| ObjectivesTo investigate the effect of metformin on the expression of tumor suppressor-related mi RNAs(mi RNA34a,mi RNA126,mi RNA143,mi RNA145,mi RNA342-3p,mi RNA342-5p)under high glucose conditions.And to explore whether metformin affects colon cancer stem cell proliferation,apoptosis and CD133 m RNA expression through mi R-342-3p.Methods1. CD133~+colon cancer stem cells were selected from SW480 cell lines by direct immunomagnetic beads method as the research object,and the content of CD133~+colon cancer stem cells was analyzed by flow cytometry.2.Colon cancer stem cells were divided into control group(common medium,glucose concentration 3.1g/L),MET group(common medium,20mmol/L metformin(MET)intervention),high glucose group(high glucose medium,glucose concentration 5.0g/L),and high glucose-MET group(high glucose medium,20mmol/L MET intervention),after 24 hours of MET intervention,the expression of tumor suppressive-related mi RNAs in each group were detected by q RT-PCR.mi R-342-3p with significantly different expression was selected as the target mi RNA.Colon cancer stem cells were infected by lentivirus has-mi R-342-3p inhibitor and LV16-NC in high glucose environment,and the successful inhibition of target mi R-342-3p was verified by q RT-PCR.3.CCK-8 assay:Colon cancer stem cells were divided into NC group(high glucose medium,LV16-NC transfection),NC MET group(high glucose medium,LV16-NC transfection+20mmol/LMET intervention),and inhibition group(high glucose medium,LV16-has-mi R-342-3p transfection),intervention MET group(high glucose medium,LV16-has-mi R-342-3p transfection+20mmol/LMET intervention),lentivirus infection for 48h,and MET intervention for 24h,The OD values of each group were detected by CCK-8 assay.4.Apoptosis test:Colon cancer stem cells were divided into NC group,NC-MET group,inhibition group,and inhibition-MET group.After 24 hours of intervention,flow cytometry was used to detect the apoptosis in different groups.5.Colon cancer stem cells were divided into NC group,NC-MET group,inhibition group,and inhibition-MET group.After 24 hours of intervention,the expression of CD133 m RNA in each group were detected by q RT-PCR.Results1.The flow cytometer showed that the content of CD133~+colon cancer stem cells in the cell population sorted by immunomagnetic beads was 82.37%;after cultured in serum-free medium,the cells showed stem cell-like characteristics:the cells grew in suspension,and the cell volume gradually increased and rounded,and formed into stem cell spheres.2.(1)expression of mi RNAs in each group:Compared with the control group,the expression of tumor suppressor-related mi RNAs(mi R-34a,mi R-126,mi R-143,mi R-145,mi R-342-3p,mi R-342-5p)in the MET group were significantly increased(P<0.05);the expression of mi R-126,mi R-143,and mi R-342-3p in high glucose group were decreased(P<0.05),the expression of mi R-34a and mi R-342-5p were increased(P<0.05),and there was no statistical difference in the expression of mi R-145.Compared with high glucose group,the expression of mi R-126 and mi R-342-3p were increased in high glucose-MET group(P<0.05),the expression of mi R-145 and mi R-342-5p were decreased(P<0.05),and there was no statistical difference in the expression of mi R-34a and mi R-143.(2)The expression of mi R-342-3p in the inhibition group was significantly lower than that in the NC group(P<0.05),suggesting that mi R-342-3p was successfully inhibited in colon cancer stem cells.3. Compared with the NC group,the OD value and the cell viability of the NC MET group was decreased significantly(P<0.05),and there was no significant change in the OD value and cell viability of the inhibition group.Compared with the inhibition group,the OD value and cell viability of the inhibition MET group were significantly decreased(P<0.05).4. Compared with the NC group,the apoptosis rate of the NC MET group was significantly increased(P<0.05),and the apoptosis rate of the inhibition group was not significantly changed.Compared with the inhibition group,the apoptosis rate of the inhibition-MET group was increased(P<0.05).5.Compared with the NC group,the expression of CD133 m RNA in the NC MET group was reduced(P<0.05),and the expression of CD133 m RNA in the inhibition group was increased(P<0.05).Compared with the inhibition group,the expression of CD133 m RNA in the inhibition-MET group was reduced(P<0.05).Conclusion1.Metformin can up-regulate the expression of mi R-34a,mi R-126,mi R-143,mi R-145,mi R-342 in colon cancer stem cells.2.Under high glucose environment,the expression of mi R-34a,mi R-342-5p was up-regulated in colon cancer stem cells,and the expression of mi R-126,mi R-143,mi R-342-3p was down-regulated,and mi R-145 expression was not significantly changed.3.In high glucose environment,metformin can up-regulate the expression of mi R-126and mi R-342-3p in colon cancer stem cells and inhibit the expression of mi R-145 and mi R-342-5p,while there was no significant change in the expression of mi R-34a and mi R-143.4.In high glucose environment,the pro-apoptotic and anti-proliferative effects of metformin on colon cancer stem cells may not be achieved by down-regulating the expression of mi R-342-3p.5. In high glucose environment,metformin can reduce the expression of CD133 m RNA in colon cancer stem cells by up-regulating the expression of mi R-342-3p. |
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