Research On The Mechanism Of PIN1 High Expression And Its Role In Gastric Cancer Metastasis

Cancer is one of the greatest threats to human health.The incidence and mortality of gastric cancer occupies one of the top position in the world and it even takes the first place in the digestive system cancer in our country.Recent years,with the improvement of diagnosis and treatment technology of gastric cancer,its morbidity and mortality have been decreasing.However,majority of gastric cancer patients are still found in the advanced stages,which leads to poor prognosis and survival time.Metastasis is an important phenotype of advanced cancers and is one of the leading cause of cancer-related death.Therefore,it is urgent to study the mechanism of gastric cancer progression,especially metastasis,for exploring new therapeutic strategies.For the special side chain structure of proline,the peptide-prolinyl motif uniquely adopts cis and trans conformations.The peptide-prolinyl cis-trans isomerase(PPIases)family can catalyze its conformational change to regulate protein folding,stability,subcellular localization and interaction.PIN1 is the only known PPIase that mediates the conformational change of phosphorylated serine/threonine-proline motif(p Ser/Thr-Pro).It plays an important role in many cellular processes for the widely existent of Ser/Thr-Pro motif in proteins.Abnormal expression or activity of PIN1 leads to many diseases including alzheimer’s disease,parkinson’s disease,huntington’s disease,systemic lupus erythematosus and variety of cancers.Recent studies have shown that PIN1 is highly expressed in gastric cancer,and it promotes the progression of gastric cancer by upregulating BRD4,PI3K/AKT,and Wnt/β-catenin.However,the mechanism of PIN1 high expression in gastric cancer and its role in the metastasis of gastric cancer have been rarely reported.Accordingly,this study intends to elucidate the role of PIN1 in gastric cancer metastasis and reveal the mechanism of PIN1 high expression at the post-transcriptional level.Purpose: to elucidate the mechanism of PIN1 high expression and its role in the invasionand migration of gastric cancer.Methods:1.Detected the background expression level of PIN1 by q RT-PCR and Western Blot in a series of gastric cancer cell lines,followed by overexpressing PIN1 in low-expression cell lines and interfering PIN1 in high-expression cell lines.Meanwhile,the expression of PIN1 in clinical gastric cancer and adjacent samples was detected.2.Explored the role of PIN1 in the proliferation,invasion and migration of gastric cancer by CCK-8 and transwell experiments.3.Predicted potential PIN1-targeted microRNAs by different databases,including mir Tarbase,mi Randa,Target Scan,mi RNApath and starbase and screened out candidates by taking the intersection.4.Screened out microRNA inhibiting PIN1 expression by q RT-PCR and Western Blot.5.Verified direct bond of the target microRNA and PIN1 m RNA by RNA-binding Protein Immunoprecipitation(RIP)and clarified the binding site by Dual-Luciferase reporter assay.6.Explored the role of the target microRNA in the proliferation,invasion and migration of gastric cancer by CCK-8 and transwell experiments.7.CCK-8 and transwell experiments were carried out to determine the target microRNA performing its function by inhibiting PIN1.8.Detected the expression level of target microRNA in clinical gastric cancer samples and tissue microarrays,and analyzed its correlation with PIN1 expression,TNM stage and tumor metastasis.Meanwhile,the receiver operating characteristic curve(ROC)about target microRNA expression and patient survival time was calculated and the relationship between the expression level of target microRNA and patient survival was also calculated based on ROC.Result:1.PIN1 is highly expressed in most gastric cancer specimens and cell lines.2.PIN1 significantly promotes the proliferation,invasion and migration of gastric cancer in vitro.3.MiR-628-5p directly inhibits PIN1 expression in gastric cancer.4.MiR-628-5p suppresses the proliferation,invasion and migration of gastric cancer by inhibiting PIN1.5.MiR-628-5p is generally low-expressed in gastric cancer tissues,and its expression level is negatively correlated with PIN1 expression,TNM stage and lymph node metastasis,while positively correlated with survival time of patients.The area under the ROC curve was 0.7369.Conclusion: Our experiment verified the high expression of PIN1 in gastric cancer at the cell line and clinical sample level.The high-expressed PIN1 promotes the proliferation,invasion and migration of gastric cancer cells significantly,while mi R-628-5p can inhibit the above procession of gastric cancer cells by inhibiting PIN1.The mi R-628-5p expression level is negatively correlated with PIN1 expression,TNM stage and lymph node metastasis,while positively correlated with survival time of patients.The area under the ROC curve about mi R-628-5p expression and gastric cancer patient survival time is 0.7369,suggesting that the expression level of mi R-628-5p is comparatively accurate to predict the prognosis of gastric cancer.However,the expression of mi R-628-5p in gastric cancer tissues is generally lower than that in adjacent nontumorous tissues,indicating that deficient mi R-628-5p expression facilitates gastric cancer progression by up-regulating PIN1.These results demonstrated that mi R-628-5p and PIN1 are potential targets for the treatment and prognostic prediction of gastric cancer.