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Role Of E3 Ubiquitin Ligase MKRN1 In Different Pathological Type Of Colorectal Cancer Cells And The Intervention Mechanism Of Apatinib

Posted on:2021-05-18Degree:MasterType:Thesis
Country:ChinaCandidate:H L YangFull Text:PDF
GTID:2404330632957515Subject:Clinical Laboratory Science
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Objective:1.To investigate the expression difference of E3 ubiquitin ligase MKRN1 in colon cancer cells of different types and whether MKRN1 plays a role by affecting the expression of SNIP1 and p53;2.To explore whether apatinib exerts its inhibits effect on colorectal cancer cells through MKRN1/p53/NF-?B pathway and whether colorectal cancer cells are resistant to apatinib through p53.Methods:Part one:1.Western Blot was used to explore the differences in MKRN1protein expression of normal colonic epithelial cells CCD-18Co,Dukes A colon cancer cells HCT116,Dukes B colon cancer cells HT29,Dukes C colon cancer cells HCT15 and RKO;2.CCK-8 was used to detect the proliferation activity of HT29,HCT116,HCT15 cells;clone formation was used to detect colony formation ability,Transwell cell migration was used to detect migration ability;3.Real-time PCR and Western Blot was used to detect MKRN1,SNIP1,p53 m RNA and protein expression.Part two:1.Western Blot was used to detect the p53 knockout efficiency in HCT116;2.CCK-8 was used to detect the proliferation activity of apatinib on HCT116 p53+/+and HCT116 p53-/-,and Annexin V-APC/PI was used to detect apoptosis rate;3.Real-time PCR was used to detect the effect of apatinib on MKRN1,Caspase-3,NF-?B p65 m RNA of HCT116 and p53 knockout HCT116 cells,while Western Blot was used to detect its protein expression.Results:Part one:1.The expression of MKRN1 was different from normal colon cells and colorectal cancer cells.MKRN1 was basically not expressed in CCD-18Co,while MKRN1 was high expression of HCT116 and HT29 and low expression in RKO,and the expression of MKRN1 in HT29 was higher than HCT116?P<0.05?.2.CCK-8 showed that the cell proliferation activity was HT29>HCT116>HCT15?P<0.001?.3.The colony formation showed that the colony formation ability of HT29>HCT116>HCT15?P<0.001?.4.Transwell migration showed that the migration capacity of HCT15>HT29and HCT116?P<0.001?,but the number of cells that migrated in HT29 and HCT116was not statistically different.5.Real-time PCR showed that MKRN1 m RNA was correlated with SNIP1 and p53 m RNA.6.Western Blot results show that MKRN1protein was correlated with SNIP1 and p53 protein.Part two:1.Western Blot showed that p53 was successful knockout in HCT116.2.CCK-8 showed that apatinib inhibited HCT116 and p53 knockout HCT116 cells in a time-and dose-dependent manner.Colorectal cancer cells were resistant to apatinib after p53 knockout.3.Flow cytometry showed that apatinib can increase apoptosis ratio of two cells in a dose-dependent manner?P<0.01?.The apoptosis ratio was reduced after p53knockout.4.Real-time PCR showed that after treated with apatinib,the expression of NF-?B p65 and Caspase-3 m RNA in both cells were increased,and?P<0.05?;two The level of MKRN1 m RNA expression in these cells decreased.5.Western Blot showed that after treated with apatinib,the expression of p53 in HCT116 cells was decreased?P<0.01?;the expression of MKRN1 in both cells was decreased;the expression of Caspase-3 was increased,while the expression of Caspase-3 was higher in HCT116 than p53 knockout HCT116 cells?P<0.01?;the expression of NF-?B p65in HCT116 cells was gradually decreases with increasing dose of apatinib?P<0.01?,but expression of NF-?B p65 in p53 knockout HCT116 cells was gradually increased?P<0.01?.Conclusion:1.MKRN1 is highly expressed in HT29 and HCT116 cells,MKRN1 is positively correlated with tumor cell proliferation;2.MKRN1 is lowly expressed in HCT15 and RKO colon cancer cells,MKRN1 is negatively correlated with tumor cell migration ability;3.MKRN1 may play a regulatory role in tumor cells by affecting the expression of SNIP1 and p53.4.Apatinib can inhibit colon cancer by reducing the expression of MKRN1 and p53 and inhibiting the activation of NF-?B pathway.Knockout of p53 gene causes colon cancer cells to develop resistance to apatinib.
Keywords/Search Tags:Colorectal Cancer, Dukes typing, MKRN1, apatinib, drug-resistance
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