| Liver fibrosis is a chronic liver disease caused by sustained stimulation of the factors, such as virus, toxic, drug, alcohol, immunization, et al. The sustained stimulation can induce excessive deposition of extracellular matrix(ECM), which is the basis of liver fibrogenesis. Meanwhile, the activation of hepatic stellate cell(HSCs) plays key role in liver fibrogenesis. HSCs usually stays in a quiescent statement in normal liver, but can be activated when liver suffers from stimulation of various pathogenic factors. This activation of HSCs induces a series of changes including disappearance of the large lipid droplets, acceleration of cell proliferation and increased deposition of collagens. Meanwhile, the activation of HSCs is often accompanied by an autophagic flux, and autophagy inhibitors can, to some extent, suppress the transformation of HSCs to myofibroblast.Autophagy is a self-eat phenomenon that widely exists in biological world. When cells suffer from starvation or stress, some dysfunctional or overproduced organelles are endocytosed by autophagosome which in succession fused with lysosome to form autolysosome. The organelles are degraded in autolysosome to provide energy to deal with foreign stress.mi RNA is a small non-coding single-stranded RNA. mi RNA can inhibit the translation of its target gene by binding with 3′- UTR of target m RNA via complementary base pairing, and hence regulate the expression of the genes in post-transcriptional level. Up to now, 35828 of mi RNAs have been identified,which regulate almost one third human genes expression.The biological role of mi RNA-30 is very wide. Recently, many studies have shown that mi RNA-30 can regulate cell proliferation, differentiation and immune. mi RNA-30 involves in the regulation of the body’s physiological development, growth, aging, and plays a very important role in regulating the occurrence and developent of human disease, especially in cancer. mi RNA-30a-5p is a member of the mi RNA-30 family. It has been proved that Beclin-1, an autophagy related protein, is one of the targets of mi RNA-30a-5p.mi RNA-30a-5p can inhibit autophagy in tumor by suppressing the expression of Beclin-1.At present, few studies focused on the relationship between mi RNA-30a-5p and liver fibrosis. This project aims to find out the change of autophagy and activation of HSCs induced by ectopic expression of mi R-30a-5p, and finally determines the function of mi R-30a-5p in liver fibrosis.Objective: To investigate the effects of mi R-30a-5p on HSCs autophagy and activation, and establish the relationship between mi R-30a-5p and liver fibrosis.Methods: Enhanced or reduced expression of mi R-30a-5p in HSCs was obtained by mi R-30a-5p mimic transfection or mi R-30a-5p inhibitor stimulation, respectively, and then autophagy and activation of HSCs was evaluated. PDGF-BB, an autophagy inducer, or 3-MA, an autophagy inhibitor was combined used with mi R-30a-5p to set up the relationship among mi R-30a-5p, HSCs autophagy and activation. Real-time PCR and western blot assay were performed to determine m RNA and protein expression respectively.The occurrence of HSCs autophagy was determined by acridine orange(AO)staining, and LC3 was detected by immunofluorescence.Results:(1) Western blot was used to detect the protein expressions ofα-SMA, Collagen-I, Beclin-1 and the relative expression of LC3 BII to LC3 BI after PDGF treatment with different concentrations. The result showed that the expression of α-SMA in 20ng/ml group was significantly increased than other groups(0.86±0.02, 0.95±0.04, 1.5±0.14 and 1.00±0.09)(P<0.05) and the same result was observed in Beclin-1(0.14 ± 0.02, 0.17 ± 0.02, 0.40 ± 0.03 and 0.24 ±0.01)(P<0.05), Collagen-I(0.39± 0.02, 0.41±0.02, 0.51±0.02 and 0.42±0.02)(P<0.05), LC3BII/LC3BI(3.22±0.38, 3.88±0.30, 8.65±0.31 and 4.41± 0.43)(P<0.05). These results demonstrated that 20 ng/ml of PDGF is the most effective concentration to induce HSCs autophagy and activation. Therefore, we use 20 ng/ml as the optimal PDGF intervention concentration in the following experiments.(2) Western blot was used to detect the protein expressions of α-SMA and Beclin-1 after PDGF treatment with different time. The results showed that the expression of α-SMA in 12 h treatment group was significantly increased than other groups(0.70 ± 0.03,0.35 ± 0.03 and 0.25 ± 0.02)(P<0.05), the expression of Beclin-1 had the same change(1.35±0.21, 0.62±0.04 and 0.30±0.02)(P<0.05). Therefore,we used 12 h as the optimal PDGF intervention time point in the following experiments.(3) Real-time PCR was used to detect the expression of mi R-30a-5p. The results showed that the expression of mi R-30a-5p in PDGF treatment group was significantly reduced as compared with control group(0.34±0.02 vs.1.00±0.00)(P<0.05).(4) Real-time PCR was used to detect the transfection efficiency in different mi R-30a-5p mimic concentration groups. According to the results, mi R-30a-5p mimic with concentration of 100nmol/L has highest transfection efficiency. Thus, in the following experiments,we use 100 nmol/L as the mi R-30a-5p mimic intervention concentration.(5)Western blot was used to detect the protein expressions among control group,mi R-30a-5p mimic group, PDGF treatment group, and mi R-30a-5p mimic combined with PDGF treatment group. The results showed that the expression of Collagen-I, Beclin-1 and the relative expression of LC3BII/LC3 BI were significantly decreased in mi R-30a-5p mimic group(1.39±0.20 vs. 2.89 ± 0.08, 10.13 ± 0.56 vs.13.19 ± 0.44, 0.15 ± 0.04 vs.0.80 ± 0.08)(P<0.05), the expression of P62 was increased in mi R-30a-5p group(1.21±0.13 vs.0.97 ± 0.01)(P<0.05). Furthermore, PDGF treatment enlarged the expression difference above. Real-time PCR was used to detect the m RNA expression of α-SMA, PDGFR-β, DDR2 and the results were consistent with western blot.(6) Real-time PCR was used to detect the transfection efficiency in different mi R-30a-5p inhibitor concentration groups. According to the results, 50 nmol/L of mi R-30a-5p inhibitor has highest transfection efficiency.Thus, in the following experiments, we use 50 nmol/L as the mi R-30a-5p inhibitor intervention concentration.(7) Western blot was used to detect the protein expressions between control group and mi R-30a-5p inhibitor group,The results showed that the expression of α-SMA, Collagen-I, Beclin-1 were significantly increased in mi R-30a-5p inhibitor group(0.07 ± 0.01 vs.0.03 ±0.01, 0.14 ± 0.06 vs.0.08 ± 0.02, 0.13 ± 0.03 vs. 0.07 ± 0.01)(P<0.01).(8)Acridine orange staining was used to detected the acidic autophagy vesicles.The results showed that the pre area of red fluorescence in mi R-30a-5p mimic group was significant lower than control group. The difference was more obvious after PDGF treatment. Instead, the pre area of red fluorescence in mi R-30a-5p inhibitor group was significant higher than control group.(9) LC3 expression was detected by immunofluorescence. The results showed that the green fluorescence in mi R-30a-5p mimic group was lower than the control group. The location showed that LC3 was diffused distributed in cytoplasm.(10) Western blot was used to detect the expression of Collagen-I and Beclin-1after 3-MA treatment. The result showed that the expression of Collagen-I and Beclin-1 in 3-MA group were significant lower than the control group.(11)Western blot was used to detect the expression of α-SMA, P62 and the relative expression of LC3BII/LC3 BI among control group, mi R-30a-5p inhibitor group, 3-MA treatment group and mi R-30a-5p inhibitor combined with 3-MA treatment group. The result showed that the expressions of α-SMA and the relative expression of LC3BII/LC3 BI in mi R-30a-5p inhibitor group were significant higher than the control group(3.05 ±0.17 vs.1.93± 0.08, 0.06 ±0.00 vs.0.04 ± 0.00)(P<0.05), while the expression of P62 lower than the control group(1.40±0.49 vs.3.20±0.30)(P<0.01). However, no significant difference was observed in 3-MA treatment group.Conclusion: mi R-30a-5p can attenuated HSCs autophagy by specifically targeting on autophagy-relate gene, Beclin-1, which further inhibited HSCs activation. |
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