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LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with <t>siRNA</t> targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.
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Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

Journal: Oncology Letters

doi: 10.3892/ol.2026.15628

LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.
Figure Legend Snippet: LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Techniques Used: Expressing, Methylation, MSP Assay, Over Expression, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.
Figure Legend Snippet: Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Techniques Used: Inhibition, Activation Assay, Functional Assay, Western Blot, Expressing, Over Expression, Derivative Assay, Control, Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.
Figure Legend Snippet: DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Techniques Used: Inhibition, Over Expression, Flow Cytometry, Cell Counting, Control, Negative Control, Small Interfering RNA

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Synthesized:

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Expressing:

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Extraction:

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Negative Control:

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Control:

Article Title: Pathogenic mechanism of Eimeria tenella Et MIC2 promotes Eimeria tenella invasion and inhibits host cell apoptosis through binding to the ITGAV receptor
Article Snippet: After centrifugation at 2200 rpm for 7 min, the pellet was resuspended in sporocyst digestion solution (0.75 % trypsin: 10 % chicken bile = 1:1) and digested with shaking at 41°C, 150 rpm for 1 h. Sporozoites were purified by filtration through a 1800-mesh sieve (10 μm) and centrifuged at 2800 rpm for 10 min. Sporozoites were resuspended and diluted to 2.0 × 105 sporozoites/mL using sporozoite inoculation medium containing 33 % DMEM, 65.5 % MEM199 (HyClone, Utah, USA), and 1.5 % FBS (referring to Zhang Yan's formulation) ( ). .. siRNA ITGAV-1/2/3 and one random non-targeting control siRNA (NC siRNA) were designed based on the ITGAV coding sequence and synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. siRNA sequences are listed in . .. According to the manufacturer's instructions for Lipofectamine® RNAiMAX (Thermo Fisher Scientific, Massachusetts, USA), three siRNAs targeting ITGAV were transfected into CECs.

Incubation:

Article Title: Codonopsis pilosula polysaccharide attenuates the inflammatory response in macrophages induced by Brucella abortus outer membrane protein 19 via regulating ATP2A1 to modulate cell adhesion and calcium signaling
Article Snippet: Following a 1 h pretreatment with 100 μM BAPTA-AM (MCE, HY-100545, China), mouse peritoneal macrophages were sequentially treated with 50 μg·mL -1 CPPS for 12 h and stimulated with 5 μg·mL -1 OMP19 for 8 h. The expression level of E-cadherin protein was then determined using Western blot analysis. .. THP-1 cells were seeded into 6-well plates at a density of 2 × 10 6 cells/mL, and siRNA transfection was performed after cell adherence. siRNA (Sangon Biotech, 113195091, China) and Lipofectamine 2000 (Thermo Fisher Scientific, 11668019, USA) were diluted separately with Opti-MEM medium (Thermo Fisher Scientific, 31985070, USA), incubated at room temperature for 5 min, then mixed thoroughly and incubated for an additional 20 min to form transfection complexes. ..



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LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with <t>siRNA</t> targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.
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LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with <t>siRNA</t> targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.
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LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with <t>siRNA</t> targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.
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LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Expressing, Methylation, MSP Assay, Over Expression, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Activation Assay, Functional Assay, Western Blot, Expressing, Over Expression, Derivative Assay, Control, Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Over Expression, Flow Cytometry, Cell Counting, Control, Negative Control, Small Interfering RNA