| Objective This study was aimed at investigating aberrant expression of JARID1 B and mi R-137,studying the functions of JARID1 B and mi R-137 in ALL,and primary discussing the possible mechanism,to provide a new target for treating ALL.Method(1)Western blot analysis of H3K4me3,H3K4me2,JARID1 B protein expression and q RT-PCR analysis of JARID1 B,mi R-137 m RNA expression were performed in 30 cases with ALL patients and 6 ALL cell lines(MOLT-4?CA46?Daudi?Jurkat?NALM-6?BALL-1),peripheral blood specimens from 12 healthy volunteers as the control.(2)JARID1B sh RNA or JARID1 B GFP was transfected to both MOLT-4 and BALL-1 cells to knockdown or promote the JARID1 B gene to study its function in ALL.q RT-PCR analysis JARID1 B m RNA expression,Western blot analysis analysis JARID1 B protein expression.The viability of cells was observed by MTT method.Apoptotic rate were measured by flow cytometry.(3)mi R-137 mimic or mi R-137 inhibitor was transfected to both MOLT-4 and BALL-1 cells to knockdown or promote the mi R-137 to study its function in ALL.q RT-PCR analysis JARID1 B m RNA expression,Western blot analysis analysis JARID1 B protein expression.The viability of cells was observed by MTT method.(4)Bioinformatics softwares were applicated to the mi RNA regulating JARID1 B,JARID1B wild-type3'UTR Luciferase plasmid(JARID1B-3'UTR-WT),and JARID1 B Mutant 3'UTR Luciferase plasmid were constructed(JARID1B-3'UTR-Mut),And transfected into MOLT-4 and BALL-1 cells,Luciferase activity was detected for target gene validation.(5)JARID1B interference and restore expression experiments was employed.Cotransfecting JARID1B-GFP with mi R-137 mimic,and cotransfecting JARID1B-sh RNA with mi R-137 inhibitor into BALL-1 cells,to test the effects on cell proliferation and apoptosis,to further verificate the relationship between mi R-13 and JARID1B.Results(1)Compared with healthy volunteers,The expression of H3K4me3 and H3K4me2 was significantly lower(P<0.0001),JARID1 B protein and m RNA was higher(P<0.0001),mi R-137 m RNA was lower in ALL patients and ALL cell lines,mi R-137 was significantly negative correlated with JARID1B(patients R=0.7277,P<0.0001;cell lines R=0.9004,P<0.0001).(2)JARID1B sh RNA reduce the expression of JARID1 B both m RNA and protein,Increase histone H3K4me3 and H3K4me2 levels,Inhibit cell proliferation,Induce apoptosis in MOLT-4 cell and BALL-1 cell,Compared with the control group,the difference was statistically significant,P<0.0001.JARID1B-GFP increase the expression of JARID1 B both m RNA and protein,Reduce histone H3K4me3 and H3K4me2 levels in MOLT-4 cell and BALL-1cell;Compared with the control group,Promote cell proliferation(P<0.0001),Inhibit apoptosis in BALL-1 cell(P=0.0325),But there was no effect on cell proliferation in MOLT-4(P=0.6646),and.no effect on cell apoptosis(P=0.2259).(3)mi R-137 mimic reduce the expression of JARID1 B m RNA and protein,Inhibit cell proliferation in MOLT-4 cell and BALL-1 cell,Compared with the control group,the difference was statistically significant,P<0.0001.mi R-137 inhibitor increase the expression of JARID1 B protein in MOLT-4 cell and BALL-1 cell,Promote cell proliferation in BALL-1 cell(P<0.0001),But there was no effect on cell proliferation in MOLT-4(P=0.5832).(4)mi Randa predicted JARID1 B may be a target genes of mi R-137.Luciferase Reporter Assay showed that,Compared with the control group,Luciferase gene activity can be suppressed by cotransfection of mi R-137 mimic and Wild-type JARID1B-3'UTR-WT(MOLT-4 P=0.0069;BALL-1 P=0.0002),but not by cotransfection with mutation type JARID1B-MT-3'UTR.(5)Over-expression JARID1 B partially cancelled effect of mi R-137 mimic on inhibiting proliferation and inducing apoptosis in BALL-1 cells;Down-regulated JARID1 B partially rescued mi R-137 inhibitor in promoting proliferation and inhibiting apoptosis in BALL-1.Conclusions(1)Low levels of histone H3K4me3 and H3K4me2 in ALL.(2)JARID1B protein and m RNA was high expressed in ALL,Increase the expression of JARID1 B can inhibit the proliferation and promote apoptosis of leukemia cells,and reducing its expression can promote cell proliferation and inhibit apoptosis,functions as carcinogenesis.(3)mi R-137 was loe expressed in ALL,functions as anti-oncogene.(4)JARID1B is a target gene of mi R-137,mi R-137 effect on cell proliferation and apoptosis by JARID1 B in ALL. |
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