The Study Of MiR-15a And MiR-16-1 Inhibiting The Growth Of Raji Cells And Enhancing Ara-C Sensitivity

Objective:To Study the growth inhibitory effect of miR-15a/16-1 to Raji cells,and to observe weather miR-15a/16-1 could ehance the sensitivity of the Raji cells to Ara-C.Methods:synthesizing miR-15a/16-1 oligonucleotides and pre-miR-15a which was contructed in plasmid vector,and then extracting plasmids from E.coli.we made Random sequence and the negative control plasmid as control,and transfected oligonucleotides or plasmids into Raji cells with Lipofectamine^TM 2000,which were respectively combined with Ara-C.GFP Expression in cells was detected by fluorescent microscopy;Flurecence quantitive PCR detected miR-15a levels in Raji cells;Bcl-2 protein expression was detected by indirect immuno- fluorescence;Semi-quantitative RT-PCR detected the expression level of Bcl-2 mRNA; The apoptotic cells were observed by Hoechst Dyeing;AnnexinV/PI double dyeing method or PI dyeing method was used to detect the cell apoptotic rate by Flow Cytometry(FCM).The growth inhibitory effect of Raji cells was measured by trypan blue dye exclusion method and CCK8 assay.Results:Eukaryotic expression vector of pre-miR-15a was successfully constructed,and the consequence of recombinant clones by Polymerase Chain Reaction and sequences analysis were both coinsided with target sequences.The electrophoresis of plasmid extracting from E.coli demonstrated that,the pre-miR-15a expression plasmid and negative control plasmid were about 7.5kb which were coinsided with expriment assume.Many green fluorescent cells were observed under fluorescent microscopy.The expression level of miR-15a was obviously increased in pre-miR-15a group,which was much more than blank group and negative control group;Bcl-2 protein expression levels obviously decreased at miR-15a/16-1 group(or pre-miR- 15a group), but which at the other groups did not change.The levels of Bcl-2 mRNA at every group had no obviously difference.Trypan blue dye exclusion method and CCK8 assay showed that transfection of mir-15a/16-1 oligonucletides or pre-miR-15a expression plasmid decreased the cell growth at 24,48 and 72 h post-transfection,apoptotic cells can be seen with Hoechst Dyeing. Anexxin V/PI double dying assays by FCM indicated that the cell apoptotic rates in earlier period and advanced stage of mir-15a group were 9.74%and 9.65%,and mir-16-1 group were 9.70%and 9.34%,which were obviously higher than blank group and control group,and the cell apoptotic rates in earlier period of the latters were 0.7%and 1.73%,in advanced stage's were 2.18%and 1.54%.PI dying assays by FCM indictaed that the cell apoptotic rate of pre-miR-15a group was 12.43%,which was obviously higher than blank group(1.26%) and negative control group(4.69%).miR-15a/16-1 oligonucletides or pre-miR-15a expression plasmid combined with Ara-C significantly decreased the IC50.IC50 at miR-15a group and miR-16-1 group were 10.41and 10.86,which were obviously lower than blank group and control group,the latters were 15.43 and 14.92.IC50 at pre-miR-15a group was 10.02,which was obviously lower than blank group and negative control group,the latters were 15.37 and 15.14.Trypan blue dying exclusion method and CCK8 assay showed that miR-15a/16-1+Ara-C group(pre-miR-15a +Ara-C group) obviously decreased the cell growth than miR-15a/16-1 group,Ara-C group and random sequence+Ara-C group(or pre-miR-15a group,Ara-C group and negative control+Ara-C group), Plenty of apoptotic cells can be seen with Hoechst dyeing.AnnexinV/PI double dying assays by FCM indicated that the cell apoptotic rates in earlier period and advanced stage of mir-15a+Ara-C group were 20.93%and 25.27%,and miR-16-1+Ara-C group were 18.69%and 23.13%,which were obviously higher than miR-15a group,miR-16-1 group,Ara-C group and control group,and the cell apoptotic rates in earlier period of the latters were 6.99%,4.73%, 10.88%and 14.39%,in advanced stage's were 10.08%,10.64%,11.83%and 11.93%o PI dying assays by FCM indictaed that the cell apoptotic rate of pre-miR-15a+Ara-C group was 32.56%, which was obviously higher than pre-miR-15 group,Ara-C group and negative control+ Ara-C group,The cell apototic rates of the latters were 12.24%,20.87%and 21.76%。Conclusion:The miR-15a/16-1 oligonucletides and pre-miR-15a expression plasmid can induce Raji cells apoptosis,inhibit Raji cells growth,And enhance Ara-C-induced apoptosis inRaji cells.