Expression And Significance Of E2F1in Clear Cell Renal Cell Carcinoma

Objective: Transcription factor E2F1exerts various functions in many types of cancers.As an upstream regulator of a host of genes, it can trigger diverse aberrant transcriptionprocesses which may dominate malignancy. Clear cell renal cell carcinoma (ccRCC) isthe most common seen subtype in renal cell carcinoma which displays high malignancyand is short of biomarker in clinics. Our study aims to explore the function of E2F1inccRCC and its correlation with clinicalpathological parameters. Materials andMethods:①Message RNA and protein levels of E2F1were detected in52clear cellrenal cell carcinoma (ccRCC) tissue samples and their corresponding adjacent normaltissue samples by real-time polymerase chain reaction (PCR) and western-blot,respectively. Then, correlation analyses were utilized between mRNA expressions ofE2F1and clinicalpathological parameters.②Western-blot assay was used to detectthe protein level expression in five cell lines, ccRCC cell lines presented higher E2F1expression than HKC(human kidney cell). E2F1was over-expressed using HA-taggedE2F1plasmid with the empty vector (pcDNA3.1-entry) as control. Cell cycle assaysand cell proliferation were detected by flow cytometer and MTS in ccRCC cell line786-O and A498.③Wound healing and transwell experiments were employed todetect the migration and invasion of cell lines786-O and A498. Results:①mRNAand protein levels of E2F1expression in52pairs surgical samples was significantlyhigher in Tumor group than in noncancerous tissue group (both p<0.05).E2F1mRNAlevels were significantly associated with Fuhrman tumor grade (r=-0.470，p<0.001),pTstage(r=-0.455，p=0.001),TNM stage grouping(r=-0.467，p<0.001)andMAVI(r=-0.475，p<0.001),but not age and sex;②Overexpression of E2F1proteinpromoted the proliferation of ccRCC cell lines and enhanced the number of colonyforming(p<0.05), for786-O cells, the G1ratio in E2F1plasmid group was51.97±1.66%compared with65.47±1.11%in un-transfected group and64.20±0.71%in entry grouprespectively(p<0.001). For A498cells, the G1ratio in E2F1plasmid group was53.22±0.58%versus62.65±0.79%in un-transfected group and63.47±0.86%in entry group respectively(p<0.001).③In transwell assay trans-membrane cell numbers were higher inexperimental group than control group (p<0.001),for wound healing assay, direct resultsof motility of tumor cells were obtained as entering the intermediate empty spacereflecting migratory ability of786-O(p<0.01) and A498(p<0.01), MMP2and MMP9were greatly up-regulated after E2F1plasmid interference (p<0.001) both in786-O andA498.Conclusions:①E2F1promotes tumor malignancy and correlates with TNMstages in clear cell renal cell carcinoma.②E2F1transfection contributed greatly tocancer cell proliferation and colony forming in vitro.③Overexpression of E2F1enhances migration and invasion of tumor cell and may be a key event in the local andvascular infiltration of ccRCC indicated by activation of MMP2and MMP9.