transfection,relative to negative control group(P<0.001).3.Based on CCK-8 and colony formation assay,in the cell model,A498 cells and ACHN cells presented obviously weakened and enhanced proliferation ability after POSTN expression was down-regulated and up-regulated,respectively,showing a significant difference from negative control group.4.As found by the wound healing assay,the migration ability regarding ACHN cells and A498 cells was obviously weakened and enhanced after the POSTN expression was down-regulated and up-regulated,respectively,with statistical difference from negative control group.As revealed by the Transwell assay,POSTN expression downregulation obviously suppressed the invasion ability regarding ACHN cells and A498 cells,while POSTN expression upregulation had a positive effect on the invasion ability of the two types of cells.5.Flow cytometry found the positive impact of POSTN knockdown on the apoptosis rate of ACHN cells and A498 cells,and the negative impact of POSTN overexpression on the apoptosis rate of ACHN cells and A498 cells.6.After POSTN was down-regulated,E-cadherin expression presented an obvious increase in ACHN cells and A498 cells,while the N-cadherin and vimentin level presented an obvious decrease.By contrast,after POSTN overexpression,Ecadherin,N-cadherin and vimentin presented an opposite change trend.7.POSTN knockdown inhibited ILK/Akt/m TOR pathway,and weakened the ILK expression as well as the phosphorylation of Akt and m TOR in ACHN cells and A498 cells.By contrast,POSTN overexpression led to an obvious activation of ILK/Akt/m TOR pathway,increased ILK expression,and enhanced phosphorylation of Akt and m TOR in the two types of cells.8.On the basis of the subcutaneous tumorigenic model regarding nude mice,the POSTN knockdown inhibited the tumorigenicity exhibited by ACHN cells and A498 cells,and the tumor weight and volume showed an obvious decrease relative to control group.By contrast,A498 cells and ACHN cells in vivo saw enhanced tumorigenicity due to POSTN overexpression,and tumor weight and volume showed an obvious increase relative to control group.9.The in vivo studies verified the inhibiting impact of POSTN knockdown on ILK/Akt/m TOR pathway activation in the two cell types,and on the ILK level as well as phosphorylation of Akt and m TOR.By contrast,POSTN overexpression promoted the activation of ILK/Akt/m TOR pathway,as well as the phosphorylation of Akt and m TOR.Conclusion:1.There is a significant increase in POSTN expression in RCC tissues and cell lines.POSTN expression was related to the TNM staging,the peripheral lymph node as well as the vascular infiltration in RCC patients.2.POSTN overexpression has the function of facilitating cell proliferation,enhancing cell migration and invasion,as well as inhibiting cell apoptosis in RCC.3.POSTN induces RCC proliferation via activating ILK/Akt/m TOR signaling pathway.Taken together,the study verified the close relation between the abnormal POSTN expression and RCC occurrence and development,meanwhile expounding that POSTN can be potentially used for RCC treatment as a biomarker and intervention target.