Showing papers in "International Journal of Clinical and Experimental Pathology in 2018"

LncRNA XIST knockdown attenuates Aβ25-35-induced toxicity, oxidative stress, and apoptosis in primary cultured rat hippocampal neurons by targeting miR-132.

Abstract: Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. The abnormal accumulation and deposition of amyloid-beta peptide (Aβ) in senile plaques and cerebral vasculature is widely recognized to be the most likely culprit in the pathogenesis of AD. Long non-coding RNAs (lncRNAs), a kind of evolutionarily conserved non-coding RNAs with over 200 nucleotides in length, have introduced a novel field of biology, and are involved in various human diseases, including neurological diseases. Recently, lncRNA X-inactive specific transcript (XIST) is reported to be upregulated in the rat spinal cord injury (a neurological disease) model and XIST knockdown has a prominent protective effect on the recovery of spinal cord injury. However, little is known about the expression and function of XIST in AD. Here, we showed that Aβ25-35 treatment increased XIST expression in hippocampal neurons. XIST knockdown ameliorated toxicity, oxidative stress, and apoptosis induced by Aβ25-35 treatment in hippocampal neurons. We further identified and confirmed that miR-132 was the target of XIST, and XIST functioned by targeting miR-132. Collectively, these data show that knockdown of XIST relieves Aβ25-35-induced toxicity, oxidative stress, and apoptosis in primary cultured rat hippocampal neurons by upregulation of miR-132. These findings encourage continued investigation of the potential of manipulating XIST in the treatment of AD.

miR-338-3p suppresses colorectal cancer proliferation and progression by inhibiting MACC1.

Abstract: Colorectal cancer (CRC) is one of the most common malignancies worldwide. This study aimed to elucidate the clinicopathological significance of miR-338-3p and its association with metastasis-associated in colon cancer-1 (MACC1) in CRC. We evaluated miR-338-3p and MACC1 expression in CRC cell lines and analyzed the clinicopathological features of miR-338-3p in 98 samples of CRC tissues. Subsequent Western blot and cellular biological techniques, and xenograft mouse models were performed to investigate the biological role of miR-338-3p and its association with MACC1 in CRC. Our results show that miR-338-3p expression is lower in CRC cell lines and tissues than that in a human normal colonic epithelial cell line and adjacent normal colorectal tissue, respectively. miR-338-3p expression was significantly associated with histological differentiation, UICC stage, T classification, N classification, and M classification in 98 samples of CRC. The overall survival of CRC patients was significantly less in the low miR-338-3p expression group than in the high miR-338-3p expression group (p<0.01). miR-338-3p mimics suppressed cell proliferation, colony formation, migration, and invasion, but induced apoptosis in CRC cells. miR-338-3p inhibitor reversed these biological phenotypes. miR-338-3p mimics or inhibitor suppressed or increased MACC1 expression in HCT116 and SW620. miR-338-3p mimics reversed the effect of increased MACC1 expression induced by HCT116 with MACC1 over-expression plasmid. Increased cell proliferation, colony formation, and suppressed cell apoptosis caused by MACC1 over-expression were significantly reversed in HCT116 transfected with miR-338-3p mimics, respectively. Suppressed cell proliferation, colony formation, migration, invasion, and increased cell apoptosis caused by MACC1 knockdown were significantly reversed in SW620 transfected with miR-338-3p inhibitor, respectively. In vivo, miR-338-3p agomir significantly inhibited xenograft CRC tumor growth and reversed the effect of increased xenograft tumor growth induced from HCT116 with MACC1 overexpression. In conclusion, our data suggest that miR-338-3p suppresses CRC carcinogenesis and progression by inhibiting MACC1. Targeting miR-338-3p might be a novel treatment strategy for CRC.

Hsa_circ_0007534 as a blood-based marker for the diagnosis of colorectal cancer and its prognostic value.

Abstract: Background Many studies have shown that differentially expressed circular RNA (circRNA) in plasma can serve as biomarkers in non-invasive detection of cancers during screening. However, the clinical significance of plasma circRNA in the diagnosis of colorectal cancer (CRC) is still not clear. Therefore, we examined expression of hsa_circ_0007534 in plasma to verify whether it can be utilized to diagnose and monitor CRC in routine clinical practice. Methods 112 CRC patients and 46 healthy controls were recruited to participate in our study. The levels of hsa_circ_0007534 in plasma samples and tumor tissues were identified by real-time quantitative polymerase chain reaction (RT-qPCR). The diagnostic value was evaluated using receiver operating characteristics (ROC) curves and the area under the ROC curves (AUC). The Kaplan-Meier survival curve was used to evaluate whether the expression level of hsa_circ_0007534 was associated with overall survival rate. Results Compared with the healthy control group, hsa_circ_0007534 expression was significantly increased in plasma from CRC patients. Increased hsa_circ_0007534 expression level in plasma was associated with progression of clinical classifications, metastatic phenotype, and poor differentiation in CRC patients. ROC analysis showed that hsa_circ_0007534 could distinguish CRC patients from healthy controls with high AUC (0.780), sensitivity (0.92) and specificity (0.522). Finally, high hsa_circ_0007534 expression was positively correlated with poor prognosis in CRC patients. Conclusion All of the results suggest that hsa_circ_0007534 may be a potential cancer marker of patients with CRC and may associate with poor prognosis.

Comparison of microsatellite status detection methods in colorectal carcinoma.

Abstract: There are two commonly accepted methods for detecting microsatellite status. One is to detect amplified microsatellite loci by polymerase chain reaction (PCR) and the other is to detect mismatch repair gene (MMR) protein expression by immunohistochemistry (IHC). PCR detection is considered to be accurate in clinical operations while IHC is widely used due to ease of operation and lesser expense. In order to compare IHC with PCR in detecting microsatellite status in colorectal carcinoma, a total of 569 samples of colorectal carcinoma resection were collected in the Department of Pathology, Nanjing Drum Tower Hospital, between June 2014 and June 2017. In all samples, IHC and PCR was used to detect microsatellite status and the consistency of results between the two methods was compared. We found that 48 cases of microsatellite instability (MSI) were detected by PCR including 37 cases of microsatellite instability high (MSI-H), 11 cases of microsatellite instability low (MSI-L), and 521 cases of MSS. MSI accounted for 8.44% of all cases and MSI-H accounted for 6.50%. IHC results of the 569 patients showed that 69 cases were deficient mismatch repair (dMMR) and 500 cases were proficient mismatch repair (pMMR). dMMR accounted for 12.13% of all cases. Loss expression of PMS2 protein was the most common while MSH6 was rare. The coincidence rate of the two methods for detecting microsatellite states was 91.92%. IHC and the PCR method had high consistency in microsatellite status. Compared with PCR, the IHC method is more economical and more convenient for clinical operations. When the 4 repair proteins were without deficiency detected by IHC, it could be diagnosed as MSS/MSI-L and further PCR was not necessary. When any repair protein was found to be deficient, PCR detection was needed to determine whether MSI existed. Our conclusion will save a lot of time and costs in clinical work.

The correlation between exposure to BPA and the decrease of the ovarian reserve.

Abstract: OBJECTIVE This study aimed to evaluate whether exposure to bisphenol A (BPA) affects the ovarian reserve. METHODS Follicular fluid (FF) was collected from diminished ovarian reserve (DOR) and non-DOR patients who underwent in vitro fertilization or intracytoplasmic sperm injection. ELISA was used to detect the BPA and hormones levels in 54 cases of DOR and 67 cases of non-DOR. A total of 64, five-week-old SPF C57BL/6 mice were randomly divided into four groups, of which three were exposed to 5, 50, and 500 µg/kg/day of BPA solution, and one was exposed to con oil only as the control. The weight and estrus of each mouse were recorded daily, and the E2 hormone and anti-Mullerian hormone (AMH) in the serum were detected by ELISA. The expression levels of AMH mRNA and protein were also detected. RESULTS The BPA levels in the FF of DOR patients were significantly higher than those of non-DOR patients (234.048±81.736 ng/L vs. 193.300±67.225 ng/L, P<0.01); The AMH and E2 levels in the FF of DOR patients were lower than those of non-DOR patients ([555.689+74.224] pg/ml vs. [587.178+77.731] pg/ml, P<0.05, [209.720+31.556] pg/ml vs. [221.845+32.632] pg/ml, P<0.05). The BPA concentration was correlated with the AMH and E2 levels in the FF (rBPA & AMH=-0.312, P<0.05; rBPA & E2=-0.290, P<0.05); in the animal experiment, the levels of serum AMH and E2 as well as the expression levels of the AMH gene and protein in the BPA treatment group displayed downward trends. The concentrations of the 5 and 500 µg/kg/day groups decreased significantly (P<0.05). CONCLUSION The increased BPA in the FF may promote the pathogenesis of DOR. BPA did not present a single-dose effect on the mouse ovary. Sub-chronic exposure to a low dose of BPA can reduce the ovarian reserve in female mice.

Hypoxia-inducible factor (HIF)-1alpha knockout accelerates intervertebral disc degeneration in mice.

Abstract: Introduction: The abnormality of nucleus pulposus (NP) plays a critical role in intervertebral disc (IVD) degeneration, in which NP cells show apoptosis and fibrosis, leading to the ability of the disc to transfer and distribute loads between the vertebrae is decreased. Considering that hypoxia inducible factor-1α (HIF-1α) is abundantly expressed in NP and that it mediates cell proliferation, migration and apoptosis in various cell types, we hypothesized that NP-HIF-1α plays an important role in NP and evaluate whether NP-HIF-1α is involved in IVD degeneration. Material and methods: Sonic Hedgehog-Cre+/- mice were crossed with HIF-1αflox/flox mice to generate NP specific HIF-1α-deficient (HIF-1α-/-) mice. Magnetic resonance imaging (MRI) study was used to evaluate NP dehydration and X-ray study was used to acquire the changes of disc height. Histological changese, content of glycoproteins and the in situ expression of aggrecan were evaluated by hematoxylin & eosin (H&E) staining, safranin-O/fast green staining and immunohistochemistry assay, respectively. Western bloting was used to detect the change of extracellular matrix in IVD. Results: Firstly, the results of in situ hybridization confirmed that HIF-1α in NP was successfully knocked out in HIF-1α-/- mice. Next, for HIF-1α deficiency mice, imaging study shows IVD was narrowed in X-ray and signal intensity of NP was decreased in MR T2-weight imaging. Accordingly, the size and cell number of NP and proteoglycan content was decreased in NP-HIF-1α-/- mice. Finally, Western bloting shows that protein level of collagen II and aggrecan, two main matrix in disc, were both decreased in NP-HIF-1α-/- mice. Conclusions: The present study demonstrates that HIF-1α is essential for NP development and homeostasis and the deficiency of NP-HIF-1α leads to IVD degeneration in mice.

Expression of exportin-1 in diffuse large B-cell lymphoma: immunohistochemistry and TCGA analyses.

Abstract: Exportin-1 (XPO1) is an essential nuclear export receptor that is involved in the pathogenesis of multiple tumors. However, the role of XPO1 in diffuse large B-cell lymphoma (DLBCL) requires clarification. This study aims to detect XPO1 expression in DLBCL and to explore its relationships with clinicopathologic parameters and prognoses. Methods A total of 131 cases of DLBCL and 30 cases of reactive lymphoid hyperplasia were selected for immunohistochemistry to examine XPO1 expression and analyze the relationships of XPO1 expression with clinicopathologic parameters and prognosis. DLBCL datasets downloaded from The Cancer Genome Atlas (TCGA) were used to analyze the mutations, expressions, and clinical values of XPO1 in DLBCL. Results XPO1 expression was markedly upregulated in DLBCL compared to the reactive lymphoid hyperplasia group (χ2 = 10.734, P = 0.001). High XPO1 expression was associated with an advanced clinical stage (χ2 = 4.036, P = 0.045) and a risky International Prognostic Index (IPI) score (χ2 = 5.301, P = 0.025). Moreover, high XPO1 expression was associated with a lower overall survival rate compared with low expression (P = 0.043). XPO1 was an independent prognostic factor for DLBCL (risk ratio, RR = 3.772, P = 0.006). Furthermore, XPO1 overexpression in DLBCL was correlated with a high IPI score (P = 0.024) in TCGA datasets. Conclusion High XPO1 expression in DLBCL was related to an advanced clinical stage, poor IPI score, and poor prognosis. Thus, XPO1 may be useful for condition identification and prognostic assessment.

MiR-106a-5p promotes 5-FU resistance and the metastasis of colorectal cancer by targeting TGFβR2.

Abstract: Background Colorectal cancer (CRC) is the third leading cause of cancer-related deaths. 5-Fluorouracil (5-FU)-based chemotherapy has always been the first-line treatment. However, development of 5-FU resistance seriously affects its curative effect. The aim of this study was to elucidate the molecular mechanisms of 5-FU resistance through miR-106a-5p in CRC. Methods Colorectal cancer tissues were collected to analyze miR-106a-5p and TGFβR2 expressions by qPCR. Functional experiments for evaluating cell survival and metastasis were conducted to observe the biological effects of miR-106a-5p and TGFβR2. The cell survival rate was calculated using an MTT assay; the metastasis was confirmed with a Transwell invasion assay and Western blotting, which we used to measure the expression levels of the epithelial-mesenchymal transition (EMT) markers E-cadherin and vimentin. The combination of miR-106a to TGFβR2 was predicted using Targetscan, and confirmed through the construction of the luciferase reporter plasmid pGL3-basic. The interplay between miR-106a-5p and TGFβR2 was tested with qPCR and Western blotting. A Spearman rank analysis was employed to verify the correlation of miR-106a-5p and TGFβR2 expressions. Results MiR-106a-5p was up-regulated and TGFβR2 was down-regulated in 5-FU resistant CRC tissues and HT-29 cells. MiR-106a-5p promoted cell survival and suppressed the apoptosis rate and caspase 3 activity. Additionally, cell invasion was promoted by miR-106a-5p overexpression in the HT-29 cells and was inhibited by miR-106a-5p knockdown in the 5-FU resistant HT-29 cells; miR-106a-5p overexpression contributed to migration by increasing vimentin expression and by decreasing E-cadherin expression in the HT-29 cells; miR-106a-5p functioned by directly binding to TGFβR2. The TGFβR2 knockdown conferred chemoresistance of 5-FU and metastasis in 5-FU resistant HT-29 cells, and TGFβR2 overexpression reduced cell survival, invasion numbers, vimentin expression, and increased the cell apoptosis rate and caspase 3 activity in 5-FU resistant HT-29 cells. Also, miR-106a-5p negatively regulated TGFβR2 in a linear correlation way in the CRC tissues. Conclusion The up-regulation of miR-106a-5p contributes to the pathomechanism of colorectal cancer by promoting 5-FU resistance and metastasis via inhibiting target TGFβR2. Our findings provide new promising ways for the clinical application of the TGFβR2-miR-106a axis in clinical chemotherapy for 5-FU resistant colorectal cancer.

Adiponectin inhibits NLRP3 inflammasome by modulating the AMPK-ROS pathway.

Abstract: Chronic inflammation is a key contributor to obesity-related insulin resistance and type 2 diabetes (T2D). NLRP3 inflammasome activation plays an important role in impairing insulin signaling and insulin sensitivity. Adiponectin is an adipocyte-derived cytokine that has been shown to promote insulin sensitivity and exert anti-inflammatory properties, yet the detailed mechanism is still unclear. In this study, we aimed to investigate the anti-inflammatory effect of adiponectin on lipopolysaccharide (LPS) plus palmitic acid (PA)-induced THP-1 cells and to identify the underlying mechanism. We report here that adiponectin was able to inhibit interleukin (IL)-1β and IL-18 by suppressing NLRP3 inflammasome activation. Furthermore, we, for the first time, describe that adiponectin attenuates NLRP3 inflammasome activation by modulating the AMPK-ROS signaling pathway. These findings provide insight suggesting that adiponectin and NLRP3 inflammasome be considered molecular targets for the development of new treatment for T2D and the related metabolic diseases.

miR-424 promotes cardiac ischemia/reperfusion injury by direct targeting of CRISPLD2 and regulating cardiomyocyte pyroptosis.

Abstract: As a complex pathophysiological event, myocardial ischemia/reperfusion injury (IRI) can cause heart failure, which has been associated with pyroptosis, a pro-inflammatory programmed cell death. Small endogenous non-coding RNAs have been shown to be involved in myocardial IRI. In the present study, we aimed to investigate whether miR-424 modulated pyroptosis in response to myocardial IRI and determine its underlying regulatory mechanism. An in vivo mouse model of cardiac IRI was established, and contractile function was evaluated by echography. The serum and heart tissue were harvested 24 h after reperfusion to assess the status of pyroptosis. For the in vitro study, H9C2 cells (a rat heart cell line) were subjected to 6 h of hypoxia, followed by 18 h of reoxygenation. The gene expressions at the mRNA level were assessed by real-time PCR, and the expressions at the protein level were examined by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Bioinformatic analysis was applied to predict miR-424 targets, which were then confirmed by a luciferase reporter assay. We found that the expressions of pyroptosis-related proteins, including caspase-1, caspase-11, IL-1β, and IL-18, were significantly increased upon myocardial IRI. Similarly, hypoxia/reoxygenation injury (HRI) also induced pyroptosis in H9C2 cells. Furthermore, our study revealed that the miR-424 expression was substantially increased in I/R heart tissue and H/R-challenged H9C2 cells. In addition, we found that exogenous expression of miR-424 directly targeted cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and up-regulated the expressions of caspase-1 and the pro-inflammatory cytokines IL-1β and IL-18. Taken together, our findings provided a new signaling pathway of miR-424/CRISPLD2 in cardiac pyroptosis under IRI conditions.

Circular RNA expression profiles in synovial fluid: a promising new class of diagnostic biomarkers for osteoarthritis.

Abstract: Background Previous studies suggest that circRNAs abnormally function in the progression of osteoarthritis (OA). However, little is known about the diagnostic value of circRNAs in patients with OA. To assess potential applications of circRNAs as diagnostic tools in OA, expression profiles of circRNAs in synovial fluid from OA patients and healthy subjects were obtained. Methods Microarray analysis was performed to profile the expression of circRNAs in an unbiased manner. CircRNA expression in synovial fluid was identified by real-time quantitative polymerase chain reaction (RT-qPCR). The diagnostic value was evaluated using receiver operating characteristics (ROC) curves and the area under the ROC curves (AUC). Spearman correlation analysis was performed to assess the correlation of circRNAs and clinical parameters. Results We identified five circRNAs that were significantly elevated in synovial fluid from OA patients compared with those of the healthy controls. Among these five circRNAs, hsa_circ_0104873, hsa_circ_0104595, and hsa_circ_0101251 could effectively separate patients with OA from healthy controls with high AUC (0.683, 0.708 and 0.754, respectively). Furthermore, we found that three circRNAs were positively correlated with the degree of radiographic grading and symptomatic severity of OA patients. Conclusion This study suggests that increased expression of hsa_circ_0104873, hsa_circ_0104595, and hsa_circ_0101251 in synovial fluid from OA patients may serve as potential biomarkers for OA screening.

CircRNA circFNDC3B promotes esophageal cancer progression via cell proliferation, apoptosis, and migration regulation

Abstract: Background Esophageal cancer is one of the most common malignant tumors threatening human health worldwide. Circular RNAs (circRNAs) are a large group of covalently closed continuous loops that are prevalently expressed in human cells and might be applied as novel esophageal cancer biomarkers. Purpose To investigate the expression of a novel circular RNA, circFNDC3B, in esophageal cancer, as well as determine its function in the regulation of esophageal cell proliferation, apoptosis, migration, and invasion. Methods Quantitative RT-PCR using circular RNA-specific primers was performed to analyze the existence and expressional change of circFNDC3B in esophageal cancer tissues. The esophageal cancer cell lines ECA109 and KYSE150 with inhibited circFNDC3B expression by gene silencing were subjected to proliferation analysis with the MTS method, FITC Annexin V apoptosis detection, and a proliferation and invasion evaluation using a transwell system. Results circFNDC3B was specifically up-regulated in esophageal cancer tissues. Esophageal cancer cell lines ECA109 and KYSE150 with decreased circFNDC3B expression by gene silencing showed inhibited proliferation, increased apoptosis, and weakened migration and invasion abilities. Conclusion The circFNDC3B encoded by two FNDC3B gene exons is an important regulator of esophageal cancer progression.

MicroRNA-20a-5p inhibits epithelial to mesenchymal transition and invasion of endometrial cancer cells by targeting STAT3.

Abstract: Background Endometrial cancer (EC) is the fourth most common malignancy among women. Epithelial to mesenchymal transition (EMT) and invasion have been identified as central cellular processes in tumor progression and malignant transformation. The function and molecular basis of microRNA-20a-5p (miR-20a-5p) in the development of EC are poorly defined. Methods RT-qPCR assay was used to detect the levels of miR-20a-5p and signal transducer and activator of transcription 3 (STAT3) mRNA. Western blot assay was conducted to determine protein expression of STAT3, N-cadherin, Vimentin and E-cadherin. Cell invasive capacity was examined by transwell invasion assay. Bioinformatics analysis, luciferase reporter assay and RIP assay were performed to investigate the interaction between miR-20a-5p and STAT3 3'UTR. Results miR-20a-5p expression was strikingly reduced in EC tissues and cells. Functional analysis revealed that miR-20a-5p overexpression inhibited EMT and invasion of EC cells. Further exploration showed that miR-20a-5p inhibited STAT3 expression by direct interaction. Moreover, the knockdown of STAT3 suppressed EMT and invasion of EC cells. Additionally, the depletion of STAT3 weakened miR-20a-5p downregulation-induced cell EMT and invasion in EC. Conclusion miR-20a-5p inhibited EMT and invasion of EC cells by targeting STAT3, highlighting the vital roles of miR-20a-5p and STAT3 in the metastasis and malignant transformation of EC.

Detection of gastritis-associated pathogens by culturing of gastric juice and mucosa

Abstract: It is well known that Helicobacter pylori (H. pylori) is not the only indigenous bacterium in the stomach, as numerous studies have revealed that the gastric microbiota contributes to the pathogenesis of gastric disease. However, the correlation between the gastric bacterial flora and gastritis is unclear. By comparing differences in viable gastric bacteria between a gastritis group and a healthy group, we examined the potential species related to chronic gastritis. We collected juice and mucosa samples from 103 consecutive patients and identified 81 species by culturing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The positive rates of Streptococcus and Neisseria were markedly higher in the gastritis group than those in the normal group, suggesting that certain bacterial species may play vital roles in the development of gastritis rather than acting as transient microbes. This finding can be applied to the diagnosis and treatment of chronic gastritis as evidence supporting non-Helicobacter pylori infection-related gastritis.

SK-Hep1: not hepatocellular carcinoma cells but a cell model for liver sinusoidal endothelial cells.

Abstract: SK-Hep1 cells serve as a cell model of hepatocellular carcinoma and hepatocyte biology. However, SK-Hep1 cells are markedly different from normal hepatocytes and other hepatocellular carcinoma cells in their gene expression and protein levels. Furthermore, endothelial-specific makers and morphological characteristics are found in SK-Hep1 cells, indicating an endothelial origin. To confirm their cell phenotype, we investigated and compared the surface ultrastructure, endothelial function, and molecular markers of SK-Hep1 cells in vitro and in vivo. The results revealed that SK-Hep1 cells expressed endothelial-specific makers and exhibited the endothelial function of endocytosis and tubular formation. Capillary-like structures with CD31 expression were also observed in SK-Hep1 allografts in nude mice. Moreover, SK-Hep1 cells possessed fenestrae without diaphragms, consistent with liver sinusoidal endothelial cells, as seen by electron microscopy. In conclusion, SK-Hep1 cells would be better considered a cell model for liver sinusoidal endothelial cells instead of hepatocellular carcinoma cells.

Genome-wide DNA methylation and transcriptome changes in Mycobacterium tuberculosis with rifampicin and isoniazid resistance.

Abstract: We investigated the genome-wide DNA methylation and transcriptome changes in M. tuberculosis with rifampicin or isoniazid resistance. Single-molecule real-time (SMRT) sequencing and microarray technology were performed to expound DNA methylation profiles and differentially expressed genes in rifampicin or isoniazid resistant M. tuberculosis. Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis and methylated regulatory network analysis were conducted by online forecasting databases. Integrated analysis of DNA methylation and transcriptome revealed that 335 differentially methylated genes (175 hypermethylated and 160 hypomethylated) and 132 significant differentially expressed genes (68 up-regulated and 63 down-regulated) were found to be regulated by both rifampicin and isoniazid in M. tuberculosis H37Rv. Correlation analysis showed that differential methylated genes were negatively correlated with their transcriptional levels in rifampicin or isoniazid resistant strains. KEGG pathway analysis indicated that nitrogen metabolism pathway is closely related to differentially methylated genes induced by rifampicin and isoniazid. KEGG also suggested that differentially expressed genes in rifampicin or isoniazid-resistant strains may play different roles in regulating signal transduction events. Furthermore, five differentially methylated candidate genes (Rv0840c, Rv2243, Rv0644c, Rv2386c and Rv1130) in rifampicin resistant strains and three genes (Rv0405, Rv0252 and Rv0908) in isoniazid-resistant strains were verified the existence of protein-protein interaction in STRING database. Integrated DNA methylation and transcriptome analyses provide an epigenetic overview of rifampicin and isoniazid-induced antibiotic resistance in M. tuberculosis H37Rv. Several interesting genes and regulatory pathways may provide valuable resources for epigenetic studies in M. tuberculosis antibiotic resistance.

Circulating circular RNAs hsa_circ_0001204 and hsa_circ_0001747 act as diagnostic biomarkers for active tuberculosis detection.

Abstract: In recent years, increasing evidence has suggested that circRNAs can serve as novel diagnostic markers for many diseases. However, little is known about the value of circRNAs in the diagnosis of active tuberculosis (TB). In this study, 10 circRNAs which we previously found to be involved in Mycobacterium tuberculosis infection were selected as candidate targets for subsequent circulating circRNA assay. Compared with healthy controls, plasma levels of hsa_circ_0001204 and hsa_circ_0001747 were significantly decreased (P < 0.001). Plasma levels of hsa_circ_0001204 and hsa_circ_0001747 were correlated with TB severity. Hsa_circ_0001204 and hsa_circ_0001747 were selected for further analysis in another 145 TB patients and 120 control individuals. The area under the receiver operating characteristic curve (AUC) for distinguishing TB patients was 0.928 (95% confidence interval: 0.897-0.960; sensitivity = 86.21%, specificity = 89.17%) when hsa_circ_0001204 and hsa_circ_0001747 were used in combination. Further evaluation on potential biomarkers showed that hsa_circ_0001204 and hsa_circ_0001747 may specifically identify patients with TB. Additionally, hsa_circ_0001204 and hsa_circ_0001747 plasma levels after treatment were significantly higher than that pre-treatment (P < 0.001). Our present study indicates that circulating hsa_circ_0001204 and hsa_circ_0001747 may represent novel plasma biomarkers for TB diagnosis.

Comparative immunoprofiling of polymyositis and dermatomyositis muscles.

Abstract: The morphological, immunohistochemical, and immunopathological analyses of muscle biopsy are essential for the diagnosis of idiopathic inflammatory myopathies (IIMs). However, they are also one of the most common causes of misdiagnosis. Although several diagnostic criteria have been proposed for the diagnosis of IIMs, misdiagnosis still remains common in clinical practice. The present study aims to characterize the inflammatory profile of IIMs, including the expression of MHC-I, MHC-II, MAC and infiltrating cells. We also investigated the sensitivity and specificity of MHC-I and MHC-II immunostaining for the diagnosis of IIMs. We found that the expression of MHC-I and MHC-II was both higher in IIMs than in non-inflammatory myopathies (NIMs). The distribution of MHC-I in IIMs is different from that of MHC-II. MHC-I is mainly located in the sarcoplasms, while MHC-II is located mostly on the sarcolemmas. Moreover, our findings suggest that MAC may be a potential marker to diagnose DM, and the combination of MHC-I and MHC-II immunostaining results in a higher sensitivity and specificity for IIM diagnosis, especially for DM. In addition, infiltrating cells in PM were mainly CD8+ cells, but we found in DM and NIMs they were primarily CD4+ cells, which is consistent with previous studies. Lastly, glucocorticoid treatment and disease duration have little effect on the MHC-I and MHC-II expression pattern. Our findings indicate that the immunostaining of inflammatory markers such as MHC-I, MHC-II, CD4, CD8, CD303 and MAC are of diagnostic value for IIMs regardless of the immunosuppression regime and disease duration.

miR-181a-5p is downregulated and inhibits proliferation and the cell cycle in prostate cancer

Abstract: Prostate cancer (PCa) is one of the most common cancers in men worldwide. However, the detailed molecular mechanisms underlying PCa tumorigenesis and progression remain largely unclear. MicroRNAs are key regulators of gene post-transcriptional expression in human cancer. In this study, we used public datasets, including {"type":"entrez-geo","attrs":{"text":"GSE21036","term_id":"21036"}}GSE21036, {"type":"entrez-geo","attrs":{"text":"GSE14857","term_id":"14857"}}GSE14857 and {"type":"entrez-geo","attrs":{"text":"GSE45604","term_id":"45604"}}GSE45604 to analyze the expression of miR-181a in PCa. We also explored the potential role of miR-181a by using bioinformatics analysis and gain of function assay. miR-181a was down-regulated in PCa. Bioinformatics analysis revealed miR-181a was significantly involved in regulating cell metabolic process and gene expression. Of note, gain of function assay results showed overexpression of miR-181a could significantly inhibit cell proliferation by inducing G1 cell cycle arrest. Our results suggest miR-181a-5p may be adiagnostic and therapeutic biomarker for prostate cancer.

Overexpression of miR-153 promotes oxidative stress in MPP+-induced PD model by negatively regulating the Nrf2/HO-1 signaling pathway.

Abstract: Increasing evidence points to oxidative stress as a chief mediator of Parkinson's disease (PD) characterized by progressive loss of dopamine neurons in the pars compacta of the substantia nigra. At present, microRNAs (miRNAs) have been recognized as important regulators in oxidative stress. Furthermore, miRNAs were also involved in the neuropathology of neurodegenerative disorders, including PD. In this study, we aimed to explore the influences of miR-153 and Nrf2 in oxidative stress during the development of PD. It was found that the expression of miR-13 and Nrf2 detected by qRT-PCR were significantly increased and decreased, respectively, in serum of PD patients and MPP+-induced SH-SY5Y cells. The target relationship between miR-153 and Nrf2 was determined by dual-luciferase assay. Moreover, after transfecting with miR-153 mimics and inhibitor, the expressions of Nrf2 in mRNA and protein were down-regulated and up-regulated, respectively. The indexes of oxidative stress were examined by biochemical methods. The data revealed that miR-153 could facilitate oxidative stress by negatively regulating Nrf2 in MPP+-treated SH-SY5Y cells. Finally, it was observed that miR-153 could suppress the Nrf2/HO-1 signaling pathway in MPP+-treated SH-SY5Y cells. Therefore, these findings indicated that overexpression of miR-153 could promote oxidative stress in PD by targeting the Nrf2/HO-1 signaling pathway, possibly providing a new way to treat PD.

Curcumin suppresses gastric cancer biological activity by regulation of miRNA-21: an in vitro study.

Abstract: Objective: The aim of this study was to explain the effects of curcumin to depress gastric cancer biological activity by regulation miRNA-21 an in in vitro study. Methods: Collecting 30 pairs of adjacent and cancer tissues to measure miRNA-21 expression by ISH, evaluating pathology by H&E staining and measuring PTEN protein expression by IHC. Evaluating curcumin anti-tumor and correlation between curcumin and miRNA-21 in gastric cancer cell line (AGS) biological activities by CCK-8, flow cytometry, transwell, scratch test, transmission electron microscope, and western blot. Results: Compared with adjacent normal tissues, the miRNA-21 and PTEN expressions of gastric cancer tissues were significantly different (P < 0.001, respectively). By cell experiments, compared with NC group, the AGS cell proliferation was significantly depressed with significantly increasing cell apoptosis by keeping cell cycle in G1 phase (P < 0.001, respectively), and AGS cell invasion and migration were significantly down-regulated (P < 0.001, respectively) in Cur and Cur+BL groups. However, with miRNA-21 supplementation, the AGS cell biological activities were significantly recovered (P < 0.001, respectively). By western blot, compared with the NC group, the PTEN and P21 proteins expressions were significantly up-regulated (P < 0.001, respectively) and the PI3K, AKT, MMP-2 and MMP-9 proteins expressions were significantly down-regulated (P < 0.001, respectively). PTEN, PI3K, AKT, P21, MMP-2 and MMP-9 proteins were significantly decreased with miRNA-21 supplementation (P < 0.001, respectively). Conclusion: Curcumin had anti-tumor effects to gastric cancer via ion of miRNA-21 by regulation of the PTEN/PI3K/AKT pathway.

Expression of Tim-3 in breast cancer tissue promotes tumor progression

Abstract: T-cell immunoglobulin mucin-3 (Tim-3) plays a pivotal role in immune regulation and tolerance induction as a negative regulatory molecule on innate versus adaptive immune cells, especially in antitumor immunity. However, the mechanism of Tim-3 expression on tumor cells and the mechanism that inhibits anti-tumor immunity are obscure. In this present study, we aimed to investigate the functions of Tim-3 in breast cancer and to explore its correlation to tumor prognosis. In a total of 42 clinical samples of invasive breast cancer, Tim-3 was semiquantitatively scored based on both distribution and intensity of immunohistochemistry staining and was found to correlate with clinicopathological parameters. Western blotting was used to detect the expression of Tim-3 in breast cancer cells. Furthermore, the effect of Tim-3 in breast cancer cells was evaluated after overexpression by ADV-Tim-3 and downregulation by Tim-3-siRNA. High immunoreactivity of Tim-3 was found to be significantly correlated with clinical stage, metastasis, KI67, and a lower 5-year survival rate. We supported this finding by confirming the presence of Tim-3 protein in the breast cell lines. Downregulation of Tim-3 significantly inhibited the proliferation, migration, and invasion of breast cancer cells while it promoted apoptosis. Overexpression of Tim-3 promoted the proliferation, migration, and invasion of breast cancer cells and inhibited apoptosis. Taken together, as a valuable marker of breast cancer prognosis, Tim-3 in breast cancer cells play an important role in the progression of breast cancer and may be an effective novel target in tumor prevention and treatment.

MicroRNA-129-3p functions as a tumor suppressor in serous ovarian cancer by targeting BZW1.

Abstract: The aberrant expression of microRNAs (miRNAs) underlies a series of human diseases, including ovarian cancers. In our previous study, we found that miR-129-1-3p and miR-129-2-3p levels were significantly decreased in serous ovarian cancer via a microarray and quantitative PCR. In this study, we investigated the pathological role of miR-129-3p in an ovarian cancer cell line, SKOV3 cells. The results demonstrated that miR-129-3p overexpression distinctively inhibited the proliferation, migration, and invasion of ovarian cancer cells. The regulator of cell cycling, BZW1 (basic leucine zipper and W2 domains 1), was validated as a novel direct target of miR-129-3p. Specifically, miR-129-3p bound directly to the 3' untranslated region of BZW1 and suppressed its expression. Our results indicate that miR-129-3p serves as a tumor suppressor by targeting BZW1 in ovarian cancer cells and highlight that the restoration of miR-129-3p might be a novel therapeutic strategy for ovarian cancer.

MMP2 is associated with glioma malignancy and patient outcome.

Abstract: Gliomas are fast growing and usually manifest in an aggressive infiltrative model. MMP2 overexpression is associated with brain tumor malignancy and metastasis formation. The aim of this study was to investigate the influence of MMP2 on glioma formation and clinical outcomes by performing analysis at the DNA, RNA, and protein levels. Methylation status and mRNA level were evaluated in 162 samples; the MMP2 protein level was analyzed in 28 patient preoperative and postoperative blood samples using protein microarray analysis and conventional ELISA. The MMP2 MSP analysis revealed a gradually increasing gene promoter demethylation frequency, and the Kaplan-Meier analysis showed that the methylated gene promoter is related to longer overall survival (Log-rank test X 2 = 12.508, df = 1, P < 0.0001). Relative mRNA expression was significantly downregulated when the promoter was methylated. Pairwise comparison analysis showed statistically significant (Mann-Whitney test, P < 0.05) differences in the MMP2 expression median when comparing different glioma grades. The Kaplan-Meier analysis revealed that low MMP2 expression was associated with better survival (Log-rank test X 2 = 7.732, df = 1, P = 0.005). At the protein level, MMP2 expression in patient sera showed no differences between malignancy grades and patient preoperative and postoperative states, while the ELISA assay showed the tendency of accumulating MMP2 protein in higher malignancy patient sera samples. The Kaplan-Meier analysis showed the tendency of having a shorter survival time with a higher MMP2 protein level in patient sera. MMP2 has a significant role in glioma pathogenesis and could be used as a potential molecular marker for tumor progression.

Serum miR-10a-5p and miR-196a-5p as non-invasive biomarkers in non-small cell lung cancer.

Abstract: Background Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 85% of all cases. MicroRNAs are stable molecules in the blood and can be used as biomarkers for early diagnosis of various malignancies. The aim of this study was to evaluate expression of miR-10a-5p and miR-196a-5p in tissue and serum of patients with NSCLC and to explore its relationship with clinicopathological characteristics. Methods A total of 20 pairs of tissues and 80 serum samples were obtained from NSCLC patients. Seventy-five serum samples from healthy individuals of the same age and gender were also collected. The expression level of miR-10a-5p and miR-196a-5p was detected by quantitative real-time PCR. The relationship between miR-10a-5p and miR-196a-5p expression level in NSCLC tissues and serum and clinicopathological characteristics was estimated respectively. The diagnostic value of miRNA-10a-5p and miR-196a-5p in NSCLC was assessed by the Receiver-operating characteristic (ROC) curve method. Results We found that miRNA-10a-5p and miR-196a-5p expression levels were increased significantly in NSCLC tissues compared with non-tumor adjacent normal tissues. Serum miR-10a-5p and miR-196-5p were over-expressed in NSCLC patients compared with healthy controls. The higher miR-10a-5p or miR-196-5p expression levels were positively correlated with advanced tumor stage and positive lymph node metastasis. The area under the curve (AUC) of serum miR-10a-5p and miR-196-5p to diagnose NSCLC were 0.709 and 0.785. Optimal sensitivity and specificity were 65.98% and 72.71%, 67.86% and 77.57%, respectively in differentiating NSCLC patients from healthy controls. The combination of these two miRNAs with carcinoembryonic antigen (CEA) further increased the diagnostic value, with an area under the curve (AUC) of 0.801 (sensitivity, 76.34%; specificity, 79.26%) using logistic regression model analysis. Conclusions Serum miR-10a-5p and miR-196a-5p may be useful noninvasive biomarkers for the clinical diagnosis of NSCLC.

BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism.

Abstract: Branched-chain amino acid aminotransferase 1 (BCAT1) enzyme is an aminotransferase of glutamate and branched-chain amino acids (BCAAs), which is required for survival of various cancers. However, the role of BCAT1 in human endometrial cancer (EC) remains unknown. We analyzed the expression of BCAT1 in endometrial lesions using IHC. After BCAT1 gene knockdown and activity inhibition, cell proliferation, apoptosis, and metabolism were detected using CCK-8 assay, flow cytometry, and LC-MS/MS analysis. We analyzed molecular signature characteristics to understand how BCAT1 promotes cell proliferation. In this study, we demonstrated a significant increase in BCAT1 expression from normal endometrium to atypical endometrial hyperplasia (AEH) and then to EC, and the expression of BCAT1 in EC samples was related to tumor grade, FIGO stage and lymph node metastasis. Next, cell proliferation was markedly inhibited by lentiviral BCAT1 knockdown or Gbp treatment, but this had little effect on apoptosis rate. Further, BCAT1 knockdown resulted in 31.2% and 33.3% decreases in the amount of intracellular isoleucine and leucine produced, respectively, relative to a control. BCAT1 knockdown or activity inhibition resulted in a decrease of pS6K, a downstream target kinase of mTORC1. In conclusion, our study showed that BCAT1 is essential for EC progression and to increase EC cell proliferation through the production of BCAAs to activate the mTORC1 pathway, providing ideas for clinicians to identify metabolism-based targeted approaches for patients with EC.

Decreased expression of TIGIT in NK cells correlates negatively with disease activity in systemic lupus erythematosus.

Abstract: It is well-known that decreased levels of NK cells are found in patients with systemic lupus erythematosus (SLE). However, the mechanism of deregulation of NK cells in SLE is largely unknown. In this study, expression of T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT) on NK cells was determined by flow cytometry and correlation with markers of autoimmune response, inflammation, disease activity and severity of SLE was further analyzed. Moreover, the function of TIGIT on NK cells in SLE was investigated. We have found that the frequency of TIGIT-expressing NK cells was significantly decreased in SLE patients. The frequency of TIGIT-expressing NK cells in patients with SLE was decreased significantly in subjects with low complement, positive anti-ribosomal RNP (anti-rRNP), and high SLE Disease Activity Index (SLEDAI) score. Furthermore, the frequency of TIGIT-expressing NK cells was significantly increased in SLE patients after regular treatment. In addition, the activation marker CD69, degranulation marker CD107a and cytokine IFN-γ production potential of TIGIT+ NK cells were significantly lower than those of TIGIT- NK cells. Blocking the TIGIT pathway by functional anti-TIGIT monoclonal antibody restored IFN-γ secretion of NK cells. In conclusion, TIGIT expression was significantly decreased on NK cells in patients with SLE and correlated negatively with disease activity and severity of SLE. Additionally, the functional potential of TIGIT+ NK cells was significantly decreased compared with TIGIT- NK cells. This study reveals that TIGIT is a powerful negative regulator of NK cells in SLE.

Long non-coding RNA MEG3 mediates high glucose-induced endothelial cell dysfunction.

Abstract: Long noncoding RNAs (lncRNAs) are implicated in the progression of diabetes mellitus (DM) and diabetes-induced endothelial dysfunction. Maternally expressed gene 3 (MEG3) encodes an lncRNA which is suggested to function as a tumor suppressor. Therefore, the aim of the present study was to investigate whether MEG3 is a potential regulator and molecular biomarker of high glucose-induced endothelial dysfunction. LncRNA Meg3-specific small interfering RNA (siRNA) and scrambled (Scr) siRNA were transfected for MEG3 dysfunction studies. RNA and protein expression were examined by quantitative RT-PCR (qPCR) and Western blot, respectively. The percentage of apoptotic cells was measured by flow cytometry. Cell viability was determined through MTT assay. This study demonstrates involvement of lncRNA MEG3 in high glucose-induced endothelial dysfunction. MEG3 is significantly downregulated in an endothelial cell model of hyperglycemia. In addition, MEG3 knockdown could exacerbate inflammatory damage in endothelial cells. Interestingly, MEG3 knockdown in HUVECs significantly induced proliferation and inhibited apoptosis by upregulating Bcl-2 and downregulating Bax, caspase-3, and P53. It should be noted that MEG3 knockdown could activate the TGF-β signaling pathway via upregulating TGF-β1, SMAD2, and SMAD7 and activate the Wnt/β-catenin signaling pathway via upregulating β-catenin and Cyclin D1 and downregulating TCF7L2. Our results indicate that MEG3 can be regarded as a novel therapeutic target and molecular biomarker for high glucose-induced endothelial dysfunction.

Up-regulation of Wnt7b rather than Wnt1, Wnt7a, and Wnt9a indicates poor prognosis in breast cancer.

Abstract: Aberrant activation of Wnt/β-catenin signaling is one of the most frequent abnormalities in human cancer, including breast cancer. The prognostic value of Wnt ligands has never been fully characterized. In this study, we focused on four Wnt ligands, namely Wnt1, Wnt7a, Wnt7b and Wnt9a, which were commonly studied and found pivotal in Wnt/β-catenin signaling, but seldom explored for their prognostic value. We investigated the expression of Wnt1, Wnt7a, Wnt7b and Wnt9a in breast cancer tissues by using real-time PCR and immunohistochemical analysis, and further identified their prognostic significance. Results demonstrated that only Wnt7b expression level in breast cancer was significantly higher than that of benign breast. Spearman rank-correlation analysis revealed the expression level of Wnt1, Wnt7b and Wnt9a, but not Wnt7a, were all significantly associated with positive lymph nodes. The Kaplan-Meier survival curve demonstrated that patients with high Wnt7b expression had a shorter overall survival (OS) and recurrence-free survival (RFS) than those with low Wnt7b expression. Moreover, the univariate and multivariate analysis revealed that Wnt7b expression was an independent prognostic factor for both OS and RFS of breast cancer patients. In addition, the high expression of Wnt7b in breast cancer and its prognostic role were further validated by GENT (Gene Expression database of Normal and Tumor tissues) database and the Kaplan-Meier plotter database. Taken together, we identified that high expression of Wnt7b, rather than Wnt1, Wnt7a and Wnt9a, may serve as a prognostic biomarker for breast cancer.

Fibroblast growth factor 21 attenuates inflammation and oxidative stress in atherosclerotic rat via enhancing the Nrf1-ARE signaling pathway.

Abstract: Inflammation and oxidative stress are associated with atherosclerotic progression. Fibroblast growth factor 21 (FGF21), a regulator of energy metabolism, has been reported to suppress the pathogenesis of atherosclerosis. However, the mechanism of anti-atherosclerotic effects of FGF21 remains unclear and needs to be further investigated. Transcription factor NF-E2-related 2 (Nrf2), a sensitive regulator of oxidative stress, is also associated with atherosclerotic progression. In this study, we investigated whether up-regulation of FGF21 affected inflammation and oxidative stress in atherosclerotic rats and whether the Nrf2-signaling pathway was involved in FGF21-mediated effects. Pathological changes were detected in arterial tissues of rats, and the expression of inflammatory and oxidative stress indicators, vascular endothelial markers, and Nrf2-signaling related protein were measured in the serum or/and arterial tissues of rats. As a result, expression of FGF21 and Nrf2-ARE signaling related proteins were markedly suppressed in arterial tissues of model rats. Thickness of endarteria and infiltrating cells obviously increased in atherosclerotic rats, whereas the increase of FGF21 expression could decrease thickness of endarteria. Moreover, the levels of ET-1, MDA, MCP-1, ICAM-1 and VCAM-1 were significantly higher in model rats than that in normal rats, whereas the levels of NO, GSH and T-AOC were significantly lower. Compared with model rats, up-regulation of FGF21 could increase the expression of Nrf2-ARE signaling related proteins and the level of anti-oxidative indicators, decrease the levels of endothelial dysfunction, and reduce inflammatory indicators. Down-regulation of FGF21 could reverse these actions. Therefore FGF21 reduces inflammation and oxidative stress in atherosclerotic rats via Nrf2-ARE signaling pathway.