| Objectives:1.To investigate the expression difference of miR-4505 between)human mesangial cells cultured with high glucose and low glucose;2.To explore whether miR-4505 regulates the proliferation of human mesangial cells through Wnt/β-catenin signaling pathway.Methods:1.qRT-PCR was used to detect the expression level of miR-4505 in human mesangial cells(HMCs)cultured with high glucose and low glucose;2.miR-4505 mimic,mimic NC,miR-4505 inhibitor and inhibitor NC were transfected into HMCs in vitro,and the expression level of miR-4505 in HMCs was detected by qPT-PCR to evaluate the transfection efficiency;3.CCK-8 was conducted to evaluate the effect of miR-4505 on the proliferation of HMCs cultured with high glucose or low glucose;4.We detected the effect of miR-4505 on the expression of Wnt/β-catenin signaling pathway related proteins(Wnt5a,Axin1,c-Myc,CyclinD1,β-catenin)in HMCs cultured with high glucose or low glucose by western blot and qRT-PCR.5.SPSS software was employed to make the statistical analysis of experimental results.We used One-way analysis of variance for comparison among multiple groups,LSD-t test was used for pairwise comparison,and P<0.05 was considered to be statistically significant.Results:1.CCK-8 was conducted to detect the proliferation ability of HMCs cultured with high and low glucose:the proliferation effect of HMCs stimulated by high glucose was more significant(p<0.05)than that by low glucose;The mRNA expressions and protein expressions of Wnt/β-catenin signaling pathway related factors(Wnt5a,Axin1,Dvl3,β-catenin,c-Myc and CyclinD1)in HMCs cultured with high glucose and low glucose were detected by qRT-PCR and western blot,and the results showed that:the mRNA expressions of Wnt5a,Axin1,Dv13,β-catenin,c-Myc and CyclinD1 in HMCs cultured with high glucose were significantly increased compared with those cultured with low glucose(P<0.001);The protein expression of Wnt5a,Axin1,Dvl3,c-Myc and CyclinD1 in HMCs stimulated with high glucose increased compared with those cultured with low glucose(P<0.001).While there was no significant difference in the expression of β-catenin protein)2.The expressions of miR-4505 in HMCs cultured with high glucose and low glucose were detected by qRT-PCR.It was found that the expression level of miR-4505 in HMCs cultured with high glucose was significantly lower than that in low glucose group(P<0.05).3.The results of CCK-8 showed that compared with the control grouop,overexpression of miR-4505 could inhibit the proliferation of HMCs(P<0.01=4.The results of qRT-PCR showed that the expression levels of Wnt5a,Axin1,Dvl3,β-catenin,c-Myc,and CyclinDl were higher in low glucose+miR-4505 mimic group than that in low glucose group(p<0.001);the expression levels of Wnt5a,Axin1,c-Myc,and CyclinD1 were also higher in low glucose+miR-4505 inhibitor group than that in low glucose group(P<0.001),while Dvl3 expressed less(P<0.001),and there was no significant difference in the expression of β-catenin(P>0.05).The expression levels of Wnt5a,Axin1,Dvl3,β-catenin,c-Myc,and CyclinD1 were lower in high glucose+miR-4505 mimic group than that in high glucose group(p<0.001);the expression levels of Wnt5a,Axin1,Dvl3,β-catenin,c-Myc,and CyclinD1 were also lower in high glucose+miR-4505 inhibitor group than that in high glucose group(P<0.001).5.The results of western blot indicated that:The expression quantities of Wnt5a,Axin1,Dv13,and c-Myc showed more in low glucose+miR-4505 mimic group than that in low glucose group(P<0.001),there was no significant difference inβ-catenin protein expression and CyclinDl protein expression between the two groups(P>0.05);the expression leves of Wnt5a,Axin1,Dv13,and c-Myc were higher in low glucose+miR-4505 inhibitor group than that in low glucose group(P<0.001),while CyclinDl showed less expression(P<0.001=,and there was no significant difference in β-catenin protein expression(P>0.05);The protein expression levels of Wnt5a and Dvl3 were higher in high glucose+miR-4505 mimic group than that in high glucose group(P<0.001=,while the protein expression levels of Axin1,c-Myc and β-catenin showed lower in high glucose+miR-4505 mimic group than that in high group(P<0.001),no significant difference was found in the expression of CyclinD1 between the two groups(P>0.05);the protein expression quanyities of Wnt5a,Dvl3,and CyclinDl were larger in high glucose+miR-4505 inhibitor group than that in high glucose group(P<0.001),Axin1,c-Myc,and β-catenin protein expressed less in high glucose+miR-4505 inhibitor group than that in high glucose group(P<0.001).Conclusion:In high-glucose environment,miR-4505 can inhibit the proliferation of HMCs by negatively regulating the Wnt/β-catenin signaling pathway. |
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