Additional file 2: Table S1. of miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer

Dysregulated miRNAs in TNBC patients. (DOCX 13 kb

A Novel Strategy for TNF-Alpha Production by 2-APB Induced Downregulated SOCE and Upregulated HSP70 in <i>O</i>. <i>tsutsugamushi</i>-Infected Human Macrophages

<div><p><i>Orientia</i> (<i>O</i>.) <i>tsutsugamushi</i>-induced scrub typhus is endemic across many regions of Asia and the Western Pacific, where an estimated 1 million cases occur each year; the majority of patients infected with <i>O</i>. <i>tsutsugamushi</i> end up with a cytokine storm from a severe inflammatory response. Previous reports have indicated that blocking tumor necrosis factor (TNF)-α reduced cell injury from a cytokine storm. Since TNF-α production is known to be associated with intracellular Ca²⁺ elevation, we examined the effect of store-operated Ca²⁺ entry (SOCE) inhibitors on TNF-α production in <i>O</i>. <i>tsutsugamushi</i>-infected macrophages. We found that 2-aminoethoxydiphenyl borate (2-APB), but not SKF96365, facilitates the suppression of Ca²⁺ mobilization via the interruption of Orai1 expression in <i>O</i>. <i>tsutsugamushi</i>-infected macrophages. Due to the decrease of Ca²⁺ elevation, the expression of TNF-α and its release from macrophages was repressed by 2-APB. In addition, a novel role of 2-APB was found in macrophages that causes the upregulation of heat shock protein 70 (HSP70) expression associated with ERK activation; upregulated TNF-α production in the case of knockdown HSP70 was inhibited with 2-APB treatment. Furthermore, elevated HSP70 formation unexpectedly did not help the cell survival of <i>O</i>. <i>tsutsugamushi</i>-infected macrophages. In conclusion, the parallelism between downregulated Ca²⁺ mobilization via SOCE and upregulated HSP70 after treatment with 2-APB against TNF-α production was found to efficiently attenuate an <i>O</i>. <i>tsutsugamushi</i>-induced severe inflammatory response.</p></div

Schematic diagram of 2-APB activity in strategically regulating <i>O</i>. <i>tsutsugamushi</i>-induced and LPS-stimulated TNF-α production in macrophages.

<p>(A) Pathogenic infection induced increased level of TNF-α, which was attenuated by 2-APB interrupting Ca²⁺ signaling activity; suppressed Ca²⁺ mobilization also suppressed release and expression of TNF-α. Mitogen-activated protein kinase (MAPK) pathway is involved in regulating TNF-α production under <i>O</i>. <i>tsutsugamushi</i> infection; however, 2-APB restrains signal pathway of JNK and p38 but activates ERK pathway to promote upregulation of HSP70, which facilitates downregulation of TNF-α expression by blocking NF-κB translocation to nucleus. (B) Although 2-APB attenuates LPS-stimulated TNF-α production, different mechanism displays in regulating TNF-α expression. The importance of 2-APB in LPS-stimulated macrophages is to block TNF-α release rather than inducing ERK pathway to activate upregulation of HSP70.</p

Increased level of HSP70 expression by treatment with 2-APB in <i>O</i>. <i>tsutsugamushi</i>-infected or LPS-stimulated macrophages.

<p>(A) Effect of 2-APB on expressions of HSP10, HSP40, HSP70, HSP90, and Actin in <i>O</i>. <i>tsutsugamushi</i> (OT)-infected macrophages. (B) Western blot analysis was used to analyze MAPK signal pathways in <i>O</i>. <i>tsutsugamushi</i>-infected macrophages at indicated time point. Treatments included phosphorylated JNK (p-JNK), JNK, phosphorylated p38 (p-p38), and p38, which were blocked by co-treatment with 50 μM of 2-APB for 24 h (24+). Further measurements were made of expression of phosphorylated ERK (p-ERK), ERK, HSP70, and Actin, which were treated with MAP kinase (MEK) inhibitor, 50 μM PD98059, 2-APB (50, 50 μM and 100, 100 μM) or combination for 24 h in (C) <i>O</i>. <i>tsutsugamushi</i>-infected and (D) LPS-stimulated macrophages. (E) Western blot analysis of expression of ERK pathway, HSP70, and Actin without pathogen infection in macrophages. Survival probability is shown for each condition in panel with (F) <i>O</i>. <i>tsutsugamushi</i>-stimulated macrophages and (G) LPS-stimulated macrophages for 24 h (**, <i>p</i> < 0.01; ***, <i>p</i> < 0.001).</p

Additional file 1: Figure S1. of miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer

Association between identified miRNAs and overall survival in TNBC and non-TNBC patients. A Identification of dysregulated miRNAs in TNBC compared with non-TNBC using Student t test. B Elevated expression of miR-301b, miR-181a-3p, miR-105, and miR-93 were individually associated with poor overall survival in 1095 non-triple negative breast cancer patients by Kaplan-Meier analysis. The correlation between indicated miRNAs and overall survival in (C) 204 TNBC patients and (D) 1095 non-TNBC patients was analyzed by Kaplan-Meier analysis. E Indicated miRNAs expression levels were examined in the independent cohort (GSE40267, N = 173), which contained 94 TNBC, 79 non-TNBC. Figure S2 miR-105 and miR-93-3p promote cellular migration but not proliferation. The effect of ectopic overexpression or silencing of indicated miRNAs on cell proliferation as determined by (A) colony-forming assay and (B) MTT assay. C The effect of ectopic overexpression or silencing of indicated miRNAs on cell migration ability as measured by the Boyden chamber transwell migration assay. D The miRNA-overexpressing HCC70 cells and miRNA silenced-BT-549 cells were seeded into matrigel-coated transwells to evaluate cell invasion in vitro. Figure S3 miR-105 and miR-93-3p confer cisplatin resistance. Cisplatin was administered with the indicated dose to cells with (A) ectopic overexpression or (B) silencing of indicated miRNAs prior to determining cell viability by the MTT assay. C Co-transfection with miR-105 and miR-93-3p antagomiRs in HCC1937 followed by measurement of the cisplatin response by MTT assay. D miR-105/93-3p co-silenced-BT-549 cells were seeded at 10,000 cells/ml into an ultra-low attachment plate for 10 days to evaluate mammosphere formation, as an indicator of stemness. Relative efficiency of mammosphere formation was measured in control and miR-105/93-3p-knockdown BT-549 cells. Figure S4 miR-105 and miR-93-3p activate Wnt/β-catenin signaling. A Bioinformatic analysis to identify potential miR-105 and miR-93-3p target genes. B The miR-105 and miR-93-3p associated mRNA profiling, paired with miRNA microarrays from the same patients were analyzed by IPA. C Ectopic overexpression of miR-105 or miR-93-3p and subsequent determination of β-catenin activity by the TOP/FOP reporter assay. D Immunoblotting was performed to measure levels of the indicated proteins in miR-105 or miR-93-3p manipulated TNBC cells. Figure S5 miR-93-3p/105 target to SFRP1 and decrease SFRP1 expression level. A Bioinformatic prediction of target genes for miR-105/93-3p involved in Wnt/b-catenin signaling. B IPA was performed to identify potential upstream regulators of Wnt/β-catenin that are modulated in TNBC with high expression of miR-105 or miR-93-3p. C RNA hybrid was performed to examine the binding energy between miR-105 or miR-93-3p with 3′-UTR of SFRP1. D Ectopic overexpression or silencing of indicated miRNAs followed by determination of SFRP1 protein levels by immunoblotting. Figure S6 miR-93-3p/105 can serve as biomarker for early TNBC patients and inversely correlated with SFRP1 in TNBC but not in non-TNBC patients. A The SFRP1 levels were determined in 12 non-TNBC N-T paired tissues by immunoblotting. B The contingency plot showed the distribution of SFRP1 in the indicated circulating miRNA levels. C Overall survival of 249 TNBC patients and 93 TNBC patients with chemotherapy, those were obtained from Kaplan-Meier plotter website were stratified with SFRP1 by Kaplan-Meier analysis. D Combination of circulating miR-93-3p/105 to evaluate the predictive power for stage I/II (N = 46) or stage III/IV (N = 21) stage of TNBC by ROC curve. (PDF 29884 kb

Effect of 2-APB on profiles of inflammatory cytokines in <i>O</i>. <i>tsutsugamushi</i>-infected and LPS-activated macrophages.

<p>Cytokine levels of production were determined with ELISA system (eBioscience) analysis during 24 h of co-treatment (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001).</p

Additional file 3: Table S2. of miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer

Univariate and multivariate analysis of clinical features and four oncomiRs associated with overall survival. (DOCX 12 kb

2-aminoethoxydipheny (2-APB) reduced pathogen-activated Ca²⁺ signaling in macrophages.

<p>(A) Fluorescence images of <i>O</i>. <i>tsutsugamushi</i> (OT)-infected macrophages at indicated time point with staining by Celltrackers (cytoplasm, green; OT, red) and DAPI (uncles, blue). (B) In <i>O</i>. <i>tsutsugamushi</i>-infected macrophages co-treatment with various Ca²⁺ inhibitors, 50 μM 2-APB (OT+2-APB), 50 μM SKF96365 (OT+SKF), or 25 μM BAPTA (OT+BAPTA) at indicated time points. (C) Effect of Ca²⁺ inhibitors, 2-APB (10 μM, 30 μM, 50 μM and 100 μM), SKF96365 (SKF) and BAPTA on intracellular Ca²⁺ concentration of <i>O</i>. <i>tsutsugamushi</i>-infected macrophages after 24 h of co-incubation. OT+10, OT+30, OT+50 and OT+100 indicate <i>O</i>. <i>tsutsugamushi</i> co-incubation with different concentration of 2-APB. Ca²⁺ imaging analysis of TG-induced Ca²⁺ response after application of 1 μM TG (small black arrow) (n = 3). (D) Quantification of area (above dotted line) of intracellular Ca²⁺ responses shown in (C) (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001). (E) CaCl₂ was extracellularly applied (large black bar) to enter Ca²⁺ via store-operated calcium channel after application of TG (small black bars) in Ca²⁺-free BSS solution (open bar) (n = 3). Effect of 2-APB on (F) Ca²⁺dynamics and (G) intracellular Ca²⁺ elevation in LPS-stimulated macrophages during 24 h of co-incubation (***, <i>p</i> < 0.001). LPS co-incubated with 50 μM 2-APB (LPS+50) and 100 μM 2-APB (LPS+100) respectively. (H) Orai1 expression was decreased by 2-APB, but not STIM1, TRPV1, or Actin in <i>O</i>. <i>tsutsugamushi</i>-infected macrophages, as determined by western blot analysis.</p