| Objectives:1,To clarify the interaction,expression correlation and relationship with the clinical data of the isomerase Pinl and the transcription factor Twistl in the development of colorectal cancer.2,To clarify how isomerase Pinl regulates transcription factor Twistl and its biological role in the development of colorectal cancer.The aim is to provide scientific basis for the diagnosis,prognosis and targeted therapy of colorectal cancer,and to seek new targets for the prevention and treatment of colorectal cancer.Methods:1,Immunohistochemical staining was used to detect the expression of isomerase Pinl and transcription factor Twistl in adjacent tissues and colorectal cancer tissues of patients with colorectal cancer.The clinical data of patients were collected.The correlation between the expression of isomerase Pinl and transcription factor Twistl and clinical parameters such as tumor development,pathological grade,clinical stage and survival prognosis were analyzed by SPSS software.2,Pin1+/+ and Pinl-/-of mouse fibroblasts,colorectal cancer cells(RKO cells,Caco2 cells,SW1116 cells)were cultured.Western blot was used to detect the protein expression level of isomerase Pinl and transcription factor Twistl in different cells.And RNA was extracted,RT-PCR was used to detect the correlation between the expression of the isomerase Pinl and the transcription factor Twistl at the transcription level.3,Through software(http://www.cbs.dtu.dk/services/NetPhos/)prediction and literature analysis,the regulation of Pin1 on the expression of Twistl may be achieved by regulating the phosphorylation of Twistl.4,The direct interaction between isomerase Pinl and transcription factor Twistl was verified by protein immunoprecipitation and confocal experiments.5,The Pinl expression vector and siRNA vector and Twistl expression vector were constructed and transfected into colorectal cancer cells.The expressions of Pinl,Twistl and bcl2 and MMP9 were observed by Western blot.CCK8 was used to examine cell viability,cell scratch test and cell migration experiment to detect changes in cell migration ability,cell invasion test to detect changes in cell invasion ability,and flow cytometry to detect cell cycle changes,and as well as the relevant functional proteins bcl2/bax and MMP9 were detected.Results:1,The results of immunohistochemistry showed that:(1)The expression of Isomerase Pinl and Twistl in colorectal cancer was higher than that in adjacent tissues;(2)There was a significant positive correlation between there two expressions(P<0.05).(3)The expression of isomerase Pinl is not related to TNM staging and clinical stage,as well as the transcription factor Twistl.2,The results of WB and RT-PRC showed that the expression of the isomerase Pin1 and the transcription factor Twistl was consistent in different cell lines of different species.The expression of the isomerase Pinl and the transcription factor Twistl in human colorectal cancer cell SW1116 was lower than that in the colorectal cancer RKO.The expression of the isomerase Pinl was consistent with the transcription factor Twistl in mouse epithelial fibroblasts Pin1+/+ and Pin1-/-cells,and the expression of Pinl and Twistl in Pin1+/+ cells was higher than that in Pin1-/-cells.3,WB results showed that the expression of Twist1-1,Bcl-2 and MMP-9 increased significantly after overexpression of isomerase Pin-1 in SW1116 cells.After inhibiting the expression of isomerase Pinl in RKO cells,the expression of transcription factor Twistl,Bcl2 and MMP9 decreased significantly.After overexpression of Twistl in SW480 cells,the expression of Twistl was increased in cells infected with Twistl expression plasmid,and the expression of Pinl was also increased,indicating that Twistl also affected Pinl at the protein level.4,Flow cytometry showed that after inhibiting the expression of isomerase Pinl in RKO cells,the cells decreased in G1 and G2 phases and increased in S phases compared with the control group,suggesting that inhibiting the expression of Pin1 caused S phase arrest in cell cycle.5,The CCK8 experiment showed that overexpression of Pinl could significantly promote the proliferation of Caco2 cells,and inhibit the expression of Pinl could significantly inhibit the proliferation of Caco2 cells.6,The results of cell scratch test showed that overexpression of Pin1 could significantly promote the migration ability of SW116 cells.7,The results of Transwell migration experiment showed that overexpression of Pin1 could significantly promote the migration of colorectal cancer Caco2 cells.8,Through software(http://www.cbs.dtu.dk/services/NetPhos/)prediction and literature analysis,we analyzed the protein sequence of Twist1 1 and predicted its phosphorylation sites.It was found that there were direct action motifs(serine 8 and 68)of Pinl,which could be directly regulated by Pinl.9,Endogenous co-immunoprecipitation results showed that there was interaction between isomerase Pinl and pTwist1(Ser68).In the endogenous co-immunoprecipitation experiment,pTwistl(Ser68)was found in the immune complexes of Pin1 when pTwist1(Ser68)was precipitated by isomerase Pin1.And after Ser68 of Twist1 was mutated into Ala and that pTwist1(Ser68)was precipitated by isomerase Pin1,no pTwist1(Ser68)was detected.The results showed that there was endogenous interaction between isomerase Pin1 and pTwist1(Ser68).10,Laser confocal experiments showed that isomerase Pin1 and transcription factor Twistl were co-localized in RKO cells and they were mainly located in the nucleus.Conclusion:In colorectal cancer,Pinl is associated with high expression of Twist1.Pinl binds to serine at 68 of Twistl phosphorylation,which in turn affects Twistl activity,regulates transcriptional expression,and promotes tumor development.And Twistl also has a reverse regulation effect on Pin1. |
