Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: The expression level of WTAP in GC patients by IHC.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: The relationship between the relative expression level of WTAP and clinicopathological variables in 50 patients with fresh GC tissues.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: The relationship between the relative expression level of WTAP and clinicopathological variables in 50 patients with paraffin GC tissues.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Overexpressed WTAP promoted the cell proliferation, wound healing, migration and invasion ability. The overexpression efficiency of WTAP in HGC-27 and AGS cell lines was detected by qRT-PCR (A, B) and Western blot analysis (C, D) . (E, F) The cell viability of HGC-27 and AGS cell lines were determined. (G, H) The scratch healing ability of HGC-27 and AGS cell lines was detected. Scale bar, 100 μm. (I, J) Transwell migration and invasion assays were used to evaluate HGC-27 and AGS cell migration and invasion ability. Scale bar, 100 μm.** P <0.01; *** P <0.001; **** P <0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Migration, Over Expression, Quantitative RT-PCR, Western Blot

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Knockdown of WTAP inhibited the cell proliferation, wound healing, migration and invasion ability. The knockdown efficiency of WTAP in HGC-27 and AGS cell lines was detected by qRT-PCR (A, B) and Western blot analysis (C, D) . (E, F) The cell viability of HGC-27 and AGS cell lines were determined. (G, H) The scratch healing ability of HGC-27 and AGS cell lines was examined. Scale bar, 100 μm. (I, J) Transwell migration and invasion assays were used to evaluate HGC-27 and AGS cell migration and invasion ability. Scale bar, 100 μm.* P <0.05; *** P <0.001; **** P <0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

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

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: WTAP regulates the tumorigenesis and growth of GC cells in nude mice. (A) The expression level of WTAP mRNA in stable cell lines was detected by qRT-PCR. (B) The expression level of WTAP protein in stable cell lines was detected by WB. (C) The tumor size was measured every 2 days and the tumor growth curve was plotted. (D) Tumors of the two groups of nude mice were dissected and photographed on the 18th day after transplantation. (E) The tumor was weighed after dissection. (F) Representative images of HE staining of the 2 groups of xenograft tumors. Scale bar, 20 μm. (G) Representative immunohistochemical results and quantitative analysis of Ki-67 positive staining in 2 groups of xenograft tumors. Scale bar, 20 μm. Data are shown as means ± S.D. *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing, Stable Transfection, Quantitative RT-PCR, Transplantation Assay, Dissection, Staining, Immunohistochemical staining

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Analysis of transcriptome sequencing and m 6 A sequencing results. (A-C) The sequencing results identified the genes significantly changed in Sh-WTAP group compared with Sh-ctrl group. (D, E) m 6 A sequencing identified the diminished m 6 A peaks in Sh-WTAP group. (F) The m 6 A consensus sequence motif was identified in HGC-27 cells. (G, H) Distribution of m 6 A IP signal in mRNA transcripts in HGC-27 cells.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Sequencing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: MAP2K6 is a direct downstream target gene of WTAP. (A) The intersection of genes corresponding to the downregulated m 6 A peaks in m 6 A sequencing and downregulated genes in transcriptome sequencing. (B, C) The expression levels of CNTN1, LHX9, SYT1, MAP2K6 and PRUNE2 genes were detected by qRT-PCR. (D) Attenuation of WTAP diminishes m 6 A modification of MAP2K6 mRNA in HGC-27 cells. (E) MeRIP-qPCR was used to detect the change of MAP2K6 mRNA enrichment in HGC-27 cells after knocking down WTAP expression level. (F) MeRIP-qPCR was used to detect the change of MAP2K6 mRNA enrichment in AGS cells after up-regulating WTAP expression level. (G) Diagram illustrating the fusion of the wild-type MAP2K6 sequence and the MAP2K6 sequence with a mutation at the 1409th m 6 A site with the luciferase reporter gene, respectively. (H, I) Relative luciferase intensity of HGC-27/AGS cells under different treatments. (J) Schematic diagram of the catRAPID website predicting the binding site between WTAP protein and MAP2K6 mRNA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Modification, Mutagenesis, Luciferase, Binding Assay

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: WTAP positively regulates MAP2K6. (A, B) The mRNA expression levels of MAP2K6 upon WTAP knockdown or overexpression were measured by qRT-PCR. (C, D) The protein expression levels of MAP2K6 upon WTAP knockdown or overexpression were measured by WB. (E) Co-expression of WTAP and MAP2K6 in GC cells was determined by confocal immunofluorescent assay (600X). Scale bar, 50 μm. (F) The mRNA expression levels of MAP2K6 in 50 GC tissues and paired adjacent normal tissues were determined by qRT-PCR. (G) WTAP was positively correlated with MAP2K6 expression in 50 GC tissues. (H) The colocalization of WTAP and MAP2K6 in GC tissues were confirmed by immunofluorescent assay (200X). Scale bar, 150 μm.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing, Knockdown, Over Expression, Quantitative RT-PCR

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Oncogenic function of WTAP depends on MAP2K6. (A, B) CCK-8 and colony formation assay were used to determine the effect of down-regulation of MAP2K6 expression on cell proliferation induced by overexpression of WTAP. (C, D) Wound healing assay and Transwell migration assay were used to investigate the effect of down-regulation of MAP2K6 expression on cell migration potency induced by WTAP overexpression. (E) Transwell invasion assay was used to clarify the effect of down-regulation of MAP2K6 expression on cell invasion potency induced by overexpression of WTAP. (F-J) The effects of MAP2K6 on WTAP in HGC-27 cells were detected by CCK-8 assay (F) , colony formation assay (G) , Wound healing assay (H) , Transwell migration assay (I) , Transwell invasion assay (J) .* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: CCK-8 Assay, Colony Assay, Expressing, Over Expression, Wound Healing Assay, Transwell Migration Assay, Migration, Transwell Invasion Assay

Journal: American Journal of Cancer Research

Article Title: Ultrasound coupled RES-loaded ultrasound microbubble inhibits the proliferation of ovarian cancer cells by expression of long non-coding RNA (lncRNA) involved in apoptosis using real-time PCR

doi:

Figure Lengend Snippet: A comparison of the siRNA levels against control and ING5.

Article Snippet: Small interfering RNAs (siRNA) targeting WTAP, BRCA1, and negative control RNAs (siNC) were purchased from GenePharma (Shanghai, China).

Techniques: Comparison, Control

Journal: MedComm

Article Title: Osthole regulates N6‐methyladenosine‐modified TGM2 to inhibit the progression of rheumatoid arthritis and associated interstitial lung disease

doi: 10.1002/mco2.219

Figure Lengend Snippet: OS impairs the activity of a TGM2/Myc/WTAP‐positive feedback loop mediated by NF‐κB signaling. (A) A Venn diagram analysis revealing genes associated with TNF‐mediated N6‐methyladenosine (m6A) modification. (B, C) methylated RNA immunoprecipitation (MeRIP) assays combined with qPCR (B) and PCR (C) were conducted to detect m6A modification of TGM2 mRNA. (D) Western blot analyses were adopted to explore the impact of OS on WTAP protein levels in RA‐FLS. (E) qPCR assays were applied to reveal the impact of WTAP knockdown on TGM2, IL1A, IL1B, and Myc mRNA levels in RA‐FLS. (F) Western blot analyses were adopted to detect the impact of WTAP depletion on TGM2, p‐NF‐κB, NF‐κB, and Myc protein levels in RA‐FLS. (G) MeRIP assays combined with PCR displaying the effect of WTAP knockdown on m 6 A modification of TGM2 mRNA. (H) Bioinformatics analyses elucidating a Myc binding site within the promoter region of WTAP based on Cistrome Data Browser. (I) qPCR assays were applied to reveal the impact of Myc knockdown on TGM2, IL1A, IL1B, and WTAP mRNA levels in RA‐FLS. (J) Western blot analyses were adopted to detect the impact of Myc depletion on WTAP, TGM2, p‐NF‐κB, and NF‐κB protein levels in RA‐FLS. (K, L) Chromatin immunoprecipitation (ChIP) assays combined with qPCR (K) and PCR (L) were applied to measure Myc‐mediated WTAP transcription. (M) ChIP assays combined with PCR showing the impact of TGM2 and RELA on Myc‐mediated WTAP transcription. (N) MeRIP assays combined with PCR displaying the impact of OS on TGM2 mRNA m 6 A modification. (O) ChIP combined with PCR assays showing the impact of OS and TGM2 on Myc‐mediated WTAP transcription. ** p < 0.01 and *** p < 0.001 versus TNF‐treated group.

Article Snippet: TGM2 siRNAs (si‐TGM2), WTAP siRNAs (si‐WTAP), and Myc siRNAs (si‐Myc) were designed by RiboBio Corporation (Table ).

Techniques: Activity Assay, Modification, Methylation, RNA Immunoprecipitation, Western Blot, Knockdown, Binding Assay, Chromatin Immunoprecipitation

Journal: MedComm

Article Title: Osthole regulates N6‐methyladenosine‐modified TGM2 to inhibit the progression of rheumatoid arthritis and associated interstitial lung disease

doi: 10.1002/mco2.219

Figure Lengend Snippet: OS cooperates with MTX to inhibit RA progression in vivo. The collagen‐induced arthritis (CIA) mice model was constructed and randomly divided into control, CIA, MTX, OS, and OS combined with MTX treatment groups ( n = 10 per group). (A, B) The impacts of OS and MTX on arthritis score (A) and incidence of arthritis (B) in CIA models. (C) The measurement of mice body weight in CIA and OS group. (D) Representative histopathological images of mice livers and kidneys in OS‐treated and the control groups. Scale bar: 50 μm. (E) Gross and H&E staining view of mice joints in CIA models exhibiting the impacts of OS and MTX on arthritis. Synovial inflammation, synovial hyperplasia, cartilage damage, and bone erosion were evaluated in the mice joints. Scale bar: 125 μm. (F) Representative images (upper scale bar: 125 μm and below scale bar: 12.5 μm) of immunohistochemistry staining exhibiting the impacts of OS and MTX on TGM2, WTAP, Myc, ABCG2, CCNA2, and vimentin expression in synovial tissue of joints. * p < 0.05, and *** p < 0.001 versus CIA group. ### p < 0.001 versus MTX‐treated group.

Article Snippet: TGM2 siRNAs (si‐TGM2), WTAP siRNAs (si‐WTAP), and Myc siRNAs (si‐Myc) were designed by RiboBio Corporation (Table ).

Techniques: In Vivo, Construct, Control, Staining, Immunohistochemistry, Expressing

Journal: MedComm

Article Title: Osthole regulates N6‐methyladenosine‐modified TGM2 to inhibit the progression of rheumatoid arthritis and associated interstitial lung disease

doi: 10.1002/mco2.219

Figure Lengend Snippet: OS suppresses RA‐associated interstitial lung disease (ILD) by downregulating the aggregation of M2 macrophages. (A) Representative images (left scale bar: 125 μm and right scale bar: 12.5 μm) of H&E staining displaying the impact of OS and MTX on the generation of subpleural inflammation in CIA models. (B) Representative images (left scale bar: 125 μm and right scale bar: 12.5 μm) of immunofluorescence staining displaying the impact of OS and MTX on the aggregation of CD11b + interstitial macrophage in the subpleural inflammation area of the CIA mouse model. (C) Representative images (scale bar: 12.5 μm) of immunofluorescence staining displaying the impact of OS on TGM2, Myc, and WTAP expression of CD11b + interstitial macrophage in the subpleural inflammation area of the CIA mouse model. (D) Flow cytometry indicating the effect of OS on the percentage of CD163 + CD11b + and CD206 + CD11b + RAW 246.7 cells. (E) RAW 246.7 cells were exposed to OS (0, 2.5, 5, 10, and 20 μg/mL) for 48 h, and cell viability was measured by CCK8 assays. (F) Western blot analyses were applied for elucidating the impact of OS on CCNB1, CD163, and TGM2 protein levels in RAW 246.7 cells. (G, H) qPCR assays were applied to reveal the impact of OS on CD206 (G) and TGM2 (H) in IL4 and IL13‐induced M2 macrophage differentiated from THP‐1. * p < 0.05 and *** p < 0.001 versus control group. # p < 0.05 and ## p < 0.01 versus M2 group.

Article Snippet: TGM2 siRNAs (si‐TGM2), WTAP siRNAs (si‐WTAP), and Myc siRNAs (si‐Myc) were designed by RiboBio Corporation (Table ).

Techniques: Staining, Immunofluorescence, Expressing, Flow Cytometry, Western Blot, Control

Journal: MedComm

Article Title: Osthole regulates N6‐methyladenosine‐modified TGM2 to inhibit the progression of rheumatoid arthritis and associated interstitial lung disease

doi: 10.1002/mco2.219

Figure Lengend Snippet: Bioinformatics analysis emphasizes the significance of the molecular regulatory network regulated by OS. (A) The differential distribution of FLS infiltration calculated by single sample gene set enrichment analysis (ssGSEA) between RA patients and normal controls based on GSE89408 dataset. (B) The differential distribution of FLS infiltration calculated by ssGSEA in RA patients with low and high TGM2 levels based on GSE89408 dataset. (C) The correlation analysis displaying the relationship between TGM2 levels and FLS infiltration calculated by ssGSEA in RA patients as per GSE89408 dataset. (D) The correlation analyses displaying the relationship among TGM2, Myc, WTAP, TNF, CCNA2, CCNB1, CDK1, CDK2, IL1A, IL1B, IL6, and IL8 levels as per GSE89408 dataset. (E) The correlation analyses displaying the relationship among TGM2, Myc, WTAP, ABCG2, CCNA2, CCNB1, CDK2, IL1B, Twist1, vimentin, and N‐ca levels as per GSE109449 dataset. (F) The correlation analyses presenting the relationship between the severity of RA disease (DAS28‐CRP, DAS28‐ESR, CRP, ESR, and joint swollen), therapy response (delta DAS28‐CRP, delta DAS28‐ESR, delta CRP, and delta ESR), and the levels of TGM2, Myc, WTAP, IL1A, IL1B, CCNA2, and CCNB1 based on PEAC RNA‐seq database. (G) Working model indicating the modulation of OS‐downregulated TGM2/Myc/WTAP‐positive feedback circuit in anti‐RA effect of MTX as well as proliferation and metastasis of RA‐FLS through modulating NF‐κB signaling. This graphical abstract was generated by applying the Biorender website ( https://biorender.com/ ).

Article Snippet: TGM2 siRNAs (si‐TGM2), WTAP siRNAs (si‐WTAP), and Myc siRNAs (si‐Myc) were designed by RiboBio Corporation (Table ).

Techniques: RNA Sequencing, Generated