MicroRNA-98 Reduces Drug Chemosensitivity And Regulates Mitochondrial Dynamics By Targeting LASS2 In Bladder Cancer Cells

Objectives:1.To investigate the expression of miR-98 in bladder cancer cells and its effect on proliferation,apoptosis,chemosensitivity of bladder cancer cells.2.To investigate the effects of miR-98 on mitochondrial dynamics and mitochondrial membrane potential changes in bladder cancer cells,and to examine the expression of mitochondrial dynamics-related protein effected by miR-98.3.Different 3SS-UTR plasmids of LASS2 gene were constructed to verify the targeted binding ability of miR-98 to LASS2.To investigate the effects of LASS2 on proliferation,apoptosis and chemosensitivity of bladder cancer cells,and studying possible interaction between miR-98 and LASS2.Methods:1.The expression of miR-98 in different bladder cancer cell lines(J825 T24,5637,RT4,BIU-87)and normal urothelial cells was detected by real-time PCR.The bladder cancer cell lines with high-expression and low-expression were transfected into miR-98 inhibitors and mimics,respectively.CCK-8 assay and colony formation assay were used to detect the effect of miR-98 on the proliferation of bladder cancer cells.The effect of miR-98 on the chemosensitivity of bladder cancer cells was detected by CCK-8,and the effect of miR-98 on the apoptosis of bladder cancer cells was detected by flow cytometry.2.MitoTrackerTM Red mitochondrial staining,Hoechst 33258 nuclear staining and laser confocal microscopy were used to observe the effect of miR-98 on mitochondrial dynamics of bladder cancer cells.The changes of mitochondrial membrane potential in bladder cancer cells was detected by JC-1 staining and flow cytometry.The effect of miR-98 on the expression of mitochondrial dynamics-related proteins was detected by Western blot.3.In order to detect whether LASS2 is a direct target of miR-98,qRT-PCR was used to detect the expression of LASS2 mRNA in bladder cancer cells firstly,reporter plasmid having LASS2 wild type(CUACCUC)or mutant(CUAAAUC)3’-UTR binding site was transfected into T24 eells together with miR-98 mimic,and a dual luciferase reporter assay was used to detect luciferase activity.Simultaneously,LASS2 plasmid was transfected into bladder cancer cells,the effects of LASS2 on chemosensitivity and mitochondrial dynamics of bladder cancer cells were detected by CCK-8 assay and laser confocal microscopy,the changes of mitochondrial membrane potential were detected by JC-1 staining,and the expression of mitochondrial dynamics related proteins was detected by Western blot.Results:1.Compared with the SV-HUC-1 cell line,the expression of miR-98 was higher in bladder cancer cell lines tested.T24 cells showing the lowest endogenous miR-98 levels and BIU-87 with relatively high miR-98 levels were transfected with miR-98 mimic and miR-98 inhibitor,respectively.The results of CCK-8 assay showed that miR-98 inhibitor attenuated the proliferation of BIU-87 cells,while enhancing the expression of miR-98 promoted the proliferation of T24 cells.The colony formation assay detected the long-term proliferation of miR-98 on bladder cancer cell lines have similar result.CCK-8 was used to detect the response of bladder cancer cells to cisplatin or doxorubicin treatment.It was found that cell viability of T24 cells was enhanced after 48 and 72 hours of treatment with cisplatin or doxorubicin compared with the control group,while the decrease of miR-98 expressi.on down regulated the cell viability of bladder cancer cell line BIU-87.Flow cytometry analysis showed that the decrease of miR-98 expression induced apoptosis of BIU-87 cells,while the increase of miR-98 expression decreased the apoptosis of T24 cells.2.Confocal microscopy showed enhanced expression of miR-98,which promoted mitochondrial division into punctate,while inhibiting the expression of miR-98,the mitochondria in BIU-87 cells were more easily fused into a linear or rod-like shape.Flow cytometry revealed that mitochondrial membrane potential was down-regulated in BIU-87 cells with miR-98 inhibitor,and mitochondrial membrane potential was increased after increasing miR-98 expression in T24 cells.Detection of changes in mitochondrial-associated protein expression revealed that increased expression of miR-98 up-regulated cyclin D1,p-Drpl,Drpl and Fisl proteins,while down-regulating LASS2 protein expression,whereas miR-98 inhibitors decreased cyclin D1,p-Drpl,Drp1 and Fisl protein expression,while up-regulating LASS2 protein expression.3.Increased expression of miR-98 significantly inhibited the expression of LASS2 mRNA.Dual luciferase reporter assay found that luciferase intensity was inhibited in T24 cells transfected with miR-98 mimic and wild-type LASS2 3’-UTR,however no significant change was observed in the fluorescence intensity of cells transfected with the mutant plasmid.LASS2 plasmid was transfected into BIU-87 and T24 cells to enhance the expression level of LASS2 in both cells.Overexpression of LASS2 could inhibit the proliferation of bladder cancer cells and increase the sensitivity of bladder cancer cells to cisplatin.LASS2 overexpression also induced mitochondrial tropism in cells,decreased mitochondrial membrane potential,inhibited Drpl phosphorylation and total Drpl protein expression.Using the transfection of LASS2 siRNA into T24 cells to knock down the expression of LASS2 and transfecting the miR-98 mimic,it was found that in cells transfected with LASS2 siRNA,the role of miR-98 mimic in p-Drp1/Drp1 was unpaired.CCK-8 and flow cytometry results also showed that T24 cells treated with LASS2 siRNA and miR-98 did not significantly regulate the chemosensitivity of cisplatin.LASS2 knockdown impaired the inhibitory effect of miR-98 on T24 cells..Conclusions:1.miR-98 is up-regulated in bladder cancer cell lines,promoted the proliferation,inhibitted the apoptosis,and reduced the chemosensitivity of bladder cancer cells to cisplatin and doxorubicin.2.High expression of miR-98 increases mitochondrial division,inhibits △Ψm reduction,affects mitochondrial dynamics and mitochondrial apoptosis pathway.lt may be caused by up-regulation of p-Drpl and Fisl expression.miR-98 acts as a regulator of mitochondrial function and inhibitor of mitochondrial apoptosis in bladder cancer cells.3.The expression of LASS2 mRNA and protein can be inhibited by miR-98.LASS2 acts as a direct target of miR-98,allowing miR-98 to directly bind to LASS2 3-UTR.miR-98 affects proliferation,apoptosis and reduces chemosensitivity of bladder cancer cells by targeting and down-regulating LASS2.Mitochondrial division and mitochondrial membrane potential can be regulated by miR-98 targeting LASS2.