| Objective:This study aims to explore the changes of biological characteristics of CML cells and the sensitivity of mi R-15a-5p to TKIs by regulating YAP1,so as to provide a new theoretical basis and basis for enriching the molecular biological mechanism of drug resistance of existing CML cells to TKIs,and to provide a new idea for reversing the drug resistance of CML cells to TKIs.Methods:1.CCK-8 kit was used to detect the IC50 of K562 and K562 G cells.The expression of mi R-15a-5p in CML-resistant cell lines,non-resistant cell lines and healthy donor PBMC was detected by q RT-PCR.2.To verify the effect of down-regulation of mi R-15a-5p expression on drug resistance of K562 cells.K562 cells were transfected with empty vector control group(NC)and mi R-15a-5p inhibitor respectively for 48 hours,and the transfection efficiency was detected by q RT-PCR.K562 cells were transfected with NC and mi R-15a-5p inhibitor,and their cell viability and IC50 were detected by CCK-8 method 48 hours after transfection.The mi R-15a-5p inhibitor group and NC control group were treated with0.1μM IM and without IM for 48 hours,respectively.the apoptosis rate was detected by flow cytometry,and the expression differences of Cyclin D1 and Caspase3 were detected by Western Blot.3.To verify the effect of up-regulation of mi R-15a-5p expression on drug resistance of K562 G cells.K562G cells were treated with empty vector control group(NC)and mi R-15a-5p mimic agent for 48 hours,and the transfection efficiency was detected by q RT-PCR.K562 G cells were transfected with NC and mi R-15a-5p mimic,and their cell viability and IC50 were detected by CCK-8 method 48 hours after transfection.The mimic group transfected with mi R-15a-5p and NC control group were treated with 5μM IM and without im for 48 hours respectively.the apoptosis rate was detected by flow cytometry,and the expression difference of Cyclin D1 protein and Caspase3 protein was detected by Western Blot.4.The target genes of mi R-15a-5p are predicted by mi RDB,mi RTarbase and Targetscan,and verified by double luciferase reporting system.5.The YAP1 expression of K562 G cells was knocked down by si RNA,and the low knock efficiency was detected by Western Blot and q RT-PCR.CCK-8 kit was used to detect cell proliferation 48 hours after si RNA transfection.Western Blot was used to detect the expression of proliferation and apoptosis-related proteins.c Results:1.the IC50 of K562 cells is 0.16μM,and that of K562 G cells is 10.94μM.The results of q RT-PCR showed that mi R-15a-5p had the highest expression in PBMC of healthy donors and the lowest expression in K562 G cells,and there were statistical differences between them.2.Down-regulating the expression of mi R-15a-5p can reduce the sensitivity of K562 to IM.The expression of mi R-15a-5p in each group was detected by q RT-PCR.Compared with NC group,mi R-15a-5p inhibitor can significantly reduce the expression of mi R-15a-5p in K562 cells(P<0.01).CCK-8 results showed that after the transfection group was treated with the same concentration of IM,the vitality of K562 cells in mi R-15a-5p inhibitor group increased compared with the control group,and the IC50 value of IM increased.Compared with NC group,the apoptosis rate of mi R-15a-5p inhibitor group decreased(p< 0.05).Compared with NC+IM group,the apoptosis rate of mi R-15a-5p inhibitor+IM group decreased(p< 0.05).Compared with NC group,the apoptosis rate of NC+IM increased by 2.4 times,while that of mi R-15a-5p inhibitor+IM group only increased by 2.1 times,indicating that inhibition of mi R-15a-5p expression reduced the sensitivity of K562 cells to im.The results of Western Blot showed that compared with NC group,the expression of proliferating protein Cyclin D1 increased and the expression of apoptotic protein Caspase-3 decreased in mi R-15a-5p inhibitor group.Compared with NC+IM group,mi R-15a-5p inhibitor+IM group increased the expression of Cyclin D1 protein and decreased the expression of Caspase-3 protein,which indicated that down-regulation of mi R-15a-5p could reverse the decrease of Cyclin D1 protein and increase the expression of Caspase-3 protein caused by IM application.3.Up-regulation of mi R-15a-5p can increase the sensitivity of K562 G cells to IM.qRT-PCR was used to detect the expression of mi R-15a-5p in each group.the results showed that mi R-15a-5p mimetic could significantly up-regulate the expression of mir-15a-5p in K562 G compared with NC group(P<0.001).The results of CCK-8 experiment showed that the viability of K562 G cells in mi R-15a-5p mimic group was significantly inhibited compared with that in control group,and IC50 of im decreased.Compared with NC,the apoptosis rate of mir-15a-5pmic group increased(p < 0.05).Compared with NC+IM group,the apoptosis rate of mi R-15a-5p mimic+IM group increased(p < 0.05).The apoptosis rate of NC+IM increased by 1.7times compared with NC group,while that of mi R-15a-5p mimic +IM group increased by 1.9 times compared with mi R-15a-5p mimic group,suggesting that K562 G cells were more sensitive to im after overexpression of mi R-15a-5p.The results of Western Blot showed that compared with NC,the expression of Cyclin D1 protein decreased and Caspase-3 protein increased in mi R-15a-5p mimic group.Compared with NC+IM group,mi R-15a-5p mimic+im group decreased Cyclin D1 protein expression and increased Caspase-3 protein expression.overexpression of mir-15a-5p in K562 G cells can promote the decrease of Cyclin D1 protein expression and increase of Caspase-3 protein expression caused by im application.4.YAP1 is the target gene of mi R-15a-5p.it is predicted by mi RDB,mi RTarbase and Targetscan database that there are 47 target genes of mi R-15a-5p,and YAP1 is one of them.q RT-PCR and Western Blot results show that YAP1 expression in K562 G is higher than that in K562.The difference was statistically significant(P<0.05),and it was negatively correlated with the expression of mi R-15a-5p.The results of double luciferase report experiment showed that compared with the control group,the reporter fluorescence expression of h-YAP1-MUT was significantly higher than that of h-YAP1-WT after transfection of hsa-mi R-15a-5p(P<0.05)The above results indicate that hsa-mi R-15a-5p has obvious interaction with this site on h-YAP1 3’UTR,and YAP1 is the direct target of mi R-15a-5p.5.YAP1 promotes apoptosis and inhibits proliferation of K562 G cells.After 48 hours of transfection of si-YAP1 and empty vector(control group),CCK-8 method was used to detect the proliferation of transfected and control groups.It was found that the proliferation of transfected si-YAP1 group was significantly inhibited(P<0.01)compared with control group.Western Blot showed that the expression of Cyclin D1 protein decreased.The expression of Caspase-3 protein increased,the above results indicated that up-regulation of mi R-15a-5p expression or inhibition of YAP1 expression in K562 G could promote apoptosis and inhibit proliferation of K562 G cells,suggesting that mi R-15a-5p could play a role by targeting YAP1Conclusions:The low expression of mi R-15a-5p promoted the expression of YAP1,inhibited the apoptosis of K562 cells,promoted their proliferation,and promoted the resistance of K562 cells to IM.Mi R-15a-5p may become a new target for the reversal of CML drug resistance. |
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