DataSheet_1_Small RNA-modulated anaerobic respiration allows bacteria to survive under antibiotic stress conditions.pdf

Despite extensive knowledge of antibiotic-targeted bacterial cell death, deeper understanding of antibiotic tolerance mechanisms is necessary to combat multi-drug resistance in the global healthcare settings. Regulatory RNAs in bacteria control important cellular processes such as cell division, cellular respiration, metabolism, and virulence. Here, we investigated how exposing Escherichia coli to the moderately effective first-generation antibiotic cephalothin alters transcriptional and post-transcriptional dynamics. Bacteria switched from active aerobic respiration to anaerobic adaptation via an FnrS and Tp2 small RNA-mediated post-transcriptional regulatory circuit. From the early hours of antibiotic exposure, FnrS was involved in regulating reactive oxygen species levels, and delayed oxygen consumption in bacteria. We demonstrated that bacteria strive to maintain cellular homeostasis via sRNA-mediated sudden respiratory changes upon sublethal antibiotic exposure.</p

Complex Behavior of ALDH1A1 and IGFBP1 in Liver Metastasis from a Colorectal Cancer

<div><p>Using our data set (GSE50760) previously established by RNA sequencing, the present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) and verify their biological behavior. The potential roles of candidate genes in tumors were assessed using cell proliferation and invasion assays. Tissue samples were collected from 18 CRC patients with synchronous CLM and two CRC cell lines (SW480 and SW620) were used for transfection and cloning. The roles of the genes identified in CLM were verified using immunohistochemistry in 48 nude mice after intrasplenic transplantation of CRC cells. mRNA and protein expression was determined by quantitative real-time reverse transcription polymerase chain reaction and western blot, respectively. Nine genes were initially selected according to the relevance of their molecular function and biological process and, finally, <i>ALDH1A1</i> and <i>IGFBP1</i> were chosen based on differential mRNA expression and a positive correlation with protein expression. The overexpression of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (<i>p</i> ≤ 0.001–0.003) and suppressed invasiveness by ≥3-fold over control cells (<i>p</i> < 0.001) in the SW480 cell line, whereas they had a slight effect on reducing SW620 cell proliferation. The protein expression levels of E-cadherin, N-cadherin, claudin-1, and vimentin were significantly higher in CLM than in primary tumor tissues (<i>p</i> < 0.05). However, the cadherin switch, namely, N-cadherin overexpression with reduced E-cadherin expression, was not observed in CLM tissues and transfected CRC cells. Irrespective of reduced proliferation and invasion found on <i>in vitro</i> cell assays, persistent overexpression of β-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells possibly contributed to CLM development in mice implanted with IGFBP1-overexpressing SW480 cells (CLM occurrences: SW480/<i>IGFBP1</i>-transfected mice <i>vs</i>. SW480/vector- and SW480/<i>ALDH1A1</i>-transfected mice, 4/8 <i>vs</i>. 0/10, <i>p</i> = 0.023). In conclusion, ALDH1A1 and IGFBP1 are differentially overexpressed in CLM and may play a dual role, functioning as both tumor suppressors and metastasis promoters in CRC.</p></div

Proliferation and cell cycle assays using <i>ALDH1A-</i> and <i>IGFBP1-</i>transfected cells.

<p>ALDH1A1and IGFBP1 specifically decrease CRC cell proliferation (left column) and accumulation of cells in the S and G2/M phases (right column) in SW480 cells (A and C) and SW620 cells (B and D). The cell proliferation rates were measured using the CCK8 cell proliferation assay and expressed as daily proliferation rate. Cell-cycle phases were measured on a flow cytometer with propidium iodide staining. *<i>p</i> < 0.05 between vector and clone #1, and <i>p</i> < 0.05 **between vector and clone #2, respectively.</p

Expressions with relative densities of epithelial-mesenchymal transition/CRC stem cell (EMT/CSC)-related molecules.

<p>CRC patients with CLM (A), vector, ALDH1A1- and IGFBP1-overexpressing CRC cells (B). *<i>p</i> < 0.05 between PCC and CLM or between vector and ALDH1A1- or IGFBP1-overexpressing clones. NCE (N), normal colic epithelium; PCC (P), primary colorectal cancer; CLM (M), colorectal cancer liver metastasis.</p

Invasion and migration assays using <i>ALDH1A-</i> and <i>IGFBP1-</i>transfected cells.

<p>Invasiveness was measured by the number of CRC cells invading into the lower chamber in the well (A and C) and by the gelatinolytic activity of MMP-2 and MMP-9 (B and D). All treated cells showed significantly reduced invasiveness in these assays except IGFBP1-overexpressing SW620 cells on gelatin zymography. *<i>p</i> < 0.05 *between vector and clone #1 and **<i>p</i> < 0.05 between vector and clone #2, respectively.</p

Primary tumors at the spleen (transplant site) and liver metastases.

<p>Arrows indicated tumors (the spleen and liver were set at the upper and lower parts, respectively) and histological views (H & E, ×100; photomicrographs of the spleen and liver were set at the left and right sides, respectively, from red squared specimens). CLM, colorectal cancer liver metastasis. The mice with CLM was in order (right and downward) with long diameter: A, no CLM; B, no CLM; C, #2 (2.7 mm) #3 (2–5.3 mm) #5 (4.4–6.6 mm) #7 (4.4 mm); D, #1 (16.0 mm) #2 (2.2–8.0 mm); E, no CLM; F, no CLM.</p

The mRNA and protein expressions of ALDH1A1and IGFBP1 in ten CLM patients.

<p>The expression levels of <i>ALDH1A1</i> (A) and <i>IGFBP1</i> (B) were higher in CLM than in PCC tissues (specifically in 4 patients: #30, 38, 43, 47). *<i>p</i> < 0.001–0.05 between PCC and CLM. NCE (N), normal colic epithelium; PCC (P), primary colorectal cancer; CLM (M), colorectal cancer liver metastasis.</p

Expression of <i>PDGFR</i> isoforms in bladder cancer (NMIBC and MIBC) patients.

<p>FDR, false discovery rate; NMIBC, non-muscle invasive bladder cancer; MIBC, muscle invasive bladder cancer; BT, bladder tumor</p><p>Expression of <i>PDGFR</i> isoforms in bladder cancer (NMIBC and MIBC) patients.</p

<i>AXL</i> mRNA expression in bladder cancer (NMIBC and MIBC) patients and normal controls.

<p>FDR, false discovery rate; NMIBC, non-muscle invasive bladder cancer; MIBC, muscle invasive bladder cancer; BT, bladder tumor</p><p><i>AXL</i> mRNA expression in bladder cancer (NMIBC and MIBC) patients and normal controls.</p

MMP2 and MMP9 may be downstream effectors of <i>c-MET</i> knockdown, leading to suppression of migration in T24 bladder cancer cells.

<p>(A) Wound-healing assay showing that knockdown of c-MET inhibitsthe migration of T24 cells. (B) Loss of <i>c-MET</i> downregulated the expression of matrix metalloproteinases (MMP)-2 and MMP-9. All experiments were performed using two <i>c-MET</i> knockdown cell lines (si<i>c-MET</i>-1 and si<i>c-MET</i>-2) transfected with different MET siRNAs, and two control cell lines (Ctrl and NT). Ctrl, control; NT, non-transfected.</p