Regulatory Mechanisms Of EIF3D On Malignant Proliferation Of Renal Cell Carcinoma

[INTRODUCTION]Renal cell carcinoma(RCC)is the most common type of kidney cancer,accounting for the third genitourinary malignancies.With the popularity of abdominal imaging,more and more of early renal cell carcinoma are diagnosed,but most of the kidney is still found in advanced stages.Currently,radical nephrectomy is gold standard of treatment for limitations of early renal cell carcinoma.However,there are still some patients developing tumor recurrence or distant metastasis after surgery,and its underlying mechanisms are still unknown.Despite the recent development of molecular biology and therapeutic targeting VEGFR progression to advanced renal cell carcinoma has brought hope,but about 50% of patients are not sensitive to targeted therapy,or a majority of the patients after taking the drug for about 1 year are tolerance to targeted drug.Therefore,exploring new therapeutic target to increase the understanding of the development of renal cell carcinoma complicated molecular mechanism and then providing the basis for early clinical diagnosis and malignant progression of renal cell has become an important research focus of the Department of Urology.EIF3D is a key member of translation initiation factor.As a relatively new molecule,the present study reported less,and is mostly limited to the tumor phenotype.The study found that EIF3D is associated with anti-apoptotic process of tumor cells,in addition to knocking EIF3D gene can inhibit the proliferation of mesothelioma cells.The change of proteome was detected after hepatitis D virus infection HEK-293 cells.The results found that EIF3D upregulation is associated with liver cancer.There are also reports indicating that EIF3D is related with malignant proliferation of colon cancer,melanoma and glioma,but not yet in depth its downstream mechanisms.In kidney cancer,the function of EIF3D has not been elucidated.To further illustrate the mechanism of EIF3D in malignant proliferation of renal cell carcinoma,we used gene database,pairing fresh renal cell carcinoma,renal cell carcinoma microarray to test the mRNA and protein levels of EIF3D.Subsequently,after lentivirus-mediated knock-down model to silence EIF3D in renal cell carcinoma cell lines,a series of studies were carried out to expound the biological characteristics of tumor cells and its downstream mechanisms.[OBJECTIVE]To explore EIF3D expression level in renal cell carcinoma and its biological characteristics and the molecular mechanisms of renal cell cancer cells.[MATERIALS and METHODS]The expression mRNA and protein levels of EIF3D in RCC fresh tissue,RCC tissue sections and ONCOMINE database20 fresh tissue specimens of renal cell carcinoma were collected at urologic department of Shanghai Changzheng Hospital from January 2015 to June 2015.All specimens were stored in two ways: 1,made immunohistochemistry slides;2,fresh tissue preserved in liquid nitrogen.A renal cell carcinoma tissue microarray including 102 cases was provided from urologic department of Shanghai Changhai Hospital.These samples were used to detect EIF3D protein expression levels by immunohistochemistry and Western blot(Western Blot),then analysis of clinical indicators of relevance.ONCOMINE database was applied to analyze EIF3D mRNA expression levels in renal cell carcinoma,and the correlation with clinical parameters.Lentivirus construction and screening stable EIF3D silencing in renal cell carcinoma cellsSh RNA sequences of targeting EIF3D was designed,inserted into a plasmid containing the green fluorescent protein(GFP)to build a stable lentiviral vectors to silence EIF3D.Six renal cell carcinoma cell lines were detected the expression level of EIF3D by Western Blot analysis,and 2 cell lines with high EIF3D expression were selected to silence EIF3D.Two renal cell carcinoma cells were infected lentivirus containing sh EIF3D,infection efficiency was judged by the expression of GFP,and knockdown efficiency was analyzed using Real-time PCR and Western Blot.Detecting the change of tumor biological characteristics including cell proliferation and cell colony formation ability after EIF3D silencing in renal cell carcinoma cellsOn the basis of steady in renal cell carcinoma cell lines,MTT experiment was carried out to analyze the growth of renal cell carcinoma cells with EIF3D silencing,representing cell proliferation.Colony formation assay was carried out to analyze cell clones situation after EIF3D silencing.Exploring the molecular mechanism of cell proliferation growth change with EIF3D silencing in renal cell carcinoma cellsFlow cytometry was used to detect cell cycle distribution in renal cell carcinoma cells treated with EIF3D silencing.After statistical analysis,downstream molecules affecting the cycle distribution were detected by Western Blot to explain the molecular mechanisms.[RESULTS]The protein expression level of EIF3D in renal cell carcinoma was up-regulationFresh renal cell carcinoma tissues and paired adjacent normal tissues were detected by Western Blot and the results found that EIF3D protein levels in renal cell carcinoma tissues significantly increased comparing to normal tissues(P = 0.005).Immunohistochemical staining was also found that the staining level of renal cell carcinoma tissues were observably high compared with normal tissue,there are significant differences in the number of positive cells(P <0.001).The expression level of EIF3D positively correlated with the clinical features of patientsRenal cell carcinoma microarray detected EIF3D expression levels correlating with tumor size(P = 0.004)and TNM stage(P = 0.03),which showed a significant positive correlation.use ONCOMINE microarray database EIF3D mRNA expression levels in renal cell carcinoma and normal kidney tissue meta-analysisMaking meta-analysis using five renal cell carcinoma microarray database,the mRNA expression levels of EIF3D in renal clear cell carcinoma was significantly increased compared to normal kidney tissue(P = 0.004).Through the analysis of pathological types of renal cell carcinoma,CCRCC and PRCC with high malignant was presents higher EIF3D expression levels than and less than CRCC with low malignant(P = 0.002;P = 0.010).Renal cell carcinoma cells were successfully knockdown of EIF3D gene by lentivirus-mediated sh RNAEIF3D expression was up-regulated in786-O and ACHN cells comparing to other cells.786-O and ACHN cells were infected with lentivirus-mediated sh RNA to silence EIF3D.Real-time PCR and Western Blot confirmed knockdown efficiency and EIF3D silencing model successfully established.EIF3D silencing significantly inhibited proliferation and colony formation of renal cell carcinoma786-O and ACHN cells after silencing EIF3D,significantly inhibited cell growth curve than the control group(P <0.001).Cloned size in silencing and control groups was significantly different,and the number of clone in knockdown group was significantly less than the control group(786-O: P = 0.0002;ACHN: P <0.0000).EIF3D silencing induced renal cell carcinoma G2 / M phase arrest786-O and ACHN cells after silencing EIF3D,induced G2 / M phase arrest through flow cytometry analysis,and cells in sub-G1 phase accumulated.To verify the molecular mechanism of G2 / M phase arrest,Western Blot was carried to detect the downstream molecules.The results showed that cycle-related proteins Cyclin B1 and CDK1 were down-regulation in the silencing group and apoptosis-related proteins p21 and RARP cleavage products were up-regulation.By ONCOMINE gene database analysis,EIF3D expression showed a positive correlation with Cyclin B1(r2 = 0.2354,P <0.0001)and CDK1(r2 = 0.1332,P = 0.0003),and the proliferation-associated molecular markers PCNA(r2 = 0.1687,P <0.0001)showed a positive correlation.[CONCLUSION] 1.EIF3D significantly up-regulated in renal cell carcinoma,and was positively correlated with TNM staging.2.EIF3D silencing in renal cell cancer cells induced G2 / M phase arrest through down-regulated Cyclin B1 / CDK1 signaling pathway,thereby significantly inhibit malignant proliferation of renal cell cancer.