PRAF2 Can Promote The Proliferation,Invasion And Metastasis Of Esophageal Cancer Cells,influence The Apoptosis Through The Mitochondrial Pathway

Objective Analyse The relationship between the expression and the survival of the patients through the study of the differences of oncogene PRAF2 in esophageal squamous cell carcinoma,to evaluate PRAF2 as a biomarker for prognosis of esophageal squamous cell carcinoma prediction;further study of the effect of PRAF2 on proliferation,invasion and metastasis of esophageal squamous cell carcinoma and apoptosis at the cellular level,evaluate its application value as a potential drug target for blocking.Methods 1.Clinical retrospective study:we uesd qPCR to detect the expression of PRAF2 in 77 ESCC tissues and corresponding adj acent tissues,and then analyze the relationship between the expression level and the clinicopathological features and the patient’s survival period.2.Cellular research:transfect siRNA-PRAF2 into ESCC(esophageal squamous cancerous cells)cells to down-regulated the expression of PRAF2,using qPCR to detect the expression of PRAF2 in ESCC after interference;the differential proliferation rate of transfected cells and untreated cells was decteded by CCK-8 and cloning formation assay,transwell assay was used to detect the invasion and metastasis ability,cell apoptosis rate was decteded by flow cytomery.3.Molecular mechanism research:western blot was used to detect the differential expression of apoptosis related protein BCL-2 and caspase family protein after transfecting siRNA into ESCC cells;the ESCC cell lines EC9706 was treated with the ER stress inducer,which interfered with siRNA or overexpressed PRAF2 and used western blot to detect the protein expression of ER stress marker protein GRP78,GADD153 and PRAF2;information analysis was performed to predict the binding proteins with PRAF2 and to validate it by COIP(Co-Immunoprecipitation).Results 1.The results of clinical experiment:1.1 qPCR showed that the mRNA expression levelof PRAF2 in ESCC tissues was significantly higher than that of corresponding adjacent tissues(P<0.001);1.2 The median value was employed as the cut-off points,Samples with mRNA expression above the median were considered as high expression,whereas those with value below or equal to the median as low expression.PRAF2 mRNA expression was significantly associated with clinical features including patients’ differentiation grade(P=0.009)and tumor TNM grade(P=0.01);1.3High PRAF2 expression was associated with poor outcome(33 months:20 months,P<0.001).Univariate and multivariate analyses demonstrated that high PRAF2(HR 2.05;95%CI 1.10-3.85;P=0.03)mRNA expression emerged as independent prognostic factors.2.The cell lines of ESCC:downregulated the expression level of PRAF2 in ESCC cells leaded to a significant decrease in the proliferation,migration and invasion,accompanied by a significant rise in the rate of apoptosis.The cycle analysis strongly indicate that PRAF2 downregulation inhibits the proliferation of ESCC cells by causing a G1 or S cell cycle arrest.3.Mechanism research:3.1 The expression of Bcl-2,caspase9,caspase3 and PARP were decreased after the downregulation of PRAF2 in esophageal cancer cell line TE1,the expression of cleaved caspase-3 and the changes in BAX and cleaved caspase-9 were not significant.3.2 The expression of GADD153 and PARP increased with the increase of drug concentration,but PRAF2 protein did not change significantly.3.3 There was no significant change in ER stress marker protein GRP78 between the interference group and the control group.There was no significant change in the GRP78 between the control group and the over expression group.3.4 COIP revealed that there was no association between PRAF2 and PARP but there was a binding between PRAF2 and BCL-XL.Conclusion 1.The expression of oncogene PRAF2 may be an independent prognostic factor in patients with esophageal squamous cell carcinoma;2.PRAF2 can affect the proliferation,invasion,apoptosis and cell cycle of esophageal squamous cell carcinoma,and may be a potential target for the treatment of esophageal squamous cell carcinoma;3.PRAF2 may enhance the activity of caspase protein and induce apoptosis by inhibiting the expression of BCL-XL and BCL-2.