Integrated bioinformatics analysis of microarray data from non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), with its high mortality rate, lack of early diagnostic markers and prevention of distant metastases are the main challenges in treatment. To identify potential miRNAs and key genes in NSCLC to find new biomarkers and target gene therapies. The GSE102286, GSE56036, GSE25508, GSE53882, GSE29248 and GSE101929 datasets were obtained from the Gene Expression Omnibus (GEO) database and screened for differentially co-expressed miRNAs (DE-miRNAs) and lncRNAs (DElncRs) by GEO2R and R software package. Pathway enrichment analysis of DE-miRNAs-target genes was performed by String and Funrich database to construct protein-protein interaction (PPI) and competing endogenous RNA (ceRNA) network and visualized with Cytoscape software. Nineteen co-expressed DE-miRNAs were screened from five datasets. The 7683 predicted up- and down-regulated DE-miRNAs-target genes were significantly concentrated in cancer-related pathways. The top 10 hub nodes in the PPI were identified as hub genes, such as MYC, EGFR, HSP90AA1 and TP53, MYC, and ACTB. By constructing miRNA-hub gene networks, hsa-miR-21, hsa-miR-141, hsa-miR-200b and hsa-miR-30a, hsa-miR-30d, hsa-miR-145 may regulate most hub genes and hsa-miR-141, hsa-miR-200, hsa-miR-145 had higher levels in the miRNA and ceRNA regulatory networks, respectively. In conclusion, the identification of hsa-miR-21, hsa-miR-141, hsa-miR-200b hsa-miR-30a, hsa-miR-30d and hsa-miR-145 provides a new theoretical basis for understanding the development of NSCLC.

Accumulated cytotoxicity of CDK inhibitor dinaciclib with first-line chemotherapy drugs in salivary adenoid cystic carcinoma cells.

Adenoid cystic carcinoma (ACC) is one of the most common salivary gland malignant tumors. Its treatment failure is partly due to the limitations of chemotherapeutic agents and their adverse effects. The objective of this study was to determine the potential additive anti-cancer effect of a novel CDK inhibitor dinaciclib with first-line chemotherapy drugs in ACC. Protein expression of phosphorylated CDK2 (p-CDK2) in paraffin-embedded tissue specimens of ACC from 17 patients was investigated by immunohistochemistry (IHC). Cell Counting Kit (CCK-8), clone formation assay, and flow cytometry were used to test the proliferation and apoptosis of ACC-2 cells treated with dinaciclib with or without other first-line chemotherapy drugs. Protein expression was also determined by Western blot. Interestingly, we discovered that p-CDK2 protein was expressed in both cytoplasmic and nucleus in salivary ACC tissues, which was higher than that in normal salivary tissues, indicating that agents targeting CDK2 may be potential therapeutic strategies against this type of tumor. As expected, CDK inhibitor dinaciclib significantly induced ACC-2 cells apoptosis. Moreover, it sensitized cells to the chemotherapeutic agents such as cisplatin, pemetrexed, and etoposide (VP-16), and this effect by dinaciclib may induce cell cycle arrest via abrogating CDK2 activity. Therefore, combinational therapy of CDK inhibitor dinaciclib with first-line chemotherapy drugs may be a promising strategy in the treatment of salivary ACC.

PFMG1 promotes osteoblast differentiation and prevents osteoporotic bone loss.

Nacre is a widely used mineral medicine that has been reported to have beneficial effects in bone remodeling without an increase in inflammation. Water-soluble nacre matrix has been demonstrated to be responsible for the effect, yet core active ingredients are unknown. Pinctada fucata mantle gene 1 (PFMG1) was first discovered in the mantle tissue of Pinctada fucata. The protein has 2 EF-hands, a calcium-binding domain. PFMG1 protein can affect the growth of calcium carbonate crystals in vitro. Here, we demonstrate that PFMG1 affects cell-cycle distribution and promotes preosteoblast proliferation. PFMG1 accelerates preosteoblast differentiation and extracellular matrix mineralization. During the differentiation process, PFMG1 increases the expression level of osteoblastic marker genes and activates the Erk signaling pathway. PFMG1 also accelerates calcium crystal aggregation in culture medium and suppresses osteoclast formation. Moreover, PFMG1 prevents bone loss caused by ovariectomy. RNA sequencing analysis demonstrated that PFMG1 stimulates genes that are associated with tissue development and ossification, which indicated new genes that function in bone remodeling. Our findings demonstrate the therapeutic potential of PFMG1 from nacre as a novel medicine for osteoporosis.-Li, L., Wang, P., Hu, K., Wang, X., Cai, W., Ai, C., Liu, S., Wang, Z. PFMG1 promotes osteoblast differentiation and prevents osteoporotic bone loss.

A novel mutation of adenomatous polyposis coli (APC) gene results in the formation of supernumerary teeth.

Supernumerary teeth are teeth that are present in addition to normal teeth. Although several hypotheses and some molecular signalling pathways explain the formation of supernumerary teeth, but their exact disease pathogenesis is unknown. To study the molecular mechanisms of supernumerary tooth-related syndrome (Gardner syndrome), a deeper understanding of the aetiology of supernumerary teeth and the associated syndrome is needed, with the goal of inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary teeth and colonic polyp lesion biopsies revealed that these patients exhibited significant pathological characteristics. APC gene mutations were detected by PCR and direct sequencing. We revealed the pathological pathway involved in human supernumerary tooth development and the mouse tooth germ development expression profile by RNA sequencing (RNA-seq). Sequencing analysis revealed that an APC gene mutation in exon 15, namely 4292-4293-Del GA, caused Gardner syndrome in this family. This mutation not only initiated the various manifestations typical of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this case. Furthermore, RNA-seq analysis of human supernumerary teeth suggests that the APC gene is the key gene involved in the development of supernumerary teeth in humans. The mouse tooth germ development expression profile shows that the APC gene plays an important role in tooth germ development. We identified a new mutation in the APC gene that results in supernumerary teeth in association with Gardner syndrome. This information may shed light on the molecular pathogenesis of supernumerary teeth. Gene-based diagnosis and gene therapy for supernumerary teeth may become available in the future, and our study provides a high-resolution reference for treating other syndromes associated with supernumerary teeth.

Synergistic action of 5Z-7-oxozeaenol and bortezomib in inducing apoptosis of Burkitt lymphoma cell line Daudi.

Treatment failure in cancer chemotherapy is largely due to the toxic effects of chemotherapeutic agents on normal cells/tissues. The proteasome inhibitor bortezomib has been successfully applied to treat multiple myeloma (MM), but there are some common adverse reactions in the clinic including peripheral neuropathy (PN). The TAK1 selective inhibitor 5Z-7-oxozeaenol has been widely studied in cancer therapy. Here, we investigated the potential synergy of bortezomib and 5Z-7-oxozeaenol in Burkitt's lymphoma (BL) cell lines. Cell viability assay showed that co-treatment of bortezomib at 8 nM, representing a one-eighth concentration for growth arrest, and 5Z-7-oxozeaenol at 2 µM, a dose that exhibited insignificant cytotoxic effects, synergistically induced apoptosis in the cell line Daudi. In parallel with the increasing dose of the bortezomib, and 5Z-7-oxozeaenol at 0.5 µM, lower colony formation efficiencies were seen in the cell line Daudi. Western blotting analysis verified that TAK1 inhibition by 5Z-7-oxozeaenol completely blocked JNK, p38, Erk, IKK, and IκB phosphorylation, which was almost instantly activated by TAK1 both directly or indirectly. Both agents synergistically prevented nuclear translocation of NF-κB, a characteristic of NF-κB inactivation. Moreover, a synergistic effect of bortezomib and 5Z-7-oxozeaenol on Western blotting analysis and flow cytometry was disclosed. Collectively, our results indicated that the proteasome inhibitor bortezomib and the TAK1 inhibitor 5Z-7-oxozeaenol displayed synergy on inhibiting BL cell apoptosis by inhibiting NF-κB activity.

Statin Function as an Anti-inflammation Therapy for Depression in Patients With Coronary Artery Disease by Downregulating Interleukin-1ß.

It is well known that inflammation contributes to the development of coronary artery disease (CAD) and depressive symptoms. Previous studies have shown that long-term application of statin reduces the occurrence of depression in patients with CAD. However, the mechanism remains unclear. We hypothesized that inflammation contributes to depression in patients with CAD and statin function as an anti-inflammation therapy for those depressive patients. Patients with confirmed CAD hospitalized in the Department of Cardiology of Tongji Hospital in Shanghai, China, were enrolled. Depression was identified as none (ND), mild (MiD), moderate (MoD), or severe (SD) on the basis of scores of the patient health questionnaire with 9 items. Inflammatory factors in peripheral blood were measured using a chemiluminescence immunoassay and Bio-plex. Luciferase expression level was detected using the Dual-Luciferase Reporter Assay System for IL-1ß or NF-κB expression by transfection in human umbilical vein endothelial cells, and patient serum was added. Data obtained from 217 patients with CAD were analyzed. The IL-1ß level of CAD with SD was 14.70, which was significantly higher than that of CAD with ND 7.52, MiD 7.73, or MoD 8.63. Luciferase reporter gene analysis showed that IL-1ß or NF-κB expression level was upregulated by the serum of CAD and depression patients. After the addition of atorvastatin, IL-1ß or NF-κB luciferase reporter expression level decreased. It suggested that depression in patients with CAD is associated with inflammation. Statin may function as an anti-inflammation therapy for depression in patients with CAD by downregulation of IL-1ß.

Current approaches to the diagnosis and treatment of white sponge nevus.

White sponge nevus (WSN) in the oral mucosa is a rare autosomal dominant genetic disease. The involved mucosa is white or greyish, thickened, folded and spongy. The genes associated with WSN include mutant cytokeratin keratin 4 (KRT4) and keratin 13 (KRT13). In recent years, new cases of WSN and associated mutations have been reported. Here, we summarise the recent progress in our understanding of WSN, including clinical reports, genetics, animal models, treatment, pathogenic mechanisms and future directions. Gene-based diagnosis and gene therapy for WSN may become available in the near future and could provide a reference and instruction for treating other KRT-associated diseases.

Effects of Seipin on Mouse Mesenchymal Stem Cell Osteo-Adipogenic Balance.

Seipin deficiency is an important cause of type 2 Berardinelli-Seip congenital dyslipidemia (BSCL2). BSCL2 is a severe lipodystrophy syndrome with lack of adipose tissue, hepatic steatosis, insulin resistance, and normal or higher bone mineral density. Bone marrow mesenchymal stem cells (BMSCs) are believed to maintain bone and fat homeostasis by differentiating into osteoblasts and adipocytes. We aimed to explore the role of seipin in the osteogenic/adipogenic differentiation balance of BMSCs. Seipin loxP/loxP mice are used to explore metabolic disorders caused by seipin gene mutations. Compared with wild-type mice, subcutaneous fat deficiency and ectopic fat accumulation were higher in seipin knockout mice. Microcomputed tomography of the tibia revealed the increased bone content in seipin knockout mice. We generated seipin-deficient BMSCs in vitro and revealed that lipogenic genes are downregulated and osteogenic genes are upregulated in seipin-deficient BMSCs. In addition, peroxisome proliferator-activated receptor gamma (PPARγ) signaling is reduced in seipin-deficient BMSCs, while using the PPARγ activator increased the lipogenic differentiation and decreased osteogenic differentiation of seipin-deficient BMSCs. Our findings indicated that bone and lipid metabolism can be regulated by seipin through modulating the differentiation of mesenchymal stem cells. Thus, a new insight of seipin mutations in lipid metabolism disorders was revealed, providing a prospective strategy for MSC transplantation-based treatment of BSCL2.

Genetic effects have a dominant role on poor responses to infant vaccination to hepatitis B virus.

The objective of this study was to investigate the overall contribution of genetic and environmental effects on poor response to hepatitis B virus (HBV) vaccination in Chinese infants. One-year-old healthy twins were recruited from child-care settings. Parental factors, neonates' condition at birth, postnatal infant feeding history and growth measurements during the 12 months were investigated by conducting an interview and checking the medical records. HBV-related markers were examined at 1 year of age. Heritability of surface antibody to HBV (anti-HBs) concentrations (ordinal variable) among twins was estimated using MX software. The role of perinatal environmental factors on poor vaccine response (anti-HBs<100 mIU ml(-1)) was analyzed using XTGEE (fit population-averaged panel-data models by using GEE) programs. Overall, 172 out of 225 recruited twin pairs were analyzed for heritability, including 82 pairs (47.67%) of monozygotic twins and 90 pairs of dizygotic twins, which consisted 43 pairs of (25.0%) opposite sex twins, 29 pairs of male twins and 18 pairs of female twins. Seventy-one (19.9%) of 370 twins showed poor responses to HBV vaccine. An additive genetic (0.91 of the variance)-random environmental (0.09 of the variance)-model best fit the variation of anti-HBs response. Risk factor analysis showed that with a smoking father and low birth weight, the infants were associated with an increased risk of poor response to HBV vaccination (odds ratio (OR)=4.50, 95% confidence interval (CI): 2.52-8.03 and OR=2.55, 95% CI: 1.33-4.87, respectively). Higher Apgar score and gaining more body weight in the first year of life reduced this risk. Genetic factors have a dominant role in determining infant HBV vaccination responses (91%) compared with perinatal environmental factors.

Expression of Different Isoforms of Versican During the Development of Mouse Mandibular First Molars.

Versican is a large chondroitin sulfate proteoglycan enriched in the extracellular matrix, and it has at least four different isoforms, termed V0, V1, V2, and V3. Although several studies have demonstrated that versican is stably expressed in various developing organs, the expression of versican isoforms during tooth development has not been elucidated yet. Therefore, the present study was to investigate the expression of versican isoforms in the developing mouse molars. The mandibular first molars from embryonic day (E) 11.5 to postnatal day (PN) 21 were used to investigate the expression of versican isoforms by immunohistochemistry, and the gene expressions of versican (Vcan) isoforms from E13.5 to PN7 were analyzed by quantitative real-time PCR. The results exhibited different expressing patterns of versican isoforms-the stellate reticulum (SR) and the dental mesenchymal cells adjacent to Hertwig's Epithelial Root Sheath (HERS) only expressed V1 and the mature odontoblasts mainly expressed V2, while the dental papilla and the ameloblasts might both express V0/V1/V2. These results suggested that different versican isoforms may act different roles in the tooth development, and we speculated that V0/V1 might be intimately involved in the cell proliferation while V2 was associated in the cytodifferentiation.

RNA-Sequencing Analyses Demonstrate the Involvement of Canonical Transient Receptor Potential Channels in Rat Tooth Germ Development.

Tooth development depends on multiple molecular interactions between the dental epithelium and mesenchyme, which are derived from ectodermal and ectomesenchymal cells, respectively. We report on a systematic RNA sequencing analysis of transcriptional expression levels from the bud to hard tissue formation stages of rat tooth germ development. We found that GNAO1, ENO1, EFNB1, CALM1, SIAH2, ATP6V0A1, KDELR2, GTPBP1, POLR2C, SORT1, and members of the canonical transient receptor potential (TRPC) channel family are involved in tooth germ development. Furthermore, Cell Counting Kit 8 (CCK8) and Transwell migration assays were performed to explore the effects of these differentially expressed genes (DEGs) on the proliferation and migration of dental pulp stem cells. Immunostaining revealed that TRPC channels are expressed at varying levels during odontogenesis. The identified genes represent novel candidates that are likely to be vital for rat tooth germ development. Together, the results provide a valuable resource to elucidate the gene regulatory mechanisms underlying mammalian tooth germ development.

The epidemiology of supernumerary teeth and the associated molecular mechanism.

Supernumerary teeth are common clinical dental anomalies. Although various studies have provided abundant information regarding genes and signaling pathways involved in tooth morphogenesis, which include Wnt, FGF, BMP, and Shh, the molecular mechanism of tooth formation, especially for supernumerary teeth, is still unclear. In the population, some cases of supernumerary teeth are sporadic, while others are syndrome-related with familial hereditary. The prompt and accurate diagnosis of syndrome related supernumerary teeth is quite important for some distinctive disorders. Mice are the most commonly used model system for investigating supernumerary teeth. The upregulation of Wnt and Shh signaling in the dental epithelium results in the formation of multiple supernumerary teeth in mice. Understanding the molecular mechanism of supernumerary teeth is also a component of understanding tooth formation in general and provides clinical guidance for early diagnosis and treatment in the future.

Drug-induced premature senescence model in human dental follicle stem cells.

Aging is identified by a progressive decline of physiological integrity leading to age-related degenerative diseases, but its causes is unclear. Human dental pulp stem cells (hDPSCs) has a remarkable rejuvenated capacity that relies on its resident stem cells. However, because of the lack of proper senescence models, exploration of the underlying molecular mechanisms has been hindered. Here, we established a cellular model utilizing a hydroxyurea (HU) treatment protocol and effectively induced Human dental pulp stem cells to undergo cellular senescence. Age-related phenotypic changes were identified by augmented senescence-associated-ß-galactosidase (SA-ß-gal) staining, declined proliferation and differentiation capacity, elevated G0/G1 cell cycle arrest, increased apoptosis and reactive oxygen species levels. Furthermore, we tested the expression of key genes in various DNA repair pathways including nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. In addition, our results showed that Dental pulp stem cells from young donors are more resistant to apoptosis and exhibit increased non-homologous end joining activity compared to old donors. Further transcriptome analysis demonstrate that multiple pathways are involved in the HU-induced Dental pulp stem cells ageing, including genes associated with DNA damage and repair, mitochondrial dysfunction and increased reactive oxygen species levels. Taken together, the cellular model have important implications for understanding the molecular exploration of Dental pulp stem cells senescence and aging.

Interleukin 37 promotes angiogenesis through TGF-ß signaling.

IL-37 is a novel pro-angiogenic cytokine that potently promotes endothelial cell activation and pathological angiogenesis in our previous study, but the mechanisms behind the pro-angiogenic effect of IL-37 are less well understood. Extending our observations, we found that TGF-ß interacts with IL-37, and potently enhances the binding affinity of IL-37 to the ALK1 receptor complex, thus allowing IL-37 to signal through ALK1 to activate pro-angiogenic responses. We further show that TGF-ß and ALK1 are required in IL-37 induced pro-angiogenic response in ECs and in the mouse model of Matrigel plug and oxygen-induced retinopathy. The result suggests that IL-37 induces pro-angiogenic responses through TGF-ß, which may act as the bridging molecule that mediates IL-37 binding to the TGF-ß receptor complex.

Hyaluronan and hyaluronan synthases expression and localization in embryonic mouse molars.

Hyaluronan (HA) and hyaluronan synthases (HASs) have been shown to play critical roles in embryogenesis and organ development. However, there have not been any studies examining HA and HAS expression and localization during tooth development. The present study was designed to investigate the expression of HA and three isoforms of HASs (HAS1, 2, 3) in embryonic mouse molars. The first mandibular embryonic mouse molars were examined by immunohistochemistry at E11.5, E13.5, E14.5, E16.5, and E18.5. PCR and western blot analyses were performed on RNA and proteins samples from E13.5 to E18.5 tooth germs. At the initial stage (E11.5), HA and HASs were expressed in the dental epithelium but not the underlying dental mesenchyme. HA immunostaining gradually increased in the enamel organ from the bud stage (E13.5) to the late bell stage (E18.5), and HA and HASs were highly expressed in the stellate reticulum and stratum intermedium. HA immunostaining was also enhanced in the dental mesenchyme and its derived tissues, but it was not expressed in the ameloblast and odontoblast regions. The three HAS isoforms had distinct expression patterns, and they were expressed in the dental mesenchyme and odontoblast at various levels. Furthermore, HAS1 and HAS2 expression decreased, while HAS3 expression increased from E13.5 to E18.5. These results suggested that HA synthesized by different HASs is involved in embryonic mouse molar morphogenesis and cytodifferentiation.

[Yeast expression and application of an antifreeze protein from the desert beetle Microdera punctipennis].

Insect antifreeze protein (AFP) has high antifreeze activity. Antifreeze proteins can be used in cryopreservation of biological tissues and cells. We expressed an antifreeze protein from the desert beetle Microdera punctipennis in yeast and determined the function of the protein at low temperatures. Yeast expression vector, pPIC9K-Mpafp698, was constructed and transformed into Pichia pastoris GS115. The expression of MpAFP698 was induced by methanol, and identified by tricine SDS-PAGE and Western blotting. Mpafp698 gene was inserted into the genome of the host yeast strain GS115, and correctly expressed. Hardly any yeast's own protein was secreted into the media. Cryoprotective experiments showed that MpAFP698 can significantly protect mouse liver as well as other mouse organs from cold damage compared with those in the control of Bovine serum albumin (BSA) addition. Besides, the hemolysis of blood cells protected by MpAFP698 at 4 degrees C was reduced and the survival rate of SF9 cells protected by MpAFP698 after freezing and thawing was increased compared to those of the control with BSA addition. Our results showed that MpAFP698 can be expressed in yeast, which allows a convenient purification of the MpAFP protein that has the cryoprotective effect.

Deubiquitinases in cancer.

Deubiquitinases are deubiquitinating enzymes (DUBs), which remove ubiquitin from proteins, thus regulating their proteasomal degradation, localization and activity. Here, we discuss DUBs as anti-cancer drug targets.

Expression profiling of white sponge nevus by RNA sequencing revealed pathological pathways.

BACKGROUND: White sponge nevus (WSN) is a rare periodontal hereditary disease. To date, almost all WSN studies have focused on case reports or mutation reports. Thus, the mechanism behind WSN is still unclear. We investigated the pathogenesis of WSN using expression profiling. METHODS: Sequence analysis of samples from a WSN Chinese family revealed a mutation (332 T > C) in the KRT13 gene that resulted in the amino acid change Leu111Pro. The pathological pathway behind the WSN expression profile was investigated by RNA sequencing (RNA-seq). RESULTS: Construction of a heatmap revealed 24 activated genes and 57 reduced genes in the WSN patients. The ribosome structure was damaged in the WSN patients. Moreover, the translation rate was limited in the WSN patients, whereas ubiquitin-mediated proteolysis was enhanced. CONCLUSIONS: Our results suggest that the abnormal degradation of the KRT13 protein in WSN patients may be associated with keratin 7 (KRT7) and an abnormal ubiquitination process.

Keratin 13 mutations associated with oral white sponge nevus in two Chinese families.

White sponge nevus (WSN) is an autosomal dominant hereditary disease. Keratin 4 (KRT4) and Keratin 13 (KRT13) gene mutations were involved in the WSN. We recruited two WSN Chinese families, and oral lesion biopsy with hematoxylin and eosin staining showed that patients had significant pathological characteristics. The mutations of KRT4 and KRT13 gene were detected by PCR and direct sequencing. The multiple alignments of KRT13 from 23 diverse species homology analyses were performed by the ClustalW program. The KRT13 expression was measured by Real-Time RT-PCR and Western blot analysis. Sequencing analysis revealed two mutations of KRT13 gene: one mutation was 332T>C and amino acid change was Leu111Pro. Another mutation was 340C>T and amino acid change was Arg114Cys. The sequence of KRT13 was highly conserved. Real-Time RT-PCR and Western blot analysis results show that KRT13 expression level is lower in patient but keep almost no change in mRNA level. When cells were treated with MG132, KRT13 protein level was increased and kept almost the same in normal and patient cells. We identified two heritable mutations in the KRT13 gene, which were associated with the development of WSN. The abnormal degradation of KRT13 protein of WSN may probably associate with the abnormal ubiquitination process.