| Objective: The incidence rate and mortality rate of colorectal cancer have always been high.In many clinical trials,long-term use of celecoxib can effectively inhibit the occurrence and recurrence of colorectal cancer,but the mechanism of celecoxib inhibiting colorectal cancer is not fully elucidated.The microRNA200 family plays an important role in regulating the process of a variety of tumors,and clinical detection of miR-200s expression can help to judge the development and prognosis of the disease.Clinical trial results show that miR-200s are decreased in colorectal cancer,suggesting that miR-200s may participate in the regulation of the development of colorectal cancer.The purpose of this study is to explore that celecoxib regulates the microRNA200s to regulate the expression level of tumor stemness-related proteins in human colon cancer cells,and explore that celecoxib can affect the expression of EMT,autophagy,apoptosis and other related proteins to inhibit the development of colorectal cancer.Methods:This experiment consists of two parts.1.The first part detects the effect of celecoxib-mediated miR-200s on the expression of tumor stem proteins by colorectal cancer cell experiment.(1)After celecoxib intervened in human colon cancer HT29 and SW480 cells,a CCK-8 reagent was selected to evaluate cancer cell proliferation.(2)After celecoxib intervened with human colon cancer HT29 and SW480 cells,the expression level of microRNA200 family members(miR-200s)was detected by qRT-PCR technology.(3)After overexpression of miR-200b in HT29 and SW480 cells,qRT-PCR was used to verify the transfection efficiency.Western blot was used to detect the expression of tumor stemness-related proteins(including Oct4,CD133,Lgr5,Sox2,Nanog,and CD44).At the same time,after inhibition of the miR-200b in cancer cells,the inhibition effect was verified by qRT-PCR,and the expression of tumor stemness-related proteins was detected by Western blot.After inhibition of the miR-200b in cancer cells,then celecoxib was added,the expression of miR-200b was detected by qRT-PCR,and the expression of tumor stemness-related proteins was detected by Western blot.2.The second part is about the effect of celecoxib intervention or overexpression of miR-200b on the expression of EMT,autophagy,apoptosis,programmed cell death ligand 1 and other related proteins in colorectal cancer cells.(1)After celecoxib interfered with human colon cancer HT29 and SW480 cells,the expression of EMT-related proteins was detected by Western blot.After overexpression or inhibition of the miR-200b,the expression of EMT-related proteins in the transfected cells was detected by Western blot.(2)After celecoxib interfered with HT29 and SW480 cells,the content of reactive oxygen species(ROS)in each group of cancer cells was detected by flow cytometry.(3)After celecoxib interfered with HT29 and SW480 cells,the expression of autophagy-related proteins was detected by Western blot.After overexpression of the miR-200b in cancer cells,the expression of autophagy-related proteins was detected by Western blot in the transfected cells.(4)After celecoxib interfered with HT29 and SW480 cells,the expression of the apoptotic-related proteins was detected by Western blot.After overexpression or inhibition of the miR-200b,the expression of apoptotic-related proteins in the transfected cancer cells was detected by Western blot.(5)After celecoxib interfered with HT29 and SW480 cells,the number of apoptotic cells(Annexin V-FITC/PI principle)was detected by flow cytometry.(6)After celecoxib interfered with HT29 and SW480 cells,the expression of Programmed death-ligand 1(PD-L1)protein was detected by Western blot.After overexpression of the miR-200b in cancer cells,the expression of PD-L1 protein in the transfected cells was detected by Western blot.(7)The expression of EMT-related proteins and apoptotic-related proteins in the modeled rat large intestine tissue was detected by the Western blot experiment.Results:1.Part 1:(1)The results of CCK8 showed that with the extension of the intervention dose and time of celecoxib,which significantly inhibited the proliferation of HT29 and SW480 cells(p<0.05).(2)Celecoxib intervention in HT29 and SW480 cells could significantly increase the expression of miR-200s and miR-200b/200c/141 compared with the control group(p<0.05).(3)After overexpression of the miR-200b in cancer cells,the results of q PCR experiment showed that the expression of miR-200b was up-regulated(p<0.05).After overexpression of the miR-200b in cancer cells,the expression of tumor stemness-related proteins such as Oct4,CD133,Lgr5,Sox2,Nanog,and CD44 was decreased,compared with the control group(p<0.05).After inhibition of the miR-200b in cancer cells,the results of q PCR experiments showed that the expression of miR-200b was decreased,compared with the control group(p<0.05),while the expression of tumor stemness-related proteins was increased,compared with the control group(p<0.01).Compared with the control group,after inhibition of the miR-200b in cancer cells,the expression of miR-200b in the celecoxib group was increased,which the expression of tumor stemness-related proteins in cancer cells was decreased,and the expression of tumor stemness-related proteins in this group was increased than that in the celecoxib group alone(p<0.05).2.Part 2:(1)Compared with untreated cells,the effect of celecoxib on HT29 and SW480 cells increased the protein expression of epithelial phenotype marker E-cadherin,and decreased the protein expression of mesenchymal phenotype marker N-cadherin(p<0.05).After overexpression of the miR-200b in cancer cells,the expression of E-cadherin protein was increased,while the expression of Vimentin and Snail proteins was decreased compared with the control group(p<0.05).After inhibition of the miR-200b in cancer cells,the expression of E-cadherin protein was decreased,while the expression of Vimentin protein was increased(p<0.05).(2)Celecoxib intervention in HT29 and SW480 cells could significantly increase ROS levels,compared with the control group(p<0.05).(3)Celecoxib intervention in HT29 and SW480 cells increased the expression of autophagy proteins LC3 Ⅱ and P62(p<0.05).After overexpression of the miR-200b in cancer cells,the expression of LC3Ⅱ protein was increased,but the expression of P62 protein was decreased(p<0.05).(4)Celecoxib intervention in HT29 and SW480 cells increased the expression of pro-apoptotic proteins Bax and cytochrome C,and decreased the expression of anti-apoptotic protein Bcl2(p<0.05).After overexpression of the miR-200b in cancer cells,the expression of pro-apoptotic proteins Bax and Caspase-9 was increased,while the expression of anti-apoptotic protein Bcl2 was decreased,compared with the control group.After inhibition of the miR-200b in cancer cells,there was observed that the expression of Bax protein was decreased,while the expression of anti-apoptotic proteins Bcl2 was increased,compared with the control group(p<0.05).(5)Celecoxib intervention in HT29 and SW480 cells for 48 hours could significantly increase the apoptosis rate of cancer cells,compared with untreated cancer cells(p<0.05).(6)Celecoxib intervention in HT29 and SW480 cells decreased the expression of PD-L1 protein(p<0.05).After overexpression of the miR-200b in cancer cells,the expression of PD-L1 protein was decreased,compared with the control group(p<0.05).(7)Rats with celecoxib treatment for 36 weeks,the expression of E-cadherin protein was increased,and the expression of N-cadherin and vimentin proteins was decreased in large intestine specimen of rats(p<0.01).Moreover,the expression of Bax protein was increased and the expression of Bcl2 protein was decreased,compared with large intestine specimen of the model rats(p<0.05).Conclusion: Celecoxib could regulate miR-200b to regulate the expression of tumor stemness indicators of colorectal cancer cells.At the same time,celecoxib intervention or overexpression of miR-200b can regulate the expression of related proteins such as EMT,autophagy,apoptosis and immune escape in colorectal cancer cells. |
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