cNPAS2 induced ß cell dysfunction by regulating KANK1 expression in type 2 diabetes.

BACKGROUND: Diabetes mellitus type 2 (T2DM) is formed by defective insulin secretion with the addition of peripheral tissue resistance of insulin action. It has been affecting over 400 million people all over the world. AIM: To explore the pathogenesis of T2DM and to develop and implement new prevention and treatment strategies for T2DM. METHODS: Receiver operating characteristic (ROC) curve analysis was used to conduct diagnostic markers. The expression level of genes was determined by reverse transcription-PCR as well as Western blot. Cell proliferation assays were performed by cell counting kit-8 (CCK-8) tests. At last, T2DM mice underwent Roux-en-Y gastric bypass surgery. RESULTS: We found that NPAS2 was significantly up-regulated in islet ß cell apoptosis of T2DM. The ROC curve revealed that NPAS2 was capable of accurately diagnosing T2DM. NPAS2 overexpression did increase the level of KANK1. In addition, the CCK-8 test revealed knocking down NPAS2 and KANK1 increased the proliferation of MIN6 cells. At last, we found that gastric bypass may treat type 2 diabetes by down-regulating NPAS2 and KANK1. CONCLUSION: This study demonstrated that NPAS2 induced ß cell dysfunction by regulating KANK1 expression in type 2 diabetes, and it may be an underlying therapy target of T2DM.

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

HADHA promotes ovarian cancer outgrowth via up-regulating CDK1.

BACKGROUND: Ovarian cancer, a prevalent cause of cancer-related mortality among gynecological cancers, still lacks a clear understanding of its pathogenesis. In this study, our objective was to investigate the functional roles and pathogenic mechanisms of HADHA in ovarian cancer. METHODS: We utilized an ovarian cancer tissue microarray and three ovarian cancer cell lines (HO-8910, A2780, and SK-OV-3) for our analysis. Lentiviral-mediated short hairpin RNA (shRNA) was employed to interfere with HADHA expression in ovarian cancer cells. Various cellular events associated with tumor development were assessed using techniques such as Celigo cell counting assay, wound healing assay, Transwell assay, and flow cytometry analysis. Additionally, xenograft tumor models were developed to visualize the impacts of HADHA/CDK1 on ovarian cancer progression. RESULTS: Our data revealed significant HADHA overexpression in both ovarian cancer tissues and cell lines. Patients with elevated HADHA levels tended to experience poor survival outcomes. Moreover, HADHA upregulation correlated with several pathological parameters, including pathological stage, tumor size, tumor infiltrate, metastasis, and recurrence. Loss-of-function experiments targeting HADHA demonstrated that its suppression in ovarian cancer cells hindered cell growth and migration, while promoting apoptosis. To elucidate the underlying mechanism by which HADHA regulates ovarian cancer, we identified CDK1 as a target of HADHA. HADHA upregulated CDK1 expression by inhibiting its ubiquitination-dependent proteasomal degradation. Significantly, the overexpression of CDK1 reversed the impaired cell development caused by HADHA depletion, both in vitro and in vivo. CONCLUSION: Our study highlights the involvement of HADHA in ovarian cancer tumorigenesis and suggests its potential as a promising prognostic marker in ovarian cancer. Through its regulation of CDK1, HADHA influences critical cellular processes in ovarian cancer, providing insights into its pathogenic mechanism.

Protein kinase C pathway mediates the protective effects of glucagon-like peptide-1 on the apoptosis of islet ß-cells.

The incidence of diabetes has been increasing over previous years. It is hypothesized that promoting the survival of islet ß-cells is a key direction for the treatment of diabetes. Although gastric bypass surgery improves certain types of diabetes and attenuates its progression, there are certain associated disadvantages (including intestinal obstruction and anastomotic leakage), and quality of life and physical status (such as malnutrition) are significantly affected by gastric bypass surgery. Therefore, it is important to determine the mechanisms underlying the improvement of diabetes by gastric bypass surgery and identify novel gene targets for diabetes therapeutics. In the present study, glucagon­like peptide­1 (GLP­1), whose secretion was markedly increased following gastric bypass surgery, increased the activity of protein kinase C (PKC) in islet ß­cells in a dose­dependent manner. Additionally, treatment with GLP­1 boosted cell viability and decreased cell death in starved islet ß­cells, and inhibited mitochondria­dependent apoptosis by regulating the expression levels of Bcl­2/Bax. These effects were reversed by inhibiting the PKC pathway using hypericin. Therefore, the present study concluded that GLP­1 may promote the survival and inhibit the apoptosis of islet ß­cells at least in part by activating the PKC pathway, which is an important underlying mechanism and may be exploited in the treatment of diabetes.

Proliferating pancreatic beta-cells upregulate ALDH.

High levels of aldehyde dehydrogenase (ALDH) activity have been regarded as a specific feature of progenitor cells and stem cells. Hence, as an indicator of ALDH activity, aldefluor fluorescence has been widely used for the identification and isolation of stem and progenitor cells. ALDH activity was recently detected in embryonic mouse pancreas, and specifically and exclusively in adult centroacinar and terminal duct cells, suggesting that these duct cells may harbor cells of endocrine and exocrine differentiation potential in the adult pancreas. Here, we report the presence of aldefluor+ beta-cells in a beta-cell proliferation model, partial pancreatectomy. The aldefluor+ beta-cells are essentially all positive for Ki-67 and expressed high levels of cell-cycle activators such as CyclinD1, CyclinD2, and CDK4, suggesting that they are mitotic cells. Our data thus reveal a potential change in ALDH activity of proliferating beta-cells, which provides a novel method for the isolation and analysis of proliferating beta-cells. Moreover, our data also suggest that aldefluor lineage-tracing is not a proper method for analyzing progenitor or stem activity in the adult pancreas.

Downregulation of a putative tumor suppressor BMP4 by SOX2 promotes growth of lung squamous cell carcinoma.

SOX2 is a transcription factor essential for self-renewal and pluripotency of embryonic stem cells. Recently, SOX2 was found overexpressed in the majority of the lung squamous cell carcinoma (SQC), in which it acts as a lineage-survival oncogene. However, downstream targets/pathways of SOX2 in lung SQC cells remain to be identified. Here, we show that BMP4 is a downstream target of SOX2 in lung SQC. We found that SOX2-silencing-mediated inhibition of cell growth was accompanied by upregulation of BMP4 mRNA and its protein expression. Meta-analysis with 293 samples and qRT-PCR validation with 73 clinical samples revealed an inversely correlated relationship between levels of SOX2 and BMP4 mRNA, and significantly lower mRNA levels in tumor than in adjacent normal tissues. This was corroborated by immunohistochemistry analysis of 35 lung SQC samples showing lower BMP4 protein expression in tumor tissues. Cell-based experiments including siRNA transfection, growth assay and flow cytometry assay, further combined with a xenograft tumor model in mice, revealed that reactivation of BMP4 signaling could partially account for growth inhibition and cell cycle arrest in lung SQC cells upon silencing SOX2. Finally, chromatin immunoprecipitation analysis and luciferase reporter assay revealed that SOX2 could negatively regulate BMP4 promoter activity, possibly through binding to the promoter located in the first intron region of BMP4. Collectively, our findings suggest that BMP4 could act as a tumor suppressor and its downregulation by elevated SOX2 resulting in enhanced growth of lung SQC cells.

Increased Cdc7 expression is a marker of oral squamous cell carcinoma and overexpression of Cdc7 contributes to the resistance to DNA-damaging agents.

Cdc7-Dbf4 kinase (Dbf4-dependent kinase, DDK) is an essential factor of DNA replication and DNA damage response (DDR), which is associated with tumorigenesis. However, Cdc7 expression has never been associated to the outcome of oral squamous cell carcinoma (OSCC) patients, and the mechanism underlying cancer cell survival mediated by Cdc7 remains unclear. The Cdc7 protein expression of 105 OSCC tumor and 30 benign tissues was examined by immunohistochemistry assay. Overall survival rates of 80 OSCC patients were measured using Kaplan-Meier estimates and the log-rank tests. Cdc7 overexpression by adenovirus system was used to scrutinize the underlying mechanism contributed to cancer cell survival upon DDR. In silico analysis showed that increased Cdc7 is a common feature of cancer. Cdc7 overexpression was found in 96 of 105 (91.4%) studied cases of OSCC patients. Patients with higher Cdc7 expression, either categorized into two groups: Cdc7 high expression (2+ to 3+) versus Cdc7 low expression (0 to 1+) [hazard ratios (HR)=2.6; 95% confidence interval (CI)=1.28-5.43; P=0.0087] or four groups (0 to 3+) [HR=1.71; 95% CI=1.20-2.44; P=0.0032], exhibited a poorer outcome. Multivariate analysis showed that Cdc7 is an independent marker for survival prediction. Overexpressed Cdc7 inhibits genotoxin-induced apoptosis to increase the survival of cancer cells. In summary, Cdc7 expression, which is universally upregulated in cancer, is an independent prognostic marker of OSCC. Cdc7 inhibits genotoxin-induced apoptosis and increases survival in cancer cells upon DDR, suggesting that high expression of Cdc7 enhances the resistance to chemotherapy.

Inflammation-related gene expression profiles of endocervical polyps.

The roles of inflammation-associated genes in the pathogenesis of endocervical polyps remain unclear. We thus compared the expression levels of 509 inflammation-associated genes between endocervical polyp tissues and endocervical canal membrane tissues using a gene microarray. Sixteen inflammation-related genes were differentially expressed in endocervical polyps compared with those of normal endocervical canal membrane tissues. Expression of 8 of these 16 genes was further validated biochemically. The protein expression levels of IL-12P40, IL-17, IFN-γ, TNF-α, CCR2, and IL-11 were significantly higher in endocervical polyps than those in endocervical canal tissues, while expression of TGF-ß1 and IL-10 was significantly lower (P<0.05). In addition, endocervical polyp tissues expressed IL-12P40, IL-17, IFN-γ, TNF-α, CCR2, IL-11, TGF-ß1, and IL-10 mainly in the cytoplasm of the inflammatory cells and, to a lesser extent, in the acinus of the serous gland. Endocervical polyp is a polygenic disease and aberrantly expressed genes may play roles in its pathogenesis.

Knockdown of HMGA1 expression by short/small hairpin RNA inhibits growth of ovarian carcinoma cells.

The aim of the current study was to investigate the influence of downregulating high-mobility group protein A1 (HMGA1) on the tumor gene and the mechanisms underlying the antitumor of HMGA1. The efficient short/small hairpin RNAs (shRNAs) of HMGA1 were constructed and transfected into human ovarian carcinoma OVCAR cells. The changes were identified by reverse transcription PCR (RT-PCR), Western blotting, methyl thiazolyl tetrazolium, and invasion assay. The knockdown of HMGA1 expression in OVCAR cells could obviously change cell morphology, decrease cell proliferation, and reduce invasion in vitro. BALB/C nude mice injected with OVCAR cells transfected HMGA1 shRNA showed a significantly lower tumor weight and volume than those in the control group. Taken together, HMGA1 knockdown could reduce the growth and metastasis potentials of OVCAR cells.

Expression of tumor suppressor gene ING4 in ovarian carcinoma is correlated with microvessel density.

PURPOSE: Angiogenesis, estimated by microvessel density (MVD), has been shown to predict poor progression-free survival in women with advanced epithelial ovarian cancer. Inhibitor of growth (ING) family proteins inhibit angiogenesis in a number of cancers. We evaluated the role of ING4 in regulation of angiogenesis in patients with epithelial ovarian cancer. METHODS: Semi-quantitative RT-PCR was used to determine ING4 mRNA levels in 40 ovarian cancer patients and 40 normal controls. Also, we used immunohistochemistry to evaluate (1) ING4 protein expression levels and (2) the level of MVD by staining CD34, a microvessel marker, in these patients. Through statistical analysis, the possible correlation between the ING4 expression and angiogenesis was explored. RESULTS: ING4 mRNA and protein were significantly downregulated in all ovarian cancer patients compared to normal controls (P < 0.001). Endometrioid carcinoma tissue had significantly lower ING4 levels compared to serous or mucinous ovarian cancer. ING4 expression correlated negatively with stage and histological grade of ovarian cancers. MVD correlated negatively with ING4 protein and mRNA levels (ρ = -0.865; P < 0.001 and ρ = -0.724; P < 0.001, respectively). CONCLUSIONS: Loss of ING4 may promote microvessel formation and plays a role in facilitating the development of ovarian cancer. Although the specific mechanisms are not yet understood, our data suggest that ING4 may be a promising target for the treatment for ovarian cancer.

Chemotherapeutic effects of different paclitaxel plus poldine combination methods for treatment of ovarian carcinoma.

The study aimed to evaluate the curative effects and toxicity of different paclitaxel (PTX) plus poldine chemotherapeutic combination methods for treatment of advanced ovarian carcinoma. A total of 27 patients with ovarian epithelial carcinoma were divided into four groups: A1, taxotere plus poldine intravenous chemotherapy (n=5); A2, taxotere intravenous chemotherapy combined with poldine intraperitoneal chemotherapy (n=7); B1, paclitaxel plus poldine intravenous chemotherapy (n=6); B2, paclitaxel intravenous chemotherapy combined with poldine intraperitoneal chemotherapy (n=9). Toxic side effects were observed after chemotherapy, and the short-term effects were assessed. Some 25 (25/27) cases completed a four-course treatment, the remaining two stopping halfway due to anaphylactic shock. The total effective rate for the A1 Group was 60% (3/5) and that of A2 group was 71.4% (5/7), Figuires for the B1 and B2 groups were 50% (3/6) and 66.7% (6/9), respectively. In comparisons of toxic side reactions, there were significant differences between taxotere groups and paclitaxel groups, and between intravenous chemotherapy alone groups and intravenous plus intraperitoneal combination chemotherapy groups (p<0.05). Chemotherapy of toxol plus poldine was effective in treatment of advanced ovarian cancer, the toxicities of intravenous plus intraperitoneal combination chemotherapy was lower than that of intravenous chemotherapy alone, and the heart toxicity with taxoere was lower than with paclitexal.

Upregulation of SOX9 in lung adenocarcinoma and its involvement in the regulation of cell growth and tumorigenicity.

PURPOSE: SOX9 is an important transcription factor required for development and has been implicated in several types of cancer. However, SOX9 has never been linked to lung cancer to date. Here, we show that SOX9 expression is upregulated in lung adenocarcinoma and show how it is associated with cancer cell growth. EXPERIMENTAL DESIGN: Data mining with five microarray data sets containing 490 clinical samples, quantitative reverse transcription-PCR validation assay in 57 independent samples, and immunohistochemistry assay with tissue microarrays containing 170 lung tissue cores were used to profile SOX9 mRNA and protein expression. Short interference RNA suppression of SOX9 in cell lines was used to scrutinize functional role(s) of SOX9 and associated molecular mechanisms. RESULTS: SOX9 mRNA and protein were consistently overexpressed in the majority of lung adenocarcinoma. Knockdown of SOX9 in lung adenocarcinoma cell lines resulted in marked decrease of adhesive and anchorage-independent growth in concordance with the upregulation of p21 (CDKN1A) and downregulation of CDK4. In agreement with higher SOX9 expression level in lung adenocarcinoma, the p21 mRNA level was significantly lower in tumors than that in normal tissues, whereas the opposite was true for CDK4, supporting the notion that SOX9 negatively and positively regulated p21 and CDK4, respectively. Finally, whereas SOX9-knockdown cells showed significantly attenuated tumorigenicity in mice, SOX9 transfectants consistently showed markedly stronger tumorigenicity. CONCLUSIONS: Our data suggest that SOX9 is a new hallmark of lung adenocarcinoma, in which SOX9 might contribute to gain of tumor growth potential, possibly acting through affecting the expression of cell cycle regulators p21 and CDK4.

[Effects of small interfering RNA specific to stably transfected HMGA1 gene on biological characters of ovarian carcinoma cells].

OBJECTIVE: To investigate the effects of biological characters of the human ovarian cell line (OVCAR) by stable transfection short hairpin RNA into the target HMGA1 gene. METHODS: Experiments were divided into two groups: transfected the OVCAR cells with pSilence4.1-CMV-Hs plasmid as group A, while transfected OVCAR cells with pSilence4.1-CMV-Hn plasmid as group B, in which stably transfected cells were gained by antibiotic screening. The comparative expressions of HMGA1 were detected by RT-PCR and western blot. Methyl thiazolyl tetrazolium (MTT) method was applied to measure cell proliferation and at the same time, the cell growth curve was also be mapped. Vitro invasion assay was used to observe the invasion ability of the cancer cells, and the tumor growth of the nude mice inoculated of tumor cells were compared with before and after transfection. RESULTS: In group A, the expression level of mRNA and protein HMGA1 gene in OVCAR cells were remarkably reduced before and after the stable transfected with HMGA1 siRNA, in which the percents of mRNA expression were [(86.3 ± 2.7)% vs. (35.8 ± 3.1)%, P < 0.05], the expression of protein were [(68.6 ± 2.8)% vs. (22.3 ± 4.2)%, P < 0.05)]. The OVCAR cell growth in stable transfection status was more significantly decreased than that in non-transfection status (P < 0.05). In group B, there were no statistical difference in the expression of HMGA1 siRNA, protein and the cell growth between before and after transfection states (P > 0.05). The invasion cell numbers were reduced from before to after transfection state in group A [(53 ± 6) vs. (21 ± 6), P < 0.05], while there was no significant difference in group B [(51 ± 6) vs. (47 ± 8), P > 0.05]. After inoculated transfected cells into nude mice, it took (6.0 ± 0.9) days to grow the planed tumors in group A, which was much shorter than that (12.3 ± 3.9) days in group B (P < 0.05). After 5 weeks, the tumor weight and volume in group A was were significantly lower than those in group B [(0.8 ± 0.3) g vs. (2.1 ± 0.4) g, (205 ± 34) mm(3) vs. (987 ± 82) mm(3), all P < 0.05]. CONCLUSION: HMGA1 siRNA could remarkably reduce the expression of HMGA1 gene in ovarian cell and also inhabit the ovarian cell growth.

Sensitive and quantitative detection of severe acute respiratory syndrome coronavirus infection by real-time nested polymerase chain reaction.

A quantitative, real-time, nested polymerase chain reaction (PCR) method, combining the high sensitivity of nested PCR with time-saving real-time instrumentation, was developed for large-scale screening for severe acute coronavirus (SARS) coronavirus. Forty-six clinical specimens were analyzed by this method, and results were compared with those obtained by conventional, single-round, real-time reverse-transcriptase PCR (RT-PCR) performed in parallel. Of the 17 positive results, 2 identified by our method were not detected by single-round, real-time RT-PCR, which suggests that real-time nested PCR has the potential for increased sensitivity, leading to earlier detection of SARS.

[Arsenic trioxide induced apoptosis and expression of p53 and bcl-2 genes in human small cell lung cancer cells].

OBJECTIVE: To study the arsenic trioxide (As(2)O(3)) induced apoptosis in a human small cell lung cancer cell line (NeI-H cells) and its possible mechanisms. METHODS: Apoptotic cells were detected by the TUNEL method. The expression of p53 and bcl-2 was analyzed with immunohistochemical staining. RESULTS: NeI-H cells showed the sub-G(1) peak after treatment with As(2)O(3) (0.5 micromol/L, 1.0 micromol/L, and 2.0 micromol/L) for 72 hours. The ratio of apoptotic cells increased with the increasing concentrations of the drug and the time of culture. Immunohistochemical staining of NeI-H cells showed increased expression of p53, but decreased expression of bcl-2 with the increasing concentrations of the drug. CONCLUSION: The anti-carcinogenic effect of As(2)O(3) is due to the induction of cell apoptosis. Up-regulation of the p53 gene and down-regulation of the bcl-2 gene may be an underlining mechanism.