Micrornas - 21 Through Pten/akt Pathway Regulating Human Hepatic Stellate Cells The Expression Of Matrix Metalloproteinases - 2

ObjectiveOur study was to investigate whether microRNA-21regulates the mRNA level of matrix metalloproteinase2(MMP-2) in hepatic stellate cells and the activity of MMP-2in cell culture supernatant. Meanwhile, the PTEN/Akt signaling pathway involved in the above process was also explored.Methods1. Treated LX-2cells with5ng/ml PDGF-BB1) Expression of microRNA-21and MMP-2mRNA were analyzed by real-time PCR.2) Activity of MMP-2in cell culture supernatant was detected by zymography.2. Transfected LX-2cells with pre-microRNA-21or anti-microRNA-21at a finalconcentration of50nM1) Detected the microRNA-21and MMP-2mRNA levels by real-time PCR.2) Observed the activity of MMP-2in cell culture supernatant by zymography.3) Observed the expression of PTEN protein in the above different groups by western blot.3. Blocked the PI3K-Akt signaling pathway in the LX-2cells1) Expression of Akt and P-Akt protein in different groups were observed by western blot.2) mRNA level of MMP-2in different groups was analyzed by real-time PCR.3) Activity of MMP-2in cell culture supernatant in different groups was detected by zymography.Results1. The expression levels of microRNA-21and MMP-2mRNA in PDGF-BB treated group were significantly higher than the control group (P<0.05). Activity of MMP-2in cell culture supernatant of PDGF-BB treated group was higher than that of the control one.2.1) Comparing with negative control group, the expression levels of microRNA-21and MMP-2mRNA were upregulated in pre-microRNA-21transfected group, that were similar to the stimulation effect of PDGF-BB (P<0.05); However, transfected anti-microRNA-21into LX-2cells reduced the activity of MMP-2upregulated by PDGF-BB (P<0.05). The trend of activity of MMP-2in cell culture supernatant in different groups was in parallel with the aforementioned mRNA levels.2) Comparing with negative control group, the expression of PTEN protein was downregulated in the pre-microRNA-21transfected group, while upregulated in the anti-microRNA-21transfected group. PDGF-BB acted in synergy with pre-microRNA-21, nevertheless, opposite of anti-microRNA-21.3.1) Enhanced expression of P-Akt were found in PDGF-BB treated group comparing with LY294002+PDGF-BB treatment group. LY294002could significantly dropped the stimulus effect of pre-microRNA-21on P-Akt. The total levels of Akt were no significant difference in different groups.2) The mRNA level of MMP-2was significantly downregulated in group cotreated with LY294002+PDGF-BB than group treated with PDGF-BB alone (P<0.05); microRNA-21overexpression mimicked PDGF-BB mediated effect (P<0.05). The similar effect on MMP-2activity in the above-mentioned groups was observed.ConclusionsPDGF-BB upregulates the expression of microRNA-21in LX-2cells, microRNA-21mediates the upregulation of MMP-2mRNA and the activity of extracellular MMP-2via the PTEN/Akt signaling pathway.