| Objective:This study by using sodiumvalproic acid in vitro culture of multiple myeloma cell lines RPMI8226 and U266,combined with a variety of autophagy flux detection methods,observe the two cell lines of autophagy flux,thus accurately reflects the overall process of autophagy,explore autophagy process involved in multiple myeloma diseases occurrence and progress of molecular biology mechanisms,as valproic acid sodiumis used to provide theoretical basis for clinical treatment.Methods:Multiple myeloma cell lines RPMI8226 and U266 were cultured and logarithmic growth phase cells were taken for follow-up experiments.The experimental cells were divided into 4 groups and treated with different treatments: blank control group,sodiumvalproate group(8mmol/L),chloroquine group(8ug /ml),and combination of the two drugs.Cell count kit 8(CCK8)was used to detect the inhibition of cell proliferation during 6h,12 h,24h and 48 h of drug treatment.According to the inhibition rate,the drug treatment time was selected as 24 hours to complete the follow-up experiments.The mRNA expression levels of autophagy signature protein LC3 B were analyzed by real-time PCR.Western Blot was used to verify the LC3 B turnover assay,namely,the expression of LC3 B protein in different drug treatment groups.The CYTO-ID autophagy assay kit was used for fluorescence staining,and the average fluorescence intensity of the autophagy-positive cells in each group was detected by flow cytometry.The changes of autophagosome formation and autophagy flow in monomer red fluorescentprotein(mRFP)-greenfluorescentprotein(GFP)-microtubule-associat ed protein light chain 3(LC3)after transfection with dual standard autophagy adenovirus in RPMI8226 and U266 cells were observed by fluorescence microscopy.Results:1 Proliferation inhibition of human multiple myeloma RPMI8226 and U266 cell lines in different experimental treatment groups was detected by cck-8 method: proliferation inhibition rate of the above MM cell lines was detected after different drug treatments at different times(6,12,24 h,48h),and the proliferation inhibition rate of the drug increased with the extension of the duration of action(P<0.05).The duration of drug action was 24 hours,and the observation of autophagy was better.2 mRNA and protein expression of the signature molecule LC3 of autophagy: real-time quantitative rt-qpcr indicated that the mRNA and protein expression of LC3 B of RPMI8226 and U266 cells were compared with that of the blank control group(1.00±0.00,1.00±0.00)after 24 hours of action in the 8mmol/L VPA group,8mug /ml CQ group and the combination of the two drugs,and the differences were statistically significant(all P values were <0.05).The cyto-ID autophagy assay kit was used for fluorescence staining,and the average fluorescence intensity of the autophagy-positive cells in each group was detected by flow cytometry.The average fluorescence intensity of autophagy positive cells in the combination group was significantly higher than that in other groups.Monomer red fluorescent protein(mRFP)-green fluorescent protein(GFP)--microtubule-associated protein light chain 3(LC3)autophagy double-labeled adenovirus transfection experiment suggested that the total number of spots in the VPA group was increased compared with the blank control group,and the number of red spots was significantly increased,while the number of yellow spots was increased compared with the control group.Conclusion:1 Valproic acid induces the generation of autophagy flux,causes the increase of autophagosomes,and activates the autophagy process.2 Valproic acid can inhibit the proliferation of multiple myeloma cell lines RPMI8226 and U266,and the induction of increased autophagy expression in multiple myeloma cell lines may be one of the molecular basis for valproate to play an anti-tumor role. |
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