Changes Of MicroRNA And Inflammatory Factors In Human Peripheral Blood Mononuclear Cells Induced By MC-LR And Their Correlation Study

Purposes: To investigate the effect of microcystin-leucine-arginine(MC-LR)on the expressions of micro RNA(mi RNA)and pro/anti-inflammatory cytokines in human peripheral blood mononuclear cells(PBMCs)and to further explore the possible mechanism of MC-LR causing inflammatory response.Methods: In this study,human PBMCs were used as experimental objects.ELISA was used to detect the content of Microcystins(MCs)in serum samples of hospital physical examination subjects.According to the detection results,the samples are divided into high exposure group(100-150 ng/m L),medium exposure group(20-30ng/m L),and low exposure group(4-6 ng/m L),and the inflammatory factor tumor necrosis factor-alpha(TNF-α),Interleukin-1beta(IL-1β),IL-6,IL-4,and IL-10 in each group were detected.Fresh peripheral blood of these selected samples were collected again at a later stage for PBMC extraction and q RT-PCR was used to detect the expression of hsa-mi R-146a-5p.The correlation between them was analyzed by Spearman correlation analysis.In vitro experiments,PBMCs were stimulated with MC-LR to establish an in vitro cell inflammation model,and the exposure concentration of MC-LR was determined by cell Counting Kit-8(CCK-8).The expression of inflammatory factors(TNF-α,IL-10,IL-4,IL-6,IL-1β)in the supernatant of the cell culture medium were detected by ELISA to explore whether MC-LR exposure caused inflammatory response.Meanwhile,total RNA was extracted and the expression of related mi RNAs was detected by q RT-PCR.Statistical analysis was used to analyze whether there was statistical correlation between the expression of inflammatory factors and mi RNAs.Finally,the statistical association was verified by in vitro transfection of mi RNA inhibitors.Results: In population samples,the expression of inflammatory factors and mi R-146a-5p were increased with increasing MCs concentration(P< 0.05).Meanwhile,correlation analysis results showed a high positive correlation between them.In vitro experiments,the exposure concentrations of MC-LR were 0,0.1,1.0,10μg/m L,and the exposure times were 6,24 h.The expressions of hsa-mi R-146a-5p,hsa-mi R-21-3p and hsa-mi R-150-3p were increased with the increasing of exposure time when any exposure concentration.However,the expression of hsa-mi R-155-3p was increased with the increasing of exposure time only at 1.0 and 10.0 μg/m L groups(P<0.05);When the exposure time was fixed,the expressions of hsa-mi R-21-3p,hsa-mi R-150-3p and hsa-mi R-155-3p were only increased in the 10.0 μg/m L dose group compared with the control group no matter at 6h or 24 h.However,the expression of hsa-mi R-146a-5p was increased in the 10.0 μg/m L dose group compared with the control group at 6h,but it was gradually increased with the increasing of dose at 24h(P<0.05).For inflammatory cytokines,ELISA results were consistent with q RT-PCR results.At a given dose,the expressions of TNF-α,IL-1β,IL-6 and IL-4 were increased with the increase of exposure time,while the expression of IL-10 was increased with the increase of exposure time in 0.1 and 1.0μg/m L groups,and decreased with the increase of exposure time in 10 μg/m L group;At a given time 6h,only TNF-α expression was increased in 10.0 μg/m L dose group compared with the control group.At 24 h,the expressions of IL-1β,IL-6 and IL-4 were increased significantly in 1.0 and 10 μg/m L dose groups compared with the control group,and the expression of TNF-α was increased significantly only in 10 μg/m L dose group compared with the control group.The expression of IL-10 was significantly increased in 0.1 and 1.0 μg/m L groups compared with control group(P<0.05).Correlation analysis showed that the expression of each mi RNA was positively correlated with the expressions of various inflammatory factors to varying degrees.The intracellular transfection of mi RNA inhibitors experiments found that the addition of a mi RNA inhibitor could inhibit the expression of some inflammatory factors,further proving the correlation between the two(P<0.05).Conclusions:1.MC-LR can induce changes in the expression of mi R-146a-5p,mi R-21-3p,mi R-150-3p,mi R-155-3p and inflammatory factors TNF-α,IL-10,IL-4,IL-6 and IL-1β in PBMC cells;2.The expressions of mi R-146a-5p,mi R-21-3p and mi R-150-3p were positively correlated with the expressions of TNF-α,IL-10,IL-4,IL-6 and IL-1β to varying degrees;3.mi R-146a-5p、mi R-21-3p、mi R-150-3p may play a certain positive regulatory role in the changes of inflammatory factors caused by MC-LR.