Differential expression of cyclic RNA in peripheral blood mononuclear cells of patients with rheumatoid arthritis / 中华风湿病学杂志

Objective:To investigate the differences in the expression profiles of cyclic RNA (circRNA) in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) and its clinical significance.Methods:Venous blood were collected from 4 patients with RA (group T) and 4 healthy subjects (group C). The expression profiles of circRNA in PBMCs of the two groups were detected by Arraystar circRNA microarray, and the differentially expressed circRNA was analyzed by clustering analysis. The binding sites for interaction between differentially expressed circRNA and miRNA were predicted, and functional analysis such as geneontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed. quantitative real-time polymerase chain reaction (RT-qPCR) was used to verify the expression of partially differentially expressed circRNA in the two groups of PBMCs, and a circRNA-miRNA-mRNA regulatory network (ceRNA network) was constructed for the target circRNA with significantly differential expression. A receiver operating characteristic curve [receiver operating characteristic curve (ROC)] was established to analyze the potential diagnostic value of target circRNA. SPSS Statistics 23.0 and Graphpad Prism 8.0 were used to analyze the data, and the independent t test was used to analyze the difference between the two groups. Results:① Microarray results showed that, compared with group C, a total of 399 [fold of difference (FC)>1.5, and P<0.05] circRNA were abnormally expressed in PBMCs of group T; including 149 up-regulated and 250 down-regulated. ② Bioinformatics analysis: The prediction of the binding site of circRNA and miRNA suggested that the differentially expressed circRNA in RA might affect the inflammatory response by targeting miR-140-5p, miR-338-5p, and miR-9-5p. GO analysis showed that the differentially expressed circRNA was mainly involved in the intimal-binding organelles, protein metabolism and binding, etc. KEGG pathway analysis showed that most of the involved pathways were related to infection and human immune dysregulation. ③ The results of multi-sample RT-qPCR validation showed that the expression level of hsa_circRNA_009012 in group T was significantly higher than that in group C ( t=-4.417, P<0.01), the expression level of hsa_circRNA_101328 was significantly lower than that in group C ( t=-1.042, P<0.01), and the expression of hsa_circRNA_058230 had no significant change ( t=4.691, P>0.05). ④ ROC curve analysis indicated that hsa_circRNA_009012 had potential value in the diagnosis of RA [area under curve=0.96]. Conclusion:The expression of circRNA in PBMCs of patients with RA is imbalanced, and it may participate in the regulation of the development of RA. Among them, hsa_circRNA_009012 is expected to become a new biological marker for the diagnosis and treatment of RA.

Role of EZH2 Inhibitor Combined with Gefitinib in EGFR-TKIs Resistant Lung Cancer Cells / 中国肺癌杂志

BACKGROUND@#Lung cancer is one of the common malignant tumors that impair human health. With the development of epigenetics, the researchers found that enhancer of Zeste homolog 2 (EZH2) is highly expressed in lung cancer tissue and its expression is closely related to the prognosis. EZH2 inhibitor can also enhance the sensitivity of tumor cells to a variety of anti-tumor drugs. The purpose of this study is to investigate the effect of combination of EZH2 inhibitor and gefitinib on the proliferation, apoptosis and migration of Gefitinib-resistant lung cancer cells.@*METHODS@#PC9 and PC9/AB2 cells were used for this study. CCK-8 and EdU experiment were used to detect combined treatment on cell viability and proliferation activity; Wound healing assay and Transwell chamber experiment were used to determine the effects of combination therapy on cell migration ability; Flow cytometry was used to detect the effect of combination therapy on EZH2 and apoptosis; Western blot was used to observe the effect of combination therapy on epidermal growth factor receptor (EGFR) signaling pathway-related proteins expression.@*RESULTS@#In gefitinib-resistant cell line PC9/AB2, gefitinib combined with EZH2 inhibitor GSK343 can significantly inhibit cell viability, reduce cell migration and increase cell apoptosis. At the same time, combination therapy can significantly inhibit the expression of EZH2 and phosphorylation EGFR proteins.@*CONCLUSIONS@#The combination of EZH2 inhibitor GSK343 and gefitinib sensitize PC9/AB2 cell to gefitinib response. This study also suggests that synergistic therapy plays a role in the reversal of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) resistance in lung cancer.

Effect of Apatinib on Invasion and Migration of Lung Cancer Cells and Its Mechanism / 中国肺癌杂志

BACKGROUND@#Lung cancer is one of the most deadly cancers in the world for human. In recent years, the effect of targeted therapy has become increasingly significant. Apatinib is a multi-target anti-tumor drug that is currently under study. The purpose of this study is to investigate the effects of Apatinib on the biological characteristics of lung cancer cells and its possible mechanism.@*METHODS@#Lung cancer cell lines H1299 and H3255 were cultured in vitro. The effects of Apatinib on proliferation, migration and invasion of H1299 and H3255 cells were detected by cell proliferation assays wound healing assays and Transwell assays. The protein expression related to cancer angiogenesis and invasion was detected by Western blot.@*RESULTS@#Apatinib significantly inhibited the proliferation, migration and invasion of H1299 and H3255 in a concentration-dependent manner. Western blot showed that with the increasing of drug concentration, VEGF, VEGFR2, N-cadherin, MMP9, MMP2 and Vimentin were down-regulated, and E-cadherin were up-regulated.@*CONCLUSIONS@#Apatinib can inhibit the invasion and migration of lung adenocarcinoma cells H1299 and H3255. By regulation of epithelial-mesenchymal transition and the expression of matrix metalloproteinase-related proteins.

Study on the Difference of Gene Expression between Central and Peripheral Lung Squamous Cell Carcinoma Based on TCGA Database / 中国肺癌杂志

BACKGROUND@#Lung cancer is a malignant tumor disease with high morbidity and high mortality. The non-small cell lung cancer (NSCLC) is the most common type, among them, lung squamous cell carcinoma own special pathological type and specific treatment, is a subtype of non-small cell lung cancer and can be divided into peripheral type and central type according to clinical phenotype. This study explores the differences in gene levels and their potential values based on clinical differences between central and peripheral in lung squamous cell carcinoma.@*METHODS@#The lung squamous cell carcinoma dataset was collected from The Cancer Genome Atlas (TCGA) database, clinical information and the corresponding gene expression profiles were downloaded. Then we further sort and analyze all these data.@*RESULTS@#In clinical characteristics analysis, result showed that central lung squamous cell carcinoma was more likely to metastasis with lymph node than peripheral lung squamous cell carcinoma (46.2%, 67/145 vs 28.9%, 26/90; P=0.019), while there were no significant differences in gender, age, tumor size, distant metastasis, tumor node metastasis (TNM) stage, and EGFR mutation. Gene expression analysis showed 1,031 differentially expressed genes between central and peripheral lung squamous cell carcinoma, of which 629 genes were up-regulated and 402 genes were down-regulated (peripheral vs central). Further enrichment analysis showed differentially expressed genes were mainly riched in 6 signaling pathways. Among them, the neuroactive ligand-receptor interaction pathway was the main enrichment pathway of differentially expressed genes, and other differential expressed genes were mainly involved in lipid metabolism and glucose metabolism. The analysis of interaction network showed that hepatocyte nuclear factor 1 homeobox A (HNF1A) and cytochrome p450 family, Cytochrome P450 3A4 (CYP3A4) own widely effect in up-regulated genes, while ALB and APOA1 at the key positions of the network in down-regulated genes were CONCLUSIONS: Central and peripheral lung squamous cell carcinoma showed clinical phenotype difference not only reflected in the incidence of lymph node metastasis, but also in gene expression profiles. Among them, HNF1A, CYP3A4, ALB, APOA1 at the key position of the differential gene interaction network and maybe as regulatory factors in the phenotypic difference.

The clinical expression and significance of inhibitory receptor TIGIT gene on peripheral NK cells in rheumatoid arthritis / 中华检验医学杂志

Objective@#To investigate the expression of inhibitory receptor TIGIT gene in peripheral NK cells of patients with rheumatoid arthritis (RA) and its clinical significance.@*Methods@#A case control study was conducted of 58 RA patients(30 patients with active RA disease, 28 patients with remission of RA) and 22 healthy controls (HC) in the department of rheumatology and immunology from Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine during December 2018 to June 2019, the related clinical data were collected. Flow cytometry was used to compare the expression of TIGIT gene on peripheral NK cells. The IFN-γ secretion level of cytokines in peripheral blood was detected by ELISA, and analyzed the correlations between TIGIT gene and disease activity and IFN-γ level. T-test or non-parametric test was used for comparison between the two groups, and Pearson correlation analysis was used for correlation between the two variables.@*Results@#Compared with HC group, the number of NK cells in RA disease group was reduced, which was (13.88±4.56) ×107 cells/L in RA disease group and (25.69± 2.48) ×107 cells/L in HC group (t=-2.036, P=0.041). The percentage of TIGIT gene expression in peripheral blood NK cells was not statistically different between RA disease group (39.73±9.37)% and HC group (45.64±9.91)% (t=-1.241, P=0.218). However, the average fluorescence intensity (MFI) of TIGIT gene expression was decreased, which was (7.21±2.03) in RA group and (9.01±3.29) in HC group (t=-2.947, P=0.004).MFI of TIGIT gene in NK cells of the disease active subgroup was (6.72±2.01), lower than that of the disease remission subgroup (8.75±2.64), (t=-3.316, P=0.002), and MFI of TIGIT gene in NK cells of the disease active subgroup was negatively correlated with DAS28 score (r2=0.649 6, P<0.000 1).The secretion level of IFN-γ cytokine in the RA disease group was (67.13±14.84) pg/ml, higher than that in the HC group (57.21±14.23) pg/ml (t=2.757, P=0.017), and the secretion level of IFN-γ cytokine in the disease active subgroup was negatively correlated with the MFI of TIGIT gene on NK cells (r2=0.662 2, P<0.000 1). Experimental results of peripheral blood mononuclear cell stimulation in the RA disease activity subgroup showed that the secretion level of cytokines IFN-γ was reduced after stimulation compared with that before stimulation (t=11.38, P<0.000 1).@*Conclusion@#The abnormal expression of TIGIT gene on peripheral NK cells are observed in patients with RA, which correlate with disease activity and IFN-γ secretion level.

The clinical expression and significance of inhibitory receptor TIGIT gene on peripheral NK cells in rheumatoid arthritis / 中华检验医学杂志

Objective To investigate the expression of inhibitory receptor TIGIT gene in peripheral NK cells of patients with rheumatoid arthritis (RA) and its clinical significance. Methods A case control study was conducted of 58 RA patients(30 patients with active RA disease, 28 patients with remission of RA) and 22 healthy controls (HC) in the department of rheumatology and immunology from Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine during December 2018 to June 2019, the related clinical data were collected. Flow cytometry was used to compare the expression of TIGIT gene on peripheral NK cells. The IFN-γ secretion level of cytokines in peripheral blood was detected by ELISA, and analyzed the correlations between TIGIT gene and disease activity and IFN-γ level. T-test or non-parametric test was used for comparison between the two groups, and Pearson correlation analysis was used for correlation between the two variables. Results Compared with HC group, the number of NK cells in RA disease group was reduced, which was (13.88 ± 4.56) × 107 cells/L in RA disease group and (25.69 ±2.48) ×107 cells/L in HC group (t=-2.036, P=0.041). The percentage of TIGIT gene expression in peripheral blood NK cells was not statistically different between RA disease group (39.73 ± 9.37) ％ and HC group (45.64 ± 9.91) ％ (t=-1.241, P=0.218). However, the average fluorescence intensity (MFI) of TIGIT gene expression was decreased, which was (7.21±2.03) in RA group and (9.01±3.29) in HC group (t=-2.947,P=0.004).MFI of TIGIT gene in NK cells of the disease active subgroup was (6.72±2.01), lower than that of the disease remission subgroup (8.75 ± 2.64), (t=-3.316, P=0.002), and MFI of TIGIT gene in NK cells of the disease active subgroup was negatively correlated with DAS28 score (r2=0.6496, P<0.0001).The secretion level of IFN-γcytokine in the RA disease group was (67.13±14.84) pg/ml, higher than that in the HC group (57.21 ± 14.23) pg/ml (t=2.757, P=0.017), and the secretion level of IFN-γ cytokine in the disease active subgroup was negatively correlated with the MFI of TIGIT gene on NK cells (r2=0.6622, P<0.0001). Experimental results of peripheral blood mononuclear cell stimulation in the RA disease activity subgroup showed that the secretion level of cytokines IFN-γwas reduced after stimulation compared with that before stimulation (t=11.38, P<0.0001). Conclusion The abnormal expression of TIGIT gene on peripheral NK cells are observed in patients with RA, which correlate with disease activity and IFN-γsecretion level.

Role of PD 0332991 on the Proliferation and Apoptosis of Vascular Endothelial Cells / 中国肺癌杂志

BACKGROUND@#Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells.@*METHODS@#EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991.@*RESULTS@#PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells.@*CONCLUSIONS@#PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.

Expression and Clinical Significance of LC-3 and P62 in Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#LC-3 and P62, two of important autophagy-related proteins, were reported highly expressed in many kinds of human malignancies and associated with outcomes of the patients. The purpose of this study was to investigate the expression status of LC-3 and P62 in non-small cell lung cancer patients and define the clinical-pathologic features.@*METHODS@#66 cases of non-small cell lung cancer patients were employed. The expression of LC-3 and P62 were detected by immunohistochemistry.@*RESULTS@#LC-3 was positive stained in 27 out of 66 cases (40.9%) and P62 was positive stained in 43 out of 66 cases (65.2%). LC-3 positive staining was more frequently in squamous cell carcinoma patients (P<0.05); while P62 positive staining was more frequently in late-stage adenocarcinoma patients with metastasis (P<0.05). There was a significant negative correlation between LC-3 and P62 expressions in non-small cell lung cancer tissues (rs=-0.065, P<0.001). Kaplan-Meier analysis showed that patients with positive LC-3 expression had favorable clinical outcomes compared with the patients with negative LC-3 expression (P<0.05).@*CONCLUSIONS@#LC-3 and P62 showed abnormal expression in non-small cell lung cancer tissues, suggesting that autophagy is involved in the occurrence and development of NSCLC.

The roles of high-resolution computer tomography and bronchoalveolar lavage in the diagnosis of connective tissue diseases associated with interstitial lung disease / 中国综合临床

Objective To evaluate the roles of high-resolution computer tomography (HRCT) and bron- choalveolar lavage (BALF) in the diagnosis of connective tissue diseases associated with interstitial lung disease (CTD-ILD). Methods Clinical data of chest HRCT and BALF of patients with CTD-ILD from January 1997 to December 2007 in in-patient department of Peking University First Hospital, were retrospectively analyzed. Results ①Among 46 cases with the picture of chest HRCT, 19 (41.3%)showed usual interstitial pneumonia(UIP) -like pattern and 18 (39.1%) showed lobular and interlobular septa thickening. 8 (8/17) of ANCA vasculitis (AASV) and 5 (5/9) of rheumatoid arthritis (RA) manifested as UIP-like patterns respectively. In polymyositis/dermatomyositis(PM/DM) and Sjogren's syndrome (KS) patients, the organizing pneumonia(OP)-like pattern and lymphocytic interstitial pneumonia(LIP)-like pattern were 2/4 and 2/4 respectively. ②Among 32 cases undergoing BAL, 10/10 patients with AASV-ILD all showed that neutrophils were dominant in BALF, while, the other 22 patients showed that the ratio of neutrophils elevation (14/22, 63.6%) and the ratio of lymphocytes elevation (18/22, 81.8%) were comparable, and there were 12/22(54.5%)patients with both types of cell elevation. Among 13 cases with iymphocytes elevation in BALF who performed analysis of sub-type lymphocytes, 10/13 cases showed decreased CD4/CD8 ratio, 3/13 cases showed increased CD4/CD8 ratio which were all related with SS. ③Among 15 patients undertaken HRCT and BALF detection together, 7/7 UIP-like cases showed the ratio of neutrephils elevation in BALK While in non-UIP-like cases, 5/8 showed the ratio of lymphocytes elevation. Conclusion ①UIP-like patterns and patterns of lobular and interlobular septa thickening are the most common imaging features of HRCT in CTD-ILD, the former are mostly seen in AASV and RA. OP-like patterns and LIP-like patterns are commonly seen in PM/DM and SS respectively. ②Tbe increased neutrophil percentage is dominant in BAL fluid of patients with AASV-ILD, while the others show that the ratio of neutrophil and lymphocyte elevation are comparable, lymphocytes subtype analysis shows decreased CD4/CD8 ratio is dominant in CTD-ILD patients with lymphocytes increased. There is a significant relationship between increased CD4/CD8 ratio and SS-LIP. ③All of the cases with UIP-like patterns show the ratio of neutrophils elevation in BALF. While the ratio of lymphocytes elevation is dominant in non-UIP-like cases.