The Effects And Mechanisms Of PDK1 By Regulating PI3K/AKT Signaling Pathway In The Tumorigenesis Of Renal Cell Carcinoma

Renal cell carcinoma(RCC)is one of the most popular malignant tumors in the urinary system.The incidence rate of RCC has been increasing in recent years.In anatomical structure,the kidney is located in the retroperitoneal surrounded by loose connective tissue.Therefore,the renal tumor tends to be large or metastatic when appearing clinical symptoms.It is estimated that approximately 20-30% of patients are metastatic diseases at first diagnosis,and 20% of patients will progress or metastasis after radical nephrectomy.As metastatic RCC is not sensitive to either radiotherapy or chemotherapy,RCC would remain incurable if not resectable for surgical treatment.With the rapid development of the targeted therapy and immunotherapy,the clinical effect of advanced RCC has been significantly improved.However,even with the robust combination of immunotherapy and targeted therapy,the efficacy of the treatment of advanced RCC is still unsatisfactory.Therefore,it still remains a challenge to further explore the mechanism of the occurrence and development of RCC for new therapeutic drug strategies.Receptor tyrosine kinase(RTK)/P13K/AKT signal pathway contains many proto-oncogenes.Researches have confirmed the abnormal expression of numerous genes in this pathway in tumor cells.RTK/P13K/AKT signaling pathway has become one of the research hotspots in many tumors including RCC.Previously,we downloaded the data set of RCC by searching Gene Expression Omnibus(GEO)public database.Gene expression profiles were identified to screen the related gene of RCC by using bioinformatics method.Interestingly,a potential gene named 3-phosphateinositol-dependent protein kinase 1(PDK1),which may play an important role in RCC,was screen out and also confirmed to be potentially connected with PI3K-AKT through signal pathway interaction network analysis.In this study,we studied the expression of PDK1 in RCC and its adjacent normal tissues.The correlation between PDK1 and clinicopathological features was also analyzed.Furthermore,we explored the effect of si-PDK1 on the biological behavior of RCC involving the PI3K-AKT pathway by using the small interfering RNA(si RNA)technology to specifically inhibit the function of PDK1.Chapter One: Expression of PDK1 in RCC and its relationship with clinicopathological featuresObjective: To explore the expression characteristics of PDK1 in RCC and its adjacent normal tissues,and to analyze the relationship between PDK1 expression and clinicopathological characteristics of RCC patients.Materials and methods: 1.Bioinformatics analysis was adopted to identify differentially expressed genes related to RCC from GEO public database.The targeted genes were analyzed for interaction relationship through network analysis tool.2.The screened targeted gene PDK1 was detected.A total of 112 paraffin specimens of RCC confirmed by pathologists in our hospital were selected for analysis of expression of PDK1 by immunohistochemical staining.The expression of PDK1 was analyzed according to the standard procedure kit.The expression of PDK1 was correlated with the clinicopathological parameters of RCC patients.3.In addition,35 cases of fresh frozen RCC and adjacent normal tissues preserved in liquid nitrogen were collected.The expression of PDK1 was detected by q RT-PCR and Western Blot.Results: 1.RCC expression chips GSE6344,GSE53757,GSE14762 and GSE781 in GEO database were identified.An intersecting gene PDK1 which was significantly increased in RCC tissues was screened out.Gene interaction network analysis revealed that PDK1 might closely relate to the development of RCC through PI3K/AKT pathway.2.Immunohistochemical staining results showed that PDK1 was mainly expressed in cytoplasm and cell membrane in tumor cells.The expression of PDK1 was significantly higher in RCC than in adjacent normal tissues(p<0.05).The expression of PDK1 was positively correlated with TNM stage,pathological grade and lymphatic metastasis(p<0.05),but not with sex,age and tumor diameter.3.The expression of PDK1 detected by Quantitative RT-PCR and Western Blot was both higher in 35 RCC tissues than in adjacent normal tissues(p<0.05).Conclusions: 1.PDK1 gene was highly expressed in RCC.The expression level of PDK1 was positively correlated with TNM grade,pathological grade and lymph node metastasis in RCC patients.Chapter Two: Effect of silencing of PDK1 gene on biological behavior of renal cell carcinoma and its mechanismObjective: To study the effect of PDK1 gene knockdown on biological behavior of RCC and explore the underlying mechanism of PDK1 on biological behavior of RCC.Materials and methods: 1.QRT-PCR was used to analyze the expression of PDK1 for the common use of renal cell lines.The relatively high expressions of PDK1 of two RCC cell lines were selected for subsequent experiment.Si RNA technique was applied to knockdown the PDK1 expression.Si RNA plasmid with the best silencing effect was screened for the cell biological function test.2.The selected RCC cell lines were transfected with either a si-PDK1 or IGF-1,an activator of the PI3K-AKT pathway for grouping purposes.The m RNA and protein expressions of PDK1,the PI3K-AKT pathway-,EMT-and apoptosis-related genes were evaluated.3.RCC cell lines selected were transfected with either a si-PDK1 or/and IGF-1 to study the cellular biological function.The specific methods were as follows.: MTT and flow cytometry were used to analyzed cell proliferation;Flow cytometry was applied to examine cell apoptosis;Scratch-test and Transwell assay were adopted to detect the cell migration,invasion and metastasis.Results: 1.PDK1 expression increased significantly in RCC cell lines,especially in 786-O and A498 cell lines when compared with normal renal cell line(p<0.05).Therefore,RCC cell lines 786-O and A498 were selected for subsequent cell biological function experiments.Three PDK1 si RNA plasmids were established.Results showed that si-PDK1-1 was the best silenced plasmid and therefore been selected for subsequent use.2.Compared with the negative control group,the expressions of PDK1,PI3 K,AKT,N-cadherin,Vimentin,Bcl-2,p-PI3 K,p-AKT/PI3 K,p-AKT/AKT and PCNA in the si-PDK1 group were significantly down-regulated,while the expressions of E-cadherin,Bax,caspase-3 increased significantly in 786-O and A498 cell lines(p<0.05).However,the combination use of si-PDK1 and IGF-1 could antagonize the effect of PDK1 silencing(p<0.05).3.Compared with the negative control group,the proliferation,migration and invasion ability of cells in the si-PDK1 group decreased significantly,and the apoptosis of cells increased significantly in 786-O and A498 cell lines(p<0.05).Further,combining si-PDK1 with IGF-1 could antagonize the above biological behavior(p<0.05).Conclusions: 1.Knockdown the epxreesion of PDK1 in 786-O and A498 cell lines could inhibit RCC cell proliferation,migration,invasion and EMT by inhibiting the PI3K-AKT pathway.2.PDK1 would play an oncogene role in the occurrence and development of RCC.PDK1 may serve as a potential novel marker and a therapeutic target for RCC.