Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: MiR-106a-5p induces ferroptosis by targeting STAT3 in breast cancer cells. ( A ) The interaction of miR-106a-5p and STAT3 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). ( B – D ) The MDA-MB-231 and T47D cells were treated with the miR-106a-5p mimic or control mimic. ( B ) The luciferase activities of wild type STAT3 (STAT3 WT) and STAT3 with the miR-106a-5p-binding site mutant (STAT3 MUT) were determined by luciferase reporter gene assays in the cell. ( C ) The mRNA expression of STAT3 was analyzed by qPCR in the cells. ( D ) The protein expression of STAT3 and β-actin was tested by Western blot analysis in the cells. ( E ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor. The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the cells. ( E , F ) The MDA-MB-231 and T47D cells were treated with 5 mmol/L erastin, co-treated with 5 mmol/L erastin and miR-106a-5p mimic, or o-treated with 5 mmol/L erastin, miR-106a-5p mimic, and pcDNA.1-STAT3. The cell growth was analyzed by MTT assays. ( G – I ) The MDA-MB-231 and T47D cells were treated control shRNA, miR-106a-5p mimic, or co-treated with miR-106a-5p mimic and pcDNA.1-STAT3. ( G ) The levels of iron were analyzed by Iron Assay Kit. ( H ) The levels of ROS were measure by flow cytometry analysis in the cells. ( I ) The expression of GPX4, SLC7A11, and β-actin was measured by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: Luciferase, Binding Assay, Mutagenesis, Expressing, Western Blot, shRNA, Iron Assay, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 contributes to breast cancer progression by miR-106a-5p/STAT3 axis. ( A – D ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor or pcDNA.1-STAT3. ( A , B ) The cell viability was measured by MTT assays in the cells. ( C , D ) The cell apoptosis was measure by flow cytometry analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: shRNA, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 promotes the tumor growth of breast cancer in vivo . ( A – E ) The effect of circRHOT1 on tumor growth of breast cancer cells in vivo was analyzed by nude mice tumorigenicity assay by injected with the MDA-MB-231 cells treated with control shRNA or circRHOT1 shRNA. ( A ) Representative images of dissected tumors from nude mice were presented. ( B ) The average tumor volume was calculated and shown. ( C ) The average tumor weight was calculated and shown. ( D ) The expression levels of miR-106a-5p were measured by qPCR in the tumor tissues of the mice. ( E ) The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the tumor tissues of the mice. N = 5. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: In Vivo, Tumorigenicity Assay, Injection, shRNA, Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: YAP1 is upregulated in HBZY-1 cells treated with high glucose. (A) RT-qPCR was used to measure the expression of YAP1 in HBZY-1 cells treated with 30 mmol/l serum-free DMEM medium for 0, 6, 12, 24 and 48 h, respectively. (B) Western blotting was used to measure the expression of YAP1 in HBZY-1 cells treated with 30 mmol/l serum-free DMEM medium for 0, 6, 12, 24 and 48 h, respectively. (C) Transfection efficiency of YAP1 small interfering RNA was detected by RT-qPCR. *** P<0.001 vs. HG 0 h; ### P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; RT-qPCR, reverse transcription-quantitative PCR; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Small Interfering RNA, Real-time Polymerase Chain Reaction, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 inhibited the proliferation of HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the viability of HBZY-1 cells treated with high glucose was detected by Cell Counting Kit-8 assay. (B) Effect of YAP1 knockdown on the expression of PCNA in the HBZY-1 cells treated with high glucose was measured by western blotting. * P<0.05 vs. HG + si-NC. YAP1, Yes-associated protein 1; PCNA, proliferating cell nuclear antigen; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Cell Counting, Expressing, Western Blot, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 suppresses the inflammation of HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the expressions of pro-inflammatory factors (TNF-α, IL-1β and IL-6) was detected by enzyme-linked immunosorbent assay. (B) Effect of YAP1 knockdown on the expression of MCP-1 was detected by reverse transcription-quantitative PCR. (C) Effect of YAP1 knockdown on the expression of MCP-1 was detected by western blotting. ** P<0.01 and *** P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; TNF, tumor necrosis factor; IL, interleukin; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 inhibits the deposition of extracellular matrix and fibrosis in HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the expressions of collagen IV, α-SMA and FN was detected by western blotting. (B and C) Effect of YAP1 knockdown on the expressions of collagen IV was detected by immunofluorescence. Scale bars, 50 µm. (D) Effect of YAP1 knockdown on the expressions of fibrogenic factors CTGF and TGF-β1 was detected by western blotting. * P<0.05, ** P<0.01 and *** P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; SMA, smooth muscle actin; FN, fibronectin; CTGF, connective tissue growth factor; TGF, transforming growth factor.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Western Blot, Immunofluorescence, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 inhibited NF-κB/JMJD3 signal pathway in HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the phosphorylation levels of IκBα and p65 were detected by western blotting. (B) Effect of YAP1 knockdown on the expression of JMJD3 was detected by western blotting. * P<0.05, ** P<0.01 and *** P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; NF-κB, nuclear factor-κB; JMJD3, Jumonji domain-containing protein D3; p-, phosphorylated-; IκBα, inhibitor of NF-κB.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Inhibitors of NF-κB/JMJD3 signal pathway attenuates the facilitatory effects of YAP1 on the proliferation and inflammation of HBZY-1 cells induced by high glucose. (A) The efficiency of YAP1 overexpression was determined by RT-qPCR. (B) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of IκBα and p65 were detected by western blotting. (C) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the viability of HBZY-1 cells treated with high glucose was detected by Cell Counting Kit-8 assay. (D) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression of PCNA in the HBZY-1 cells treated with high glucose was measured by western blotting. (E) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expressions of pro-inflammatory factors (TNF-α, IL-1β and IL-6) was detected by enzyme-linked immunosorbent assay. (F) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression of MCP-1 was detected by RT-qPCR. (G) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression of MCP-1 was detected by western blotting. ** P<0.01 vs. HG + Oe-NC. # P<0.05, ## P<0.01 and ### P<0.001 vs. HG + Oe-YAP1. YAP1, Yes-associated protein 1; RT-qPCR, reverse transcription-quantitative PCR; Oe, overexpression vector; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; NF-κB, nuclear factor-κB; JMJD3, Jumonji domain-containing protein D3; p-, phosphorylated-; IκBα, inhibitor of NF-κB; TNF, tumor necrosis factor; IL, interleukin; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Cell Counting, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Inhibitors of NF-κB/JMJD3 signal pathway attenuates the facilitatory effects of YAP1 on the deposition of extracellular matrix and fibrosis of HBZY-1 cells induced by high glucose. (A) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of collagen IV, α-SMA and FN was detected by western blotting. (B and C) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of collagen IV was detected by immunofluorescence. Scale bars, 50 µm. (D) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of fibrogenic factors CTGF and TGF-β1 was detected by western blotting. # P<0.05 and ## P<0.01 vs. HG + Oe-YAP1. YAP1, Yes-associated protein 1; Oe, overexpression vector; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; NF-κB, nuclear factor-κB; JMJD3, Jumonji domain-containing protein D3; SMA, smooth muscle actin; FN, fibronectin; CTGF, connective tissue growth factor; TGF, transforming growth factor.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Expressing, Western Blot, Immunofluorescence, Over Expression, Plasmid Preparation, Negative Control

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH inhibits the proliferation and induces apoptosis of NP cells. ( A ) The expression levels of circITCH were measured by qPCR in the NP tissues of IDD patients (n=90) and normal cases (n=90). ( B – F ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( B ) The expression levels of circITCH were examined by qPCR in the cells. ( C , D ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( E ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( F ) The expression of Bax, caspase3, cleaved caspase3 (c-caspase3), caspase9, and cleaved caspase9 (c-caspase9) was measured by Western blot analysis. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH promotes ECM degradation of degenerative NP cells. ( A – E ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( A – D ) The mRNA expression of collagen II ( A ), aggrecan ( B ), MMP13 ( C ), and ADAMTS4 ( D ) was measured by qPCR in the cells. ( E ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was tested by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Infection, shRNA, Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH serves as a miR-17-5p sponge in NP cells. ( A ) The potential interaction between circITCH and miR-17-5p was identified by the bioinformatic analysis using ENCORI ( http://starbase.sysu.edu.cn/index.php ). ( B , C ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type circITCH (circITCH WT) and circITCH with the miR-17-5p-binding site mutant (circITCH MUT) were determined by luciferase reporter gene assays in the cells. ( D ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. The expression of miR-17-5p was analyzed by qPCR in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Luciferase, Binding Assay, Mutagenesis, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: MiR-17-5p targets SOX4 in NP cells. ( A ) The interaction of miR-17-5p and SOX4 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). ( B – E ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type SOX4 (SOX4 WT) and SOX4 with the miR-17-5p-binding site mutant (SOX4 MUT) were determined by luciferase reporter gene assays in the cell. ( D ) The mRNA expression of SOX4 was analyzed by qPCR in the cells. ( E ) The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( F ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the NP cells treated with control mimic, miR-17-5p mimic, or co-treated with miR-17-5p mimic and pcDNA3.1-SOX4 overexpression vector. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Luciferase, Binding Assay, Mutagenesis, Western Blot, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH activates Wnt/β-catenin signaling by targeting miR-17-5p/SOX4 axis. ( A ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and miR-17-5p inhibitor. The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( B , C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, lentiviral plasmids carrying circITCH shRNA, pcDNA3.1-SOX4 overexpression vector, and miR-17-5p mimic, or lentiviral plasmids carrying circITCH shRNA and LiCl. The expression of Wnt1, β-catenin, c-Myc, Cyclin D1, and β-actin was analyzed by Western blot analysis in the cells. The results of Western blot analysis were quantified by ImageJ software. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, shRNA, Expressing, Western Blot, Over Expression, Plasmid Preparation, Software

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH contributes to ECM degradation of degenerative NP cells by modulating miR-17-5p/SOX4/Wnt/β-catenin signaling. ( A – C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, miR-17-5p inhibitor, or LiCl. ( A ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( B ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( C ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

Journal: Cancer Management and Research

Article Title: The Maturation of Tumor Suppressor miR-497 in Hepatocellular Carcinoma is Inhibited by Oncogenic circRNA SCARB1

doi: 10.2147/CMAR.S304125

Figure Lengend Snippet: The expression of circRNA SCARB1, mature miR-497 and miR-497 precursor was altered in HCC. HCC and paired non-tumor tissues were collected from the 64 HCC patients included in this study, and the expression of circRNA SCARB1 ( A ), mature miR-497 ( B ) and miR-497 precursor ( C ) in these tissue samples was determined by RT-qPCR. Average values of three technical replicates were used to express gene expression data in paired HCC and non-tumor tissues, ** p < 0.01.

Article Snippet: To overexpress circRNA SCARB1, pcDNA3.1(+) CircRNA Mini Vector (Addgene) was used as the backbone to construct the expression vector of circRNA SCARB1.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: Cancer Management and Research

Article Title: The Maturation of Tumor Suppressor miR-497 in Hepatocellular Carcinoma is Inhibited by Oncogenic circRNA SCARB1

doi: 10.2147/CMAR.S304125

Figure Lengend Snippet: The Correlations Between Clinicopathological Variables and the Expression of circRNA SCARB1 in HCC

Article Snippet: To overexpress circRNA SCARB1, pcDNA3.1(+) CircRNA Mini Vector (Addgene) was used as the backbone to construct the expression vector of circRNA SCARB1.

Techniques: Expressing, Infection

Journal: Cancer Management and Research

Article Title: The Maturation of Tumor Suppressor miR-497 in Hepatocellular Carcinoma is Inhibited by Oncogenic circRNA SCARB1

doi: 10.2147/CMAR.S304125

Figure Lengend Snippet: CircRNA SCARB1 and mature miR-497 were inversely correlated across HCC samples. Pearson’s correlation coefficient analysis was used to analyze the correlations between circRNA SCARB1 and mature miR-497 ( A ) or miR-497 precursor ( B ) across HCC tissue samples.

Article Snippet: To overexpress circRNA SCARB1, pcDNA3.1(+) CircRNA Mini Vector (Addgene) was used as the backbone to construct the expression vector of circRNA SCARB1.

Techniques:

Journal: Cancer Management and Research

Article Title: The Maturation of Tumor Suppressor miR-497 in Hepatocellular Carcinoma is Inhibited by Oncogenic circRNA SCARB1

doi: 10.2147/CMAR.S304125

Figure Lengend Snippet: Overexpression of circRNA SCARB1 downregulated mature miR-497 in HCC cells. To explore the effects of overexpression of circRNA SCARB1 on the maturation of miR-497, SNU-423 and SNU-387 cells were transfected with either circRNA SCARB1 expression vector or miR-497 mimic, followed by the confirmation of transfections at 48 h post-transfection by RT-qPCR ( A ). The effects of overexpression of circRNA SCARB1 of the expression of mature miR-497 ( B ) and miR-497 precursor ( C ), as well as the effects of the transfection of miR-497 mimic on circRNA SCARB1 expression ( D ) were also analyzed by RT-qPCR. Mean ± SD values were used to express data of 3 biological replicates of in vitro cell experiments. * p < 0.05.

Article Snippet: To overexpress circRNA SCARB1, pcDNA3.1(+) CircRNA Mini Vector (Addgene) was used as the backbone to construct the expression vector of circRNA SCARB1.

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, In Vitro

Journal: Cancer Management and Research

Article Title: The Maturation of Tumor Suppressor miR-497 in Hepatocellular Carcinoma is Inhibited by Oncogenic circRNA SCARB1

doi: 10.2147/CMAR.S304125

Figure Lengend Snippet: Overexpression of circRNA SCARB1 increased HCC cell proliferation through miR-497. CCK-8 assay was performed to analyze the effects of overexpression of circRNA SCARB1 and miR-497 on the proliferation of SNU-423 ( A ) and SNU-387 cells ( B ). Mean ± SD values were used to express data of three biological replicates of in vitro cell experiments. * p < 0.05.

Article Snippet: To overexpress circRNA SCARB1, pcDNA3.1(+) CircRNA Mini Vector (Addgene) was used as the backbone to construct the expression vector of circRNA SCARB1.

Techniques: Over Expression, CCK-8 Assay, In Vitro

Journal: Cancer Management and Research

Article Title: The Maturation of Tumor Suppressor miR-497 in Hepatocellular Carcinoma is Inhibited by Oncogenic circRNA SCARB1

doi: 10.2147/CMAR.S304125

Figure Lengend Snippet: Overexpression of circRNA SCARB1 increased HCC cell migration through miR-497. Transwell assay was conducted to analyze the effects of overexpression of circRNA SCARB1 and miR-497 on the migration of SNU-423 ( A and C ) and SNU-387 cells ( B and D ). Mean ± SD values were used to express data of three biological replicates of in vitro cell experiments. * p < 0.05.

Article Snippet: To overexpress circRNA SCARB1, pcDNA3.1(+) CircRNA Mini Vector (Addgene) was used as the backbone to construct the expression vector of circRNA SCARB1.

Techniques: Over Expression, Migration, Transwell Assay, In Vitro

Journal: The Journal of International Medical Research

Article Title: miR-384 targets metadherin gene to suppress growth, migration, and invasion of gastric cancer cells

doi: 10.1177/0300060518817171

Figure Lengend Snippet: MTDH was a direct target of miR-384 in GC. (a) Predicted binding site for miR-384 in MTDH 3′-UTR. (b) Relative luciferase activities of SGC-7901 cells co-transfected with MTDH -wt or MTDH -mut and miR-384 mimic or NC miRNA. (c) MTDH protein expression in SGC-7901 cells transfected with miR-384 mimic, miR-384 inhibitor, or NC miRNA. *** P < 0.001, ns: not significant. MTDH: metadherin; GC: gastric cancer; miR-384: microRNA-384; NC: negative control; wt: wild-type; mut: mutant; UTR: untranslated region; GAPDH: glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: Constructs overexpressing MTDH and NC vector were purchased from GenScript (Nanjing, China).

Techniques: Binding Assay, Luciferase, Transfection, Expressing, Negative Control, Mutagenesis

Journal: The Journal of International Medical Research

Article Title: miR-384 targets metadherin gene to suppress growth, migration, and invasion of gastric cancer cells

doi: 10.1177/0300060518817171

Figure Lengend Snippet: Overexpression of MTDH impaired miR-384-induced inhibition of GC cell proliferation, migration, and invasion. (a) MTDH and MMP-9 expression, (b) cell proliferation, (c) cell migration (×200), and (d) cell invasion in SGC-7901 cells transfected with MTDH construct or miR-384 mimic (crystal violet stain; ×200). ** P < 0.01, ns: not significant. MTDH: metadherin; GC: gastric cancer; miR-384: microRNA-384; NC: negative control; OD: optical density; MMP-9: matrix metallopeptidase 9

Article Snippet: Constructs overexpressing MTDH and NC vector were purchased from GenScript (Nanjing, China).

Techniques: Over Expression, Inhibition, Migration, Expressing, Transfection, Construct, Staining, Negative Control