Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Primer sequences used for reverse transcription polymerase chain reaction analysis of NELIN.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Primer sequences used for reverse transcription polymerase chain reaction analysis of SM22α.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Reverse transcription polymerase chain reaction analysis revealed weak mRNA expression of NELIN in the vascular smooth muscle cells (VSMCs) of the experimental group, but strong expression in the control group. (A) Expected length of the PCR products was 611 bp (NELIN) and 312 bp (β-actin). Lanes: N, control group; X, DNA marker DL 2000; and P, experimental group. (B) Downregulation of NELIN mRNA expression in the VSMCs from the varicose vein tissue. Data are presented as the mean ± standard deviation.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Reverse transcription polymerase chain reaction analysis revealed weak mRNA expression of SM22α in the vascular smooth muscle cells (VSMCs) of the experimental group, but strong expression in the control group. (A) Expected length of the PCR products was 232 bp (SM22α) and 472 bp (GADPH). Lanes: N, control group; X, DNA marker DL 2000; and P, experimental group. (B) Downregulation of SM22α mRNA expression in the VSMCs from the varicose vein tissue. Data are presented as the mean ± standard deviation.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Standard Deviation

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