Fig 2: LINC00628 Recruits Methylase to Promote the DNA Methylation of LAMA3 Promoter(A) Binding sites between LINC00628 and LAMA3 predicted by the bioinformatics website. (B) The binding of LINC00628 to LAMA3 promoter confirmed by dual-luciferase reporter gene assay. (C) Binding of LINC00628 to DNMT1, DNMT3A, and DNMT3B in H1650 cells detected by RIP assay. (D) Enrichment of DNMT1, DNMT3A, and DNMT3B in the promoter region of LAMA3 upon oe-LINC00628 treatment determined using ChIP assay. (E) Methylation of LAMA3 in the H1650 cell line with silenced DNMT1, DNMT3A, and DNMT3B treatment determined using MSP. The experiment was performed in triplicate. The statistical values were measurement data, which were expressed as mean ± SD and compared with t test; *p < 0.05; **p < 0.01; ***p < 0.001. The experiment was performed in triplicate.

Fig 3: Inhibition of LAMA3 Promoter Methylation Hinders the Proliferation, Migration, and Invasion of Lung Adenocarcinoma Cells(A) The protein expression of LAMA3 in H1650 cells and H1299 cells after the addition of 5-Aza (5 µM) examined by western blot analysis. (B) Proliferation of H1650 cells and H1299 cells after the addition of 5-Aza (5 µM) detected using MTT assay. (C) Migration of H1650 cells and H1299 cells after the addition of 5-Aza (5 µM) examined by scratch assay. (D) Invasion of H1650 cells and H1299 cells after the addition of 5-Aza (5 µM) examined by Transwell assay. (E) Western blot analysis of protein expression of invasion-related markers (MMP2 and MMP9) and EMT-related makers (N-cadherin, Vimentin, and E-cadherin) in H1650 cells and H1299 cells. The experiment was performed in triplicate. The statistical values were measurement data, which were expressed as mean ± SD and compared with one-way ANOVA; *p < 0.05; **p < 0.01; and ***p < 0.001 compared with cells served as control and NC or the cells that served as NC treated with LAMA3.

Fig 4: Low Expression of LAMA3 Is Found in Lung Adenocarcinoma Tissues and Cells(A) TCGA database analysis of the expression of LAMA3 in lung adenocarcinoma tissues and adjacent normal tissues. (B) qRT-PCR detection of LAMA3 expression in adjacent normal tissues and lung adenocarcinoma tissues (n = 70). (C) Western blot analysis of LAMA3 protein expression in lung adenocarcinoma cell lines. (D) Positive expression rate of LAMA3 protein in lung adenocarcinoma tissues and adjacent normal tissues detected by IHC. The statistical values were measurement data, which were expressed as mean ± SD and compared with t test, n = 70; *p < 0.05; **p < 0.01; and ***p < 0.001 versus the adjacent normal tissues.

Fig 5: Low Expression of LAMA3 Is Associated with High Expression of LINC00628 in Lung Adenocarcinoma(A) Bioinformatics analysis of LAMA3 and LINC00628, hsa04151:PI3K-Akt signaling pathway. (B) TCGA database analysis of LINC00628 expression in lung adenocarcinoma tissues and adjacent normal tissues. (C) Expression of LINC00628 in lung adenocarcinoma tissues and adjacent normal tissues, which was normalized to GAPDH expression. (D) LINC00628 expression in normal cell BEAS-2B and lung adenocarcinoma cell lines A549, H1650, HCC827, H1975, and H1299. (E) Correlation of LINC00628 and LAMA3 in human lung adenocarcinoma tissues analyzed with the Pearson correlation coefficient. (F) The location of LINC00628 in cells analyzed by sub-cell location website. (G) The location of LINC00628 in cells analyzed by FISH (400×) and DAPI-represented nucleic localization. The statistical values were measurement data, which were expressed as mean ± SD and compared with t test, n = 70; *p < 0.05; **p < 0.01; and ***p < 0.001 versus the adjacent normal tissues.