Regulation Of Mcl-1 Expression By HGF In SMAD4 Deficient Colorectal Cancer Cells

Colorectal cancer(CRC)is the third most common cancer in male and the second in female worldwide,and about 30% of the patients will develop metastasis even after curative surgery.Therefore,to further understand the molecular mechanism of the development of colorectal cancer,to explore more effective treatment is very urgent.It has suggested that the hepatocyte growth factor(HGF)/c-Met signaling pathyway has been implicated in the development and progression of colon cancer.Hepatocyte growth factor(HGF)is a multifunctional cytokine with important roles in cell proliferation,survival,motility and morphogenesis.Secreted by cells of mesenchymal origin.HGF is the specific ligand for the tyrosine-kinase receptor c-Met.c-Met is expressed in different types of epithelial,endothelial and hematopoietic progenitor cells.The HGF/MET axis is involved in several biological processes,such as embryogenesis,organogenesis,adult tissue regeneration(including wound healing and liver regeneration)and carcinogenesis,for both solid and hematopoietic malignancies.c-Met are frequently overexpressed by several tumor types,including colorectal cancer(CRC).It was binded by it specific ligands,hepatocyte growth factor(HGF),respectively,and promote signaling cascades which mediates many functions such as proliferation and protection against apoptosis,cell scattering,tumor cell motility,invasion and metastasis.According to clinical research found that according to univariate analysis,the mean survival time was shorter in the HGF-positive and c-Met-positive groups.The one-to-one ligand-receptor relationship makes the HGF/MET axis an attractive target for drug development.SMAD4 is an important part of the TGF-β signaling pathway.The data suggest that SMAD4 plays an important and complex role in the interaction with the immune system,the matrix,and the epithelium,and the interruption of its effect promotes the development of colorectal cancer.Clinical studies have found that the lack of SMAD4 is involved with advanced colorectal cancer and metastatic disease.It has been reported that mutations in SMAD4 and BMPR1 A genes have been identified in FJP patients and account for about half of FJP cases.In addition to increasing the risk of colorectal cancer,mutations in SMAD4 were also recorded in high proportion of sporadic colorectal cancer.And in microsatellite instability(MSI)and chromosomal instability in the tumor,there are SMAD4 mutation found.Therefore,SMAD4 is an important target gene in colorectal cancer research.According to the literature,HGF can transcribe the expression of Mcl-1,a member of the anti-apoptotic Bcl-2 family.In contrast,HGF did not alter the expression levels of Bcl-2 and Bcl-x L.Mcl-1 belongs to the Bcl-2 family of proteins and serves as a top molecule for the control of apoptosis,which can promote cell survival by leading to mitochondrial release of cytochrome C cascade events.The up-regulation of Bcl-2 and Mcl-1 protein expression was considered to be anisometritis in several types of tumors.More recent reports suggest that anoikies damage has a significant effect on malignant breast and colorectal cancer,and a similar effect has recently been found in lung cancer.In recent studies,it has been found that Mcl-1 stabilizes and inhibits Bim as a key event in inhibiting tumor cell anoikies.In this study,we investigated the effect of HGF on the expression of Mcl-1 and the expression of Mcl-1 in SMAD4 expression and its mutations or loss.And the relationship between the level of Mcl-1 expression and anoikies.Studies have shown that HGF may regulate the expression of Mcl-1 through ERK,Akt or STAT signaling pathway depending on the type of cell,and there are reports in the literature that anoikis is associated with ERK and Akt signaling pathways.Therefore,we discuss the molecular mechanism of HGF in the regulation of Mcl-1 expression in SMAD4-loss or mutation cells on the basis of this study.Based on the above findings,we collected 50 cases of colorectal cancer tissue,using immunohistochemical method to detect the expression of SAMD4 and Mcl-1 expression.In this study,we also selected four colorectal cancer cells,expressed SMAD4 DLD-1 and HCT15 cells,and SMAD4 mutations or loss of SW620 and HT-29 cells,the identification of SMAD4 expression,the use of lentivirus transfection method.The expression of SMAD4 was silenced in DLD-1 and HCT15 cells,and SMAD4 was overexpressed in SW620 and HT-29 cells and a stable cell line was obtained.The regulation of Mcl-1 expression by HGF was detected at the protein and gene level,and its molecular mechanism was explored.At the same time,the effect of HGF and SMAD4 expression on the anoikies of colorectal cancer was detected by cytoselect 24-well anoikies assay.Designed to inhibit colorectal cancer metastasis and deterioration,improve clinical treatment to provide new ideas.Objective:In this study,SMAD4 mutation as the background,to investigate the expression of Mcl-1 induced by HGF in CRC cells and to elucidate the mechanism of regulation and its effect on the anoikis ability of CRC cells.Methods:1.The expression of SMAD4 and Mcl-1 protein in colorectal cancer and adjacent tissues were detected by immunohistochemical staining.2.The expression of Mcl-1 in four kinds of cells after HGF incubation was detected by Western Blot.SMAD4 was overexpressed in SW620 cells and HT29 cells using lentivirus transfection,Western Blot and RT-q PCR were used to detect the expression of SMAD4,and the expression of Mcl-1 induced by HGF in negative control and transfected cells.3.The anoikis rate was measured using the CBA-080 Cyto Select 24-well Anoikis Assay kit.4.The effect of SMAD4 on HGF-induced ERK and Akt phosphorylation in four wild-type cells,DLD-1,HCT15,SW620,HT29 cells was verified by Western Blot.5.The phosphorylation levels of ERK and Akt protein in SW620 and HT29 cells were detected by Western Blot,and the specific inhibitor U0126 and LY294002 were added to detect the expression level of Mcl-1 after adding the inhibitor.,to investigate through which signal path to regulate Mcl-1.Result:1.SMAD4 was positive in the cytoplasm and nucleus of the cells,and the positive rate was higher in the adjacent tissues.Mcl-1 was positive in the cytoplasm and the positive rate was higher in colorectal cancer tissues.2.HGF had no significant effect on the expression of Mcl-1 in DLD-1 and HCT15 cells expressing SMAD4,and the expression level of Mcl-1 was up-regulated in SW620 and HT29 cells without SMAD4 expression.The overexpression of SMAD4 in SW620 and HT29 cells was achieved by lentivirus transfection,and stable cell lines were obtained.Moreover,HGF regulates the expression of Mcl-1 in SW620 and HT29 cells,and has no significant effect on the cells that overexpress SMAD4.3 SMAD4-deficient SW620 and HT29 cells were significantly enhanced in the ability of anoikis resistance.4.Western blot analysis revealed that HGF induced ERK and Akt protein phosphorylation levels in SWAD4-deficient SW620 and HT29 cells is higher than in SMD4-expressing DLD-1 and HCT15 cells.5.Western blot analysis showed that HGF could phosphorylate the ERK and Akt proteins in SW620 and HT29 cells with no expression of SMAD4,but the phosphorylation level of empty cells was higher than that of overexpression cells.The ERK pathway was specifically inhibited after the addition of ERK pathway-specific inhibitor U0126 in wild-type SW620 and HT29 cells.Mcl-1 was not expressed.After the addition of Akt pathway inhibitor LY294002,the Akt pathway was specifically inhibited and Mcl-1 was still expressed.Conclusion:1.Mcl-1 was highly expressed in colorectal cancer tissues and low in paracancer.SMAD4 was highly expressed in paracancer and low in tumor tissues.The expression of Mcl-1 induced by HGF was affected by SMAD4 expression.2.HGF can enhance the ability of SMAD4-deficient colorectal cancer cells to resist anoikis.3.HGF induced the expression of Mcl-1 through the ERK signaling pathway rather than the Akt signaling pathway in SMAD4-deficient colorectal cancer cells.