Effect of miR-29c-3p/IGF1 Molecular Axis on Activation，Proliferation and Apoptosis of Hepatic Stellate Cells
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摘要:
目的 探讨miR-29c-3p通过IGF-1对肝星状细胞（hepatic stellate cells，HSCs）活化，增殖和凋亡的影响。 方法 原代培养小鼠HSCs，并通过免疫荧光检测HSCs标志物ɑ-SMA表达。双荧光素酶报告基因实验验证miR-29c-3p和IGF-1的靶向关系。TGF-β激活HSCs，并且外源性调控miR-29c-3p和IGF-1的表达水平后，分别采用Westernbolt，CCK-8，克隆形成实验和流式细胞术检测活化HSCs中活化相关蛋白（ɑ-SMA，DDR2，FN1，ITGB1和GFAP）的表达，增殖，克隆形成数和凋亡。 结果 ɑ-SMA阳性表达表明成功分离小鼠HSCs。miR-29c-3p mimic可降低野生型IGF-1的荧光素酶活性，但是对突变型IGF-1没有影响。过表达miR-29c-3p和低表达IGF-1能减少ɑ-SMA，DDR2，FN1和ITGB1表达，增加GFAP的表达，并且降低HSCs的增殖活力和克隆形成数，上调其凋亡比例。 结论 miR-29c-3p通过靶向抑制IGF-1表达，进而抑制HSCs活化和增殖，并促进其凋亡。 Abstract:Objective To investigate the effects of miR-29c-3p on the activation, proliferation and apoptosis of hepatic stellate cells （HSCs） via IGF-1. Methods Primary mouse HSCs were cultured and the expression of HSCs marker ɑ-SMA was detected by immunofluorescence. By dual-luciferase reporter gene assay was conducted to Validate the Targeting Relationship between miR-29c-3p and IGF-1. After TGF-β activation of HSCs and exogenous regulation of miR-29c-3p and IGF-1 expression, the expression of activation-related proteins （ɑ-SMA, DDR2, FN1, ITGB1 and GFAP）, proliferation, colony-forming number and apoptosis in activated HSCs were detected by Western bolt, CCK-8, colony-forming unit assays and flow cytometry, respectively. Results Positive expression of ɑ-SMA indicated that the successful isolation of mouse HSCs. miR-29c-3p significantly reduced the luciferase activity of wild-type IGF-1, but had no effect on mutant IGF-1. Over-expression of miR-29c-3p and hypo-expression of IGF-1 significantly decreased ɑ-SMA, DDR2, FN1 and ITGB1 expression, increased GFAP expression, and decreased proliferation viability and colony-forming number of HSCs and upregulated their apoptotic ratio. Conclusion miR-29c-3p inhibits the activation and proliferation of HSCs and promotes their apoptosis by targeting IGF-1 expression. -
Key words:
- Alcoholic liver disease /
- Hepatic stellate cells /
- Activation /
- Proliferation /
- Apoptosis /
- miR-29c-3p /
- IGF-1
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图 1 成功分离HSCs，并且miR-29c-3p在不同状态的HSCs中差异表达
A：采用IF检测HSCs标志物ɑ-SMA是否表达；B：TGF-β处理HSCs前后，WB检测活化相关蛋白（ɑ-SMA，DDR2，FN1，ITGB1和GFAP）的表达；C：在静息及激活状态HSCs中，miR-29c-3p的表达差异。**P < 0.01，***P < 0.001。
Figure 1. Successful isolation of HSCs and differential expression of miR-29c-3p in HSCs of different status.
图 2 miR-29c-3p抑制HSCs的活化和增殖，并促进其凋亡
A：采用RT-qPCR检测miR-29c-3p mimic的转染效率；B：通过WB检测miR-29c-3p对活化相关蛋白（ɑ-SMA，DDR2，FN1，ITGB1和GFAP）表达的影响；C：CCK-8试剂盒检测不同组别中TGF-β激活的HSCs增殖活力；D：克隆形成实验检测miR-29c-3p对活化HSCs的克隆形成数的影响；E：流式细胞术检测活化HSCs的凋亡比例。**P < 0.01，***P < 0.001。
Figure 2. miR-29c-3p inhibits the activation and proliferation of HSCs and promotes their apoptosis.
图 3 IGF-1是miR-29c-3p下游靶标mRNA
A：Starbase数据库预测得到的miR-29c-3p与IGF-1的潜在3’ UTR结合序列（上），并且突变IGF-1的3’ UTR结合序列后，双荧光素酶报告基因实验验证miR-29c-3p与IGF-1的靶向关系（下）；B：活化的HSCs中分别转染miR-29c-3p inhibitor和miR-29c-3p mimic，采用WB检测IGF-1的表达变化。**P < 0.01。
Figure 3. IGF-1 is a downstream target mRNA of miR-29c-3p.
图 4 miR-29c-3p通过IGF-1抑制HSCs的活化和增殖，并促进其凋亡
A： miR-29c-3p inhibior的转染效率；B：通过WB检测得到的sh-IGF-1的转染效率；C：不同组别HSCs中活化相关蛋白（ɑ-SMA，DDR2，FN1，ITGB1和GFAP）表达的表达变化；D：CCK-8实验检测活化的HSCs增殖活力；E：克隆形成实验得到的不同组别中活化HSCs的克隆形成数；F：流式细胞术检测活化HSCs的凋亡比例。与sh-NC组比较，aP < 0.05，aaP < 0.01，aaaP < 0.001；与sh-IGF-1组比较，bP < 0.05，bbP < 0.01，bbbP < 0.001；*P < 0.05，***P < 0.001。
Figure 4. miR-29c-3p inhibits the activation and proliferation of HSCs and promotes their apoptosis through IGF-1.
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