The Function And Mechanism Involving In The Development And Progression Of Pancreatic Ductal Adenocarcinoma

OBJECTIVES: To investigate the aberrant expression of RPL15 and the correlation between RPL15 expression and the clinicopathological features and prognosis of PDAC patients.To explore the effect of RPL15 on PDAC cell function and its possible molecular mechanism or pathway.METHODS: By utilizing and analyzing public microarray datasets,RPL15,whose expression was not only decreased in PDAC but also significantly associated with PDAC patients' prognosis,was screened and identified.The screening results were validated by quantitative real-time PCR(q RT-PCR),Western Blot and immunohistochemistry(IHC)in Ren Ji cohort and the relationship between RPL15 expression and clinicopathological characteristics and clinical prognosis of PDAC patients was analyzed.After transfection of RPL15-si RNA or RPL15 overexpression plasmids in PDAC cells,knockdown or overexpression efficiency was validated by q RT-PCR.The changes of cell functions were detected by experiments of cell proliferation,invasion in vitro and the PDAC lung metastasis model in nude mouse in vivo.Gene Set Enrichment Analysis(GSEA)was performed to gain further insight into the biological pathways involved in PDAC progression through RPL15 pathway.The regulation of RPL15 to epithelial-mesenchymal transition(EMT)markers was detected by q RT-PCR and Western Blot.Concentrations of MMP-2 and MMP-9 in cell supernatants was examined by enzyme linked immunosorbent assay(ELISA).RESULT: The expression of RPL15 was significantly downregulated in PDAC in both PDAC public microarray datasets and Ren Ji cohort.The expression level of RPL15 in PDAC tissues was prominently correlated with TNM stage,T classification,vascular invasion and histological differentiation of PDAC patients.Kaplan-Meier survival analysis showed that RPL15 expression was dramatically associated with PDAC patients' overall survival and the overall survival was significantly superior in PDAC patients with higher RPL15 expression than in those with lower RPL15 expression.The multivariable Cox regression analysis also confirmed that RPL15 expression was an independent predictor of PDAC patients' overall survival after pancreatectomy(HR: 0.461,P=0.000).RPL15-si RNA was successfully transfected into PANC-1 cell line which exhibited the highest expression levels of RPL15 among 6 PDAC cell lines,and RPL15 overexpression plasmids were successfully transfected into SW1990 cell line which exhibited lower expression.CCK-8 assay showed no difference between RPL15 overexpression and negative control.Transwell invasion assay demonstrated that RPL15 overexpression notably blocked the invasiveness of SW1990,while RPL15-depleted PANC-1 cells had a prominently higher invasive capacity than negative control.Nude mice subcutaneous tumor experiments indicated that less metastatic PDAC foci were observed in the lungs of nude mice at 12 weeks after injection of SW1990-RPL15 overxpression stable cells,compared with SW1990-control stable and only SW1990 cells.GSEA showed that the EMT-related gene signatures were more active in patients with lower RPL15 expression than in those with higher RPL15 expression.Further q RT-PCR and Western Blot data revealed that overexpression of RPL15 significantly increased CDH1(E-cadherin)and ZO-1 expression,meanwhile reduced CDH2(N-cadherin)and FN-1 expression in SW1990 and As PC-1 cells.Besides,we have transfected SW1990 cell with 1ug,2ug and 3ug of RPL15 overexpression plasmids respectively,the results showed that E-cadherin was upregulated and N-cadherin was downregulated both in a dose dependent manner.Reductions in the concentrations of both MMP2 and MMP9 in cell supernatant were observed in RPL15 overexpression cells compared with control-plasmid transfected cells.CONCLUSION: The expression of RPL15 was significantly downregulated in PDAC.The expression level of RPL15 in PDAC tissues was prominently correlated with TNM stage,T classification,vascular invasion and histological differentiation of PDAC patients.Furthermore,higher expression of RPL15 was associated with superior prognosis.RPL15 expression status could serve as an independent prognostic biomarker among PDAC patients.RPL15 functioned as a tumor suppressor gene in PDAC progression.RPL15 could inhibit PDAC invasion and metastasis ability,but it had no impact on proliferation.RPL15 could inhibit pancreatic cancer cell invasion and metastasis ability via suppressing EMT process.