Inhibition of HIST1H2AB suppresses the proliferation of lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma is one of the common causes of cancer-related deaths worldwide. Histone cluster 1 H2A family member b (HIST1H2AB) is a member of the histone H2A family. Bioinformatic analyses have revealed that HIST1H2AB is highly expressed in some cancers and might be an oncogene. However, information on the function of HIST1H2AB in lung adenocarcinoma is limited. METHODS: The expression of HIST1H2AB was analyzed in normal lung, lung adenocarcinoma and paracancerous tissues from The Cancer Genome Atlas (TCGA) database and immunohistochemistry staining. It was further verified in the relative cell lines using real-time quantitative polymerase chain reaction (RT-qPCR). When the adenocarcinoma cells lines (A549 and H1299) were successfully transfected with shHIST1H2AB or an empty plasmid packaged into a lentivirus, cell proliferation was detected using Celigo fluorescence cell-counting, colony formation and annexin V-allophycocyanin assays. Twenty nude mice were subcutaneously injected with A549 cells transfected with shHIST1H2AB or empty plasmid; the tumor size was recorded on day 25 and then measured every 3 days thereafter. The final tumor weight was measured on day 37. Significantly differentially expressed genes were analyzed using a human gene expression array. Furthermore, the potentially relevant genes were verified using RT-qPCR and western blotting. RESULTS: HIST1H2AB was highly expressed in lung adenocarcinoma tissues from TCGA database and immunohistochemistry staining. Similar results were seen in the lung adenocarcinoma cells. When the cells were successfully transfected with shHIST1H2AB or an empty plasmid, downregulation of HIST1H2AB inhibited the growth and promoted the apoptosis of lung adenocarcinoma cells. The xenograft results suggested that HIST1H2AB downregulation delayed tumor growth and reduced tumor weight. Moreover, interferon signaling pathway and four genes (HMGB1, FOXM1, F2RL1 and SLC4A7) might be regulated by HIST1H2AB in the development of lung adenocarcinoma as indicated through gene expression array, RT-qPCR and western blotting analyses. CONCLUSIONS: HIST1H2AB acts as an oncogenic protein and HIST1H2AB inhibition suppresses the proliferation of lung adenocarcinoma cells. It may be a novel target for lung adenocarcinoma therapy.

Inhibition of AHNAK nucleoprotein 2 alleviates pulmonary fibrosis by downregulating the TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and advanced interstitial lung disease with poor prognosis. AHNAK nucleoprotein 2 (AHNAK2) is a macromolecular protein that is important for cell migration and muscle membrane repair. The protein acts via epithelial-mesenchymal transition (EMT), which is a key mechanism in the pathogenesis of IPF. However, very few studies have elucidated the effect of AHNAK2 in the development of IPF. Therefore, we aimed to determine the role of AHNAK2 in IPF development. METHODS: C57BL/6 mice were induced with bleomycin, while A549 and Beas-2b pulmonary epithelial cell lines were treated with TGF-ß1 to induce IPF model. The expression of AHNAK2 was detected using immunohistochemistry staining in vivo, and real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) in vitro. C57BL/6 mice were injected with adeno-associated virus (AAV)-sh NC or AAV-sh AHNAK2 and the pulmonary function and EMT marker expression were measured. The migratory abilities of the two transforming growth factor beta 1 (TGF-ß1)-induced cell lines were examined using wound-healing and Transwell assays after transfection with si-NC, si-AHNAK2-1 and -2. EMT marker expression was detected using RT-qPCR and WB. Smad3 and phosphorylated-Smad3 of the two cells were examined using WB. Following Smad3 inhibition by Smad3 phosphorylation inhibitor (SIS3), TGF-ß1-induced cell migration and EMT marker expression were evaluated again after different transfections. RESULTS: AHNAK2 expression was higher in the IPF model than in the normal model in vivo and in vitro. Partial inhibition of AHNAK2 suppressed the EMT process and improved pulmonary ventilation and compliance in the mouse model of IPF. Similarly, knockdown of AHNAK2 suppressed the migration of pulmonary epithelial cells and reversed EMT. Furthermore, Smad3 of the two TGF-ß1-induced cell lines was not activated when AHNAK2 was inhibited. When SIS3 inhibited the activation of Smad3, the suppression of AHNAK2 had no effect on A549 and Beas-2b, regardless of TGF-ß1 induction. CONCLUSIONS: Inhibition of AHNAK2 alleviates pulmonary fibrosis and partially reverses EMT by inhibiting the TGF-ß1/Smad3 signaling pathway. Therefore, AHNAK2 is a potential therapeutic target for IPF.

DNA damage repair gene signature model for predicting prognosis and chemotherapy outcomes in lung squamous cell carcinoma.

BACKGROUND: Lung squamous cell carcinoma (LUSC) is prone to metastasis and likely to develop resistance to chemotherapeutic drugs. DNA repair has been reported to be involved in the progression and chemoresistance of LUSC. However, the relationship between LUSC patient prognosis and DNA damage repair genes is still unclear. METHODS: The clinical information of LUSC patients and tumour gene expression level data were downloaded from the TCGA database. Unsupervised clustering and Cox regression were performed to obtain molecular subtypes and prognosis-related significant genes based on a list including 150 DNA damage repair genes downloaded from the GSEA database. The coefficients determined by the multivariate Cox regression analysis and the expression level of prognosis-related DNA damage repair genes were employed to calculate the risk score, which divided LUSC patients into two groups: the high-risk group and the low-risk group. Immune viability, overall survival, and anticarcinogen sensitivity analyses of the two groups of LUSC patients were performed by Kaplan-Meier analysis with the log rank test, ssGSEA and the pRRophetic package in R software. A time-dependent ROC curve was applied to compare the survival prediction ability of the risk score, which was used to construct a survival prediction model by multivariate Cox regression. The prediction model was used to build a nomogram, the discriminative ability of which was confirmed by C-index assessment, and its calibration was validated by calibration curve analysis. Differentially expressed DNA damage repair genes in LUSC patient tissues were retrieved by the Wilcoxon test and validated by qRT-PCR and IHC. RESULT: LUSC patients were separated into two clusters based on molecular subtypes, of which Cluster 2 was associated with worse overall survival. A prognostic prediction model for LUSC patients was constructed and validated, and a risk score calculated based on the expression levels of ten DNA damage repair genes was employed. The clinical utility was evaluated by drug sensitivity and immune filtration analyses. Thirteen-one genes were upregulated in LUSC patient samples, and we selected the top four genes that were validated by RT-PCR and IHC. CONCLUSION: We established a novel prognostic model based on DNA damage repair gene expression that can be used to predict therapeutic efficacy in LUSC patients.

Elevation of EIF4G1 promotes non-small cell lung cancer progression by activating mTOR signalling.

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), as the key component of the transcription initiation factor complex EIF4F, is significantly upregulated in multiple solid tumours, including lung cancer. However, the function and mechanism of EIF4G1 in the regulation of non-small-cell lung cancer (NSCLC) remain unclear. Here, using the clinical samples and the comprehensive survival analysis platforms Kaplan-Meier plotter, we observed aberrant upregulation of EIF4G1 in NSCLC tissues; furthermore, high expression of EIF4G1 showed association with low differentiation of lung cancer cells and poor overall survival in NSCLC patients. Non-small-cell lung cancer cell line A549 and H1703 stably infected with EIF4G1 shRNA were used to determine the function of EIF4G1 in regulating cell proliferation and tumorigenesis in vitro and in vivo. The results demonstrated that EIF4G1 promoted the G1/S transition of the cell cycle and tumour cell proliferation in non-small cell lung cancer. Mechanistically, EIF4G1 was found to regulate the expression and phosphorylation of mTOR (Ser2448), which mediates the tumorigenesis-promoting function of EIF4G1. The inhibition of mTOR attenuated the EIF4G1-induced development and progression of tumours. These findings demonstrated that EIF4G1 is a new potential molecular target for the clinical treatment of non-small cell lung cancer.

Expression profile of RNA binding protein in cervical cancer using bioinformatics approach.

BACKGROUND: It has been demonstrated by studies globally that RNA binding proteins (RBPs) took part in the development of cervical cancer (CC). Few studies concentrated on the correlation between RBPs and overall survival of CC patients. We retrieved significant DEGs (differently expressed genes, RNA binding proteins) correlated to the process of cervical cancer development. METHODS: Expressions level of genes in cervical cancer and normal tissue samples were obtained from GTEx and TCGA database. Differently expressed RNA binding proteins (DEGs) were retrieved by Wilcoxon sum-rank test. ClusterProfiler package worked in R software was used to perform GO and KEGG enrichment analyses. Univariate proportional hazard cox regression and multivariate proportional hazard cox regressions were applied to identify DEGs equipped with prognostic value and other clinical independent risk factors. ROC curve was drawn for comparing the survival predict feasibility of risk score with other risk factors in CC patients. Nomogram was drawn to exhibit the prediction model and validated by C-index and calibration curve. Correlations between differentially expressed RNA binding proteins (DEGs) and other clinical features were investigated by t test or Cruskal Wallis analysis. Correlation between Immune and DEGs in cervical cancer was investigated by ssGSEA. RESULTS: 347 differentially expressed RBPs (DEGs) were retrieved from cervical cancer tissue and normal tissue samples. GO enrichment analysis showed that these DEGs involved in RNA splicing, catabolic process and metabolism. Cox regression model showed that there were ten DEGs significantly associated with overall survival of cervical cancer patients. WDR43 (HR = 0.423, P = 0.008), RBM38 (HR = 0.533, P < 0.001), RNASEH2A (HR = 0.474, P = 0.002) and HENMT1 (HR = 0.720, P = 0.071) played protective roles in survival among these ten genes. Stage (Stage IV vs Stage I HR = 3.434, P < 0.001) and risk score (HR = 1.214, P < 0.001) were sorted as independent prognostic risk factors based on multivariate cox regression. ROC curve validated that risk score was preferable to predict survival of CC patients than other risk factors. Additionally, we found some of these ten predictor DEGs were correlated significantly in statistic with tumor grade or stage, clinical T stage, clinical N stage, pathology or risk score (all P < 0.05). Part of immune cells and immune functions showed a lower activity in high risk group than low risk group which is stratified by median risk score. CONCLUSION: Our discovery showed that many RNA binding proteins involved in the progress of cervical cancer, which could probably serve as prognostic biomarkers and accelerate the discovery of treatment targets for CC patients.

MicroRNA-550a-3-5p controls the brain metastasis of lung cancer by directly targeting YAP1.

BACKGROUND: This study aimed to explore the potential regulatory mechanisms of brain metastasis and to identify novel underlying targets of lung cancer with brain metastasis. METHODS: Exosomes were isolated from the plasma of lung cancer patients with or without brain metastasis and low or high metastatic lung cancer cells, and small RNA from plasma-derived exosomes were sequenced. Differentially expressed miRNAs (DE-miRNAs) were identified. Human brain microvascular endothelial cells (HBMECs) were transfected with miR-550a-3-5p mimics or inhibitors and exosomes. Cell viability, migration, and apoptosis/cycle were determined using Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. Western blotting was used to measure the expression of the associated proteins. Finally, a dual-luciferase reporter gene assay was performed to confirm the miR-550a-3-5p target. RESULTS: Transmission electron microscopy, NanoSight, and western blotting showed that exosomes were successfully isolated and cell-derived exosomes could be taken up by HBMECs. Sequencing identified 22 DE-miRNAs which were enriched in the MAPK, chemokine, PPAR, and Wnt signaling pathways. MiR-550a-3-5p was significantly enriched in brain metastatic exosomes. Cellular experiments showed that miR-550a-3-5p and exosome enrichment significantly inhibited cell viability and migration, promoted apoptosis, and regulated the cell cycle of HBMECs compared with the controls (P < 0.05). Compared with the controls, high levels of both miR-550a-3-5p and exosomes markedly upregulated cleaved-PARP expression, but downregulated the expression of pRB, CDK6, YAP1, CTGF, and CYR61 (P < 0.05). Finally, YAP1 was confirmed to bind directly to miR-550a-3-5p. CONCLUSION: Our results indicate that miR-550a-3-5p and YAP1 may be novel potential targets for controlling brain metastasis.

Identification the ferroptosis-related gene signature in patients with esophageal adenocarcinoma.

BACKGROUND: Ferroptosis is a recently recognized non-apoptotic cell death that is distinct from the apoptosis, necroptosis and pyroptosis. Considerable studies have demonstrated ferroptosis is involved in the biological process of various cancers. However, the role of ferroptosis in esophageal adenocarcinoma (EAC) remains unclear. This study aims to explore the ferroptosis-related genes (FRG) expression profiles and their prognostic values in EAC. METHODS: The FRG data and clinical information were downloaded from The Cancer Genome Atlas (TCGA) database. Univariate and multivariate cox regressions were used to identify the prognostic FRG, and the predictive ROC model was established using the independent risk factors. GO and KEGG enrichment analyses were performed to investigate the bioinformatics functions of significantly different genes (SDG) of ferroptosis. Additionally, the correlations of ferroptosis and immune cells were assessed through the single-sample gene set enrichment analysis (ssGSEA) and TIMER database. Finally, SDG were verified in clinical EAC specimens and normal esophageal mucosal tissues. RESULTS: Twenty-eight significantly different FRG were screened from 78 EAC and 9 normal tissues. Enrichment analyses showed these SDG were mainly related to the iron-related pathways and metabolisms of ferroptosis. Gene network demonstrated the TP53, G6PD, NFE2L2 and PTGS2 were the hub genes in the biology of ferroptosis. Cox regression analyses demonstrated four FRG (CARS1, GCLM, GLS2 and EMC2) had prognostic values for overall survival (OS) (all P < 0.05). ROC curve showed better predictive ability using the risk score (AUC = 0.744). Immune cell enrichment analysis demonstrated that the types of immune cells and their expression levels in the high-risk group were significant different with those in the low-risk group (all P < 0.05). The experimental results confirmed the ALOX5, NOX1 were upregulated and the MT1G was downregulated in the EAC tissues compared with the normal esophageal mucosal tissues (all P < 0.05). CONCLUSIONS: We identified differently expressed ferroptosis-related genes that may involve in EAC. These genes have significant values in predicting the patients' OS and targeting ferroptosis may be an alternative for therapy. Further studies are necessary to verify these results of our study.

Profiles of autophagy-related genes in esophageal adenocarcinoma.

BACKGROUND: Several studies have demonstrated autophagy was involved in the process of esophageal adenocarcinoma (EAC). The aim of this study was to explore autophagy-related genes (ARGs) correlated with overall survival (OS) in EAC patients. METHODS: Expressions of ARGs in EAC and normal samples were downloaded from TCGA database. GO and KEGG enrichment analyses were used to investigate the ARGs bioinformatics functions. Univariate and multivariate cox regressions were performed to identify prognostic ARGs and the independent risk factors. ROC curve was established to evaluate the feasibility to predict the prognosis. Finally, the correlations between ARGs and clinical features were further explored. In addition, significantly different ARGs were verified in EAC specimens and normal esophageal mucosal tissues. RESULTS: Thirty significantly different ARGs were selected from EAC and normal tissues. Functional enrichments showed these ARGs were mainly related apoptosis. Multivariate cox regression analyses demonstrated eight ARGs were significantly associated with OS. Among these eight genes, BECN1 (HR = 0.321, P = 0.046), DAPK1 (HR = 0.636, P = 0.025) and CAPN1 (HR = 0.395, P = 0.004) played protective roles in survival. Gender (HR = 0.225, P = 0.032), stage (HR = 5.841, P = 0.008) and risk score (HR = 1.131, P < 0.001) were independent prognostic risk factors. ROC curves showed better efficacy to predict survival using the risk score. Additionally, we found BECN1, DAPK1, VAMP7 and SIRT1 genes were correlated significantly with survival status, gender, primary tumor and tumor stage (all P < 0.05). The experimental results confirmed the BIRC5 was overexpressed and the ITPR1, PRKN were downregulated in the EAC tissues compared with the normal esophageal mucosal tissues (all P < 0.05). CONCLUSION: Our findings suggested that autophagy was involved in the process of EAC. Several ARGs probably could serve as diagnostic and prognostic biomarkers and may help facilitate therapeutic targets in EAC patients.

Time course analysis based on gene expression profile and identification of target molecules for colorectal cancer.

BACKGROUND: The study aimed to investigate the expression changes of genes in colorectal cancer (CRC) and screen the potential molecular targets. METHODS: The GSE37178 of mRNA expression profile including the CRC samples extracted by surgical resection and the paired normal samples was downloaded from Gene Expression Omnibus database. The genes whose expressions were changed at four different time points were screened and clustered using Mfuzz package. Then DAVID was used to perform the functional and pathway enrichment analysis for genes in different clusters. The protein-protein interaction (PPI) networks were constructed for genes in the clusters according to the STRING database. Furthermore, the related-transcription factors (TFs) and microRNAs (miRNAs) were obtained based on the resources in databases and then were combined with the PPI networks in each cluster to construct the integrated network containing genes, TFs and miRNAs. RESULTS: As a result, 314 genes were clustered into four groups. Genes in cluster 1 and cluster 2 showed a decreasing trend, while genes in cluster 3 and cluster 4 presented an increasing trend. Then 18 TFs (e.g., TCF4, MEF2C and FOS) and 18 miRNAs (e.g., miR-382, miR-217, miR-1184, miR-326 and miR-330-5p) were identified and three integrated networks for cluster 1, 3, and 4 were constructed. CONCLUSIONS: The results implied that expression of PITX2, VSNL1, TCF4, MEF2C and FOS are time-related and associated with CRC development, accompanied by several miRNAs including miR-382, miR-217, miR-21, miR-1184, miR-326 and miR-330-5p. All of them might be used as potential diagnostic or therapeutic target molecules for CRC.

Vascular endothelial growth factor B coordinates metastasis of non-small cell lung cancer.

Neovascularization is critical for the invasion and metastasis of non-small cell lung cancer (NSCLC). However, the molecular mechanism underlying the control of neovascularization of NSCLC is not completely understood. Both vascular endothelial growth factor B (VEGF-B) and matrix metalloproteinases 9 (MMP9) play essential roles in neovascularization of NSCLC. Here, we examined whether VEGF-B and MMP9 may affect each other to coordinate the neovascularization process in NSCLC. We found strong positive correlation of VEGF-B and MMP9 levels in the NSCLC from the patients. Moreover, patients that had NSCLC with metastasis had significantly higher levels of VEGF-B and MMP9 in the primary cancer. Using a human NSCLC line A549, we found that overexpression of VEGF-B increased expression of MMP9, while inhibition of VEGF-B decreased expression of MMP9. On the other hand, overexpression of MMP9 increased expression of VEGF-B, while inhibition of MMP9 decreased expression of VEGF-B. These data suggest that expression of VEGF-B and MMP9 may activate each other to enhance neovascularization. We then analyzed the underlying mechanism. Application of a specific ERK/MAPK inhibitor but not a PI3K/Akt inhibitor to VEGF-B-overexpressing A549 cells substantially abolished the effect of VEGF-B on MMP9 activation, while application of a specific PI3K/Akt inhibitor but not an ERK/MAPK inhibitor to MMP9-overexpressing A549 cells substantially abolished the effect of MMP9 on VEGF-B activation, suggesting that VEGF-B may activate MMP9 via ERK/MAPK signaling pathway, while MMP9 may activate VEGF-B via PI3K/Akt signaling pathway. Thus, our data highlight a coordinating relationship between VEGF-B and MMP9 in the regulation of neovascularization in NSCLC.

MicroRNA-125b promotes tumor metastasis through targeting tumor protein 53-induced nuclear protein 1 in patients with non-small-cell lung cancer.

BACKGROUND: Lung cancer, predominantly non-small-cell lung cancer (NSCLC), is the leading cause of cancer deaths worldwide. There is a great need to identify critical effectors involved in metastasis of NSCLC that will facilitate the development of new therapeutic strategies. Here we evaluated the potential role of miR-125b in the metastasis of NSCLC cells. METHODS: Human NSCLC cells were isolated from surgical tissues with Cancer Cell Isolation Kit. Expressions of miR-125b and TP53INP1 were detected with real-time PCR and western blot. Human miR-125b mimics, miR-125b inhibitor, TP53INP1 expression plasmid and TP53INP1 siRNA were transfected into NSCLC cells with nucleofector transfection kit. NSCLC metastasis was determined with adhesion assay, invasive assay and lung tumor metastasis model. RESULTS: The expression of miR-125b was significantly higher in poorly differentiated NSCLC cells that are endowed with high metastatic potentials. Up-regulation of miR-125b could enhance the metastatic potential of NSCLC cells in vitro and in vivo, while down-regulation of miR-125b resulted in decreased metastatic potentials in vitro and in vivo. Further, tumor protein 53-induced nuclear protein 1 (TP53INP1) was an important target of miR-125b involved in metastasis of NSCLC cells. TP53INP1 served as a negative regulator of NSCLC metastasis. Decreased expression of TP53INP1 in tumor tissues was inversely associated with their expression of miR-125b, significantly lower in poorly differentiated tumors and inversely correlated with the clinical stages in patients with NSCLC. CONCLUSIONS: These findings demonstrated that miR-125b promoted tumor metastasis via targeting TP53INP1 in human NSCLC cells, which uncovered a real clinical relevance of microRNAs in tumor biology, and provided novel potential candidates for NSCLC clinical practice.

Lack of association between -174G>C and -634C>G polymorphisms in interleukin-6 promoter region and lung cancer risk: a meta-analysis.

Evidence suggested that the -174G>C and -634C>G polymorphisms in interleukin-6 (IL6) promoter region may modulate risk of lung cancer; however, the conclusion was still inconclusive. Therefore, we performed this meta-analysis to determine the association between IL6 -174G>C and -634C>G polymorphisms and lung cancer risk. The association strength was measured by odds ratios (ORs) and 95% confidence intervals (CI). Egger's test and Begg's test were performed to detect potential publication bias. By searching PubMed, EMBASE and China National Knowledge Infrastructure, we included 16 eligible studies in this meta-analysis, involving 6,202 lung cancer cases and 7,067 controls. Five studies about -174G>C polymorphism and 11 studies about -634C>G polymorphism were analyzed. By pooling eligible studies, we found no significant association of -174G>C with lung cancer risk (C vs. G: OR = 1.029; 95% CI, 0.957-1.106; heterogeneity, P = 0.478) and no statistic association of -634C > G with lung cancer susceptibility (G vs. C: OR = 1.050; 95% CI, 0.893-1.235; Heterogeneity, P < 0.001). No significant publication bias was observed. In conclusion, we found that -634C>G and -174G>C polymorphisms in IL6 promoter region were not associated with lung cancer risk.

Essential role of IL-10/STAT3 in chronic stress-induced immune suppression.

Stress can either enhance or suppress immune functions depending on a variety of factors such as duration of stressful condition. Chronic stress has been demonstrated to exert a significant suppressive effect on immune function. However, the mechanisms responsible for this phenomenon remain to be elucidated. Here, male C57BL/6 mice were placed in a 50-ml conical centrifuge tube with multiple punctures to establish a chronic restraint stress model. Serum IL-10 levels, IL-10 production by the splenocytes, and activation of STAT3 in the mouse spleen were assessed. We demonstrate that IL-10/STAT3 axis was remarkably activated following chronic stress. Moreover, TLR4 and p38 MAPK play a pivotal role in the activation of IL-10/STAT3 signaling cascade. Interestingly, blocking antibody against IL-10 receptor and inhibition of STAT3 by STAT3 inhibitor S3I-201 attenuates stress-induced lymphocyte apoptosis. Inhibition of IL-10/STAT3 dramatically inhibits stress-induced reduction in IL-12 production. Furthermore, disequilibrium of Th1/Th2 cytokine balance caused by chronic stress was also rescued by blocking IL-10/STAT3 axis. These results yield insight into a new mechanism by which chronic stress regulates immune functions. IL-10/STAT3 pathway provides a novel relevant target for the manipulation of chronic stress-induced immune suppression.

The analysis of microRNA expression profiling for coronary artery disease.

OBJECTIVES: To investigate the underlying molecular mechanisms of coronary artery disease (CAD) using microarray expression profiles. METHODS: The microRNA (miRNA) expression-profiling dataset GSE28858 was obtained from the Gene Expression Omnibus database, including 24 samples from 12 patients with CAD and 12 age- and sex-matched healthy controls. Differentially expressed miRNAs were identified with false discovery rate (FDR) = 1% by the SAM (Significant Analysis of Microarray) algorithm. The target genes of selected differentials expressed miRNAs that were not only related to CAD, but were also in two databases (TargetScan, miRanda). Then, the interactive objects of selected target genes were predicted using the STRING database to construct an interaction network (confidence score = 0.4). These target genes and interactive objects were put into the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, and the significant signaling pathway was obtained by hypergeometric function enrichment analysis (p < 0.05). RESULTS: MiRNA-526b was the only differentially expressed miRNA that was upregulated in patients with CAD (FDR = 1%). Toll-like receptor 4 (TLR4) was the target gene of miRNA-526b that occurred with the highest frequency. The objects that interacted with TLR4 were predicted using the STRING database and the interaction network was obtained. The vascular endothelial growth factor (VEGF) signaling pathway was the only selected significant pathway related with CAD in the interaction network (p < 0.05). CONCLUSION: The miRNA-526b is significantly upregulated in patients with CAD and the target gene of miRNA-526b participates in the VEGF signaling pathway. Whether or not the miRNA-526b can be used as a biomarker remains to be elucidated in a larger prospective study.

Toosendanin Induces Lung Squamous Cell Carcinoma Cell Apoptosis and Inhibits Tumor Progression via the BNIP3/AMPK Signaling Pathway.

Lung squamous cell carcinoma (LUSC) is the second most common type of non-small cell lung cancer. Toosendanin can target critical cancer cell survival and proliferation. However, the function of toosendanin in LUSC is limited. Cancer cell proliferative capacity is detected using cell morphology, colony formation, and flow cytometry. The invasiveness of the cells is detected by a Transwell assay, western blotting, and RT-qPCR. Nude mice are injected with H226 (1×106) and received an intraperitoneal injection of toosendanin every 2 days for 21 days. RNA sequence transcriptome analysis is performed on toosendanin-treated cells to identify target genes and signaling pathways. With increasing concentrations of toosendanin, the rate of cell proliferation decreases and apoptotic cells increases. The number of migrated cells significantly reduces and epithelial-mesenchymal transition is reversed. Injection of toosendanin in nude mice leads to a reduction in tumor volume, weight, and the number of metastatic tumors. Furthermore, KEGG shows that genes related to the AMPK pathway are highly enriched. BNIP3 is the most differentially expressed gene, and its expression along with phosphorylated-AMPK significantly increases in toosendanin-treated cells. Toosendanin exerts anticancer effects, induces apoptosis in LUSC cells, and inhibits tumor progression via the BNIP3/AMPK signaling pathway.

Neoadjuvant camrelizumab (an anti-PD-1 antibody) plus chemotherapy or apatinib (a VEGFR-2 inhibitor) for initially unresectable stage II-III non-small-cell lung cancer: a multicentre, two-arm, phase 2 exploratory study.

This multicentre, two-arm, phase 2 study aimed to explore the efficacy and safety of neoadjuvant camrelizumab plus chemotherapy or apatinib in patients with initially unresectable stage II-III non-small-cell lung cancer (NSCLC). Eligible patients regardless of PD-L1 expression received neoadjuvant camrelizumab 200 mg and platinum-doublet chemotherapy every 3 weeks (arm A) or those with PD-L1-positive tumors received neoadjuvant camrelizumab and apatinib 250 mg once daily (arm B), for 2-4 cycles, followed by surgery. The primary endpoint was major pathological response (MPR) rate. Thirty patients in arm A and 21 in arm B were enrolled. Surgery rates were 50.0% (15/30) in arm A and 42.9% (9/21) in arm B, with all patients achieving R0 resections. Of these patients, the MPR and pathological complete response rates were both 20.0% (95% CI 4.3-48.1) in arm A and were 55.6% (95% CI 21.2-86.3) and 11.1% (95% CI 0.3-48.2) in arm B, respectively. The corresponding objective response rates were 33.3% (95% CI 11.8-61.6) and 55.6% (95% CI 21.2-86.3). With a median follow-up of 22.4 months (95% CI 19.0-26.0), the median event-free survival was not reached (NR; 95% CI 13.6-NR) in arm A and 16.8 months (95% CI 8.6-NR) in arm B. Grade 3 or above treatment-related adverse events occurred in eight (26.7%) patients in arm A and three (14.3%) in arm B. Biomarker analysis showed baseline TYROBP expression was predictive of treatment response in arm B. Neoadjuvant camrelizumab plus chemotherapy or apatinib exhibits preliminary efficacy and manageable toxicity in patients with initially unresectable stage II-III NSCLC.

T-cell immunoglobulin- and mucin-domain-containing molecule 3 gene polymorphisms and prognosis of non-small-cell lung cancer.

Lung cancer is the leading cause of death worldwide. Non-small-cell lung cancer (NSCLC) accounts for most of these cases. T-cell immunoglobulin- and mucin-domain-containing molecule 3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. Studies have shown that polymorphisms in TIM-3 gene can be associated with various diseases. The aim of this study was to investigate whether polymorphisms in the TIM-3 gene were associated with susceptibility to NSCLC. Three polymorphisms in TIM-3 gene (-1516G/T, -574G/T, and +4259T/G) were identified by polymerase chain reaction-restriction fragment length polymorphism in 432 NSCLC patients and 466 healthy controls. Results showed that frequencies of TIM-3 +4259TG genotype for cases and controls were 10.9 and 4.1 %, respectively; subjects carrying the +4259TG genotype had a 2.81-fold increased risk of NSCLC compared to the wild-type genotype (P < 0.0001). The TIM-3 -1516G/T and -574G/T polymorphisms did not show any correlation with NSCLC. In addition, when analyzing the survival time of NSCLC patients with TIM-3 +4259T/G polymorphism, cases with +4259TG genotype had significantly shorter survival time compared to the wild-type patients (15.2 months vs. 26.7 months, P = 0.007). These results suggested polymorphism in TIM-3 gene is associated with increased susceptibility to NSCLC and could be used as prognostic factor for this malignancy.

TMPOP2 Inhibition Suppresses Pancreatic Cancer Cell Migration and Development by Repressing The JNK/STAT3 Pathway.

Pancreatic cancer is a malignancy with a poor prognosis and high mortality. The lincRNA TMPOP2 is highly expressed in gynecological cancers and may exhibit tumor-promoting functions. However, the function of TMPOP2 in pancreatic cancer is limited. TMPOP2 expression in pancreatic cancer and adjacent tissues is analyzed from The Cancer Genome Atlas (TCGA) and GTEx database. It shows the high expression of TMPOP2 in pancreatic cancer tissues. Similar results are observed in resected pancreatic adenocarcinoma tumors and adjacent tissues from 20 patients and the relative cell lines. When the pancreatic cell lines are transfected with si-TMPOP2, it shows that TMPOP2 downregulation inhibits the cells migration and EMT. Furthermore, the potential mechanism is explored by detecting the expression of c-Jun N-terminal kinase (JNK), phosphorylated JNK, signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3. It suggests that TMPOP2 knockdown inactivates JNK and STAT3 phosphorylation. When a JNK activator (anisomycin) is added to the cells with si-NC or si-TMPOP2, it can partially reverse the migration and EMT inhibition of the cells with inhibited TMPOP2. TMPOP2 inhibition suppresses the migration and EMT of pancreatic cancer by repressing the JNK/STAT3 pathway. Thus, this may be a novel target for pancreatic cancer therapy.

Impacts of the zero mark-up policy on hospitalization expenses of T2DM and cholecystolithiasis inpatients in SC province, western China: an interrupted time series analysis.

Background: Since 2009, a series of ambitious health system reforms have been launched in China, including the zero mark-up drug policy (ZMDP); the policy was intended to reduce substantial medicine expenses for patients by abolishing the 15% mark-up on drugs. This study aims to evaluate the impacts of ZMDP on medical expenditures from the perspective of disease burden disparities in western China. Method: Two typical diseases including Type 2 diabetes mellitus (T2DM) in internal medicine and cholecystolithiasis (CS) in surgery were selected from medical records in a large tertiary level-A hospital in SC Province. The monthly average medical expenses of patients from May 2015 to August 2018 were extracted to construct an interrupted time series (ITS) model to evaluate the impact of policy implementation on the economic burden. Results: A total of 5,764 cases were enrolled in our study. The medicine expenses for T2DM patients maintained a negative trend both before and after the intervention of ZMDP. It had declined by 74.3 CNY (P < 0.001) per month on average in the pre-policy period and subsequently dropped to 704.4 CNY (P = 0.028) immediately after the policy. The level change of hospitalization expenses was insignificant (P = 0.197), with a reduction of 677.7 CNY after the policy, while the post-policy long-term trend was significantly increased by 97.7 CNY (P = 0.035) per month contrasted with the pre-policy period. In addition, the anesthesia expenses of T2DM patients had a significant increase in the level under the impact of the policy. In comparison, the medicine expenses of CS patients significantly decreased by 1,014.2 CNY (P < 0.001) after the policy, while the total hospitalization expenses had no significant change in level and slope under the influence of ZMDP. Furthermore, the expenses of surgery and anesthesia for CS patients significantly increased by 320.9 CNY and 331.4 CNY immediately after the policy intervention. Conclusion: Our study indicated that the ZMDP has been an effective intervention to reduce the excessive medicine expenses for both researched medical and surgical diseases, but failed to show any long-term advantage. Moreover, the policy has no significant impact on relieving the overall hospitalization burden for either condition.

ß-Arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways.

Our previous studies have shown that ß-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which ß-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either ß-arrestin 1 or ß-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. ß-arrestin 2 deficient-induced apoptosis was inhibited by transfection with ß-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on ß-arrestin 2. Furthermore, in the absence of either ß-arrestin 1 or ß-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either ß-arrestin 1 or ß-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of ß-arrestin 1/2. Our study thus demonstrates that ß-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.