Vimentin Affects Colorectal Cancer Proliferation,Invasion And Migration Via Regulated By Activator Protein 1

Objective:Uncontrolled recurrence and metastasis are important reasons for the high mortality rate of malignant tumors.Vimentin is positively correlated with the degree of malignancy of cancer cells.Vimentin is also highly expressed in colorectal cancer(CRC)cells,which plays a critical role in the metastasis and prognosis of CRC.However,the molecular mechanism of vimentin in the progression of CRC is incompletely understood.The promoter is necessary to regulate the expression of vimentin gene.The promoter itself does not control the activity of the gene but combines with transcription factors to control the activity of the gene.Nevertheless,the specific transcriptional regulation mechanism of the vimentin promoter is incompletely understood.The main purpose of this study is to explore the specific transcriptional regulation mechanism of vimentin promoter in colorectal cancer and to further study the effect of the transcriptional regulation mechanism of vimentin promoter on the biological functions of colorectal cancer cells.Methods:1.The full-length and varieties of vimentin promoter deletion were constructed by PCR method and the recombinant plasmids were detected by double enzyme digestion and DNA sequencing.2.The double-luciferase reporter system was used to detect the relative luciferase activity of the full length and each truncated fragment of the vimentin promoter and the effect of AP-1 on the relative luciferase activity of the vimentin promoter.3.Key transcription factors binding to vimentin promoters were analyzed by bioinformatics analysis and mutation assay.4.The expression levels of AP-1 and vimentin in colorectal cancer cells were detected by Western blot and Real-time PCR.5.Gel transfer assay(EMSA)and chromatin immunoprecipitation assay(ChIP)were used to detect the interaction between AP-1 and vimentin promoter+785nt ～ +1085nt regions in vitro and in vivo.6.Lentivirus overexpression vector of AP-1 and vimentin silencing vector after AP-1 overexpression were constructed.The protein expression of AP-1 and vimentin in CRC cells was detected by Western blot.7.The effects of AP-1 overexpression and AP-1 overexpression silenced by vimentin on the proliferation,migration,and invasion ability of colorectal cancer cells were detected by cell proliferation assay,clone formation assay,scratch assay,cell migration assay,and cell invasion assay.8.The effect of AP-1 on vimentin expression and tumor growth in vivo was detected by subcutaneous tumor-forming model in nude mice and immunohistochemical assay.Result:1.The full-length and varieties of vimentin promoter deletion vectors were verified by KpnI and XhoI double digestion experiments,and the results of double digestion experiments showed that the recombinant plasmid was constructed successfully.2.The results of the double-report luciferase assay showed that the plasmid pGL4.10-1085 had the highest luciferase activity relative to the complete promoter region(-121 nt ～ +1850nt),and the +785nt ～ +1085nt region was crucial for the complete activity of the vimentin promoter.3.The effects of AP-1 on vimentin promoter and vimentin expression were detected by bioinformatics analysis,overexpression and knockdown of AP-1.The experimental results showed that AP-1 could affect the relative luciferase activity of vimentin promoter and the expression of vimentin.4.The electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that AP-1 could recognize and specifically bind to vimentin promoters in vitro and in vivo.5.The results of cell proliferation assay,clone formation assay,scratch assay,cell migration assay,and cell invasion assay showed that AP-1 could affect the proliferation,migration,and invasion of CRC cells through vimentin.6.The results of animal experiments and immunohistochemistry showed that AP-1 could affect the expression of vimentin in vivo,and AP-1 could promote tumor growth by regulating vimentin.Conclusion:1.AP-1 is the most important transcription factor binding site on vimentin promoter in colorectal cancer cells.AP-1 regulates the transcription and translation of vimentin genes in colorectal cancer cells.2.AP-1 affects invasion and metastasis of CRC cells in vitro and tumor growth in vivo by activating vimentin.