Clinical significance and potential pathogenesis of VCAN in adult non-cystic fibrosis bronchiectasis: a retrospective study.

BACKGROUND: The pathogenesis of adult non-cystic fibrosis (CF) bronchiectasis is complex, and the relevant molecular mechanism remains ambiguous. Versican (VCAN) is a key factor in inflammation through interactions with adhesion molecules. This study constructs a stable panoramic map of mRNA, reveals the possible pathogenesis of bronchiectasis, and provides new ideas and methods for bronchiectasis. METHODS: Peripheral blood and tissue gene expression data from patients with bronchiectasis and normal control were selected by bioinformatics analysis. The expression of VCAN in peripheral blood and bronchial tissues of bronchiectasis were obtained by transcriptome sequencing. The protein expression levels of VCAN in serums were verified by the enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of VCAN in co-culture of Pseudomonas aeruginosa and bronchial epithelial cells were verified by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the biological function of VCAN was detected by the transwell assay. RESULTS: The expression of VCAN was upregulated in the bronchiectasis group by sequencing analysis (P < 0.001). The expression of VCAN in the bronchial epithelial cell line BEAS-2B was increased in P. aeruginosa (P.a), which was co-cultured with BEAS-2B cells (P < 0.05). The concentration of VCAN protein in the serum of patients with bronchiectasis was higher than that in the normal control group (P < 0.05). Transwell experiments showed that exogenous VCAN protein induced the migration of neutrophils (P < 0.0001). CONCLUSIONS: Our findings indicate that VCAN may be involved in the development of bronchiectasis by increasing the migration of neutrophils and play an important role in bronchial pathogenesis.

The clinical significance and mechanism of microRNA-22-3p targeting TP53 in lung adenocarcinoma.

BACKGROUND: At present, studies on MircoRNA-22-3p (miR-22-3p) in lung adenocarcinoma use a single method, lack multi-center validation and multi-method validation, and there is no big data concept to predict and validate target genes. OBJECTIVE: To investigate the expression, potential targets and clinicopathological significance of miR-22-3p in lung adenocarcinoma (LUAD) tissues. METHODS: LUAD formalin-fixed paraffin-embedded (FFPE) tumors and adjacent normal lung tissues were collected for real-time quantitative polymerase chain reaction (RT-qPCR). Collect miR-22-3p in LUAD and non-cancer lung tissue from high-throughput datasets, standardized mean difference (SMD) and area under the curve (AUC) of the comprehensive receiver operating curve (summary receiver operating characteristic cure, sROC curve) were calculated. Cell function experiments on A549 cells transfected with LV-hsa-miR-22-3p. Target genes were predicted by the miRwalk2.0 website and the resulting target genes were subjected to Gene Ontology (GO) pathway enrichment analysis and constructed to protein-protein interaction network. Finally, the protein expression level of the key gene TP53 was validated by searching The Human Protein Atlas (THPA) database to incorporate TP53 immunohistochemical results in LUAD. RESULTS: RT-qPCR result from 41 pairs of LUAD and adjacent lung tissues showed that miR-22-3p was downregulated in LUAD (AUC = 0.6597, p= 0.0128). Globally, a total of 838 LUADs and 494 non-cancerous lung tissues were included, and were finally combined into 14 platforms. Compared with noncancerous tissue, miR-22-3p expression level was significantly reduced in LUAD tissue (SMD =-0.32, AUC = 0.72l); cell function experiments showed that miR-22-3p has inhibitory effects on cell proliferation, migration and invasion, and has promotion effect on apoptosis. Moreover, target genes prediction, GO pathway enrichment analysis and PPI network exhibited TP53 as a key gene of target gene of miR-22-3p; at last, a total of 114 high-throughput datasets were included, including 3897 LUADs and 2993 non-cancerous lung tissues, and were finally combined into 37 platforms. Compared with noncancerous tissue, TP53 expression level was significantly increased in LUAD (SMD = 0.39, p< 0.01) and it was verified by the protein expression data from THPA. CONCLUSION: Overexpression of miR-22-3p may inhibit LUAD cell proliferation, migration and invasion through TP53, and promote cell apoptosis.

Single cell transcriptomic analysis of human amnion identifies cell-specific signatures associated with membrane rupture and parturition.

BACKGROUND: The human amnion is an intrauterine tissue which is involved in the initiation of parturition. In-depth understanding of gene expression signatures of individual cell types in the amnion with respect to membrane rupture at parturition may help identify crucial initiators of parturition for the development of specific strategies to prevent preterm birth, a leading cause of perinatal mortality. RESULTS: Six major cell types were revealed in human amnion including epithelial cells, fibroblasts and immunocytes as well as three other cell types expressing dual cell markers including epithelial/fibroblast, immune/epithelial and immune/fibroblast markers. The existence of cell types expressing these dual cell markers indicates the presence of epithelial-mesenchymal (EMT), epithelial-immune (EIT) and mesenchymal-immune (MIT) transitions in amnion at parturition. We found that the rupture zone of amnion exhibited some specific increases in subcluster proportions of immune and EMT cells related to extracellular matrix remodeling and inflammation in labor. The non-rupture zone exhibited some common changes in subcluster compositions of epithelial and fibroblast cells with the rupture zone in labor, particularly those related to oxidative stress and apoptosis in epithelial cells and zinc ion transport in fibroblasts. Moreover, we identified that C-C motif chemokine ligand 20 (CCL20) was among the top up-regulated genes in amnion epithelial cells, fibroblasts and immunocytes in the rupture zone at parturition. Studies in pregnant mice showed that administration of CCL20 induced immunocytes infiltration to tissues at the maternal-fetal interface and led to preterm birth. CONCLUSIONS: Apart from the conventional epithelial, fibroblast and immunocytes, human amnion cells may undergo EMT, EIT and FIT in preparation for parturition. Intense inflammation and ECM remodeling are present in the rupture zone, while enhanced apoptosis and oxidative stress in epithelial cells and zinc ion transport in fibroblasts are present in amnion regardless of the rupture zones at parturition. CCL20 derived from the major cell types of the amnion participates in labor onset.

PGE2 vs PGF2α in human parturition.

Prostaglandin E2 (PGE2) and F2α (PGF2α) are the two most prominent prostanoids in parturition. They are involved in cervical ripening, membrane rupture, myometrial contraction and inflammation in gestational tissues. Because multiple receptor subtypes for PGE2 and PGF2α exist, coupled with diverse signaling pathways, the effects of PGE2 and PGF2α depend largely on the spatial and temporal expression of these receptors in intrauterine tissues. It appears that PGE2 and PGF2α play different roles in parturition. PGE2 is probably more important for labor onset, while PGF2α may play a more important role in labor accomplishment, which may be attributed to the differential effects of PGE2 and PGF2α in gestational tissues. PGE2 is more powerful than PGF2α in the induction of cervical ripening. In terms of myometrial contraction, PGE2 produces a biphasic effect with an initial contraction and a following relaxation, while PGF2α consistently stimulates myometrial contraction. In the fetal membranes, both PGE2 and PGF2α appear to be involved in the process of membrane rupture. In addition, PGE2 and PGF2α may also participate in the inflammatory process of intrauterine tissues at parturition by stimulating not only neutrophil influx and cytokine production but also cyclooxygenase-2 expression thereby intensifying their own production. This review summarizes the differential roles of PGE2 and PGF2α in parturition with respect to their production and expression of receptor subtypes in gestational tissues. Dissecting the specific mechanisms underlying the effects of PGE2 and PGF2α in parturition may assist in developing specific therapeutic targets for preterm and post-term birth.

Enhancement of cortisol-induced SAA1 transcription by SAA1 in the human amnion.

Our previous studies have demonstrated that human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute phase protein of inflammation, wherein SAA1 may participate in parturition by inducing a number of inflammation mediators including interleukine-1ß, interleukine-6 and prostaglandin E2. However, the regulation of SAA1 expression in the fetal membranes remains largely unknown. In the current study, we examined the regulation of SAA1 expression by cortisol, a crucial steroid produced locally in the fetal membranes at parturition, and the interaction between cortisol and SAA1 in the feed-forward induction of SAA1 expression in human amnion fibroblasts. Results showed that cortisol-induced SAA1 expression in a concentration-dependent manner, which was greatly enhanced by SAA1 despite modest induction of SAA1 expression by itself. Mechanism studies revealed that the induction of SAA1 expression by cortisol and SAA1 was blocked by either the transcription factor STAT3 antagonist AZD0530 or siRNA-mediated knockdown of STAT3. Furthermore, cortisol- and SAA1-induced STAT3 phosphorylation in a sequential order with the induction by SAA1 preceding the induction by cortisol. However, combination of cortisol and SAA1 failed to further intensify the phosphorylation of STAT3. Consistently, cortisol and SAA1 increased the enrichment of STAT3 at the SAA1 promoter. Taking together, this study has demonstrated that cortisol and SAA1 can reinforce each other in the induction of SAA1 expression through sequential phosphorylation of STAT3. The enhancement of cortisol-induced SAA1 expression by SAA1 may lead to excessive SAA1 accumulation resulting in parturition-associated inflammation in the fetal membranes.

Involvement of serum amyloid A1 in the rupture of fetal membranes through induction of collagen I degradation.

The de novo synthesis of serum amyloid A1 (SAA1) is augmented in human fetal membranes at parturition. However, its role in parturition remains largely unknown. Here, we investigated whether SAA1 was involved in the rupture of fetal membranes, a crucial event in parturition accompanied with extensive degradation of collagens. Results showed that SAA1 decreased both intracellular and extracellular COL1A1 and COL1A2 abundance, the two subunits of collagen I, without affecting their mRNA levels in human amnion fibroblasts. These reductions were completely blocked only with inhibition of both matrix metalloproteases (MMPs) and autophagy. Consistently, SAA1 increased MMP-2/9 abundance and the markers for autophagic activation including autophagy related (ATG) 7 (ATG7) and the microtubule-associated protein light chain 3 ß (LC3B) II/I ratio with the formation of LC3 punctas and autophagic vacuoles in the fibroblasts. Moreover, the autophagic degradation of COL1A1/COL1A2 and activation of MMP-2/9 by SAA1 were blocked by inhibitors for the toll-like receptors 2/4 (TLR2/4) or NF-κB. Finally, reciprocal corresponding changes of SAA1 and collagen I were observed in the amnion following spontaneous rupture of membranes (ROM) at parturition. Conclusively, SAA1 may participate in membrane rupture at parturition by degradating collagen I via both autophagic and MMP pathways. These effects of SAA1 appear to be mediated by the TLR2/4 receptors and the NF-κB pathway.

Extracellular matrix remodeling effects of serum amyloid A1 in the human amnion: Implications for fetal membrane rupture.

PROBLEM: Rupture of fetal membranes is a crucial event at parturition, which is preceded by extensive extracellular matrix (ECM) remodeling. Our recent studies have demonstrated that the human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute phase protein, and the abundance of SAA1 in the amnion was increased at parturition. However, the exact role of SAA1 in human parturition remains to be established. METHOD OF STUDY: The effects of SAA1 on the abundance of collagenases and lysyl oxidase, the enzyme that cross-links collagens, were investigated in culture primary human amnion fibroblasts and tissue explants with an aim to examine the involvement of SAA1 in the ECM remodeling in the amnion. RESULTS: Serum amyloid A1 (SAA1) time- and dose-dependently increased the abundance of collagenases MMP-1, MMP-8, and MMP-13, while decreased the abundance of lysyl oxidase-like 1 (LOXL1). These effects of SAA1 were attenuated by siRNA-mediated knockdown of the Toll-like receptor (TLR) 4 and its antagonist CLI-095, but not by siRNA-mediated knockdown of TLR2. Furthermore, the inhibitors for NF-κB (JSH-23) and mitogen-activated protein kinases (MAPKs) p38 (SB203580) and JNK (SP600125) could also attenuate the effects of SAA1, while the inhibitor for MAPK ERK1/2 (PD 98059) could block the effects of SAA1 only on MMP-1, MMP-8, and LOXL1 but not on MMP-13. CONCLUSION: These data highlight a possible role for SAA1 in ECM remodeling preceding membrane rupture by regulating the expression of collagenases MMP-1, MMP-8, MMP-13, and LOXL1 through TLR4-mediated activation of the NF-κB and MAPK pathways in amnion fibroblasts.

Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.

Preterm premature rupture of fetal membranes precedes 30-40% of preterm births. Activation of matrix metalloproteases (MMPs) is the one of the major causes of extracellular matrix (ECM) degradation in membrane rupture. Increased cortisol, regenerated by 11ß-hydroxysteroid dehydrogenase 1 in the amnion at parturition, is known to participate in a number of parturition-pertinent events. However, whether cortisol has a role in the regulation of MMPs in the membranes is not known. Here, we addressed this issue using human amnion tissue, the most tensile layer of the membranes. RNA-sequencing revealed that cortisol induced MMP7 expression dramatically in amnion fibroblasts, which was confirmed by real-time quantitative RT-PCR and Western blotting analysis in cortisol-treated amnion explants and fibroblasts. Measurement of collagen IV α5 chain (COL4A5), a substrate for MMP-7, showed that cortisol reduced its extracellular abundance, which was blocked by an antibody against MMP-7. Moreover, increased MMP-7 but decreased COL4A5 abundance was observed in the amnion tissue following labor-initiated spontaneous rupture of membranes. Mechanistic studies showed that cortisol increased the phosphorylation of c-Jun and the expression of c-Fos, the 2 major components of activated protein 1 (AP-1), respectively. The knocking down of c-Fos or c-Jun significantly attenuated the induction of MMP7 expression by cortisol. Chromatin immunoprecipitation assays showed that cortisol stimulated the enrichment of c-Fos and c-Jun at the AP-1 binding site in the MMP7 promoter. The data suggest that induction of MMP7 by cortisol via AP-1 may be a contributing factor to ECM degradation in membrane rupture at parturition.-Wang, L.-Y., Wang, W.-S., Wang, Y.-W., Lu, J.-W., Lu, Y., Zhang, C.-Y., Li, W.-J., Sun, K., Ying, H. Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.

Downregulation of p16(ink4a) inhibits cell proliferation and induces G1 cell cycle arrest in cervical cancer cells.

Studies have suggested that p16(ink4a) may be a surrogate biomarker for the diagnosis of cervical cancer; however, the function of p16(ink4a) in human cervical cancer cells remains largely unknown. Therefore, in this study, we aimed to investigate the role of p16(ink4a) in human cervical cancer cells. Immunocytochemistry was used to examine invasive squamous cell carcinoma and its precancerous lesions. p16(ink4a)-siRNA was transfected into SiHa and HeLa cells to deplete its expression. The cellular levels of p16(ink4a) mRNA and protein were detected by qRT-PCR and western blot analysis. Proliferation rates were assessed by methyl thiazolyl tetrazolium (MTT) and plate colony formation assays. Cellular migration and invasion ability were assessed by a wound healing assay and Transwell assay. Cellular apoptosis and the cell cycle were measured by flow cytometry. The protein levels of retinoblastoma (Rb), phosphorylated Rb (phospho-Rb), cyclin D1 and caspase-3 were determined by western blot analysis. The results revealed that p16(ink4a) was overexpressed in the cervical cancer and precancerous lesions (P<0.05). The downregulation of p16(ink4a) in the SiHa and HeLa cells inhibited their proliferation, migration and invasion. In the SiHa cells, p16(ink4a)-siRNA also induced G1 cell cycle arrest and apoptosis. Western blot analysis revealed that the downregulation of p16(ink4a) in the SiHa cells markedly induced caspase-3 activation and decreased cyclin D1 expression. These data suggest that the overexpression of p16(ink4a) appears to be useful in monitoring cervical precancerous lesions, which supports that the hypothesis that p16(ink4a) is a surrogate biomarker for the diagnosis of cervical cancer. The therapeutic targeting of overexpressed p16(ink4a) in the p16(ink4a)-cyclin-Rb pathway may be a useful strategy in the treatment of cervical cancer.