The Role And Possible Mechanism Of MiR-148a On Cell Proliferation Of MMC Cells Induced By HMGB1

Objective: To investigate the role and possible mechanism of miR-148aon cell proliferation of MMC cells induced by HMGB1and analyse thepossible target gene.Methods:1Mesangial cells in mice (MMC) were used for this study, and wererandomly divided into six groups:1. normal group;2. HMGB1group;3.miR-148a mimic+HMGB1group;4. pre-miR+HMGB1group;5. miR-148ainhibitor+group;6. anti-miR+HMGB1group. Western Blot was used to detectthe expression of PTEN, p-Akt-S473, Akt, PCNA, CyclinD1, CDK4and p16protein. Immunocytochemistry technique was used to detect expression ofPCNA protein and immunofluorescence staining was used to detect BrdUexpression.2Mesangial cells in mice (MMC) was used and divided into four groups:1. normal group;2. HMGB1group;3. miR-148a mimic+HMGB1group;4.PTEN-WT+HMGB1group;5. miR-148a mimic+PTEN–WT+HMGB1group;6. NC+pre-miR+HMGB1group. Western blot was used to detcet expressionof PCNA protein.Results:1miR-148a mimic reduced the expression of PTEN protein in MMC andmiR-148a inhibitor upregulated the expression of PTEN protein in MMCinduced by HMGB1The results of western blot showed that the expression of PTEN proteinin miR-148a mimic+HMGB1group was significantly lower (0.35±0.028),and significantly higher in miR-148a inhibitor+HMGB1group (1.06±0.045), compared with HMGB1group (P <0.05). But there was no significantdifference between HMGB1group and pre-miR+HMGB1group (P<0.05). (Fig.1-2, Table1)2miR-148a mimic upregulated the expression of p-Akt-S473protein,while miR-148a inhibitor downregulated the expression of p-Akt-S473proteinin MMC induced by HMGB1After HMGB1stimulates MMC20minutes, the results of western blotshowed that the expression of p-Akt-S473protein in miR-148a mimic+HMGB1group was significantly higher (1.085±0.067) compared withHMGB1group (P <0.05). But there was no significant difference betweenHMGB1group and pre-miR+HMGB1group, and the expression ofp-Akt-S473in both group was higher than that in normal group(P <0.05; Fig.3，Table2). The expression of p-Akt-S473protein in miR-148a inhibitor+HMGB1group was significantly lower (0.467±0.013) than that in HMGB1group (P <0.05). However, the levels of total Akt among six groups were notsignificantly different.(Fig.4, Table2)3miR-148a mimic increased the proliferation of MMC induced byHMGB1By immunocytochemistry and immunofluorescence staining, the levelsof BrdU and PCNA protein in HMGB1group were both higher than that incontrol group, and the protein PCNA and the positive expression rate of BrdUin miR-148a mimic+HMGB1group were more significantly higher than thatin HMGB1group, the difference was not significantly different.（Fig.5，Fig.6）The results of western blot showed that the expression of PCNA proteinin miR-148a mimic+HMGB1group was significantly higher (1.04±0.045)compared with HMGB1group (P <0.05), and there was no significantdifference between HMGB1group and pre-miR+HMGB1group (P>0.05).(Fig.7, Table3)4miR-148a inhibitor reduced the expression of PCNA protein in MMCinduced by HMGB1The result of western blot showed that the expression of PCNA protein inmiR-148a inhibitor+HMGB1group was significantly lower （0.50± 0.035)compared with HMGB1group (P <0.05). In addition the expression ofPCNA protein in HMGB1group and anti-miR+HMGB1group were morehigher than that in normal group(P<0.05).(Fig.8, Table3)5miR-148a mimic upregulated the expression of CDK4and Cyclin D1protein, while miR-148a inhibitor reduce the expression of CDK4and CyclinD1protein in MMC induced by HMGB1The results of western blot showed that the expression of protein CDK4and Cyclin D1in miR-148a mimic+HMGB1group were significantly higher(1.08±0.024,0.89±0.025) compared with HMGB1group (P <0.05; Fig.7,Table3). While the expression of protein CDK4and Cyclin D1in miR-148ainhibitor+HMGB1group was significantly lower (0.19±0.016，0.48±0.013)than that in HMGB1group (P <0.05; Fig.8, Table3)6miR-148a mimic reduced the expression of p16protein in MMC, whilemiR-148a inhibitor can upregulate the expression of p16protein in MMCinduced by HMGB1After HMGB1stimulates MMC8hours, the results of western blotshowed that the expression of protein p16in miR-148a mimic+HMGB1group was significantly lower (0.32±0.019) compared with HMGB1group(P <0.05; Fig.7, Table3). While the expression of protein p16in miR-148ainhibitor+HMGB1group was significantly higher (0.84±0.02) than that inHMGB1group (P <0.05; Fig.8, Table3).7Transfection of miR-148a mimic and PTEN-WT at the same time,reduced the expression of PCNA protein in MMC induced by HMGB1Western blot showed that level of PCNA protein in PTEN-WT groupwere significantly lower (0.22±0.02) than that in HMGB1group (P <0.05).Once transfected with miR-148a mimic and PTEN-WT at the same time, theexpression of PCNA protein was statistically higher than that in PTEN-WTgroup(0.64±0.02; P <0.05; Fig.9, Table4).Conclusion: miR-148a might trigger the activation of the PI3K/Aktpathway by down-regulating the expression level of PTEN, thereby promotedCyclin D1activation and the proliferation of the MMC cells induced byHMGB1.