Proliferation,10 has been reported to be activated by xanthine oxidase-mediated ROS production inCell Death and Diseasemurine mesangial cells26 and to generate hydrogen peroxide in human eosinophils.27 Real-time PCR analysis and enzyme-linked immunosorbent analysis (ELISA) showed that transfection of miR-302d mimic suppressed CCL5 mRNA levels and protein levels in hADSCs (Figures 5a and b). Then, we determined the effect with the downregulation of CCL5 expression. The transfection of CCL5 siRNA in hADSCs inhibited CCL5 mRNA and protein levels (Figures 5c and d). Downregulation of CCL5 expression did not have an effect on basal and miR-302-induced raise in hADSCs proliferation (Figure 5e). We next tested irrespective of whether the protective effects of miR-302 against oxidant injury were mediated by the suppression of CCL5 expression. Cell viability assay revealed that downregulation of CCL5 by means of siR-CCL5 decreased CoCl2- and SIN-1-induced cell death like transfection of miR-302d, and cotransfection of miR-302d and CCL5 siRNA further improved cell viability (Figure 5f). Utilizing realtime PCR analysis (Figure 5g) and ELISA (Figure 5h), we confirmed that CoCl2 and SIN-1 treatment upregulated CCL5 expression in hADSCs, and that transfection of miR-302d decreased oxidant-induced boost in CCL5 expression plus the CCL5 siRNA and miR-302d-cotransfected cells showed reduce CCL5 protein level than that of miR-302dtransfected cells. Transfection with siR-CCL5 also decreased CoCl2- and SIN-1-induced ROS generation (Figure 5j). To confirm the part of CCL5 in miR-302d-induced protection of cell death, we determined the impact of recombinant CCL5 (Figure 5i). The addition of recombinant CCL5 (five mg/ml) increased CoCl2-induced cell death and inhibited miR-302induced protection of CoCl2-induced cell death in hADSCs. Bioinformatic analysis showed that 3’UTR of CCL5 mRNA consists of a putative target web-site for miR-302d, as described by Kumar et al.ten To identify whether miR-302d directly binds to 3’UTR of CCL5, we utilised luciferase reporter vector that includes a putative miR-302d binding sequence of CCL5 3’UTR. Cells were transfected using a luciferase Piceatannol site construct inmiR-302 protects oxidant-induced cell death JY Kim et alCDKN1A 1.2 1.0 0.eight 0.six 0.4 0.two 0.ol 2d ntr -30 -co miR iR mRelative mRNA expression (Fold modify)Relative mRNA expression (Fold modify)1.two 1.0 0.8 0.6 0.4 0.two 0.0 ##Relative mRNA expression (Fold change)CDKN1ACDKN1A 3.0 2.five two.0 1.5 1.0 0.five 0.+ ?+ ?+ ??+ ?+ + ??+ ?+ 3-Methylvaleric Acid Cancer miR-control miR-302d siR-control siR-CDKN1A p21 (21 kDa) GAPDH (36 kDa) + ?+ ?+ ??+ ?+ + ??+ ?+ miR-control IH miR-302d IH siR-control siR-CDKN1A p21 (21 kDa) GAPDH (36 kDa)ol 1A ntr KN -co -CD siR siRIH d IH ol 2 ntr -30 -co miR iR m160000 140000 Number of cells 120000 100000 80000 60000 40000 20000 0miR-control + siR-control miR-control + siR-CDKN1A miR-302d + siR-control miR-302d + siR-CDKN1A160000 140000 120000 Variety of cells #miR-control IH + siR-control miR-control IH + siR-CDKN1A miR-302d IH + siR-control miR-302d IH + siR-CDKN1A##100000 80000 60000 40000 20000 ## # #2 Days2 Days120 100 Cell viability ( ) 80 60 40 20 0 0 100Cell viability ( ) miR-control + siR-control miR-control + siR-CDKN1A miR-302d + siR-control miR-302d + siR-CDKN1A120 100 80 60 40 20 0miR-control + siR-control miR-control + siR-CDKN1A miR-302d + siR-control miR-302d + siR-CDKN1A five SIN-1 (mM)CoCl2 (M)Relative mRNA expression (Fold alter)25 20 15 10 5Relative mRNA expression (Fold adjust)CDKN1A miR-control miR-302d20 15CDKN1A miR-cont.
