Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Validation of the differential expression of miR-130a-3p in the livers of mice and patients. (a ) miR-130a-3p expression was decreased, and the expression of TGFBR1, TGFBR2, Col-1, and Col-4 was increased in the liver tissues of the MCD-fed mice. TGFBR1, TGFBR2, Col-1, and Col-4 expression was detected by IHC ( × 200 magnification), and miR-130a-3p expression was detected using an ISH ( × 400 magnification) assay. Positive staining is indicated by a brown color. (b) Effects of the MCD diet on serum ALT and AST levels. Values represent the mean±S.D., *** P <0.001 compared with control. (c) Hepatic mRNA and (d) protein expression of TGFBR1, TGFBR2, Smad2, Smad3, Col-1, and Col-4 were upregulated in the MCD-fed mice compared with the control mice. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control. (e) Histopathological changes were evaluated with hematoxylin and eosin staining, and Masson’s trichrome staining. miR-130a-3p expression was decreased, and TGFBR1, TGFBR2, Col-1, and Col-4 levels were increased in the liver tissues of patients with NASH-related liver fibrosis. The expression of miR-130a-3p was detected by ISH ( × 400 magnification), and TGFBR1, TGFBR2, Col-1, and Col-4 were detected using an IHC ( × 200 magnification) assay. Positive staining is indicated by a brown color

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Biomarker Discovery, Quantitative Proteomics, Expressing, Immunohistochemistry, In Situ Hybridization, Staining, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: miR-130a-3p expression was downregulated and regulated the downstream expression of genes of the TGF-β/SMAD signaling pathway in activated HSCs. ( a ) miR-130a-3p expression was examined by real-time qRT-PCR during HSC activation. Values represent the mean±S.D., (* P <0.05, ** P <0.01). ( b ) The hepatic mRNA and ( c ) protein expression of TGF- β 1 , α -SMA, Smad2, and Smad3 were increased during the process of HSC activation. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( d ) HSC-T6 cells were transfected with miR-130a-3p mimics or mimics control for 48 h. The mRNA and protein ( e ) levels of TGF- β 1 , Smad2, and Smad3 were analyzed by qRT-PCR and western blotting, respectively. Mimics control is the negative control of mimics, and control is the blank control in this group. The result showed that there was no significant difference between control and mimics control. β -actin was used as a loading control. Values represent the mean±S.D., ** P <0.01, *** P <0.001 compared with control. ( f ) The mRNA and protein ( g ) levels of MMP-2, MMP-9, Col-1, and Col-4 were analyzed by qRT-PCR and western blotting, respectively. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Control, Transfection, Western Blot, Negative Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Knockdown of either TGFBR1 or TGFBR2 inhibited the TGF- β / SMAD signaling pathway and reduced HSC activation. ( a ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( b ) of TGFBR1 and TGFBR2. β-actin was used as a loading control. Values represent the mean ±SD, *** P < 0.001 compared with control. ( c ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( d ) of α -SMA, Smad2, and Smad3. β-actin was used as a loading control. Values represent the mean ±SD, * P <0.05, ** P <0.01, *** P <0.001 compared with control. ( e ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein levels ( f ) of MMP-2, MMP-9, Col-1, and Col-4. β -actin was used as a loading control. Values represent the mean±S.D., *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Knockdown, Activation Assay, Expressing, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Overexpression of TGFBR1 and TGFBR2 rescued miR-130a-3p-impaired proliferation and cell growth in HSCs. The HSC-T6 cells were transduced with the miR-130a-3p mimics and the TGFBR1-expressing and TGFBR2-expressing vectors. The rescue efficiency of TGFBR1 or TGFBR2 in the HSC-T6 cells was confirmed by ( a ) RT-PCR and ( b ) western blotting. β -actin was used as a loading control. Values represent the mean±S.D. (*** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the (c) mRNA and ( d ) protein expression of Smad2 and Smad3. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the ( e ) mRNA and ( f ) protein expression of Col-1 and Col-4. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( g ) A schematic diagram highlighting the regulation of HSCs by miR-130a-3p during the genesis and resolution of liver fibrosis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Over Expression, Transduction, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Primers for quantitative real-time PCR analysis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Real-time Polymerase Chain Reaction