The Roles And Mechanism Of Abnormal Expression Of Tiam1 In The Development Of Pancreatic Cancer

Objective:The aim of this research was to investigate the expression of T-cell lymphoma invasion and metastasis inducing factor 1(Tiaml)in pancreatic cancer(PC)and its connections with clinicopathological features,and to elucidate the effects and underlying molecular mechanism of Tiaml on PC proliferation and EMT events.It will provide new theoretical and experimental basis for Tiaml protein as a new target for clinical treatment of PC.Materials and Methods:1.Database and tissues specimens analysis:Human Protein Atlas(HPA)database and Oncomine database were performed to analyze the protein and mRNA expression of Tiaml in PC.Tiaml protein expression levels in PC tissues and adjacent normal tissues were tested using immunohistochemistry.The correlation between Tiaml overexpression and the clinicopathological features of PC patients was analyzed.The Cox proportional hazards models and the Kaplan-Meier Curve were used to analyze the prognostic factors in PC.2.Experiments in vitro:The expression of Tiaml was confirmed by western blot in different PC cell lines.Then,the cells with high expression of Tiaml were screened for further study.Knockdown of Tiaml expression in BxPC-3 and SW1990 cells were constructed with short interfering RNA transduction.The greatest effective sequence of Tiaml siRNA was selected by western bolt analysis.Tiaml protein localization in cells was observed by immunofluorescence assay.The proliferation of cells were observed by colony formation assay.The migration and invasion viability were measured using transwell assay and cell scratch assay.The expression of EMT markers and PI3K/Akt/mTOR signaling pathway-related proteins were detected by western blot.3.Experiments in vivo:The transfected BxPC-3 cells were cultured and inoculated subcutaneously into nude mice to establish a transplantation tumor model of PC.The effect of Tiaml silencing on the formation and growth of xenografts in nude mice was observed.After mice were sacrificed,the tumors were separated and weighed.The histopathological for xenograft tissues were detected with hematoxylin&eosin(HE)staining.The expression of Ki-67,E-cadherin,Vimentin and Slug of transplanted tumors were detected by IHC.Results:1.Tiaml expression was overexpressed in PC tissues and correlated with poor prognosis of PC patients:HPA database and Qncomine database analysis showed that Tiaml was overexpression in PC tissues,which was further validated by immunohistochemistry in PC tissues and paracancerous tissues.The IHC staining showed that Tiaml protein in the cytoplasm and some nuclear of the cells staining of PC tissues.The positive rate of Tiaml in PC tissues(92.59%)was significantly higher than normal pancreatic tissues(37.04%).Moreover,Tiaml overexpression was significantly correlated with lymph node metastasis(P=0.031),histological grade(P=0.040).No significant difference was found between increased Tiaml and other tested items(P>0.05).Kaplan-Meier analysis observed that the PC patients with high Tiaml expression had significantly lower overall survival than patients with low Tiaml expression(P<0.05).Univariate and multivariate analysis suggested that Tiaml expression was significant independent prognostic factor in patients with PC.2.Knockdown of Tiaml inhibits the proliferation,migration and invasion of PC cells.The Immunofluorescence staining clearly showed that Tiaml protein in the cytoplasm and some nuclear of BxPC-3 and SW1990 cells staining was also observed,which was consistent with the results of immunohistochemistry.Colony formation showed that knockdown of Tiaml expression significantly decreased the colony formation ability of the PC cells(P<0.05),wound healing and transwell assay showed that knockdown of Tiaml expression significantly inhibited the migration and invasion of PC cells(P<0.05).Western blot showed that knockdown of Tiaml up-regulated the expression of epithelial marker(E-cadherin)(P<0.05)and down-regulated the expression of interstitial cell markers(Vimentin)and transcriptional factors(Slug,Snail)and PI3K/Akt/miOR signaling pathway-related proteins(p-mTOR、p-Akt and p-S6)(P<0.05).3.Knockdown of Tiaml inhibits PC cell growth in vivo:The mouse xenograft model showed that Tiaml silencing significantly inhibited tumor formation(P<0.05),and the tumor volume and weight were decreased(P<0.05).The HE staining results showed compared with control group,necrosis presentation,tumor cell shrinkage and other pathological morphological changes could be found in the knockdown of Tiaml group.The expression of Ki-67,Vimentin and Slug were decreased,and the expression of E-cadherin was increased in the tumor tissues of the knockdown of Tiaml group compared with the control group by IHC.Conclusions:1.Tiaml is overexpressed in PC tissues and is significantly related to the poor prognosis of PC patients.2.The downexpression of Tiaml inhibited the proliferation,migration and invasion of PC cells.3.The knockdown of Tiaml may suppress the metastasis and invasion of PC cells through the EMT process and the PI3K/Akt/mTOR signaling pathway.