Journal: Frontiers in Pharmacology

Article Title: Suppressing circ_0008494 inhibits HSCs activation by regulating the miR-185-3p/Col1a1 axis

doi: 10.3389/fphar.2022.1050093

Figure Lengend Snippet: Identification and characteristics of circ_0008494 in HF tissues and HSCs (A) circRNAs clustering heat map showed circ_0008494 was up-regulated in HF tissues. C represents HF samples ( n = 6). N represents control samples ( n = 3). High expression level is indicated by “red” and lower levels by “blue” (B) circRNA-miRNA-mRNA interaction network of circ_0008494 (C) circ_0008494 is derived from the second, third and fourth exons of the ARIDIA gene, which is located on chromosome 1p36 (D) The back-splice junction of circ_0008494 was verified by Sanger sequencing (E) ActD treatment assay showed circ_0008494 was more stable and resistant to ActD than the linear ARID1A mRNA (F) Electrophoresis on agarose gel and qRT-PCR assays showed circ_0008494 exhibited clear resistance to RNase R digestion (G) FISH assay was conducted on the HF tissue (nodular cirrhosis). Fibrotic area was confirmed by HE, Masson and immunohistochemical staining (Col1a1, α-SMA) and indicated with the black boxes (×200) (Low-magnification were shown in ). HSCs (α-SMA-positive, 400×, representative cells were indicated with the white boxes) with a spindle-shaped fibroblastic morphology were present in large numbers in fibrous septa, followed by some small bile ducts. FISH assay revealed that circ_0008494 was predominantly localized at the cytoplasm of HSCs (400×), but not expressed in adjacent cholangiocytes. Cy3-labeled-circ_0008494 appeared “red”, and DAPI staining nuclei appeared “blue” (H) qRT-PCR assay showed circ_0008494 significantly increased in human HF tissues ( n = 22) compared with paired normal tissues (I) qRT-PCR assay showed circ_0008494 significantly increased in TGF-β1 activated LX-2 cells, together with the HF indictors a-SMA and Col1a1. The RNA levels were normalized to total GAPDH. Statistical analysis: Student t -tests. ** and *** stand for p < 0.01 and p < 0.001, respectively. Experiments were repeated independently three times.

Article Snippet: Detection of miRNAs was performed using Reverse transcription and qRT-PCR kits (R10031.7, Ruibo Bio, Guangzhou, China).

Techniques: Expressing, Derivative Assay, Sequencing, Electrophoresis, Agarose Gel Electrophoresis, Quantitative RT-PCR, Immunohistochemical staining, Staining, Labeling

Journal: Frontiers in Pharmacology

Article Title: Suppressing circ_0008494 inhibits HSCs activation by regulating the miR-185-3p/Col1a1 axis

doi: 10.3389/fphar.2022.1050093

Figure Lengend Snippet: circ_0008494 knockdown inhibits the activation, proliferation, migration of HSCs and promoted their apoptosis (A) Three siRNAs targeting the junction sequence of circ_0008494 were designed (B) LX-2 cells emitted GFP fluorescence after circ_0008494-interfering viruses and NC virus infection (400×) (C) qRT-PCR showed LV-circ_0008494-KD3 exhibited the highest circ_0008494 reduction (0.30 ± 0.02) vs LV-NC (D and E) The mRNA and protein expression levels of a-SMA and Col1a1 in the LV-circ_0008494-KD and LV-NC groups (F and G) Effect of circ_0008494 knockdown on the proliferation and migration of LX-2 cells was detected by CCK8 and transwell assays. In CCK8 assay, the OD value at day1 was normalized to 1 and the data are expressed as fold (H) Apoptosis was detected by Annexin V-APC single staining combined with flow cytometry in LV-circ_0008494-KD and LV-NC groups. The RNA levels were normalized to total GAPDH. The protein levels were normalized to total GAPDH. Statistical analysis: Student t -tests. * , ** , and *** stand for p < 0.05, p < 0.01, and p < 0.001, respectively. Experiments were repeated independently three times.

Article Snippet: Detection of miRNAs was performed using Reverse transcription and qRT-PCR kits (R10031.7, Ruibo Bio, Guangzhou, China).

Techniques: Activation Assay, Migration, Sequencing, Fluorescence, Infection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Suppressing circ_0008494 inhibits HSCs activation by regulating the miR-185-3p/Col1a1 axis

doi: 10.3389/fphar.2022.1050093

Figure Lengend Snippet: circ_0008494 acts as a sponge of miR-185-3p (A) miRanda algorithms predicted the binding miRNAs of circ_0008494 (B) psiCHECK luciferase reporter plasmid was used to construct circ_0008494-WT-psiCHECK and circ_0008494-MUT-psiCHECK (C) Dual-Luciferase reporter gene assay verified the binding relationship between circ_0008494 and miR-185-3p (D) Association of circ_0008494 and miR-185-3p with AGO2. Cellular lysates of LX-2 were used for the RIP assay with an AGO2 antibody (IgG as control). circ_0008494 and miR-185-3p levels were detected by qRT-PCR assay (E) Biotin-coupled miRNA capture assay was constructed to verify the binding between circ_0008494 and miR-185-3p using a biotinylated miR-185-3p probe and a miRNA NC probe. The RNA levels were normalized to total GAPDH. Statistical analysis: Student t -tests. ** and *** stand for p < 0.01 and p < 0.001, respectively. ns, nonsignificant. Experiments were repeated independently three times.

Article Snippet: Detection of miRNAs was performed using Reverse transcription and qRT-PCR kits (R10031.7, Ruibo Bio, Guangzhou, China).

Techniques: Binding Assay, Luciferase, Plasmid Preparation, Construct, Reporter Gene Assay, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Suppressing circ_0008494 inhibits HSCs activation by regulating the miR-185-3p/Col1a1 axis

doi: 10.3389/fphar.2022.1050093

Figure Lengend Snippet: miR-185-3p inhibits HSCs activation, proliferation and migration (A and B) The mRNA expressions of α-SMA and Col1a1 were detected by qRT-PCR assay after miR-185-3p inhibitor/mimic transfection (C) Western blot assay showed the protein expression of α-SMA and Col1a1 after miR-185-3p mimic or mimic NC transfection (D and E) Proliferation and migration of LX-2 cells were detected by CCK8 and transwell assays after miR-185-3p inhibitor/mimic transfection. In CCK8 assay, the OD value at day1 was normalized to 1 and the data are expressed as fold. The RNA levels were normalized to total GAPDH. The protein levels were normalized to total GAPDH. Statistical analysis: Student t -tests. ** and *** stand for p < 0.01 and p < 0.001, respectively. Experiments were repeated independently three times.

Article Snippet: Detection of miRNAs was performed using Reverse transcription and qRT-PCR kits (R10031.7, Ruibo Bio, Guangzhou, China).

Techniques: Activation Assay, Migration, Quantitative RT-PCR, Transfection, Western Blot, Expressing, CCK-8 Assay

Journal: Frontiers in Pharmacology

Article Title: Suppressing circ_0008494 inhibits HSCs activation by regulating the miR-185-3p/Col1a1 axis

doi: 10.3389/fphar.2022.1050093

Figure Lengend Snippet: Suppressing circ_0008494 inhibits activation, proliferation, migration of HSCs and promotes their apoptosis through miR-185-3p (A and B) miR-185-3p inhibitor was transfected into stable LV-circ_0008494-KD LX-2 cells. miRNA inhibitor NC was transfected into stable circ_0008494-interfering cell line as a control or LX-2 NC cells as a normal control. 48 h after transfection, the mRNA and protein expression levels of a-SMA and Col1a1 in each group were detected qRT-PCR and western blot assays (C and D) After transfection as above, proliferation and migration of LX-2 cells in each group were detected by CCK8 and transwell assays. In CCK8 assay, the OD value at day1 was normalized to 1 and the data are expressed as fold (E) After transfection as above, apoptosis was detected by Annexin V-APC single staining combined with flow cytometry was performed in each group. The RNA levels were normalized to total GAPDH. The protein levels were normalized to total GAPDH. Statistical analysis: Student t -tests. ** and *** stand for p < 0.01and p < 0.001, respectively. Experiments were repeated independently three times.

Article Snippet: Detection of miRNAs was performed using Reverse transcription and qRT-PCR kits (R10031.7, Ruibo Bio, Guangzhou, China).

Techniques: Activation Assay, Migration, Transfection, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Suppressing circ_0008494 inhibits HSCs activation by regulating the miR-185-3p/Col1a1 axis

doi: 10.3389/fphar.2022.1050093

Figure Lengend Snippet: Suppressing circ_0008494 inhibits HSCs activation by regulating the miR-185-3p/Col1a1 axis (A) The pMIR-REPORT luciferase reporter plasmid was used to construct Col1a1 3′UTR-WT-pMIR and Col1a1 3′UTR-MUT-pMIR. TargetScan website predicted seven ribonucleotides of has-miR-185-3p were complementary to the 642–648 sites of Col1a1 3′-UTR (B) Dual-luciferase reporter gene assay verified the binding relationship between miR-185-3p and Col1a1 (C and D) miR-185-3p inhibitor or inhibitor NC was transfected into stable LV-circ_0008494-KD LX-2 cells, and mRNA and protein levels of Col1a1 was detected by qRT-PCR and western blot assays (E) After TGF-β1 stimulation, miR-185-3p inhibitor/mimic were transfected into LX-2 cells, and Col1a1 protein expression level were detected by western blot assay (F) After TGF-β1 stimulation of stable LV-circ_0008494-KD cells or LV-NC cells, Col1a1 protein expression was detected by western blot assays. Moreover, miR-185-3p inhibitor or inhibitor NC was transfected into stable LV-circ_0008494-KD LX-2 cells after TGF-β1 stimulation, and Col1a1 protein expression was detected. The RNA levels were normalized to total GAPDH. The protein levels were normalized to total GAPDH. Statistical analysis: Student t -tests. * , ** , and *** stand for p < 0.05, p < 0.01 and p < 0.001, respectively. ns, nonsignificant. Experiments were repeated independently three times.

Article Snippet: Detection of miRNAs was performed using Reverse transcription and qRT-PCR kits (R10031.7, Ruibo Bio, Guangzhou, China).

Techniques: Activation Assay, Luciferase, Plasmid Preparation, Construct, Reporter Gene Assay, Binding Assay, Transfection, Quantitative RT-PCR, Western Blot, Expressing