Study On The Function And Mechanism Of Prrx1 In The Process Of Recurrence And Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common type of malignancy in human liver.It has been shown that tumor metastasis is the leading cause of cancer-related death.The recurrence and metastasis result in the high mortality of HCC.The mechanisms underlying HCC metastasis are not yet fully understood,and there is a need in the effective treatment for HCC.Epithelial-mesenchymal transition(EMT)and mesenchymal-epithelial(MET)are one of the important mechanisms leading to tumor metastasis.EMT facilitates the invasion and migration of tumor cells which lead to the dissemination of tumor cells from the primary site.EMT is the initial step in tumor metastasis.MET grants the ability to colonize,proliferate and form metastasis to tumor cells.Paired-related homeobox 1(Prrx1)is a newly discovered EMT transcription factor.It is reported that the over-expression of Prrx1 induces EMT in gastric cancer cells.Lower Prrx1 expression is associated with poor prognosis in breast cancer,the mechanism underlying which is that the down-regulation of Prrx1 in breast cancer cells induces MET.However,the mechanism under Prrxl down-regulation-induced MET has not been revealed.The knowledge of the function of Prrx1 in HCC is limited.In this study,we aim to reveal the role of Prrxl in the recurrence and metastasis of HCC.Firstly,we analyzed the expression level of Prrx1 in different HCC cells with different cell phenotypes and invasiveness.We found higher Prrx1 level in HCC cells with mesenchymal phenotype and higher invasiveness.Then,we detected the level ofPrrx1 in HCC tissues and analyzed the relation between Prrx1 level and prognosis.We found that HCC patients with lower Prrx1 level had shorter disease-free survival and overall survival.Lower Prrx1 expression indicated poor prognosis of HCC patients.Next,we investigated the function and mechanism of Prrx1 in HCC EMT.We over-expressed Prrx1 in HCC cells and analyzed the EMT-related phenotypic changes.We found that Prrxl over-expression induced EMT in HCC cells.The activated TGF-? signaling and ?-catenin signaling were responsible for Prrx1 induced EMT.Then,we explored the reasons for the poor prognosis of HCC patients with lower Prrx1 expression.We found that the interference of Prrx1 in HCC cells induced MET.Our animal experiments showed that only HCC cells with down-regulated Prrx1 level formed metastasis in the lung.So we concluded that the induced MET by Prrx1 down-regulation might be the cause of poor prognosis in HCC patients with lower Prrxl expression.We investigated the mechanism leading to Prrx1 down-regulation induced MET.We screened the changed genes and signaling pathways in HCC cell with Prrx1 down-regulation by microarray analysis.We found that the interference of Prrxl inhibited TGF-? signaling pathway in HCC cell.The related verification experiments showed that inhibited TGF-? signaling pathway might be the reason for MET.The microarray results also showed that paired-like homeobox 2(Pitx2)increased after interference of Prrx1.It was reported that Pitx2 could induce MET by promoting the expression of miRNAs.So in the following experiments,we investigated the function of Pitx2 in Prrx1 down-regulation induced MET.We observed a direct interaction between Prrx1 and Pitx2.Prrx1 suppressed the expression of Pitx2,and the Prrx1 interference led to Pitx2 increase.Up-regulated Pitx2 activated the expression of miR-200b-3p,miR-200c-3p,miR-429 and miR-106b-5p,which inhibited Slug and TGFBR2 expression,resulting in increased E-cadherin and MET.Taken together,our study evaluated the function and mechanism of Prrxl in the recurrence and metastasis of HCC.We found that Prrx1could induce EMT in HCC cells through TGF-? and ?-catenin pathways.Lower Prrx1 in HCC tissue indicated poorer prognosis of HCC patients,and the reason was that Prrxl down-regulation induced MET in HCC cells.Our study also revealed the mechanism underlying down-regulated Prrx1 induced MET:Prrx1 down-regulation increased Pitx2-microRNA pathway which inhibited Slug and TGFBR2 and thus released E-cadherin expression from Slug suppression.Our research is expected to make Prrxl a novel marker in predicting the prognosis of HCC patients,and provide a theoretical basis for the development of interventions for HCC metastasis.