Journal: Oxidative Medicine and Cellular Longevity

Article Title: THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

doi: 10.1155/2021/5620475

Figure Lengend Snippet: The correlations between clinicopathological features and dysregulation of moleculars.

Article Snippet: Full-length human THP9-AS1 (ENST00000504520) and SOCS3 (NM_003955) cDNA expression plasmids (THAP9-AS1 and pcSOCS3) and the empty control vector (vector) were purchased from Sangon Biotech (China).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

doi: 10.1155/2021/5620475

Figure Lengend Snippet: THAP9-AS1 promoted SOCS3 promoter methylation by recruiting DNMTs. (a) Results of BLAST comparison between THAP9-AS1 and the SOCS3 promoter region. (b) The expression of THAP9-AS1 was significantly upregulated in MG63 cells after transfection with THAP9-AS1-expressing vectors. (c) The binding of THAP9-AS1 to the SOCS3 promoter was evaluated by dual-luciferase reporter gene assay. (d) Fold enrichment of DNMT1, DNMT3a, and DNMT3b in the SOCS3 promoter was determined by ChIP assay. (e) RIP was used to detect the binding between THAP9-AS1 and DNMTs. (f) MSP was conducted to verify the methylation status of SOCS3. U: unmethylation; M: methylation. (g) The results of the BSP assay validated that overexpression of THAP9-AS1 induced promoter hypermethylation of SOCS3; black circle, methylated CpG sites; white circle, unmethylated CpG sites. THAP9-AS1- and THAP9-AS1-expressing vectors; vector, empty vector control. pSOCS3-mut: mutants of the SOCS3 promoter; pSOCS3-wt: wild-type SOCS3 promoter. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Full-length human THP9-AS1 (ENST00000504520) and SOCS3 (NM_003955) cDNA expression plasmids (THAP9-AS1 and pcSOCS3) and the empty control vector (vector) were purchased from Sangon Biotech (China).

Techniques: Methylation, Comparison, Expressing, Transfection, Binding Assay, Luciferase, Reporter Gene Assay, PCR-BSP Assay, Over Expression, Plasmid Preparation, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

doi: 10.1155/2021/5620475

Figure Lengend Snippet: THAP9-AS1 negatively regulated SOCS3 in OS cells and tissues. (a) The mRNA levels of SOCS3 were downregulated in OS cells. (b) The mRNA levels of SOCS3 were restored in OS cells treated with 5-aza. (c, d) Knockdown of DNMTs in MG63 cells increased the mRNA and protein expression of SOCS3. (e) The expression of THAP9-AS1 was effectively downregulated in MG63 cells after transfection with si-THAP9-AS1. (f, g) The mRNA and protein expression of SOCS3 was inhibited by THAP9-AS1 but increased by si-THAP9-AS1. (h) The mRNA levels of SOCS3 were significantly reduced in OS tissues. (i) The mRNA levels of SOCS3 were inversely correlated with THAP9-AS1 in OS tissues. ∗∗ p < 0.01; ∗∗∗ p < 0.001. ns: no significance; si-DNMTs: siRNAs for knocking down DNMTs; si-THAP9-AS1: siRNA specific for silencing THAP9-AS1; si-ctrl: siRNA negative control.

Article Snippet: Full-length human THP9-AS1 (ENST00000504520) and SOCS3 (NM_003955) cDNA expression plasmids (THAP9-AS1 and pcSOCS3) and the empty control vector (vector) were purchased from Sangon Biotech (China).

Techniques: Knockdown, Expressing, Transfection, Negative Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

doi: 10.1155/2021/5620475

Figure Lengend Snippet: THAP9-AS1 activated the JAK2/STAT3 signaling pathway by regulating SOCS3. (a) THAP9-AS1 induced p-JAK2 and p-STAT3 (Tyr705) protein upregulation, which could be reversed by SOCS3. (b) p-STAT3 nuclear translocation in the different groups of cells was analyzed by IF assay, and representative images are shown; scale bars: 100 μ m. pcSOCS3: SOCS3 expressing vectors.

Article Snippet: Full-length human THP9-AS1 (ENST00000504520) and SOCS3 (NM_003955) cDNA expression plasmids (THAP9-AS1 and pcSOCS3) and the empty control vector (vector) were purchased from Sangon Biotech (China).

Techniques: Translocation Assay, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

doi: 10.1155/2021/5620475

Figure Lengend Snippet: THAP9-AS1 promoted cell growth and invasion in vitro via the SOCS3/JAK2/STAT3 signaling pathway. (a) The protein expression of p-STAT3 was measured. (b) THAP9-AS1 promoted cell proliferation, which could be reversed by either SOCS3 or AG490. (c) Cell apoptosis was inhibited by THAP9-S1 but promoted by either SOCS3 or AG490. (d, e) The number of migrated and invaded cells was increased in the THAP9-AS1 groups and decreased in the SOCS3-expressing and AG490-treated groups; scale bars: 50 μ m. (f) THAP9-AS1 promoted EMT progression by decreasing E-cadherin and increasing N-cadherin. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. AG490: inhibitor of JAK2/STAT3 signaling.

Article Snippet: Full-length human THP9-AS1 (ENST00000504520) and SOCS3 (NM_003955) cDNA expression plasmids (THAP9-AS1 and pcSOCS3) and the empty control vector (vector) were purchased from Sangon Biotech (China).

Techniques: In Vitro, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

doi: 10.1155/2021/5620475

Figure Lengend Snippet: Dysregulation of THAP9-AS1/SOCS3 causes mitochondrial dysfunction. (a) ROS were reduced in the THAP-AS1 groups and restored in the SOCS3 and AG490 treatment groups; scale bars: 100 μ m. (b) JC-1 staining was used to observe the mitochondrial membrane potential. The red fluorescence of the JC-1 probe indicates normal mitochondria, and the green fluorescence of the JC-1 probe indicates damaged mitochondrial potential; scale bars: 100 μ m. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Full-length human THP9-AS1 (ENST00000504520) and SOCS3 (NM_003955) cDNA expression plasmids (THAP9-AS1 and pcSOCS3) and the empty control vector (vector) were purchased from Sangon Biotech (China).

Techniques: Staining, Membrane, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

doi: 10.1155/2021/5620475

Figure Lengend Snippet: THAP9-AS1 promoted tumor growth and metastasis in vivo. (a, b) Upregulation of THAP9-AS1 increased the volumes and weights of mouse tumors. (c) The expression of THA9-AS1 in mouse tumors was determined. (d) The expression of SOCS3 was decreased in tumors originating from the THAP9-AS1 group. (e) IHC assay for the expression of Ki-67 and p-STAT3 in mouse tumors; scale bars: 100 μ m. (f) The number of lung metastatic nodes was increased by THAP9-AS1; HE staining was conducted to verify the lung metastasis foci; scale bars: 100 μ m. (g) A schematic showing that THAP9-AS1 contributed to cell proliferation and metastasis and inhibited ROS production, probably through epigenetically suppressing SOCS3, thereby activating the JAK2/STAT3 signaling pathway. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Full-length human THP9-AS1 (ENST00000504520) and SOCS3 (NM_003955) cDNA expression plasmids (THAP9-AS1 and pcSOCS3) and the empty control vector (vector) were purchased from Sangon Biotech (China).

Techniques: In Vivo, Expressing, Staining

Journal: Journal of Molecular Cell Biology

Article Title: Benzo[a]pyrene stimulates miR-650 expression to promote the pathogenesis of fatty liver disease and hepatocellular carcinoma via SOCS3/JAK/STAT3 cascades

doi: 10.1093/jmcb/mjab052

Figure Lengend Snippet: SOCS3 is a direct downstream target of miR-650. ( A ) Target gene prediction of miR-650 with three bioinformatics tools. ( B ) 7404 cells were transfected with miR-650 mimics or control. The transcription and protein expression levels of SOCS3 were determined by qRT-PCR and immunoblotting analyses. ( C ) The luciferase vectors containing wild-type (wt) or mutated (mu) sequences of the binding site between miR-650 and SOCS3 mRNA were constructed. Relative luciferase activities of 7404 cells with the indicated treatments were determined. * P <0.05, *** P <0.001.

Article Snippet: The expression vector pcDNA-3.1 SOCS3 was constructed by inserting the SOCS3 open-reading frame sequence into the pcDNA-3.1 vector (Invitrogen). pSilencer3.0 (Ambion) was used for the construction of human SOCS3 siRNA vector psi SOCS3 according to the manufacturer’s protocol.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Binding Assay, Construct

Journal: Journal of Molecular Cell Biology

Article Title: Benzo[a]pyrene stimulates miR-650 expression to promote the pathogenesis of fatty liver disease and hepatocellular carcinoma via SOCS3/JAK/STAT3 cascades

doi: 10.1093/jmcb/mjab052

Figure Lengend Snippet: miR-650 promotes tumor cell motility via SOCS3/JAK/STAT3 signaling axis. ( A ) Immunoblotting assay for SOCS3 protein levels in 7404-Bap100, 7404+siSOCS3, and 7404 cells. ( B ) Migration assay of 7404 cells transfected with siRNAs targeting SOCS3 or control. ( C ) qRT-PCR analysis of 7404 cells transfected with siRNAs targeting SOCS3 or control for indicated genes. ( D ) Immunoblotting assays of 7404 cells transfected with siRNAs targeting SOCS3 or control, miR-650 mimic, and miR-650 inhibitor for indicated proteins.

Article Snippet: The expression vector pcDNA-3.1 SOCS3 was constructed by inserting the SOCS3 open-reading frame sequence into the pcDNA-3.1 vector (Invitrogen). pSilencer3.0 (Ambion) was used for the construction of human SOCS3 siRNA vector psi SOCS3 according to the manufacturer’s protocol.

Techniques: Western Blot, Migration, Transfection, Quantitative RT-PCR

Journal: Journal of Molecular Cell Biology

Article Title: Benzo[a]pyrene stimulates miR-650 expression to promote the pathogenesis of fatty liver disease and hepatocellular carcinoma via SOCS3/JAK/STAT3 cascades

doi: 10.1093/jmcb/mjab052

Figure Lengend Snippet: Downregulation of SOCS3 by B[a]P-induced miR-650 may be associated with metastatic progression in liver cancer. ( A ) SOCS3 expression in normal liver tissues and liver cancer tissues. ( B ) SOCS3 expression in normal liver tissues and varying metastasis-grade liver cancer tissues. ( C ) Proposed schematic diagram of B[a]P exposure-stimulated miR-650 mediating SOCS3 suppression and JAK activation to promote tumor cell motility and ultimately metastasis of liver cancer.

Article Snippet: The expression vector pcDNA-3.1 SOCS3 was constructed by inserting the SOCS3 open-reading frame sequence into the pcDNA-3.1 vector (Invitrogen). pSilencer3.0 (Ambion) was used for the construction of human SOCS3 siRNA vector psi SOCS3 according to the manufacturer’s protocol.

Techniques: Expressing, Activation Assay