Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Oligonucleotide primer sets for RT-qPCR.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Sequencing

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal transfer of NEAT1 to EC cells leads to increased expression of STAT3 and YKL-40. (A) RT-qPCR analysis of NEAT1 expression level in NFs, CAFs, normal endometrial epithelial cells, and EC cells. (B) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or exosome-depleted CAFs-CM for 48 h, then NEAT1 level was assessed by RT-qPCR. HEC-1A and RL95-2 cells were maintained in control CM, CAFs-CM, or 20 μM GW4869-treated CAFs-CM for 48 h. (C) NEAT1 expression was determined by RT-qPCR. (D) The NEAT1 level was assessed by RT-qPCR. (E) STAT3, p-STAT3 and YKL-40 levels were assessed by Western blot. HEC-1A and RL95-2 cells were co-cultured with CAFs or GW4869-treated CAFs for 48 h. (F) Western blot analysis of STAT3, p-STAT3, and YKL-40 levels. (G) Western blot analysis of Rab27a protein level in CAFs infected with lentivirus expressing si-NC or si-Rab27a. (H) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or Rab27a-silenced CAFs-CM for 48 h, then NEAT1 level was detected by RT-qPCR. (I) HEC-1A cells and RL95-2 were co-cultured with CAFs or Rab27a-silenced CAFs for 48 h, and NEAT1 level was detected by RT-qPCR. (J)&(K) Protein levels of STAT3, p-STAT3, and YKL-40 were assessed by Western blot. (L) RT-qPCR analysis of NEAT1 level in exosomes isolated from CAFs infected with lentivirus expressing sh-NEAT1 or NEAT1 gene. (M) HEC-1A and RL95-2 cells were incubated with exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs for 48 h. RT-qPCR was performed to assess NEAT1 level. (N)&(O) STAT3 and YKL-40 expression was detected by RT-qPCR and Western blot assays. (P) The proliferation of HEC-1A and RL95-2 cells was detected by MTT assay. (Q) The growth of HEC-1A and RL95-2 cells was evaluated by colony formation assay. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Infection, Isolation, Incubation, Derivative Assay, MTT Assay, Colony Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal NEAT1 sponges miR-26a/b-5p to facilitate STAT3 and YKL-40 expression in EC cells. (A) RT-qPCR for miR-26a/b-5p level in exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs. (B) HEC-1A and RL95-2 cells were co-cultured with NEAT1-overexpressed or silenced CAFs, and miR-26a/b-5p level was detected by RT-qPCR. (C) Expression of miR-26a/b-5p in HEC-1A and RL95-2 cells infected with lentivirus containing miR-26a/b-5p mimics or inhibitor was detected by RT-qPCR. (D) Expression of YKL-40 in HEC-1A and RL95-2 cells after overexpression or silencing of miR-26a/b-5p was assessed by Western blot. (E) The binding sites between NEAT1 and miR-26a/b-5p. Luciferase analysis (F) and RIP assay (G) was performed to determine interaction between NEAT1 and miR-26a/b-5p. The expression of STAT3 and YKL-40 in HEC-1A and RL95-2 cells received various treatments was assessed by RT-qPCR (H) and Western blot (I). All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Cell Culture, Infection, Over Expression, Western Blot, Binding Assay, Luciferase

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: MiR-26a/b-5p suppresses YKL-40 expression via targeting STAT3 in EC cells. (A) Two binding sites of miR-26a/b-5p to the 3’-UTR of STAT3. The interaction between STAT3 and miR-26a/b-5p was detected by luciferase analysis (B) and RIP assay (C). (D) The protein level of STAT3 in HEC-1A and RL95-2 cells after the transfection of STAT3 expression plasmid was assessed by Western blot. RT-qPCR (E) and Western blot (F) were used for determining YKL-40 and STAT3 expression in HEC-1A cells from various groups. (G) Three p-STAT3-binding sites (BS1, BS2, and BS3) at YKL-40 promoter were shown. (H) ChIP assay using anti-p-STAT3 antibody was used to verify the binding between p-STAT3 and the promoter of YKL-40 under exosome treatment. (I) The interaction between p-STAT3 and YKL-40 was assessed by the dual luciferase reporter assay. All data from three independent experiments were shown as mean ± SD (n = 6). **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

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

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: CAFs-derived exosomal NEAT1 accelerates tumor growth in vivo via up-regulating YKL-40. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs. GW4869 (2 mg/kg) was intraperitoneally injected into the mice every other day. (A) The tumor growth curve was shown. (B) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (C) Immunohistochemical staining for Ki-67 expression in tumor tissues. (D) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs that were stably transfected with sh-NC or sh-Rab27a. (E) The tumor growth curve was shown. (F) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (G) Immunohistochemical staining for Ki-67 expression in tumor tissues. (H) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. HEC-1A cells with or without CAFs stably transfected with sh-NEAT1 or NEAT1 gene were subcutaneously injected into the nude mice. (I) The tumor growth curve was shown. (J) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (K) Immunohistochemical staining for Ki-67 expression in tumor tissues. (L) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Derivative Assay, In Vivo, Injection, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Western Blot, Stable Transfection, Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Overexpression of miR-26a/b-5p reverses exosomal NEAT1-mediated protumorigenic effect in vivo . HEC-1A cells stably transfected with miR-26a/b-5p mimics or NC mimics were mixed with or without CAFs stably expressing NC vector or NEAT1, which were subcutaneously injected into the nude mice. (A) The tumor growth curve was shown. (B) Immunohistochemical staining for Ki-67 expression in tumor tissues. (C) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Over Expression, In Vivo, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Injection, Immunohistochemical staining, Staining, Western Blot

Journal: Oncology Reports

Article Title: Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration‑resistant prostate cancer cells

doi: 10.3892/or.2024.8730

Figure Lengend Snippet: List of antibodies.

Article Snippet: The DU145 cells were transfected with pCMV2 control vector (CV013), pCMV2-Stat3-Myc (HG10034-CM), or pCMV2-Glo1-HA (HG12223-CY) (Sino Biological, Inc.) at 37°C for 24 h according to the manufacturer's protocol.

Techniques:

Journal: Oncology Reports

Article Title: Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration‑resistant prostate cancer cells

doi: 10.3892/or.2024.8730

Figure Lengend Snippet: The effects of NaB on Stat1, JAK2 and Stat3 expression. (A) DU145 cells were treated with NaB (2.5 or 5 mM) for 48 h and the expression of these genes was determined. (B) The DU145 cells were incubated with ruxolitinib (0.5 µM) for 2 h followed by treatment with NaB for 48 h, the expression of p-Stat3 and Glo1 was determined. (C) The relative ROS was measured in cells treated with NaB (2.5 or 5 mM) for 48 h. (D) After the cells were incubated with NAC (5 mM) or medium for 2 h followed by treatment with NaB for another 48 h, the protein level of p-Stat3 was determined in various groups. (E and F) Cells were pretreated with Colivelin (10 µM) or medium for 2 h followed by treatment with NaB for another 48 h. Then, the expression of the (E) target genes and (F) MG-H1 production were determined. Results are expressed as the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the Control group; # P<0.05, ## P<0.01 and ### P<0.001 vs. respective NaB-treated alone cells. NaB, sodium butyrate; Stat, signal transducer and activator of transcription; JAK2, Janus kinase 2; Glo1, glyoxalase1; ROS, reactive oxygen species; NAC, N-acetyl-cysteine; MG-H1, hydroimidazolone; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; p-, phosphorylated.

Article Snippet: The DU145 cells were transfected with pCMV2 control vector (CV013), pCMV2-Stat3-Myc (HG10034-CM), or pCMV2-Glo1-HA (HG12223-CY) (Sino Biological, Inc.) at 37°C for 24 h according to the manufacturer's protocol.

Techniques: Expressing, Incubation, Control

Journal: Oncology Reports

Article Title: Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration‑resistant prostate cancer cells

doi: 10.3892/or.2024.8730

Figure Lengend Snippet: The effects of MGO, Glo1, Stat3 and CaMKII phosphorylation on NaB-mediated target gene expression and cell viability. (A) After treatment with MGO (0.2-1.6 mM) for 48 h, the expression of Nrf2, Glo1 and HO-1, as well as the cell viability were determined. (B) The cell viability of the DU145 cells with overexpression of Glo1 or Stat3 was examined. (C) Cells were pretreated with KN-93 (10 µM) for 2 h followed by treatment with NaB for 48 h. Then, the expression of MAPKs and pro-apoptotic proteins were determined. Results are expressed as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the Vector or Control group; # P<0.05, ### P<0.001 vs. respective NaB-treated alone cells. MGO, methylglyoxal; Glo1, glyoxalase1; Stat3, signal transducer and activator of transcription 3; CaMKII, calcium/calmodulin dependent protein kinase II gamma; NaB, sodium butyrate; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; PARP, Poly (ADP-Ribose) polymerase.

Article Snippet: The DU145 cells were transfected with pCMV2 control vector (CV013), pCMV2-Stat3-Myc (HG10034-CM), or pCMV2-Glo1-HA (HG12223-CY) (Sino Biological, Inc.) at 37°C for 24 h according to the manufacturer's protocol.

Techniques: Targeted Gene Expression, Expressing, Over Expression, Plasmid Preparation, Control

Journal: Oncology Reports

Article Title: Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration‑resistant prostate cancer cells

doi: 10.3892/or.2024.8730

Figure Lengend Snippet: Effects of KN-93 on cell proliferation the effects of NaB on normal RWPE-1 cells. (A) The relative cell proliferation of DU145 cells and the expression of c-Myc and cyclin D1 were evaluated in NaB (5 mM) alone, and co-treatment with KN-93, NAC, or Colivelin for indicated time. (B) The cell viability and the levels of Glo1 and Stat3 in RWPE-1 cells were examined after treatment with NaB for 48 h. Results are expressed as the mean ± SEM. **P<0.01 and ***P<0.001 vs. the Control group. (C) The proposed schematic diagram of NaB-mediated Nrf2/Glo1/MGO pathway and apoptosis in PCa cells. NaB inhibits the expression and activity of Nrf2 through PERK inactivation and accumulation of nuclear Bach1. Then, Glo1expression is attenuated via suppression of Janus kinase 2/Stat3 pathway, leading to accumulation of MGO and apoptotic cell death. The elevated MGO further downregulates Nrf2, Glo1 and HO-1. The CaMKII-mediated activation of MAPKs and Stat1 also contributes to NaB-induced cell death. NaB, sodium butyrate; NAC, N-acetyl-cysteine; Glo1, glyoxalase1; Stat3, signal transducer and activator of transcription 3; Nrf2, nuclear factor erythroid 2-related factor 2; MGO, cytotoxic methylglyoxal; PERK, eukaryotic translation initiation factor 2 alpha kinase 3; Bach1, BTB domain and CNC homolog 1; HO-1, heme oxygenase-1; CaMKII, calcium/calmodulin dependent protein kinase II gamma.

Article Snippet: The DU145 cells were transfected with pCMV2 control vector (CV013), pCMV2-Stat3-Myc (HG10034-CM), or pCMV2-Glo1-HA (HG12223-CY) (Sino Biological, Inc.) at 37°C for 24 h according to the manufacturer's protocol.

Techniques: Expressing, Control, Activity Assay, Activation Assay

Journal: Neural Regeneration Research

Article Title: Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

doi: 10.4103/1673-5374.241470

Figure Lengend Snippet: Effect of STAT3 silencing on NLRP3 expression in response to H 2 O 2 in SH-SY5Y cells. (A) STAT3 protein expression was significantly downregulated in the shRNA group. SH-SY5Y cells were infected with lentiviruses carrying pFLU-EGFPshSTAT3 (shRNA) or scramble (shScr). STAT3 expression was determined by western blot assay. (B) Indicated cells were treated with or without H 2 O 2 . NLRP3 mRNA and β-actin were detected by reverse transcription-polymerase chain reaction. Graphs show the relative quantification of NLRP3 mRNA expression. Band intensities of NLRP3 mRNA were normalized to that of β-actin using the Quantity One software (NIH, Bethesda, MD, USA). Data are expressed as the mean ± SD ( n = 5). *** P < 0.001 (one-way analysis of variance). The experiments were performed in triplicate. ns: Not significant; STAT3: signal transducer and activator of transcription 3; NLRP3: nucleotide binding to the oligonucleotide receptor protein 3.

Article Snippet: Human STAT3 cDNA in pCMV was purchased from Sino Biological (Beijing, China) and transfected into cells to overexpress STAT3.

Techniques: Expressing, shRNA, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Software, Binding Assay

Journal: Neural Regeneration Research

Article Title: Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

doi: 10.4103/1673-5374.241470

Figure Lengend Snippet: Effect of the transfected STAT3 plasmid for caspase-1 expression before (without) or after (with) BAPTA-AM in an oxidative stress model of SH-SY5Y cells. Expression of caspase-1 detected by western blot assay. The relative optical density of caspase-1 was normalized to tubulin. Data are expressed as the mean ± SD ( n = 5; one-way analysis of variance followed by Tukey's post hoc test). ** P < 0.05; *** P < 0.01. The experiments were performed in triplicate. ns: not significant; STAT3: signal transducer and activator of transcription 3.

Article Snippet: Human STAT3 cDNA in pCMV was purchased from Sino Biological (Beijing, China) and transfected into cells to overexpress STAT3.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

doi: 10.4103/1673-5374.241470

Figure Lengend Snippet: Effect of STAT3 downregulation on IL-1β production. Cells were pre-treated without or with BAPTA-AM, and then with H 2 O 2 . IL-1β levels in supernatants were measured by human IL-1β enzyme-linked immunosorbent assay kit. Data are expressed as the mean ± SD (one-way analysis of variance followed by Tukey's post hoc test). * P < 0.05; ** P < 0.01. The experiments were performed in triplicate. IL: Interleukin; STAT3: signal transducer and activator of transcription 3; DMSO: dimethyl sulfoxide.

Article Snippet: Human STAT3 cDNA in pCMV was purchased from Sino Biological (Beijing, China) and transfected into cells to overexpress STAT3.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Neural Regeneration Research

Article Title: Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

doi: 10.4103/1673-5374.241470

Figure Lengend Snippet: Effect of STAT3 silencing on H 2 O 2 -induced dead and apoptotic cells. The cells were pretreated without or with 0.1 mM BAPTA-AM, and then with 1 mM H 2 O 2 as indicated. Cells were stained with Annexin V/PI. Dead and apoptotic cells were determined by flow cytometry. Data are expressed as the mean ± SD ( n = 5; one-way analysis of variance followed by Tukey's post hoc test). * P < 0.05. The experiments were performed in triplicate. ns: Not significant; STAT3: signal transducer and activator of transcription 3; PI: propidium iodide.

Article Snippet: Human STAT3 cDNA in pCMV was purchased from Sino Biological (Beijing, China) and transfected into cells to overexpress STAT3.

Techniques: Staining, Flow Cytometry

Journal: Neural Regeneration Research

Article Title: Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

doi: 10.4103/1673-5374.241470

Figure Lengend Snippet: BAPTA-AM partly inhibits H 2 O 2 -induced NLRP3 expression in cells with STAT3 overexpression. (A, A’) STAT3 or vehicle (empty vector) was overexpressed in STAT3 shRNA cells of the shRNA + vehicle group and the shRNA + STAT3 group, respectively. STAT3 protein expression levels were determined by western blot assay. (B, B’, B’’) Cells in the shRNA + STAT3 group were pretreated with 0.1 mM BAPTA-AM, then without or with 1 mM H 2 O 2 . NLRP3 and caspase-1 expression levels were determined by western blot assay. The relative optical density levels of target protein were normalized to GAPDH/tubulin. Data are expressed as the mean ± SD ( n = 5; one-way analysis of variance followed by Tukey's post hoc test). * P < 0.05, ** P < 0.01, vs mock group. The experiments were performed in triplicate. STAT3: Signal transducer and activator of transcription 3; NLRP3: nucleotide binding to the oligonucleotide receptor protein 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human STAT3 cDNA in pCMV was purchased from Sino Biological (Beijing, China) and transfected into cells to overexpress STAT3.

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

Journal: Neural Regeneration Research

Article Title: Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

doi: 10.4103/1673-5374.241470

Figure Lengend Snippet: H 2 O 2 activates the phosphorylation of STAT3 serine 727 in an oxidative stress model of SH-SY5Y cells. Cells were treated without or with 1 mM H 2 O 2 for 30 minutes. The levels of STAT3 and its serine 727 phosphorylation were determined by western blot assay. The relative optical density levels of target protein were normalized to GAPDH. Data are expressed as the mean ± SD ( n = 5; one-way analysis of variance followed by Tukey's post hoc test). ** P < 0.01, vs . mock group. The experiments were performed in triplicate. STAT3: Signal transducer and activator of transcription 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human STAT3 cDNA in pCMV was purchased from Sino Biological (Beijing, China) and transfected into cells to overexpress STAT3.

Techniques: Western Blot