Expression Of RhoCDI2in Pancreatic Cancer And The Influence Of Its Down-regulation On Biology Of This Cancer

Objective: RhoGDI2regulate a multitude of phenotypes including cell division,morphology, migration, vesicular trafficking and gene expression by controlling thelocation and activity of members of the Rho family of small GTPases. The Aberrantexpression of RhoGDI2is found in a variety of cancers, and regulates the invasion andmetastasis of cancers by a multiple of mechanisms, and is considered as a relative gene oftumor metastasis. However, the expression and biological functions of RhoGDI2inpancreatic cancer have been poorly defined. In this study, we aim to investigate theexpression and clinical significance of RhoGDI2in pancreatic cancer, evaluate theexpression of RhoGDI2and effects on cell proliferation, migration and invasion inpancreatic cancer cell lines. Moreover, we aim to identify the downstream effector ofRhoGDI2in pancreatic cancer cells.Methods:(1) Immunohistochemistry was used to assess the expression of RhoGDI2in60pairs of pancreatic cancer tissues and adjacent pancreatic tissues, and its correlationwith clinicopathological features of pancreatic cancer was also analyzed．(2) Western blotand RT-PCR were used to detect the expression of RhoGDI2and MMP2in30pairs ofpancreatic cancer tissues and non-tumor tissues, the correlation between RhoGDI2andMMP2was analysed by Spearman analysis.(3) Western blot and RT-PCR were used todetect the expression of RhoGDI2in pancreatic cancer cell lines PANC-1, BxPC3,SW1990and patu8988. siRNA-RhoGDI2or siRNA-NC was transfected into pancreaticcancer cells, then Western blot and RT-PCR were used to detect the effection of siRNA.(4)The CCK-8assay, wound scrape assay and transwell invasion assay were performed todetect the effect on the growth, migration and invasion of pancreatic cancer cellstransfected with siRNA-RhoGDI2or siRNA-NC in vitro．(5) Western blot and RT-PCR were used to detect the expression of MMP2in pancreatic cancer cells transfected withsiRNA-RhoGDI2or siRNA-NC.Results:(1) We validated the up-regulation of RhoGDI2in pancreatic cancerpatients by using RT-PCR, the relative expression of RhoGDI2in pancreatic cancer tissuesand adjacent pancreatic tissues were0.661±0.021、0.199±0.023, respectively (P<0.05).The data of IHC shows that the positive rate of RhoGDI2in tumor tissues was higer thanin nontumorous tissues, showing a significant difference (P<0.05). The analysis ofcorrelation between RhoGDI2expression and clinicopathologic features of pancreaticcarcinoma shows that RhoGDI2expression was strongly correlated with tumor size,differentiation, clinical stage, lymph node metastasis and vascular invasion, but did notshow a significant association with gender, age and tumor location at a statistically level.(2)We found that majority of pancreatic cancer tissues exhibited overexpression of RhoGDI2compared with adjacent pancreatic tissues both at mRNA and protein level (P<0.05,respectively). Similarly, MMP2was also overexpressed in pancreatic cancer tissuescompared with adjacent pancreatic tissues at mRNA and protein level (P<0.05,respectively). In further analysis, our data indicated that RhoGDI2expression waspositively correlated with MMP2expression at mRNA level (Spearman analysis, r=0.627,P<0.001). Consistent results were obtained when we compared the data at protein level(Spearman analysis, r=0.817, P<0.001).(3) High expression of RhoGDI2is detected inPANC-1and Patu8988，and negative expression is detected in BxPC3and SW1990. Themean inhibition rate (the RNAi-RhoGDI2group vs. the RNAi-NC group) was78.3%inPANC-1and82.4%in Patu8988(P<0.05, respectively) after depleting RhoGDI2expression in PANC-1and Patu8988cells by RNAi method.(4) There was no statisticalsignificance in cell proliferation between the RNAi-RhoGDI2group and the RNAi-NCgroup (P>0.05). Time course analysis of the wound closure showed that the re-establishedperiod of the monolayer was significantly shorter in the RNAi-NC group than that in theRNAi-RhoGDI2group (P<0.05). Depletion of RhoGDI2significantly reduced cellinvasion in the RNAi-RhoGDI2group compared to the RNAi-NC group (P<0.05).(4) MMP2mRNA and protein expression was significantly decreased in the RNAi-RhoGDI2group compared with the RNAi-NC group in PANC-1and Patu8988cells (P<0.05).Conclusion: RhoGDI2and MMP2are up-regulation in Pancreatic cancer, andRhoGDI2expression was positively correlated with MMP2expression. RhoGDI2expression is correlated with tumor size, differentiation, clinical stage, lymph nodemetastasis and vascular invasion. Depletion of RhoGDI2significantly reduced cellmigration and invasion, and inhibited expression of MMP2in pancreatic cancer cells.RhoGDI2may be a useful marker of progression in pancreatic cancer, and also a potentialtarget for therapy.