| Background:CREPT(Cell-cycle related and expression-elevated protein in tumor),a novel gene also called RPRD1B or C20ORF77,was involved in the development and prognosis of various tumors.However,the clinic relevance of CREPT expression and its biological role in non-small cell lung cancer(NSCLC)remains unclear.Method:In this study,we detected the expression and correlation between CREPT and Cyclin D3 in NSCLC tissues,adjacent tissues and normal lung tissues by immunohistochemistry,Western Blot and qRT-PCR.The correlation between CREPT expression and clinic pathologic features was analyzed in 342 NSCLC patients.Prognostic value of CREPT expression was evaluated by Kaplan-Meier analysis and Cox regression analysis.We also detected the expression of CREPT in NSCLC cell lines by Western Blot and qRT-PCR.CREPT was overexpressed in Calu-1 cell lines by using plasmid vector and its biological function was explored both in vitro and in vivo.CREPT was downexpressed in A549 cell lines by si-RNA and its biological function was explored in vitro.Results:1.The CREPT was expressed in 313 of 342(91.5%)NSCLC samples expression,while134 of 342(39.1%)corresponding adjacent tissues according to immunohistochemistry.The expression of CREPT in non-small cell lung carcinoma and paired normal tissues was significantly different(p<0.01).Higher levels of CREPT mRNA and protein were expressed in cancer lesions than in surrounding tumor adjacent regions(p<0.01).2.The expression of CREPT had no difference statistically either in different ages of patients(p=0.128),gender(p=0.744),smoking history(p=0.811),tumor location ((p=0.124))or patholgy(p=0.826).The differences were significant in tumor size(p=0.001),pathological grade(p=0.000),clinical stages(p=0.000),lymph node metastasis groups(p=0.008).3.Cyclin D3 mRNA and protein were significantly overexpressed in NSCLC tissue compared with paired adjacent non-tumor tissues.The expression of CREPT was significantly associated with the expression of Cyclin D3(r_s=0.527,p=0.000)。There was an obvious correlation of CREPT with Cyclin D3 in the group of differentiated,the pTNM stages and lymph node metastasis patient(p<0.05).Cyclin D3 mRNA and protein were significantly increased in pcDNA3.1-CREPT groups compared to those in the pcDNA3.1 group.The qRT-PCR and Western Blot results showed that Cyclin D3 mRNA and protein were significantly decreased in Si-CREPT groups compared to those in the NC group.3.Kaplan-Meier analysis showed that the RFS(p=0.006)and OS(p=0.01)of high CREPT expression groups were significantly shorter than those of the low CREPT expression group.CREPT high expression group had poor prognosis than those in comparative CREPT low expression group in SCC patients(p=0.03).CREPT overexpression also correlated with the poor outcomes(p=0.002)in the large tumor size patients(diameter>5cm).CREPT low expression group had longer time than those in comparative CREPT high expression group in pTNM III/IV patients(p=0.005).CREPT high expression patients led to poor prognosis both in lymph node-negative and lymph node-positive patients(p=0.01 and 0.03).However,there were no obviously significant difference in the survival rate with respect to CREPT expression among tumor diameter≤5cm group(p=0.7),pTNM I/II group(p=0.588),ADC patients(p=0.3).Multivariate and multivariate survival analysis revealed that CREPT expression(HR:1.223,p=0.013)was one of independent factor predictive of poor prognosis in NSCLC patients.4.Calu-1 cell line was selected for CREPT overexpression experiment.CCK8 assay results showed that pcDNA3.1-CREPT cells exhibited dramatically promotes growth compared with the control group(p<0.01).pcDNA3.1-CREPT cells decreased the proportion of cells in G1 phase compared with control cells pcDNA3.1-CREPT vs. pcDNA3.1;40.7±1.9%vs.54.5±1.1%),with concomitant increase in S phase compared with control cells(pcDNA3.1-CREPT vs.pcDNA3.1;32.3±1.1%vs.21.4±1.4%)(p<0.05).Scratch wound healing and Transwell migration assay showed that overexpression of CREPT enhanced the migratory ability of Calu-1 cell(p<0.05).In transwell invasion assay,more pcDNA3.1-CREPT cells invaded into the lower chamber than pcDNA3.1 cells (p<0.05).5.A549 cell line was selected for CREPT downregulation experiment.MTT assay results showed that downregulation of CREPT expression in A549 cells exhibited dramatically inhibites growth compared with the control group(p<0.01).Downregulation of CREPT expression in A549 cells increased the proportion of cells in G1 phase compared with control cells((Si-CREPT vs.NC;58.6±4.4%vs 46.3±2.1%),with concomitant decrease in S phase compared with control cells((Si-CREPT vs.NC;19.4±1.7%vs.27.6±3.3%).Scratch wound healing and Transwell migration assay showed that downregulation of CREPT decrease the migratory ability of A549 cell(p<0.05).In transwell invasion assay,less Si-CREPT group cells invaded into the lower chamber than NC group cells(p<0.05).6.The CREPT overexpressed Calu-1 xenografts showed significantly increased tumor weight.The tumor volumes in the pcDNA3.1-CREPT group were obviously increased compared to control group(p<0.05).The expression of Ki67 and Cyclin D3 in pcDNA3.1-CREPT xenograft tissues were significantly higher than that in tumors treated with control group(p<0.05).Conclusion:1.CREPT was significantly upregulated in NSCLC tissues compared with corresponding non-tumor tissues.Expression level of CREPT was correlated with tumor size,differentiation,lymph node metastasis and TNM stage.CREPT played critical roles during the development and progression of NSCLC.2.The expression of CREPT was significantly associated with the expression of Cyclin D3 in the NSCLC patients and cell lines,indicating that CREPT may affect the progression of NSCLC by increasing the expression of Cyclin D3.3.CREPT overexpression level was associated with poor prognosis of NSCLC patients.CREPT was specifically more relevant to the survival of advanced staged SCC patients with large tumor size,which can be used as a precise prognostic marker for NSCLC patients.Multivariate analysis showed that CREPT might be an independent biomarker for the prediction of NSCLC prognosis.4.CREPT potently promote NSCLC cell proliferation through regulation of cell cycle in vitro.Meanwhile,CREPT enhanced the migration and invasion ability of NSCLC cell.5.CREPT overexpression significantly promoted tumor growth in vivo.Mechanism study showed that CREPT may promote NSCLC cell proliferation and cell cycle through the regulation on Cyclin D3. |
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