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Quaternary ammonium compounds, including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC), are widely used as disinfectants. Increased use of inhalable products containing BAC or CPC has raised concerns for lung toxicity. This study sought to elucidate the microstructure of plasma membrane damage caused by BAC and CPC and the subsequent mechanism by which the damage is mediated, as assessed using two human pulmonary epithelial cell lines (A549 and BEAS-2B). Scanning electron microscopic observation showed that exposure to BAC or CPC for 3 hr reduced the length and density of microvilli on the plasma membrane in A549 cells. Analysis of cell cycle distribution following plasma membrane damage revealed that BAC and CPC promote G0/G1 cell cycle arrest in both cell lines. The protein levels of Cdc6, an essential regulator of DNA replication during G1/S transition, are decreased significantly and dose dependently by BAC or CPC exposure. CPC and BAC decreased the Cdc6 levels that had been increased by a PI3K agonist in A549 cells, and levels of phosphorylated AKT were reduced in response to BAC or CPC. Conversely, exposure to equivalent concentrations of pyridinium chloride (lacking a hydrocarbon tail) induce no changes. These results suggest that plasma membrane damage triggered by BAC or CPC causes Cdc6-dependent G0/G1 cell cycle arrest in pulmonary cells. These effects are attributable to the long alkyl chains of BAC and CPC. The reduction of Cdc6 following plasma membrane damage may be caused, at least in part, by diminished signaling via the PI3K/AKT pathway.

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INTRODUCTION: We investigated the function of cell division cycle 6 (CDC6) on the prognosis in colorectal carcinoma (CRC). METHODS: CDC6 protein expression levels in 121 patients with colorectal cancer and adjacent normal mucosa were detected by immunohistochemistry. RESULTS: Compared to adjacent normal tissues, CDC6 mRNA level was overexpressed in CRC tissues. Moreover, CDC6 protein levels were expressed up to 93.39% (113/121) in CRC tissues in the cell nucleus or cytoplasm. However, there were only 5.79% (7/121) in normal mucosal tissues with nuclear expression. CDC6 expression was significantly correlated with TNM stage and tumor metastasis. The 5-year survival rate was lower in the high CDC6 expression group than the low group. After silencing of CDC6 expression in SW620 cells, cell proliferation was slowed, the tumor clones were decreased, and the cell cycle was arrested in G1 phase. In multivariate analysis, increased CDC6 protein expression levels in colon cancer tissues were associated with cancer metastasis, TNM stage, and patient survival time. CONCLUSION: CDC6 is highly expressed in CRC, and downregulation of CDC6 can slow the growth of CRC cells in vitro. It is also an independent predictor for poor prognosis and may be a useful biomarker for targeted therapy and prognostic evaluation.

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OBJECTIVE: To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD). METHODS: ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting. RESULTS: We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6. CONCLUSIONS: 7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.

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Objective@#To analyze the differentially expressed genes of patients with oral squamous cell carcinoma (OSCC) from paracarcinoma through biological information analysis to preliminarily identify OSCC-associated genes. @*Methods@#GSE23558, GSE37991 and GSE30784 were downloaded from the Gene Expression Omnibus (GEO), which is the mRNA expression profile dataset. The differentially expressed genes (DEGs) were identified based on the gene ontology and the Kyoto Encyclopedia of Genes and Genomes. Then, the protein-protein interaction (PPI) network was constructed using the STRING online tool, and Cytoscape was used to filter the critical genes. Furthermore, key genes involved in the survival of patients with OSCC were analyzed using Kaplan-Meier analysis. The expression of hub genes was validated based on GEPIA（http://gepia.cancer-pku.cn/）. @*Results @#A total of 212 DEGs were screened, and further analysis revealed 16 core genes, among which the core genes associated with prognosis included aurora kinase A (AURKA), aurora kinase B (AURKB), apoptosis inhibiting factor 5 (BIRC5), cell division cycle 6 (CDC6), E2F transcription factor 7 (E2F7), ubiquitin-like with PHD and ring finger domains 1 (UHRF1). These key genes were highly expressed in patients with oral squamous cell carcinoma, and the survival time of patients was short; the difference was statistically significant (P < 0.05).@*Conclusion @# AURKA, AURKB, BIRC5, CDC6, E2F7 and UHRF1 may be useful as potential biomarkers for OSCC prognosis prediction.

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OBJECTIVE@#To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD).@*METHODS@#ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting.@*RESULTS@#We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6.@*CONCLUSIONS@#7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.

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BACKGROUND: Osteosarcoma is the most frequent malignant bone cancer in teenagers. In this study, cell division cycle 6 (Cdc6) was upregulated in the tumor tissues from patients with osteosarcoma according to the results of RNA sequencing. Cdc6 has been considered as a candidate prognostic marker, which was associated with cell apoptosis and cell cycle. However, the mechanisms by which Cdc6 regulates the apoptosis and cell cycle arrest in osteosarcoma remain unclear. METHODS: Firstly, 3 pairs of osteosarcoma and adjacent normal tissues were subjected to RNA sequencing. In addition, the levels of Cdc6 in 30 pairs of patients' tissues and in osteosarcoma cell lines were detected by western blotting and RT-qPCR. Moreover, the effects of Cdc6 on cell proliferation, apoptosis, cell cycle and invasion were evaluated by cell counting kit-8 (CCK-8), flow cytometric and transwell assay, respectively. RESULTS: Cdc6 was significantly upregulated in patients with osteosarcoma and in osteosarcoma cell lines. Downregulation of Cdc6 inhibited the proliferation and invasion of MG63 cells by promoting apoptosis. In addition, downregulation of Cdc6 induced G1 phase cell cycle arrest. Moreover, inhibition of Cdc6 downregulated the expression of Cyclin D1 and Cyclin A2, and upregulated the level of p21 in MG63 cells. In vivo study confirmed downregulation of Cdc6 inhibited osteosarcoma tumor growth by inducing apoptosis. CONCLUSION: Our findings indicated that Cdc6 may serve as an oncogene in osteosarcoma. Thus, inhibiting Cdc6 may be a therapeutic approach for the treatment of osteosarcoma.

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MicroRNAs (miRNA) play important regulatory roles in the occurrence and development of cancers. This study aimed to investigate the effects of miR-26a on the proliferation and apoptosis of ovarian cancer cells and explore the potential mechanism. qRT-PCR was performed to measure the miR-26a expression in 46 ovarian cancer tissues, and results showed miR-26a expression reduced significantly when compared with normal ovarian tissues (P<0.05). Moreover, miR-26a expression was related to the extent of cell differentiation and clinical stage of ovarian cancer (P<0.05). miR-26a mimic was transfected into SKOV3 cells and ES2 cells, and CCK8 assay, colony formation assay and flow cytometry showed miR-26a over-expression could significantly inhibit the proliferation of ovarian cancer cells and induce their apoptosis. Bioinformatics analysis revealed Cdc6 was a target gene of miR-26a. dual-luciferase assay and validation assay showed miR-26a could act on the 3'UTR of Cdc6 to regulate Cdc6 expression. These findings suggest that miR-26a may act on the 3'UTR of Cdc6 to regulate Cdc6 expression, which then inhibit the proliferation of ovarian cancer cells and induce their apoptosis. Our findings provide a new target for the diagnosis and therapy of ovarian cancer.

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Cell division cycle 6 (CDC6) is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle. CDC6 has been associated with the oncogenic activities in human cancers, but the biological function and clinical significance of CDC6 in EOC remain unclear. The aim of the present study is to examine the effect of CDC6 on epithelial ovarian cancer (EOC) cells proliferation. We found that CDC6 protein level was up-regulated in EOC tissues compared with the normal ovary tissues. CDC6 expression correlated significantly with FIGO stage (p<0.001), differentiation grade (p=0.002), ascites (p<0.001), malignant tumor cells in ascites (p=0.004), and lymph node status (p<0.001). In vitro, after the release of ovarian cancer cell line (HO8910) from serum starvation, the expression of CDC6, cyclinD1, and PCNA was up-regulated, whereas p16 expression was down-regulated. Furthermore, down-regulation of CDC6 in HO8910 cells decreased cell proliferation and colony formation. HO8910 cells transfected with sh CDC6#1 underwent G1 phase cell cycle arrest. Collectively, this study provides a novel regulatory signaling pathway of CDC6-regulated EOC growth and a new potential therapeutic target for EOC patients.

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The development and progression of melanoma have been attributed to independent or combined genetic and epigenetic events. There has been remarkable progress in understanding melanoma pathogenesis in terms of genetic alterations. However, recent studies have revealed a complex involvement of epigenetic mechanisms in the regulation of gene expression, including methylation, chromatin modification and remodeling, and the diverse activities of non-coding RNAs. The roles of gene methylation and miRNAs have been relatively well studied in melanoma, but other studies have shown that changes in chromatin status and in the differential expression of long non-coding RNAs can lead to altered regulation of key genes. Taken together, they affect the functioning of signaling pathways that influence each other, intersect, and form networks in which local perturbations disturb the activity of the whole system. Here, we focus on how epigenetic events intertwine with these pathways and contribute to the molecular pathogenesis of melanoma.

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Objective To investigate the expression and clinical significance of cell division cycle 6 (CDC6)and homeobox gene A5(HOXA5)in esophageal squamous cell carcinoma. Methods The expres-sion of CDC6 and HOXA5 in 51 specimens esophageal squamous cell carcinoma and 27 normal specimens esophageal tissues were evaluated by immunohistochemistry. Analyzed the relationship among the expression of CDC6 and HOXA5 protein and the clinicopathologic features of esophageal squamous cell carcinoma,along with the correlation between these two proteins. Results Immunohistochemical results showed that the positive expression rate of CDC6 and HOXA5 in esophageal squamous cell carcinoma tissue were 66. 7%(34 / 51)and 60. 8%(31 / 51),respectively,significantly higher than those in normal esophageal tissue18. 5%(5 / 27), 22. 2%(6 / 27),(χ2 = 16. 370,P = 0. 000;χ2 = 10. 528,P = 0. 001);There were significant positive correlation in the expression of CDC6 and HOXA5 and histological type(χ2 = 9. 031,P = 0. 011;χ2 = 7. 372,P = 0. 000), TNM stage(χ2 = 10. 474,P = 0. 015;χ2 = 11. 667,P = 0. 009),and there were no correlation in the expression of CDC6 and HOXA5 and age(χ2 = 0. 000,P = 1. 000;χ2 = 0. 001,P = 0. 972),sex(χ2 = 0. 049,P = 0. 824;χ2 = 0. 107,P = 0. 743),lymph node metastasis(χ2 = 3. 186,P = 0. 074;χ2 = 2. 212,P = 0. 137)in esophageal squamous cell carcinoma tissues. The expression of CDC6 and HOXA5 showed a positive correlation( r =0. 454,P = 0. 001). Conclusion The positive expression rate of CDC6 and HOXA5 in esophageal squamous cell carcinoma tissue were significantly higher than in normal esophageal tissue and close correlation with TNM stage and differentiation. High expression of CDC6 and HOXA5 may play important roles in the occurrence, development and proliferation of esophageal squamous cell carcinoma.

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Transcriptomics is increasingly used to assess biological responses to environmental stimuli and stressors such as aquatic pollutants. However, fundamental studies characterizing individual variability in mRNA levels are lacking, which currently limits the use of transcriptomics in environmental monitoring assessments. To address individual variability in transcript abundance, we performed a meta-analysis on 231 microarrays that were conducted in the fathead minnow (FHM), a widely used toxicological model. The mean variability for gene probes was ranked from most to least variable based upon the coefficient of variation. Transcripts that were the most variable in individual tissues included NADH dehydrogenase flavoprotein 1, GTPase IMAP family member 7-like and v-set domain-containing T-cell activation inhibitor 1-like while genes encoding ribosomal proteins (rpl24 and rpl36), basic transcription factor 3, and nascent polypeptide-associated complex alpha subunit were the least variable in individuals across a range of microarray experiments. Gene networks that showed high variability (based upon the variation in expression of individual members within the network) included cell proliferation, metabolism (steroid, lipids, and glucose), cell adhesion, vascularization, and regeneration while those that showed low variability (more stability) included mRNA and rRNA processing, regulation of translational fidelity, RNA splicing, and ribosome biogenesis. Real-time PCR was conducted on a subset of genes for comparison of variability collected from the microarrays. There was a significant positive relationship between the two methods when measuring individual variability, suggesting that variability detected in microarray data can be used to guide decisions on sample sizes for measuring transcripts in real-time PCR experiments. A power analysis revealed that measuring estrogen receptor ba (esrba) requires fewer biological replicates than that of estrogen receptor bb (esrbb) in the gonad and samples sizes required to detect a 50% change for reproductive-related transcripts is between 12 and 20. Characterizing individual variability at the molecular level will prove necessary as efforts are made toward integrating molecular tools into environmental risk assessments.

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Objective:To investigate the expression of minichromosome maintenance proteins 4(MCM4)and cell division cycle 6(CDC6)in uterine cervical carcinomas and its relationship with human papilloma virus(HPV)16/18 infection.Methods:The expression of MCM4 and CDC6 was examined in 50 squamous cell carcinoma specimens,20 cervical intraepithelial neoplasia(CIN)Ⅱ-Ⅲ specimens,20 CINⅠ specimens,and 20 normal cervical tissues by immunohistochemical method.The infections of HPV type 16,18 DNA were determined by PCR.Results:(1)The expression of MCM4 and CDC6 in uterine cervical carcinoma tissues was significantly higher than that in the CIN specimens and normal cervical tissues(Both P0.05).(2)The positive rates of(HPV)16/18 were significant different between cervical carcinomas,CIN and normal tissues(P0.05).(3)MCM4 expression were positively correlated with the expression of CDC6 in uterine cervical carcimonas(r=0.390,P