| BackgroundHepatocellula carcinoma is one of the common cancers worldwide. Hepatic fibrosis is the excessive repair process during all kinds of chronic liver injury diseases. Hepatic fibrosis is also known as a bidirectional course of disease, which would be reversed to normal construction and function if diagnosed and treated early enough, otherwise, it would develop into liver cirrhosis and carcinoma. The pathogenesis of hepatic fibrosis is a very complicated process associated with diverse pathological changes, among which the activation of hepatic stellated cell(HSC), leading to change of phenotype, becomes a common pathway that contributes to the progression of the disease.MiRNAs are a class of endogenous non-coding RNA molecules that are 20-25 nucleotides in length. MiRNAs are highly conserved molecules characterized by tissue specificity and chronology. MiRNA recruits a silence complex to repress or degrade target m RNA by integrated or un-integrated complementary binding its 3’-untranslated region(3’-UTR), which regulates cell growth, proliferation, differentiation and apoptosis. Recently, several studies reported miRNAs could regulate the activation and proliferation of HSCs through ROS pathway, lipid metabolism and peroxisome proliferators-activated receptors(PPARs) signaling pathway during hepatic fibrosis process.Our previous study showed that miR-34 a could be significantly increased after the mouse hepatic fibrosis was induced by dimethylnitrosamine with microarray analysis. Meanwhile, we speculated that Acsl1 might be a potential target of miR-34 a by using bioinformatics analysis. Therefore, this study aims to detect the expressions of miR-34 a and ACSL1 in clinical hepatic fibrosis specimens and HSCs, and to verify the targeting regulation between miR-34 a and Acsl1 by using luciferase assay, to further reveal the effect of miR-34a/Acsl1 lipid metabolic pathway on the phenotype of HSCs. Furthermore, this study also aims to understand the role of miR-34 a targeting Acsl1 in the development of hepatic fibrosis so as to provide a novel treatment strategy in future.Part 1 the expressions of miR-34 a and functional study in hepatic fibrosisObjective To detect the expressions of miR-34 a in clinical hepatic fibrosis specimens and HSCs. To analyze the correlation of miR-34 a with hepatic fibrosis and HSCs activation.Methods 1. QRT-PCR was used to detect the expressions of miR-34 a in clinical hepatic fibrosis specimens of different stages which were determined with HE, Masson and VG staining. 2. Primary rat HSCs were isolated from male SD rats. Then Trypan blue staining, autofluorescence and double immunofluorescence experiments were used to verify cell viability, cell purity and cell status. QRT-PCR was used to detect the expressions of miR-34 a in HSCs.Results1. Clinical hepatic fibrosis specimens were divided into five stages via HE, Masson and VG staining according to METAVIR standard. Compared with F0 stage, miR-34 a expressions of F1, F2, F3 and F4 stage were increased by 6.2 times, 21.3 times, 32.4 times and 39.8 times respectively, P < 0.05.2. Trypan blue staining and autofluorescence experiments were used to identify that the cell purity and viability of primary HSCs were both above 95%. Double immunofluorescence experiments verified that HSCs were quiescent during the first 2 days and activated after 14 days. Compared with quiescent HSCs(q HSCs), miR-34 a expressions were 48.6 times in semi-activated HSCs and 192.0 times in activated HSCs(a HSCs), P < 0.05.Conclusions1. miR-34 a expressions were increased along with hepatic fibrosis course.2. miR-34 a expressions were increased along with HSCs activation.Part 2 Prediction and identification of miR-34 a target geneObjective To analyze and screen miR-34 a target m RNA via gene bioinformatics technology. To validate the targeted relationship between miR-34 a and Acsl1. To detect the expressions of ACSL1 in clinical hepatic fibrosis and HSCs at last.Methods1. The target genes were predicted with rno-miR-34a-5p as a key word in micro RNA.org、Pic Tar、Target Scan、miRDB and miRbase database, and then gene ontologe(GO) analysis and pathway analysis were performed with Gene Ontology database and KEGG database respectively. Combined miRNA-m RNA expression profile derived from previous study in hepatic fibrosis animal models, miRNA-Gene-Network was set up and miR-34a-5p target m RNA screening was done.2. Acsl1 Wild Type(WT) plasmid and Acsl1 mutation(MUT) plasmid were construed by using p MIR-REPORT vector. Then a dual-luciferase reporter assay was performed to verify the direct effect of miR-34 a on 3’-UTR of Acsl1.3. QRT-PCR, Western Blot and Immunohistochemical staining were used to detect the expressions of ACSL1 in clinical hepatic fibrosis specimens and HSCs.Results1. The target genes were predicted with rno-miR-34a-5p as a key word in micro RNA.org、Pic Tar、Target Scan、miRDB and miRbase database, the top 50 selecting target genes were selected and then gene ontologe(GO) analysis and pathway analysis were performed with Gene Ontology database and KEGG database respectively. Combined miRNA-m RNA expression profile derived from previous study in hepatic fibrosis animal models, we found that Acsl1 was located in the centre of miRNA-Gene-Network and was regulated by miR-34a、miR-34b、miR-34c、miR-19a and miR181 a. Acsl1 may be the target m RNA of miR-34 a.2. Acsl1 WT plasmid and Acsl1 MUT plasmid were constructed by using p MIR-REPORT vector according to the predicted miR-34 a binding site on the 3’-UTR of the Acsl1 m RNA. A dual luciferase reporter system was performed to verify the direct effect of miR-34 a on 3’UTR of Acsl1.Then, HEK293 cells were transfected with either miR-34 a inhibitor or NC versions of each constructed plasmid. MiR-34 a inhibitor significantly raised luciferase reporter activity in HEK293 cells transfected with the WT constructive luciferase reporter plasmid, P < 0.05. However, there were no changes in HEK293 cells transfected with the MUT constructive luciferase reporter plasmid, P > 0.05. The results indicated that miR-34 a specifically bind the 3’-UTR of ACSL1.3. ACSL1 m RNA expressions in F1, F2 and F3 and F4 stage of hepatic fibrosis accounted for 72.2%, 44.4%, 32.0%, 44.4% of F0 stage respectively, P < 0.05. Correlation analysis showed that ACSL1 m RNA expressions were negatively correlated with miR-34 a expressions, and ACSL1 protein expressions were also decreased gradually.4、Compared with q HSCs, Acsl1 m RNA expressions were decreased by 22.3% in semi-activation HSCs and 55.4% in a HSCs, P < 0.05. Correlation analysis showed that Acsl1 m RNA expressions were negatively correlated with miR-34 a expressions in HSCs, P < 0.05. And Acsl1 protein expressions were also decreased with HSCs activation.Conclusions1. Acsl1 is the target m RNA of miR-34 a.2. ACSL1 expressions were decreased along with hepatic fibrosis course. ACSL1 m RNA expressions were negatively correlated with miR-34 a expressions.3. Acsl1 expressions were decreased along with HSCs activation. Acsl1 m RNA expressions were negatively correlated with miR-34 a expressions in HSCsPart 3 Regulation of miR-34 a on HSCs by targeting Acsl1Objective To verify the biological behavior of miR-34 a on HSCs activation via targeting Acsl1.Methods 1. QRT-PCR and Western Blot were used to detect the expressions of Acsl1 and hepatic fibrosis indexes(Col1 and α-SMA) after HSCs were transfected with miR-34 a inhibitor. 2. QRT-PCR and Western Blot were used to detect the expressions of hepatic fibrosis indexes(Col1 and α-SMA) in Acsl1 silencing cell line. 3. Rescue assay was used to further verify miR-34a/Acsl1 regulation on HSCs activation.Results1. Compared with q HSCs, the m RNA expressions of Col1 were increased by 8.7 times and α-SMA increased by 2.7 times respectively, P < 0.05. The protein expressions of Col1 and α-SMA were also increased in a HSCs compared with q HSCs.2. Compared with NC group, the m RNA expressions of Acsl1 were increased by 1.7 times, while Col I and α-SMA were decreased by 41.8% and 63.5% respectively, P < 0.05. But there were no significant variations of m RNA expressions between NC group and blank group. The protein expressions of Acsl1, Col1 and α-SMA showed the same trend as m RNA expression.3. Acsl1 silencing lentivirus vector was constructed with EGFP fluorescence and puromycin resistance gene. Then q RT-PCR was used to test the silencing efficiency in Acsl1 silencing cell lines( Y2313, Y2314, Y2315) after a HSCs were transfected with Acsl1 silencing vector. Experimental results showed that Acsl1 m RNA expressions of Y2314, Y2314 and Y2315 were decreased by 61%, 57% and 34% respectively, P < 0.05, compared with Acsl1 silencing contrast cell lines. There were no significantly variations between silencing contrast cell lines and a HSCs. So Y2313 were selected as Acsl1 silencing cell lines for further experiment. QRT-PCR results showed that compared with silencing contrast cell lines, Col I m RNA expressions were increased by 3.3 times and α-SMA m RNA expressions were increased by 1.4 times, P < 0.05. While there were no significant Col I and α-SMA m RNA expression variations between silencing contrast cell lines and a HSCs. The protein expressions of Col1 and α-SMA showed the same trend as m RNA expressions.4. There were no significant differences in m RNA expression between Col I and α-SMA after Acsl1 silencing cell lines were transfected with either miR-34 a inhibitor or NC. But compared with silencing contrast cell lines transfected with NC, the m RNA expressions of Col I and α-SMA were decreased by 25.1% and 43.9% respectively after silencing contrast cell lines were transfected with miR-34 a inhibitor. And the protein expressions of Col1 and α-SMA showed the same trend as that of m RNA expressions.Conclusion1. The m RNA and protein expressions of hepatic fibrosis indexes(Col I and α-SMA) were significantly increased along with HSCs activation.2. MiR-34 a promoted HSCs activation.3. Acsl1 inhibited HSCs activation.4. MiR-34 a can regulate HSCs activation via targeting Acsl1. |
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