Additional file 1 of Recombinant L. lactis vaccine LL-plSAM-WAE targeting four virulence factors provides mucosal immunity against H. pylori infection

Supplementary Material

Biological effect of lncRGMB-AS1 downregulation on cell cycle progression in A549 and SPC-A-1 cells.

<p>(A and B) Flow cytometric analysis showed a marked increase in the percentage of cells in the G0/G1 phase and a reduction in the percentage of cells in the S phase in the si-lncRNA group in A549 cells (<i>P</i><0.05), without significant changes in the NC and Blank groups (<i>P</i> > 0.05). (C and D) Flow cytometric analysis of SPC-A-1 cells showed a marked increase in the percentage of G0/G1 phase cells and a decrease the percentage of S phase cells in the si-lncRNA group (<i>P</i><0.05), without significant changes in the NC and Blank groups (<i>P</i> > 0.05). si-lncRNA: cells that were transfected with siRNA of lncRNA RGMB-AS1; NC: cells that were transfected with negative control oligonucleotides, and Blank: cells that were not transfected.</p

Biological effect of lncRGMB-AS1 downregulation on cell proliferation in lung adenocarcinoma in vivo.

<p>(A, C) In mice injected with A549 cells, the luciferase signal was lower in the si-RNA nude mice than in the control groups at 2, 3, and 4 weeks, and the signal intensity became even lower in the si-RNA group with time (<i>P</i> < 0.05). (B, D) In mice injected with SPC-A-1 cells, the luciferase signal was lower in the si-RNA nude mice than in the control groups at 2, 3, and 4 weeks, and the signal intensity also became lower in the si-RNA group with time (<i>P</i> < 0.05). (E, F) Western blot analysis of Ki67 and RGMB expression in xenograft tissues from the three groups of nude mice. Compared with the control groups, Ki67 was downregulated and RGMB was upregulated in the si-RNA group (<i>P</i> < 0.05). si-lncRNA: cells that were transfected with siRNA of lncRNA RGMB-AS1; NC: cells that were transfected with negative control oligonucleotides, and Blank: cells that were not transfected. *Indicated statistical significance (<i>P</i> < 0.05).</p

Potential mechanism of lncRGMB-AS1 in lung adenocarcinoma.

<p>(A) Exon1 and Exon2 of RGMB were amplified from human genomic DNA and inserted into the pEGFP-N3 vector with Hind III and BamHI to construct the recombination plasmids pEGFP-N3-Exon1 and pEGFP-N3-Exon2, respectively. The third exon of lncRNA RGMB-AS1 was also amplified from human genomic DNA and inserted into the pSilencer 3.1 vector with BamHI and Hind III to construct the recombination plasmid pSilencer-RGMB-AS1. (B) The fluorescence intensity of cells co-transfected with pEGFP-N3-Exon1 and pSilencer-RGMB-AS1 did not differ from that of cells transfected with pEGFP-N3-Exon1 only or cells co-transfected with pEGFP-N3-Exon1 and pSilencer 3.1 (<i>P</i> > 0.05). (C) The fluorescence intensity of cells co-transfected with pEGFP-N3-Exon2 and pSilencer-RGMB-AS1 was weaker than that of cells transfected with pEGFP-N3-Exon2 only or cells co-transfected with pEGFP-N3-Exon2 and pSilencer 3.1 (<i>P</i> < 0.05). The fluorescence intensity of the pEGFP-N3-Exon1 group was used as a control. *Indicates statistical significance (<i>P</i> < 0.05).</p

Biological effect of lncRGMB-AS1 downregulation on cell proliferation in A549 and SPC-A-1 cells.

<p>(A) A statistically significant downregulation of lncRGMB-AS1 expression in A549 and SPC-A-1 cells was observed in the siRNA group compared with the NC and Blank groups (<i>P</i> < 0.05). (B) A statistically significant increase of RGMB expression in A549 and SPC-A-1 cells was observed in the siRNA group compared with the NC and Blank groups (<i>P</i> < 0.05). (C, D) CCK-8 assays showed that the OD₄₅₀ values for the siRNA group on days 2, 3 and 4 were significantly decreased in both A549 and SPC-A-1 cells (<i>P</i> < 0.05) in comparison with the control groups (<i>P</i> > 0.05). (E, F) A statistically significant reduction in the numbers of A549 and SPC-A-1 cell colonies was observed in the siRNA group compared with the NC and Blank groups, as measured by colony forming assay (<i>P</i> < 0.05). si-lncRNA: cells that were transfected with siRNA of lncRNA RGMB-AS1; NC: cells that were transfected with negative control oligonucleotides, and Blank: cells that were not transfected. *Indicates statistical significance (<i>P</i> < 0.05).</p

Effects of lncRNA RGMB-AS1 silencing and RGMB overexpression on the biological behavior of A549 and SPC-A-1 cells.

<p>(A) RGMB expression was higher in cells transfected with si-lncRNA or pcDNA3.1 (+) / RGMB than in Blank cells (<i>P</i> < 0.05). (B and C) Proliferation rates were lower in cells transfected with si-lncRNA or pcDNA3.1 (+) / RGMB than in Blank cells. (D) Cell cycle arrest was more marked in the si-lncRNA and RGMB groups than in the Blank group cells. (E) Cell invasion ability was higher in the si-lncRNA group and RGMB group than in the Blank group. si-lncRNA: cells that were transfected with siRNA of lncRNA RGMB-AS1; RGMB: cells that were transfected with pcDNA3.1 (+) / RGMB vector, and Blank: cells that were not transfected.*Indicates statistical significance (<i>P</i> < 0.05).</p

Biological effect of lncRGMB-AS1 downregulation on cell migration and invasion in A549 and SPC-A-1 cells.

<p>(A, B) Wound healing assays showed that the migration ability of A549 and SPC-A-1 cells was lower in the si-lncRNA group at 24 h post-wounding than in both NC and Blank groups (<i>P</i> < 0.05). (C) Transwell assays showed that the invasive ability of A549 and SPC-A-1 cells was lower in the si-lncRNA group than in the NC and Blank groups in A549 and SPC-A-1 cells (<i>P</i> < 0.05). (D) Quantitative data of Transwell results in A549 and SPC-A-1 cells. si-lncRNA: cells that were transfected with siRNA of lncRNA RGMB-AS1; NC: cells that were transfected with negative control oligonucleotides, and Blank: cells that were not transfected. *Indicates statistical significance (<i>P</i> < 0.05).</p

Expression of lncRNA RGMB-AS1 and RGMB in lung adenocarcinoma tissues.

<p>(A) LncRNA RGMB-AS1 expression in lung adenocarcinoma tissues (Tumor group) was higher than in adjacent normal tissues (Normal group) (<i>P</i> < 0.05). (B) LncRNA RGMB-AS1 expression was successively increased in the well, moderately and poorly differentiated groups (<i>P</i> < 0.05). (C) LncRNA RGMB-AS1 expression was higher in TNM stage III than in TNM stages I and II (<i>P</i> < 0.05). (D) LncRNA RGMB-AS1 expression level was higher in cancer tissues with positive lymph node metastasis than in those with negative node metastasis (<i>P</i> < 0.05). (E) RGMB mRNA expression in lung adenocarcinoma tissues was lower than that in adjacent normal tissues (<i>P</i> < 0.05). (F) RGMB expression was successively decreased in the well, moderately and poorly differentiated groups (<i>P</i> < 0.05). (G) RGMB expression was lower in TNM stage III than in TNM stages I and II (<i>P</i> < 0.05). (H) RGMB expression level was lower in cancer tissues with positive lymph node metastasis than in those with negative node metastasis (<i>P</i> < 0.05). (I) Negative correlation between lncRNA RGMB-AS1 and RGMB mRNA expression. (J) Quantification of RGMB expression to GAPDH in lung adenocarcinoma tissues compared to normal tissues (<i>P</i> < 0.05). (K) Western blot results of RGMB expression in lung adenocarcinoma tissues (T) and normal tissues (N). *Indicates statistical significance (<i>P</i> < 0.05).</p