Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Inhibition of cell proliferation in METTL3-KO SW480 cells. (A) METTL3-KO SW480 cells showed dysregulation of several proliferation-related genes (*P<0.05, compared with the SW480 WT cells). (B) MTS assay results validated the decreased proliferation of METTL3-KO cells (*P<0.05, compared with the SW480 WT cells). (C) METTL3-KO cells showed a reduced proliferation rate, as measured by cell counting (*P<0.05, compared with the SW480 WT cells). (D) METTL3-KO cells showed a lower colony formation rate than control cells at 7 days after seeding. (E) Tumorigenic ability of METTL3-KO SW480 cells was reduced (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; SOX2, SRY-box transcription factor 2; PCNA, proliferating cell nuclear antigen; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Inhibition, MTS Assay, Cell Counting, Control, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: SOCS2 is upregulated in colon cancer and downregulated in METTL3-KO SW480 cells. (A) TCGA database analysis revealed a lower expression level of SOCS2 in colon cancer tissues than in the paired normal tissues (*P<0.05). (B) Expression levels of SOCS2 and METTL3 were negatively correlated in colon cancer tissues. (C-E) METTL3-KO upregulated SOCS2 expression at both the (C) mRNA and (D and E) protein levels in SW480 cells (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Methylation status of SOCS2 mRNA in SW480 cells. (A) Comparison of the SOCS2 mRNA methylation status between SW480 WT and METTL3-KO SW480 cells via MeRIP-qPCR (*P<0.05, compared with the SW480 WT cells). (B) Dose-dependent increase in SOCS2 mRNA levels in SW480 cells in response to demethylation treatment with DAA (*P<0.05, compared with non-treated group). (C and D) SOCS2 protein expression was upregulated after demethylation treatment with DAA (*P<0.05, compared with the SW380 WT DAA-group). (E) DAA appreciably decreased the m 6 A level in SOCS2 mRNA (*P<0.05, compared with the SW380 WT DAA-group). (F) METTL3-KO increased SOCS2 mRNA stability, showing a decreased mRNA decay rate (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells; DAA, 3-deazaadenosine.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Methylation, Comparison, Expressing, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Modulation of SOCS2 expression alters the proliferation ability of SW480 cells. (A-C) siRNA (si-SOCS2) was used to successfully knock down SOCS2 expression in SW480 cells. Knockdown of SOCS2 had minor impact on METTL3 expression but significantly increased LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D) Knockdown of SOCS2 enhanced SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the si-Ctrl group). (E-G) Transfection of the pCMV6-SOCS2 plasmid upregulated SOCS2 expression and decreased LGR5 expression in SW480 cells (*P<0.05, compared with the pCMV6-Entry group). SOCS2 overexpression had minor impact on METTL3 expression (P>0.05, compared with the pCMV6-Entry group). (H) SOCS2 overexpression decreased SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the pCMV6-Entry group). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knockdown, MTS Assay, Transfection, Plasmid Preparation, Over Expression

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: METTL3 modulates LGR5 transcription through m 6 A-mediated changes in SOCS2 expression rather than through direct regulatory mechanisms. (A) TCGA database analysis revealed an induction of LGR5 transcription in colon cancer tissues when compared with normal tissues (**P<0.01, compared with the normal tissues). (B) Expression levels of SOCS2 and LGR5 were negatively correlated in colon cancer tissues. (C) METTL3 knockout (KO) did not obviously impact LGR5 mRNA methylation (as measured by MeRIP-qPCR). (D) METTL3 KO had a minor effect on the LGR5 mRNA decay rate. (E) DAA treatment did not impact LGR5 expression in SW480 cells. (F) Luciferase reporter assay results confirmed that SOCS2 negatively controls LGR5 promoter-driven luciferase activity (*P<0.05, pGL3 basic-LGR5 promoter group vs. FULL SOCS2-LGR5 promoter group). METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; SOCS2, suppressor of cytokine signaling 2; TCGA, The Cancer Genome Atlas; SW480 A7 and SW480 A12, METTL3-KO SW480 cells; DAA, 3-deazaadenosine.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knock-Out, Methylation, Luciferase, Reporter Assay, Activity Assay

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Correlations among METTL3, SOCS2 and LGR5 expression levels in CRC. (A-C) Knockdown of METTL3 using siRNA (si-METTL3#1 and si-METTL3#2) significantly upregulated SOCS2 expression but downregulated LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D and E) Western blotting analysis of 24 paired tissues collected from CRC patients showed high protein levels of METTL3 and LGR5 and a relatively low protein level of SOCS2 in colon cancer (T, colon tumor samples; N, adjacent normal colon tissues; *P<0.05, compared to Normal tissues). (F) Visualization of expression profiles of METTL3, SOCS2 and LGR5 in CRC through the Oncomine database. METTL3 and LGR5 were upregulated in CRC datasets. In contrast, SOCS2 was downregulated in CRC datasets. CRC, colorectal cancer; SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knockdown, Western Blot

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Association between patient characteristics and gene expression in 24 paired CRC cases.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Gene Expression, Expressing

Journal: Cancer cell international

Article Title: Increased expression of miR-194-5p through the circPVRL3/miR-194-5p/SOCS2 axis promotes proliferation and metastasis in pancreatic ductal adenocarcinoma by activating the PI3K/AKT signaling pathway.

doi: 10.1186/s12935-022-02835-0

Figure Lengend Snippet: Fig. 6 SOCS2 serves as a suppressor gene and a target of miR-194-5p. A The potential targets of miR-194-5p were predicted by miRDB, TargetScan, StarBase, Tarbase and miRpathDB. Nine target genes (Gene set 2) were obtained from the intersection. B The downregulated genes in PDAC of the GSE43795 dataset (Gene set 1) and the genes of Gene set 2 were intersected to obtain the target gene of miR-194-5p. C The expression of SOCS2 was obviously reduced in PDAC tissue compared with tumor-adjacent tissue, as shown by IHC staining (n = 6). Scale bar = 50 μm. D The expression of SOCS2 was decreased in PDAC compared tissue with normal tissue based on TCGA and GTEx data. E PDAC samples from the TCGA were divided into two groups (high and low SOCS2 expression groups). Kaplan‒Meier survival analysis was used to determine the overall survival rate. F The binding sites between miR-194-5p and SOCS2 mRNA were predicted by TargetScan (top). The WT and Mut sequences of the binding sites between SOCS2 mRNA and miR-194-5p were cloned into dual-luciferase reporter vectors (bottom). G A dual-luciferase reporter assay was performed by co-transfection of PDAC cells with WT/Mut SOCS2 plasmids and miR-194-5p mimic or mimic NC. H RIP and RT‒qPCR assays were performed to confirm the binding of miR-194-5p to the AGO2 protein. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Primary antibodies against P21, E-cadherin, N-cadherin, CDK2, CDK4, Cyclin D1, Cyclin E1, p-AKT, AKT, p-PI3K, PI3K, GAPDH (1:1000; Cell Signaling Technology) and SOCS2(1:1000; ABclonal) were incubated overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Binding Assay, Clone Assay, Luciferase, Reporter Assay, Cotransfection

Journal: Cancer cell international

Article Title: Increased expression of miR-194-5p through the circPVRL3/miR-194-5p/SOCS2 axis promotes proliferation and metastasis in pancreatic ductal adenocarcinoma by activating the PI3K/AKT signaling pathway.

doi: 10.1186/s12935-022-02835-0

Figure Lengend Snippet: Fig. 7 SOCS2 inhibits the PDAC cell proliferation and negatively regulates the activity of PI3K/AKT signaling pathway. A, B EdU and colony formation assays were performed to detect the proliferation ability of SOCS2 in PDAC cells. Scale bar = 100 μm. C Transwell assays were performed to observe the migration of PDAC cells after treatment with SOCS2 overexpression plasmids or si-SOCS2. Scale bar = 200 μm. D The level of SOCS2 in the miR-194-5p overexpression or knockdown group was determined by RT‒qPCR. E The activation level of the PI3K/AKT signaling pathway was observed in the groups with miR-194-5p mimic and SOCS2 overexpression alone or combined and the groups with miR-194-5p inhibitor and si-SOCS2 alone or combined by western blotting. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Primary antibodies against P21, E-cadherin, N-cadherin, CDK2, CDK4, Cyclin D1, Cyclin E1, p-AKT, AKT, p-PI3K, PI3K, GAPDH (1:1000; Cell Signaling Technology) and SOCS2(1:1000; ABclonal) were incubated overnight at 4 °C.

Techniques: Activity Assay, Migration, Over Expression, Knockdown, Activation Assay, Western Blot

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 1. GHR expression in normal and cancerous lung tissue. (a) GHR levels in clinical samples representing 18 SCC and 40 lung adenocarcinoma analysed from microarray data (GSE10245) retrieved from Gene Expression Omnibus (GEO).24 A significant correlation was determined at P = 0.003. Gene expression analysis of four genes (b) GHR, (c) EGFR, (d) SOCS2 and (e) SOCS3 in 60 clinical samples of normal lung tissue and NSCLC from a non-smoking female cohort in accordance with microarray data (GSE19804).25 A significant difference was observed (Po0.0001). The expression levels of other genes (SOCS1, CISH and GH2) did not differ significantly (Supplementary Figure 1).

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Expressing, Microarray, Gene Expression

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 4. GHRP495T impairs SOCS2 binding to GHR. (a) No difference in SOCS2 transcript induction between WT GHR and GHRP495T. HEK293 cells transduced with WT GHR and GHRP495T were maintained in serum-starved media with sustained GH (2.3 nM) then RNA was extracted at indicated time points. SOCS2 levels were determined by real-time PCR and normalised to GAPDH reference gene. Data presented as mean ± s.e.m. analysed by one-way ANOVA (****Po0.0001, ***Po0.001). Representative of three independent experiments confirmed in three separate lines generated by independent transductions. (b) HEK293 cells stably expressing WT GHR or GHRP495T, and parental cells transfected with SOCS2 expression plasmid for 24 h and serum-starved overnight before 2.3 nM GH (+) or vehicle (−) stimulation for 15 min. Lysates were harvested and co-IP with SOCS2 antibody as described in Materials and methods. Protein complex from the immunoprecipitates were immunoblotted (IB) using anti-HA antibody for GHR (mature (m) receptor and precursor (p) receptor) and anti-SOCS2 (input). As control, parental cell line with no transduced GHR was used and a small volume of total cell lysates used for co-IP was probed for GHR levels (HA-tag) to indicate GHR levels. (c) Graph represents the signal intensity of total GHR (HA-tag) pull down in GHRP495T relative to WT GHR relative to SOCS2 input, corrected for endogenous GHR expression. Data presented as mean ± s.e.m. analysed by Student’s t-test (***Po0.001) and representative of nine independent experiments confirmed in three independently transduced cell lines. (d) CISH protein does not interact directly with GHR as compared with SOCS2. HEK293 cells stably expressing WT GHR co-transfected with CISH or SOCS2 expression plasmids for 24 h and serum-starved overnight before 2.3 nM GH (+) or vehicle (-) stimulation for 15 min. Lysates were harvested and co-IP with SOCS2 and CISH antibodies simultaneously. Protein complex from the immunoprecipitates was immunoblotted using anti-HA antibody (for GHR) and anti-SOCS2 and anti-CISH antibodies. As a control, parental cell line with no transduced GHR, but transfected with CISH was used and a small volume of total cell lysates used for co-IP was probed for GHR levels (HA-tag) to indicate endogenous GHR levels (see Supplementary Figure 2).

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Binding Assay, Transduction, Real-time Polymerase Chain Reaction, Generated, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Control

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 5. GHRP495T generates structural changes in the receptor intracellular domain. (a) Clustal Omega multiple sequence alignment of the GHR polypeptide in close proximity to Pro 495 (red). The Tyr residue, a known active STAT5 binding site is coloured blue. Symbols below indicates (*) identical residues, (:) conservation of strongly similar properties and (.) conservation of weakly similar properties. (b) Importance of residues surrounding Pro495 to SOCS2 binding. HEK293 cells stably transduced with WT GHR, GHRP495T, GHRP495K, GHRP495A and GHRT494A were subjected to co-IP analysis as described in Materials and methods. Protein complexes from the immunoprecipitates were immunoblotted for GHR (anti-HA-tag) (mature (m) receptor and precursor (p) receptor) and SOCS2. As control, a small volume of total cell lysates used for co-IP was probed to indicate endogenous levels of GHR, SOCS2, and GAPDH (loading control). (c) Graph represents the signal intensity of total GHR (HA-tag) pull down relative to SOCS2 input, corrected for endogenous GHR expression. Data presented as mean ± s.e.m. analysed by one-way ANOVA (***Po0.001) and representative of three independent experiments confirmed in three lines generated by independent transductions. (d) GHRP495T changes the structural ensemble in the GHR intracellular domain by nuclear magnetic resonance spectroscopy analysis. Secondary Cα-chemical shift (SCS) of GHR ICD455-588 WT (top panel). Consecutive positive SCS-values indicate transiently folded helices (TH) marked in grey boxes. The original TH5, as previously reported34 was re-evaluated as two interrupted transient helices as TH5.1 (E462-L475), TH5.2 (P478-S488), and are shown together with TH6 and TH7 (top panel). SCS of GHR ICD455-588 P495T (middle panel). P495T induces helicity around the mutation site and before in the C-terminal of TH5.2. Numbering includes the signal peptide. Model illustrating the change in structural ensemble around T495 (bottom panel) and the inter-conversion equilibrium between disordered and helical structures, where the helicity is increased in the GHR ICD455-588 P495T .

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Sequencing, Residue, Binding Assay, Stable Transfection, Transduction, Co-Immunoprecipitation Assay, Control, Expressing, Generated, Nuclear Magnetic Resonance, Spectroscopy, Mutagenesis

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 6. GHRP495T degradation is impaired. (a) Effect of BFA treatment on WT GHR and GHRP495T. Time course analysis on HEK293 cells transduced with WT GHR and GHRP495T subjected to 2.3 nM GH stimulation for 15 min in the presence of BFA and harvested at indicated time points. Immunoblot demonstrating GHR (HA-tag) levels (mature (m) receptor and precursor (p) receptor) and β-TUBULIN (loading control). (b) Graph indicating fold change in mature GHR levels normalised to β-TUBULIN relative to ‘0 min’ time point after GH removal. (c) Immunoblot indicating GHR (HA-tag) following BFA treatment, as above (a) in the presence of SOCS2. (d) Graph indicating fold change in mature GHR levels in the presence of SOCS2, normalised to β-TUBULIN relative to ‘0 min’ time point after GH removal. (b, d) Data presented as mean ± s.e.m. analysed by two-way ANOVA (**Po0.01, *Po0.05) and representative of at least three independent experiments confirmed in two independently transduced lines (see Supplementary Figure 3); (e) GHRP495T is less amenable to degradation owing to SOCS2 as evident from TIRF microscopy (allows detection of fluorescent proteins only at, or near the cell membrane) images of HEK293 cells transduced with WT GHR-GFP and GHRP495T-GFP transfected with SOCS2-mCherry in the absence of exogenous GH. Colocalisation (yellow) of SOCS2 (red) and receptor (green) was more pronounced in GHRP495T (See Supplementary Figure 4). Separate channel images shown below. (f) GHR levels on cell surface of HEK293 cells expressed as normalised fluorescent intensity in the presence or absence of SOCS2. Data presented as mean ± s.e.m. from 30 cells per condition across two independently transduced cell lines and analysed by one-way ANOVA (**Po0.01) relative to WT GHR-GFP no SOCS2. Student’s t-test (#Po0.05) between WT GHR-GFP + SOCS2 and GHRP495T-GFP + SOCS2. (g) SOCS2 levels at the cell surface expressed as normalised fluorescent intensity for the cells analysed above. Data presented as mean± s.e.m. from 30 cells per condition from three independent experiments across two independently transduced lines and analysed by Student’s t-test (ns, not significant). (h) HEK293 cells transduced with WT GHR and GHRP495T treated with proteasomal inhibitor MG-132 (10 μM) for 2 h before GH (2.3 nM) addition for 15 min. Immunoblot indicating GHR (HA-tag) levels with mature and precursor receptor and two remnant bands observed at ~ 60 and ~ 43 kDa only in WT GHR lysates and undetectable in GHRP495T (see Supplementary Figure 5). Blot representative of four independent experiments.

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Transduction, Western Blot, Control, Microscopy, Membrane, Transfection

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 7. GHRP495T increases transcripts associated with tumour progression. BEAS-2B cells transduced with WT GHR and GHRP495T stimulated with GH (2.3 nM) for 15 min, then RNA was harvested at 60 (a) and 120 min (b) post-initial treatment. (a) Transcript levels of early GH-response genes c-FOS, EGR1, JUN at 60 min in WT GHR and GHRP495T cells normalised to B2M. (b) Transcript levels of genes associated with epithelial–mesenchymal transition (EMT) (TWIST1, SNAI2 and TGFB1), proliferation (CCND1, MYC), and elevated GHR signalling (GHR, EGFR, SOCS2 and SOCS3) at 120 min in WT GHR and GHRP495T cells normalised to B2M (see Supplementary Figure 6). Data presented as mean ± s.e.m. analysed by one-way ANOVA (***Po0.001, **Po0.01, *Po0.05) and representative of three independent experiments confirmed in three independently transduced lines.

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Transduction

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Inhibition of cell proliferation in METTL3-KO SW480 cells. (A) METTL3-KO SW480 cells showed dysregulation of several proliferation-related genes (*P<0.05, compared with the SW480 WT cells). (B) MTS assay results validated the decreased proliferation of METTL3-KO cells (*P<0.05, compared with the SW480 WT cells). (C) METTL3-KO cells showed a reduced proliferation rate, as measured by cell counting (*P<0.05, compared with the SW480 WT cells). (D) METTL3-KO cells showed a lower colony formation rate than control cells at 7 days after seeding. (E) Tumorigenic ability of METTL3-KO SW480 cells was reduced (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; SOX2, SRY-box transcription factor 2; PCNA, proliferating cell nuclear antigen; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells.

Article Snippet: The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Inhibition, MTS Assay, Cell Counting, Control, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: SOCS2 is upregulated in colon cancer and downregulated in METTL3-KO SW480 cells. (A) TCGA database analysis revealed a lower expression level of SOCS2 in colon cancer tissues than in the paired normal tissues (*P<0.05). (B) Expression levels of SOCS2 and METTL3 were negatively correlated in colon cancer tissues. (C-E) METTL3-KO upregulated SOCS2 expression at both the (C) mRNA and (D and E) protein levels in SW480 cells (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells.

Article Snippet: The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Methylation status of SOCS2 mRNA in SW480 cells. (A) Comparison of the SOCS2 mRNA methylation status between SW480 WT and METTL3-KO SW480 cells via MeRIP-qPCR (*P<0.05, compared with the SW480 WT cells). (B) Dose-dependent increase in SOCS2 mRNA levels in SW480 cells in response to demethylation treatment with DAA (*P<0.05, compared with non-treated group). (C and D) SOCS2 protein expression was upregulated after demethylation treatment with DAA (*P<0.05, compared with the SW380 WT DAA-group). (E) DAA appreciably decreased the m 6 A level in SOCS2 mRNA (*P<0.05, compared with the SW380 WT DAA-group). (F) METTL3-KO increased SOCS2 mRNA stability, showing a decreased mRNA decay rate (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells; DAA, 3-deazaadenosine.

Article Snippet: The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Methylation, Comparison, Expressing, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Modulation of SOCS2 expression alters the proliferation ability of SW480 cells. (A-C) siRNA (si-SOCS2) was used to successfully knock down SOCS2 expression in SW480 cells. Knockdown of SOCS2 had minor impact on METTL3 expression but significantly increased LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D) Knockdown of SOCS2 enhanced SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the si-Ctrl group). (E-G) Transfection of the pCMV6-SOCS2 plasmid upregulated SOCS2 expression and decreased LGR5 expression in SW480 cells (*P<0.05, compared with the pCMV6-Entry group). SOCS2 overexpression had minor impact on METTL3 expression (P>0.05, compared with the pCMV6-Entry group). (H) SOCS2 overexpression decreased SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the pCMV6-Entry group). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Knockdown, MTS Assay, Transfection, Plasmid Preparation, Over Expression

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: METTL3 modulates LGR5 transcription through m 6 A-mediated changes in SOCS2 expression rather than through direct regulatory mechanisms. (A) TCGA database analysis revealed an induction of LGR5 transcription in colon cancer tissues when compared with normal tissues (**P<0.01, compared with the normal tissues). (B) Expression levels of SOCS2 and LGR5 were negatively correlated in colon cancer tissues. (C) METTL3 knockout (KO) did not obviously impact LGR5 mRNA methylation (as measured by MeRIP-qPCR). (D) METTL3 KO had a minor effect on the LGR5 mRNA decay rate. (E) DAA treatment did not impact LGR5 expression in SW480 cells. (F) Luciferase reporter assay results confirmed that SOCS2 negatively controls LGR5 promoter-driven luciferase activity (*P<0.05, pGL3 basic-LGR5 promoter group vs. FULL SOCS2-LGR5 promoter group). METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; SOCS2, suppressor of cytokine signaling 2; TCGA, The Cancer Genome Atlas; SW480 A7 and SW480 A12, METTL3-KO SW480 cells; DAA, 3-deazaadenosine.

Article Snippet: The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Knock-Out, Methylation, Luciferase, Reporter Assay, Activity Assay

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Correlations among METTL3, SOCS2 and LGR5 expression levels in CRC. (A-C) Knockdown of METTL3 using siRNA (si-METTL3#1 and si-METTL3#2) significantly upregulated SOCS2 expression but downregulated LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D and E) Western blotting analysis of 24 paired tissues collected from CRC patients showed high protein levels of METTL3 and LGR5 and a relatively low protein level of SOCS2 in colon cancer (T, colon tumor samples; N, adjacent normal colon tissues; *P<0.05, compared to Normal tissues). (F) Visualization of expression profiles of METTL3, SOCS2 and LGR5 in CRC through the Oncomine database. METTL3 and LGR5 were upregulated in CRC datasets. In contrast, SOCS2 was downregulated in CRC datasets. CRC, colorectal cancer; SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Knockdown, Western Blot

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Association between patient characteristics and gene expression in 24 paired CRC cases.

Article Snippet: The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Gene Expression, Expressing