Studies On The Role Of MST1 In The Proliferation And Apoptosis Of Colorectal Cancer Cells

Mammalian STE20-like kinase 1/2(MST1/2) is the encoded protein of MST1/2,the mammalian homolog of Drosophila hippo.As a central role of this pathway,it has functions of adjusting the organ size and maintaining the dynamic balance between cell proliferation and apoptosis.Here,we begin the experiment from the molecular, cellcular and organic level to explore the role and molecular mechanism of MST1 in colorectal cancer, aiming at providing a potential target for the gene treatment of colorectal cancer.Methods:1. Double enzyme digested identification,purification and amplification of the plasmid.A small amount of plasmid was amplificated by Trans5α Chemically Competent Cell and use AgeI and EcoRI double enzyme digested system to identify the plasmid p-EGFP-MST1 and p-EGFP-N1. After identification of the plasmid, a large number of amplification, purification and detection of the concentration are needed.2. Effects of different concentrations and different treatment time of 5-FU on the proliferation and the levels of MST1, Phospho-YAP1(Ser127), PUMA and other proteins of SW480 cells.Treat the SW480 cells with 5-FU in different concentrations and time, detect the cell proliferation by MTS assay, detect MST1, YAP, Phospho-YAP1(Ser127), p73,p53, PUMA and caspase-3 proteins with different concentrations of 5-FU by western blot.3. The study of the role of MST1 in SW480 cells in vitro.Western blot and RT-qPCR detection of siRNA silencing efficiency of three fragments of MST1 in SW480 cells, choose a more efficiency fragment. Transfect p-EGFP-MST1 or siRNA with DNA in vitro transfection reagent into SW480 cells and combined the treating with 5-FU,use MTS assay, flow cytometry and Hoechst33342 staining to dectect the proliferation and apoptosis after upregulation or silencing of MST1; detect the mRNA level of MST1, yap, p73, p53, caspase3, CTGFand AREG by RT-qPCR, use western blot to detect the expression of MST1, YAP,Phospho-YAP1(Ser127), p73, p53, PUMA and caspase-3 proteins in order to explore the molecular mechanisms of proliferation and apoptosis after the change of MST1 in SW480 cells in vitro and the relationship between the expression and activation of MST1 protein and 5-FU-induced cell death.4. Effects of MST1 overexpression in human colorectal cancer grown from SW480 cells in nude miceConstruct the human colorectal cancer model formated by SW480 cells in nude mice to simulated MST1 gene therapy and combined with 5-FU to observe the growth of xenografts.Then use immunohistochemistry to observe the expression of MST1,Phospho-YAP1(Ser127) and PUMA protein in order to study the preliminary molecular mechanisms of MST1 in tumor growth inhibition.Results:1. With the treatment of 5 FU in SW480 cells in vitro, the inhibition rate of proliferation was significantly increased with the increasing of the 5-FU concentration and the time of treatment.Along with the increasing of 5-FU concentration,the expression of MST1 also increased, prompting the phosphorylation of YAP forming into Phospho-YAP1(Ser127).Besides,the levels of proapoptotic related proteins p73, p53, PUMA and caspase3 were also significantly increased.2. Overexpression of MST1 can obviously inhibit the growth and promote apoptosis of SW480 cells.After transfection of p-EGFP-MST1 plasmid into SW480 cells in vitro,the cell growth inhibition rate and apoptosis rate was significantly increased.3. The level of MST1 was increased and the downstream YAP was phosphorylated to Phospho-YAP1(Ser127) and the levels of proapoptotic related proteins p73, p53, PUMA and caspase3 were increased and all the levels of the corresponding mRNA were also significantly increased after overexpression of MST1.4. Overexpression of MST1 significantly downregulated the mRNA expression levels of CTGF and AREG.After transfection of p-EGFP-MST1 plasmid into SW480 cells in vitro,the expression levels of yap,CTGF and AREG mRNA were significantly downregulated.The treatment that transfection of p-EGFP-MST1 combined with5-FU can promote the function of MST1.5. The silencing of MST1 can increase the cell growth and decrease the apoptosis rate of SW480 cells.The cell growth inhibition rate and apoptosis rate of SW480 cells was significantly decreased after the transfection of MST1-siRNA3 fragment.6. The silencing of MST1 can downregulate the mRNA levels of p73, p53, puma and caspase3 and upregulate the mRNA levels of yap, AREG and CTGF.The phosphorylation level of YAP into Phospho-YAP1(Ser127) decreased and the levels of downstream proapoptotic related protein p73, p53, PUMA and caspase3 and the corresponding mRNA levels of them were also significantly decreased after the silencing of MST1.However,YAP protein expression level and mRNA expression levels of yap,CTGF and AREG were significantly increased.7. After the treatment of MST1, 5-FU and 5-FU combined with MST1 in 27 days,both the average tumor volume and the weight of the three groups were significantly lower than the group of Control or negative control,and the group that MST1 combined with 5-FU has better treatment effect on the inhibition of the growth in the xenografts.Besides,the positive rate of MST1, Phospho-YAP1(Ser127), PUMA proteins in the xenografts tissues detected by immunohistochemistry were significantly increased along with the treatment.Conclusion:1.Overexpression of MST1 can inhibit proliferation and promote apoptosis of SW480 colorectal cancer cells, and the inhibition rate is strengthen with the time of treatment; besides,when MST1 is used in combination with 5-FU, the proliferation inhibition rate and apoptosis rate of the cells is higher.2. Overexpression of MST1 can promote YAP phosphorylating to YAP1(Ser127)and increase protein expression levels of p73, p53, PUMA, caspase3 in SW480 cells,and the level of MST1 also increased accordingly. In addition, it reduces the mRNA expression levels of cell CTGF, AREG. This may be an important mechanism for MST1 in the growth inhibition of SW480 cells.3. Overexpression of MST1 can increase the sensitivity of colorectal cancer SW480 cells to 5-FU, and improve the efficacy of therapy.4.Overexpression of MST1 can inhibit the growth of SW480 colorectal cancer cells in nude mice and have better results when combined with 5-FU.These results suggest that MST1 can be used as a new target of gene therapy in colorectal cancer.