Dynamic changes of throat swabs RNA and serum antibodies for SARS-CoV-2 and their diagnostic performances in patients with COVID-19.

Dynamic changes of RNA and antibodies in SARS-CoV-2 infected patients remain largely unknown, and influence factors for antibody production have not been fully clarified. In this study, consecutive throat swabs specimens (n = 1875) from 187 patients were collected to analyse the dynamic changes of RNA. Moreover, 162 serial serum samples from 31 patients were tested for seroconversion of IgM and IgG. Meanwhile, IgM and IgG were also detected in 409 COVID-19 patients and 389 controls. Additionally, the logistic regression analysis was executed to identify the possible influence factors for antibody production. The median positive conversion time for RNA was day 7 (IQR, 3-11), and the positive rate was highest in day 1-5 (74.59 %) and then gradually decreased. The median time of seroconversion for IgM and IgG were both day 12 (IQR, 10-15). The sensitivity and specificity for IgM (or IgG) was 87.04% and 96.92%, respectively. Multivariate logistic regression indicated that reduced lymphocytes and short positive conversion time for SARS-CoV-2 RNA were independent factors for negative results of IgM and IgG. In conclusion, RNA and antibodies should be combined for COVID-19 diagnosis, and delayed seroconversion was influenced by the decreased lymphocytes and short positive conversion time for RNA.

TGF-ß1 and TNF-α synergistically induce epithelial to mesenchymal transition of breast cancer cells by enhancing TAK1 activation.

TGF-ß1 is a main inducer of epithelial to mesenchymal transition (EMT). However, many breast cancer cells are not sensitive to the EMT induction by TGF-ß1 alone. So far, the mechanisms underlying the induction of TGF-ß1-insensitive breast cancer cells remains unclear. Here we report that TNF-α can induce EMT and invasiveness of breast cancer cells which are insensitive to TGF-ß1. Intriguingly, TGF-ß1 could cooperate with TNF-α to promote the EMT and invasiveness of breast cancer cells. The prolonged co-stimulation with TGF-ß1 and TNF-α could enhance the sustained activation of Smad2/3, p38 MAPK, ERK, JNK and NF-κB pathways by enhancing the activation of TAK1, which was mediated by the gradually up-regulated TßRs. Except for JNK, all of these pathways were required for the effects of TGF-ß1 and TNF-α. Importantly, the activation of p38 MAPK and ERK pathways resulted in a positive feed-back effect on TAK1 activation by up-regulating the expression of TßRs, favoring the activation of multiple signaling pathways. Moreover, SLUG was up-regulated and required for the TGF-ß1/TNF-α-induced EMT and invasiveness. In addition, SLUG could also enhance the activation of signaling pathways by promoting TßRII expression. These findings suggest that the up-regulation of TßRs contributes to the sustained activation of TAK1 induced by TGF-ß1/TNF-α and the following activation of multiple signaling pathways, resulting in EMT and invasiveness of breast cancer cells.

The prognostic significance of long noncoding RNAs in non-small cell lung cancer: a meta-analysis.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. The overall 5-year survival rate of patients is extremely low and to find a new marker is urgently needed. Numerous studies indicate that long noncoding RNAs (lncRNAs) abnormally express in cancers. However, the results have been disputed, especially in the aspects of tumor prognosis. Therefore, we performed this meta-analysis to systematically summarize the relationship between lncRNAs expression and NSCLC. A total of 34 eligible studies including 30 on overall survival, 10 on progression-free survival and 23 on clinicopathological features were identified from the databases. Our results indicated that the levels of lncRNAs were associated with the overall survival (OS; hazard ratios [HR], 1.43; 95% confidence interval [95% CI], 1.17-1.76; P < 0.001). However, there was no relationship between lncRNAs and progression-free survival (PFS; hazard ratios [HR], 1.55; 95% confidence interval [95% CI], 0.91-2.63; P = 0.11). Moreover, lncRNAs were related to lymph node metastasis (odds ratios [OR], 1.70; 95% confidence interval [95% CI], 1.03-2.80; P = 0.04), while no association was observed with other characteristics. In conclusion, our present meta-analysis indicated that lncRNAs transcription levels may serve as a promising marker for prognosis of patients with NSCLC.

Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells.

Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface.

TLR4 ligand/H2O2 enhances TGF-ß1 signaling to induce metastatic potential of non-invasive breast cancer cells by activating non-Smad pathways.

TGF-ß1 has the potential to activate multiple signaling pathways required for inducing metastatic potential of tumor cells. However, TGF-ß1 was inefficient in inducing metastatic potential of many non-invasive human tumor cells. Here we report that the enhancement of TGF-ß1 signaling is required for inducing metastatic potential of non-invasive breast cancer cells. TGF-ß1 alone could not efficiently induce the sustained activation of Smad and non-Smad pathways in non-invasive breast cancer cells. TLR4 ligand (LPS) and H2O2 cooperated with TGF-ß1 to enhance the sustained activation of non-Smad pathways, including p38MAPK, ERK, JNK, PI3K, and NF-κB. The activation of MAPK and PI3K pathways resulted in a positive feed-back effect on TGF-ß1 signaling by down-regulating Nm23-H1 expression and up-regulating the expression of TßRI and TßRII, favoring further activation of multiple signaling pathways. Moreover, the enhanced TGF-ß1 signaling induced higher expression of SNAI2, which also promoted TßRII expression. Therefore, the sustained activation levels of both Smad and non-Smad pathways were gradually increased after prolonged stimulation with TGF-ß1/H2O2/LPS. Consistent with the activation pattern of signaling pathways, the invasive capacity and anoikis-resistance of non-invasive breast cancer cells were gradually increased after prolonged stimulation with TGF-ß1/H2O2/LPS. The metastatic potential induced by TGF-ß1/H2O2/LPS was sufficient for tumor cells to extravasate and form metastatic foci in an experimental metastasis model in nude mice. The findings in this study suggested that the enhanced signaling is required for inducing higher metastatic capacity of tumor cells, and that targeting one of stimuli or signaling pathways might be potential approach in comprehensive strategy for tumor therapy.

IL-6 cooperates with G-CSF to induce protumor function of neutrophils in bone marrow by enhancing STAT3 activation.

Neutrophils are known to have antitumor potential. However, in recent years the tumor-promoting effect of neutrophils has been well demonstrated. So far, it remains unclear what causes the conversion of neutrophil function from tumor suppressive to tumor promoting. In this article, we report that the conversion of murine neutrophil function occurs in bone marrow, and that IL-6 cooperation with G-CSF is required for this conversion. IL-6 cooperated with G-CSF to modulate neutrophils in bone marrow, altering the activation potential of signaling pathways in neutrophils, especially that of STAT3. Costimulation with G-CSF and IL-6 induced a higher level of phospho-STAT3 in neutrophils, which was further increased by upregulation of STAT3 expression in neutrophils owing to downregulation of IFN-ß expression in bone marrow macrophages by IL-6. Augmented STAT3 activation was crucial for upregulating the expression of Mmp9 and Bv8 genes and downregulating the expression of Trail and Rab27a genes in neutrophils. Moreover, G-CSF/IL-6-modulated neutrophils could not efficiently release azurophilic granules because of downregulation of Rab27a and inefficient activation of PI3K and p38 MAPK pathways. Because of premodulation by G-CSF and IL-6, neutrophils in response to complex stimuli in tumor released much less myeloperoxidase, neutrophil elastase, and TRAIL, but showed much higher expression of Mmp9 and Bv8 genes. Taken together, these results demonstrate that G-CSF and IL-6, despite their well-known physiological functions, could modulate the activation potential of signaling pathways in neutrophils, resulting in the production or release of the above-mentioned factors in a way that favors tumor angiogenesis and tumor growth.

Targeting nuclear factor-κB suppresses the negative effect of toll-like receptor 4 signaling on antimetastasis therapy based on targeting αvß3.

The targeting of αvß3 is a promising therapeutic strategy for suppressing tumor metastasis. However, it is unclear whether the therapeutic efficacy could be influenced by metastasis-promoting factor(s) in vivo. Here we report that Toll-like receptor 4 (TLR4) ligand released from damaged tumor cells or bacteria had a negative effect on the therapeutic effect of a recombinant CBD-HepII polypeptide of fibronectin (CH50) that suppresses tumor metastasis by targeting αvß3. The TLR4 ligand could antagonize the inhibitory effect of CH50 on tumor cell adhesion and invasion by promoting the expression and activity of αvß3 in tumor cells. The TLR4 ligand also reduced the antimetastasis effect of CH50 by promoting tumor cell survival in circulation. Moreover, TLR4 ligands released by tumor cells in circulation could increase the survival and proliferation capacity of tumor cells after extravasation, resulting in the formation of more metastatic nodules. The effect of TLR4 signaling was mainly mediated by nuclear factor-κB (NF-κB). Inhibiting NF-κB could abrogate the negative effect of TLR4 ligand, and augment the inhibitory effect of CH50 on tumor metastasis. Consistently, the combination of NF-κB inhibitor and CH50 significantly inhibited metastasis of tumor cells in vivo and prolonged the survival of mice. The findings in this study suggest that the combination of NF-κB inhibitor and αvß3 antagonist would be a novel therapeutic option for the prevention of tumor metastasis.

Extracellular HSPA1A promotes the growth of hepatocarcinoma by augmenting tumor cell proliferation and apoptosis-resistance.

Intracellular HSP70 has been implicated as a cytoprotective protein, whereas the effect of extracellular HSP70 on tumor cells has not been fully understood to date. Here we report that extracellular HSPA1A, a stress-inducible member of HSP70 family, could promote tumor growth. HSPA1A promoted the proliferation of H22 hepatocarcinoma cells through TLR2 and TLR4 signaling. The effect of HSPA1A was abolished by inhibiting NF-κB. HSPA1A also augmented the apoptosis-resistance of H22 cells by activating NF-κB, thus to promote the proliferation of H22 cells in presence of mitomycin C. Furthermore, the promoting effect of HSPA1A on tumor cell proliferation was existent after the removal of HSPA1A, which might involve HSPA1A-promoted upregulation of TLR4 expression in tumor cells and release of HMGB1 from tumor cells. These findings suggest that extracellular HSPA1A functions as endogenous ligand for TLR2 and TLR4 to facilitate tumor growth.

SCF and TLR4 ligand cooperate to augment the tumor-promoting potential of mast cells.

Mast cells may have either antitumor or tumor-promoting potential. Nevertheless, mast cells in tumor microenvironment have been found to promote tumor growth. So far the mechanisms underlying the modulation of mast cell function in tumor microenvironment remains to be fully elucidated. Here, we report that tumor-promoting potential of mast cells could be augmented by molecules released from damaged tumor cells through cooperative stimulation of stem cell factor (SCF) and ligand for Toll-like receptor 4 (TLR4). Co-simulation with SCF and TLR4 ligand inhibited mast cell degranulation, but efficiently induced the production and secretion of VEGF, PDGF, and IL-10. Although TLR4 ligand alone may induce IL-12 expression in mast cells, co-stimulation with SCF and TLR4 ligand induced the expression of IL-10, but not IL-12, in mast cells. The phosphorylation of GSK3ß was crucial for the effect of SCF and TLR4 ligand. In addition to inducing phosphorylation of GSK3ß at Ser9 through PI3K pathway, SCF and TLR4 ligand cooperated to induce phosphorylation of GSK3ß at Tyr216 by simultaneous activation of ERK and p38MAPK pathways. Both phospho-Ser9 and phospho-Tyr216 of GSK3ß were required for IL-10 expression induced by SCF/TLR4 ligand, whereas suppressive effect of SCF/TLR4 ligand on mast cell degranulation was related to phospho-Tyr216. Importantly, the effect of SCF and TLR4 ligand on mast cells could be abrogated by inhibiting phosphorylation of GSK3ß at Tyr216. These findings disclose the mechanisms underlying the modulation of mast cell function in tumor microenvironment, and suggest that inhibiting GSK3ß in mast cells will be beneficial to the treatment of cancer.

Triggering of Toll-like receptor 4 on metastatic breast cancer cells promotes αvß3-mediated adhesion and invasive migration.

Triggering of Toll-like receptor 4 (TLR4) on tumor cells has been found to promote tumor progression by promoting tumor cell proliferation and survival. So far, however, the effect of TLR4 signaling on tumor metastasis has not been well elucidated. Here, we report that triggering of TLR4 on metastatic breast cancer cells could reciprocally regulate the expression of αvß3 and the expressions of TPM1 and maspin, and promote αvß3-mediated adhesion and invasive migration of the cells. In metastatic breast cancer cells, TLR4 signaling increased the expression of integrin αvß3 by activating NF-κB, resulting in the increased adhesion capacity of tumor cells to the ligand for αvß3, and the increased polymerization of actin and production of MMP-9 in tumor cells in response to ECM. HoxD3 was required for the up-regulation of αv and ß3 expressions by NF-κB. Moreover, TLR4 signaling increased the expression of miR-21 in breast cancer cells by activating NF-κB. Accordingly, the expressions of TPM1 and maspin were decreased at protein level, whereas the transcription activity of these genes was not influenced. Consistent with the promoting effect on αvß3-mediated adhesion and invasive migration, TLR4 signaling promoted the arrest of metastatic breast cancer cells in circulation and following invasion. The effect of TLR4 signaling could be abrogated by inhibiting NF-κB. These findings suggest that metastatic breast cancer cells could acquire higher metastatic potential due to triggering of TLR4 and activation of NF-κB in the cells, and that both TLR4 and NF-κB could be therapeutic targets for preventing metastasis of breast cancer cells.

Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.