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: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol represses signal transducer and activator of transcription (STAT)3 expression by upregulating miR-125b-5p in gastric cancer cells. A: SGC7901 and BGC823 cells were treated with propofol (10 µmol/L). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measured expression of miR-125b-5p. B: The binding site of miR-125b-5p and STAT3 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). C–E: SGC7901 and BGC823 cells were treated with the miR-125b-5p mimic or control mimic. C and D: Luciferase reporter gene assays determined the luciferase activities. E: qRT-PCR analyzed mRNA expression of STAT3. F: SGC7901 and BGC823 cells were treated with propofol, or cotreated with propofol and miR-125b-5p inhibitor. Western blotting assessed protein expression of STAT3 and β-actin. n = 3, mean ± SD, b P < 0.01.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Luciferase, Western Blot

Journal: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol enhances ferroptosis by targeting signal transducer and activator of transcription (STAT)3 in gastric cancer cells. A: SGC7901 and BGC823 cells were treated with 5 mmol/L erastin, cotreated with 5 mmol/L erastin and propofol, or cotreated with 5 mmol/L erastin, propofol, and pcDNA.1-STAT3. MTT assays measured cell growth. B–D: SGC7901 and BGC823 cells were treated with propofol, or cotreated with propofol and pcDNA.1-STAT3. B: Iron Assay Kit analyzed the levels of iron; C: Flow cytometry analysis tested the levels of ROS; and D: Iron Assay Kit analyzed the levels of Fe 2+ . n = 3, mean ± SD, b P < 0.01, c P < 0.001.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: Iron Assay, Flow Cytometry

Journal: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol attenuates gastric cancer progression by miR-125b-5p/STAT3 axis. A–C: SGC7901 and BGC823 cells were treated propofol, or cotreated with propofol and miR-125b-5p inhibitor or pcDNA.1-STAT3. A and B: MTT assays analyzed the cell viability; C: Flow cytometry measured apoptosis. n = 3, mean ± SD, b P < 0.01.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: Flow Cytometry

Journal: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol attenuates growth of gastric cancer cells in vivo . The nude mice were injected with SGC7901 cells and intraperitoneally treated with propofol (50 mg/kg). A: Tumor tissues; B: Tumor volume; and C: Tumor weight; D: Expression of miR-125b-5p was analyzed by quantitative reverse transcription polymerase chain reaction. E: Protein expression of STAT3 was detected by western blotting. F: Protein expression of GPX4 and SLC7A11 was measured by western blotting. n = 5, mean ± SD, b P < 0.01.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: In Vivo, Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-140-5p regulates the proliferation, apoptosis and inflammation of RA FLSs by repressing STAT3

doi: 10.3892/etm.2020.9602

Figure Lengend Snippet: STAT3 is a direct target of miR-140. (A) Graphical representation of the conserved miR-140 binding motif at the STAT3 3'-UTR. The complementary sequences to the seed regions of miR-140 and corresponding sequence of the 3'-UTR of STAT3. (B) The luciferase activity exhibited by the reporter constructs, containing either the WT or MUT human STAT3 3'-UTR after miR-140 transfection. The observed luciferase activity was normalized to that of β-galactosidase. Overexpression of miR-140 markedly decreased the relative luciferase activity in the WT 3'-UTR, but not the MUT 3'-UTR, of STAT3 mRNA. All groups were normalized to the WT + control group (100%). (C) Western blotting and (D) RT-qPCR analysis were used to evaluate the STAT3 protein and mRNA expression levels after transfection of RA FLSs with miR-140 or miR-negative control. All groups were normalized to the control group (100%). (E) Increased expression levels of STAT3 in RA FLSs was observed by RT-qPCR, compared to that of healthy FLSs. All groups were normalized to the normal FLS group (100%). Data represents the mean ± SD. * P<0.05, ** P<0.01 vs. the corresponding control. FLS, fibroblast-like synoviocyte; miR, microRNA; MUT, mutant; RA, rheumatoid arthritis; RT-qPCR, reverse transcription-quantitative PCR; UTR, untranslated region; WT, wild-type.

Article Snippet: The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11.

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Transfection, Over Expression, Western Blot, Quantitative RT-PCR, Expressing, Negative Control, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-140-5p regulates the proliferation, apoptosis and inflammation of RA FLSs by repressing STAT3

doi: 10.3892/etm.2020.9602

Figure Lengend Snippet: STAT3 was overexpressed in cells. Cells were transfected with or without STAT3-expressing vector or empty vector. Overexpression of STAT3 in the cells was then detected using western blotting at 48 h post-transfection. NC, negative control.

Article Snippet: The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11.

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

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-140-5p regulates the proliferation, apoptosis and inflammation of RA FLSs by repressing STAT3

doi: 10.3892/etm.2020.9602

Figure Lengend Snippet: STAT3 restored the regulatory role of miR-140 in RA FLSs. STAT3 expression levels were detected in cells using (A) western blotting and (B) RT-qPCR following miR-140 and STAT3 transfections. All groups were normalized to the control group (100%). The proliferative ability of cells expressing STAT3 and miR-140, miR-140 alone, or control was measured at 48 h post-transfection using the (C) BrdU incorporation assays and (D) MTT assays. (E) The apoptotic rate of RA FLSs in each group was determined by annexin V-FITC/PI flow cytometry. Early apoptotic cells and later apoptotic cells are indicated in the top right and bottom right quadrants, respectively, in each plot. Quantitative analysis of the apoptotic rate of RA FLSs in two groups is displayed in the right panel. (F) Western blotting was carried out to assess the expression levels of pro-inflammatory cytokines regulated by the miR-140 and STAT3 plasmid transfections. (G) RT-qPCR was performed to examine the gene expression of cytokines from the RA FLSs after transfection. All groups were normalized to the control group (100%). Data represents the mean ± SD. * P<0.05, ** P<0.01 vs. the control group; # P<0.05, ## P<0.01 vs. the miR-140 group. FLS, fibroblast-like synoviocyte; IL, interleukin; miR, microRNA; PI, propidium iodide; RA, rheumatoid arthritis; RT-qPCR, reverse transcription-quantitative PCR; TNF, tumor necrosis factor.

Article Snippet: The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, BrdU Incorporation Assay, Cytometry, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: Shikonin blocks human lung adenocarcinoma cell migration and invasion in the inflammatory microenvironment via the IL-6/STAT3 signaling pathway

doi: 10.3892/or.2020.7683

Figure Lengend Snippet: Effects of shikonin on IL-6-induced STAT3 activation in A549 and H1299 cells. (A) Expression levels of phosphorylated (p)-STAT3 and STAT3 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. Protein expression levels were semi-quantified by densitometric analysis. *P<0.05, **P<0.01, compared with the IL-6-treated group. (B) Immunofluorescence assays were performed to determine the effects of shikonin on the nuclear translocation of p-STAT3 after treatment with shikonin (1.25 µM) and IL-6 (50 ng/ml) for 24 h. Upper panels, representative immunofluorescence images of p-STAT3 staining (magnification ×800). Lower panel, the percentage of cells with p-STAT3 nuclear translocation. **P<0.01, compared with the IL-6-treated group. (C) Transactivation activities of p-STAT3 in A549 and H1299 cells co-transfected with pSTAT3-TA-luc and pRL-TK Renilla, and treated with shikonin and IL-6 (50 ng/ml) for 24 h. Luciferase activity was detected by the Dual Luciferase Reporter kit and normalized against the values for the corresponding pRL-TK Renilla activity. *P<0.05, **P<0.01, compared with the IL-6-treated group. (D) The protein expression levels of p-JAK2 and JAK2 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. β-actin was used as an internal control. Protein expression levels were semi-quantified by densitometry analysis. (E and F) After transfection with STAT3 plasmids for 24 h, the cells were treated with shikonin (1.25 µM) and IL-6 (50 ng/ml) for another 24 h. (E) Expression levels of p-STAT3, Flag-STAT3, and STAT3 proteins were detected by western blotting. (F) mRNA expression levels of epithelial-mesenchymal transition (EMT)-related genes (E-cadherin, N-cadherin, vimentin and Snail) were detected by real-time PCR. Data are shown as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, compared with the IL-6-treated group; # P<0.05, ## P<0.01, compared with the combined shikonin (1.25 µM) and IL-6 (50 ng/ml) treatment group. IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; JAK, Janus kinase.

Article Snippet: PcDNA3.1-Flag-STAT3 (Bioworld Technology, Inc.) was transfected into cells (A549 and H1299) using Lipofectamine 2000™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Translocation Assay, Staining, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: Shikonin blocks human lung adenocarcinoma cell migration and invasion in the inflammatory microenvironment via the IL-6/STAT3 signaling pathway

doi: 10.3892/or.2020.7683

Figure Lengend Snippet: Effects of shikonin on tumor metastasis and growth in vivo . (A) Representative images of lung tissue and hematoxylin and eosin (H&E)-stained metastatic lung nodules. (B) Quantification of the number of metastatic nodules on lung surface. (C) Tumor volume and (D) weight of the control and shikonin treatment groups. (E) Representative immunohistochemistry images of p-STAT3, E-cadherin, N-cadherin and vimentin expression in the tumor tissues derived from the A549 ×enografts. Data are presented as mean ± SD, *P<0.05, **P<0.01, compared with the control group. p-STAT3, phosphorylated signal transducer and activator of transcription 3.

Article Snippet: PcDNA3.1-Flag-STAT3 (Bioworld Technology, Inc.) was transfected into cells (A549 and H1299) using Lipofectamine 2000™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Techniques: In Vivo, Staining, Immunohistochemistry, Expressing, Derivative Assay