miR-140-3p Knockdown Suppresses Cell Proliferation and Fibrogenesis in Hepatic Stellate Cells via PTEN-Mediated AKT/mTOR Signaling
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Purpose: Liver fibrosis is a major cause of morbidity and mortality and the outcome of various chronic liver diseases. Activationof hepatic stellate cells (HSCs) is the key event in liver fibrosis. Studies have confirmed that miR-140-3p plays a potential regulatoryeffect on HSC activation. However, whether miR-140-3p mediates the liver fibrosis remains unknown. Materials and Methods: Expression of miR-140-3p was detected by real-time quantitative PCR (qPCR). Cell proliferation wasmeasured by MTT, while cell apoptosis rate was determined via flow cytometry. Western blot assay was used to detect the expressionof cleaved PARP. The fibrogenic effect was evaluated by expression of α-smooth muscle actin and desmin. Functional experimentswere performed in transforming growth factor β1 (TGF-β1)-induced HSC-T6 cells with transfection of anti-miR-140-3pand/or siPTEN. Target binding between miR-140-3p and PTEN was predicted by the TargetScan database and identified usingluciferase reporter assay and RNA immunoprecipitation. Results: TGF-β1 induced the activation of HSC-T6 cells, and miR-140-3p expression varied according to HSC-T6 cell activationstatus. Knockdown of miR-140-3p reduced cell proliferation and the expressions of α-SMA and desmin, as well as increasedapoptosis, in TGF-β1-induced HSC-T6 cells, which could be blocked by PTEN silencing. Additionally, inactivation of the AKT/mTOR signaling pathway stimulated by miR-140-3p knockdown was abolished when silencing PTEN expression. PTEN was negativelyregulated by miR-140-3p via direct binding in HSC-T6 cells. Conclusion: miR-140-3p is an important mediator in HSC-T6 cell activation, and miR-140-3p knockdown suppresses cell proliferationand fibrogenesis in TGF-β1-induced HSC-T6 cells, indicating that miR-140-3p may be a potential novel molecular targetfor liver fibrosis.
Abstract
Purpose: Liver fibrosis is a major cause of morbidity and mortality and the outcome of various chronic liver diseases. Activationof hepatic stellate cells (HSCs) is the key event in liver fibrosis. Studies have confirmed that miR-140-3p plays a potential regulatoryeffect on HSC activation. However, whether miR-140-3p mediates the liver fibrosis remains unknown. Materials and Methods: Expression of miR-140-3p was detected by real-time quantitative PCR (qPCR). Cell proliferation wasmeasured by MTT, while cell apoptosis rate was determined via flow cytometry. Western blot assay was used to detect the expressionof cleaved PARP. The fibrogenic effect was evaluated by expression of α-smooth muscle actin and desmin. Functional experimentswere performed in transforming growth factor β1 (TGF-β1)-induced HSC-T6 cells with transfection of anti-miR-140-3pand/or siPTEN. Target binding between miR-140-3p and PTEN was predicted by the TargetScan database and identified usingluciferase reporter assay and RNA immunoprecipitation. Results: TGF-β1 induced the activation of HSC-T6 cells, and miR-140-3p expression varied according to HSC-T6 cell activationstatus. Knockdown of miR-140-3p reduced cell proliferation and the expressions of α-SMA and desmin, as well as increasedapoptosis, in TGF-β1-induced HSC-T6 cells, which could be blocked by PTEN silencing. Additionally, inactivation of the AKT/mTOR signaling pathway stimulated by miR-140-3p knockdown was abolished when silencing PTEN expression. PTEN was negativelyregulated by miR-140-3p via direct binding in HSC-T6 cells. Conclusion: miR-140-3p is an important mediator in HSC-T6 cell activation, and miR-140-3p knockdown suppresses cell proliferationand fibrogenesis in TGF-β1-induced HSC-T6 cells, indicating that miR-140-3p may be a potential novel molecular targetfor liver fibrosis.
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