The Function Of Wnt3 Gene In Colorectal Cancer

BackgroundWnt3 is one of 19 members of the Wnt signaling protein family.A large number of reports have reported that aberrant expression of Wnt3 regulates the development of various human malignancies by activating the canonical Wnt signaling pathway.Colorectal cancer is the most common gastrointestinal malignancy worldwide and poses the greatest threat to human health,whereas the mechanism of its occurrence and development is not very clear.Therefore,it is necessary to explore the molecular mechanism of the Wnt3 gene in the development of colorectal cancer.ObjectiveThe expression of Wnt3 gene in CRC tissues and adjacent normal tissues was detected.To explore the effects of Wnt3 gene on the proliferation,invasion and migration,tumorigenesis in vivo and in vitro,as well as on the cell cycle and drug sensitivity of CRC cells.To explore the intrinsic molecular mechanism by which the Wnt3 gene promotes the development of CRC.Method1.The expression of Wnt3 gene in CRC tissues and adjacent normal tissues was analyzed by qRT-PCR and Western blot.2.Constructing CRC cell lines with stable overexpression or knockdown of Wnt3 genea)Transfecting cells: shRNA plasmids targeting Wnt3 mRNA and pEX4 plasmids that can overexpress Wnt3 were constructed and amplified for preservation to transfect HCT-116 cells.b)Screening cells: Continuous screening with high concentration G418.c)Cell identification: The expression level of Wnt3 mRNA was consistent with the expectation,and the protein expression level was also the same as the expectation,which was considered as the success of the screening.d)Maintenance screening: After successful cell identification,low concentration G418 was used to maintain the screening for further experiments.3.Monoclonal HCT-116 cells were screeneda)Cell preparation: Expanded culture of HCT-116 cells with stable overexpression or knockdown of Wnt3 gene,single cell clonal screening was performed on cells with good growth status.b)Flow screening: Flow cytometry was used to inject single cells into the 96-well plate and cell expansion.c)Identification and screening: The expression of Wnt3 gene was identified by qRT-PCR,and the increase or decrease of Wnt3 expression was considered as a successful screening.4.HCT-116 cells with stable overexpression or knockdown of Wnt3 gene and control cells were selected;Cell proliferation test,invasion and migration test,cell colony formation test and tumor formation test in nude mice were conducted.5.To detect the effect of knockdown of Wnt3 gene on the CRC cell cycle.HCT-116 cells with stable overexpression or knockdown of Wnt3 gene and their controls were treated with the same dose of cisplatin,and the apoptosis rate was detected by flow cytometry.HCT-116 cells with stable knockdown of Wnt3 gene and control cells were treated with cisplatin at different concentrations to detect the changes of CRC cell activity and apoptosis-related proteins.6.Through qRT-PCR and Western blot,the influence of overexpression or knockdown of Wnt3 gene on the classical Wnt signaling pathway and the expression of downstream effector genes was evaluated,thus to reveal the molecular mechanism of the development of CRC.Result1.Compared with adjacent normal tissues,the mRNA and protein levels of Wnt3 gene in CRC tissues were significantly increased.The mRNA levels of the Wnt3 gene were also significantly higher in the four commonly used CRC cell lines than in normal tissues.2.The mRNA level and protein level of Wnt3 gene in HCT-116 cells were significantly increased after stably overexpressing Wnt3 gene.3.The mRNA level and protein level of Wnt3 gene in HCT-116 cells were significantly reduced after inhibiting endogenous Wnt3 gene.4.Real Time Cellular Analysis was used to determine the cell proliferation capacity.The results showed that overexpression of Wnt3 gene could increase the proliferation capacity of HCT-116 cells.Conversely,knockdown of Wnt3 gene reduced the proliferation of HCT-116 cells.5.Cell scratch test results showed that overexpression of Wnt3 gene could enhance the invasion and migration ability of HCT-116 cells.On the contrary,knockdown of Wnt3 gene reduced the invasion and migration ability of HCT-116 cells.6.Results of cell colony formation showed that overexpression of Wnt3 gene could enhance the tumorigenic ability of HCT-116 cells in vitro.Conversely,knockdown of the Wnt3 gene reduced the tumorigenic ability of HCT-116 cells in vitro.7.Cell cycle results showed that cells were stagnated in G0/G1 phase after knockdown of Wnt3 gene,and there were fewer cells in S phase and G2/M phase compared with the control group,suggesting that the silencing of Wnt3 gene delayed cell cycle.8.Cisplatin sensitivity test showed that overexpression of Wnt3 gene could reduce the sensitivity of HCT-116 cells to cisplatin.In contrast,HCT-116 cells were more sensitive to cisplatin after knockdown of Wnt3 gene.9.Tumor formation in nude mice showed that overexpression of Wnt3 gene promoted tumor growth and enhanced tumorigenesis in vivo.On the contrary,knockdown of Wnt3 gene inhibited tumor growth and the tumorigenic ability in vivo.10.Identify the molecular mechanism of Wnt3 gene in promoting CRCa)The protein level of Cyclin D1 was increased after overexpressing Wnt3 gene,indicated that the overexpression of Wnt3 gene could activate the classical Wnt/β-catenin signal pathway to promote the occurrence of CRC.b)The mRNA levels of CTNNB1,CCND1 and c-Myc in classical Wnt/β-catenin signal pathway were decreased after silencing Wnt3 gene,indicated that the knockdown of Wnt3 gene inhibited the occurrence of CRC by suppressing the classical Wnt/β-catenin signal pathway.c)The mRNA levels of crucial genes in glycolysis pathway were significantly down-regulated after the stable knockdown of Wnt3 gene,including FBP1,GLUT1,HK2,LDHA and PKM2,indicated that the knockdown of Wnt3 gene could also inhibit the occurrence of CRC by inhibiting the glycolysis pathway.Conclusion1.The expression level of Wnt3 gene was significantly up-regulated in both CRC tissues and cell2.Overexpression of Wnt3 gene promotes the growth of CRC cells and tumors by activating the classical Wnt signaling pathway,and reduces the sensitivity of CRC cells to cisplatin.3.Inhibition of endogenous Wnt3 gene inhibits the growth of CRC cells and tumors by inhibiting the classical Wnt signaling pathway and glycolysis pathway,and enhances the sensitivity of CRC cells to cisplatin.4.It was determined that knockdown of Wnt3 gene could inhibit the occurrence of CRC,thus to provide new drug targets for the treatment of CRC.