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Objective Recently, fibulin-1 (FBLN1) has been shown to be downregulated in various cancers via promoter hypermethylation. Our study aimed to determine the expression and methylation status of FBLN1 in tongue squamous cell carcinoma (TSCC) tissues and cells. Methods Methylation-specific PCR was implemented to detect the methylation status of the FBLN1 gene in TSCC tissues and Western blot analysis was used to detect the expression of FBLN1 protein. The human TSCC cell lines, CAL27 and SCC9, were cultured in vitro and treated with 5-aza-deoxycytidine (5-Aza-dC). CCK-8, colony formation, and Transwell assays were performed to test TSCC cell proliferation, migration, and invasion following 5-Aza-dC treatment or overexpression of FBLN1, which was further verified in in vivo experiments. Results FBLN1 was hypermethylated and the protein expression was reduced in TSCC tissues. After human TSCC cell lines (CAL27 and SCC9) were treated with 5-Aza-dC or overexpressed FBLN1, FBLN1 expression was upregulated and the TSCC cell proliferation, migration, and invasion abilities were suppressed. In vivo experiments further showed that demethylation or overexpression of FBLN1 slowed tumor growth in nude mice. Conclusion This study demonstrated that 5-Aza-dC treatment or overexpression of FBLN1 inhibited the growth of human TSCC.

Summary The Drosophila testis has been fundamental to understanding how stem cells interact with their endogenous microenvironment, or niche, to control organ growth in vivo. Here, we report the identification of two independent alleles for the highly conserved tumor suppressor gene, Retinoblastoma-family protein (Rbf), in a screen for testis phenotypes in X chromosome third-instar lethal alleles. Rbf mutant alleles exhibit overproliferation of spermatogonial cells, which is phenocopied by the molecularly characterized Rbf11 null allele. We demonstrate that Rbf promotes cell-cycle exit and differentiation of the somatic and germline stem cells of the testes. Intriguingly, depletion of Rbf specifically in the germline does not disrupt stem cell differentiation, rather Rbf loss of function in the somatic lineage drives overproliferation and differentiation defects in both lineages. Together our observations suggest that Rbf in the somatic lineage controls germline stem cell renewal and differentiation non-autonomously via essential roles in the microenvironment of the germline lineage., Graphical Abstract, Highlights • Rbf null testes exhibit failure of germline stem cells to differentiate • Rbf expression in somatic cells of L3 testes rescues the GSC differentiation defect • Somatic Rbf RNAi disrupts cyst stem cell and germline stem cell differentiation • Somatic depletion of E2f1 rescues Rbf germline proliferation and differentiation, The Drosophila testis contains germline and somatic cyst stem cell populations. Progeny from these populations cross regulate the differentiation of each lineage. In this article, Hime, Quinn, and colleagues show that the Drosophila Retinoblastoma ortholog, Rbf, is cell autonomously required by somatic cells of the testis to facilitate cyst stem cell differentiation and subsequent germline stem cell differentiation.

Long noncoding RN (IncRNA) placenta-specific 2 (PLAC2) plays a critical role in many cancer types. A previous study found that PLAC2 expression dysregulated in non-small cell lung cancer (NSCLC). However, its function in LSCC is not entirely known. The present study enrolled 68 lung squamous cell carcinoma (LSCC, a major subtype of NSCLC) patients (gender: 39 males and 29 females; age: 36 to 67 years old; mean age: 53.2 ± 5.5 years old). The expression levels of PLAC2 in two types of tissue (non-tumor and LSCC) were measured by quantitative PCR. LSCC cells H1581 and H1993 were transfected with PLAC2 and cyclin-dependent kinase 6 (CDK6) expression vectors and small interfering RNAs (siRNAs), as well as microRNA-29C (miR-29C) mimic and inhibitor to perform overexpression and knock-down experiment, respectively. PLAC2 was upregulated in LSCC and positively correlated with CDK6 but negatively correlated with miR-29C. During a 5-year follow-up, high expression levels of PLAC2 were found to be closely associated with poor survival. PLAC2 and CDK6 were significantly upregulated in LSCC cells, while miR-29C was remarkably downregulated. miR-29C was predicted to be a potential target of PLAC2, and RNA pull-down assay confirmed their direct interaction. Overexpression of PLAC2 led to upregulation of CDK6 and downregulation of miR-29C, while knock-down of PLAC2 showed opposite effects. Overexpression of miR-29C downregulated CDK6, while knock-down of miR-29C increased the expression levels of CDK6. However, the expression of PLAC2 was not affected by overexpression or knock-down of miR-29C. Overexpression of PLAC2 and CDK6 enhanced LSCC cell proliferation and cell cycle progression, while knock-down showed opposite effects. In addition, overexpression of miR-29C inhibited cell proliferation and cell cycle progression, while its knockdown display opposite effects. Moreover, overexpression of miR-29C suppressed the role of overexpression of PLAC2 in cell proliferation and cell cycle progression. In conclusion, PLAC2 upregulates CDK6 by downregulating miR-29C to promote LSCC cell proliferation.

We report here that CBX3 (Chromobox 3) may be an independent prognostic biomarker in lung adenocarcinoma (LUAD). High expression levels of CBX3 are implicated in the cell cycle and p53 pathway, as well as tumor proliferation in LUAD. Copy number alterations are correlated with aberrant CBX3 expression in LUAD ., Chromobox (CBX) family members are vital epigenetic regulators that repress the transcription of target genes through chromatin modification. Several studies have investigated the role of CBX family members in cancer. However, the function and prognostic value of diverse CBX family members in non‐small‐cell lung cancer remain largely unknown. In this study, we reveal that CBX family members are overexpressed in non‐small‐cell lung cancer tissue compared with normal lung tissue, with the exception of CBX6. Kaplan–Meier analysis demonstrated that high expressions of CBX1 and CBX3 are correlated with overall survival, disease‐specific survival, disease‐free interval, and progression‐free interval for patients with lung adenocarcinoma (LUAD). Furthermore, regression model analysis suggests that CBX3 may be suitable as an independent prediction factor for overall survival and progression‐free interval in patients with LUAD. In addition, CBX3 mRNA expression was found to be associated with tumor diameter and lymph node metastasis. Gene enrichment analysis suggests that CBX3 is involved in the cell cycle and P53 signaling pathways. Aberrant expression of CBX3 in LUAD is correlated with DNA copy number alteration. In summary, our data imply that CBX3 plays an important role in the promotion of LUAD and may thus have potential as a prognostic biomarker and molecular therapeutic target for the disease.

Progranulin (PGRN) mediates cell cycle progression and cell motility as a pleiotropic growth factor and acts as a universal regulator of cell growth, migration and transformation, cell cycle, wound healing, tumorigenesis, and cytotoxic drug resistance as a secreted glycoprotein. PGRN overexpression can induce the secretion of many inflammatory cytokines, such as IL-8, -6,-10, TNF-α. At the same time, this protein can promote tumor proliferation and the occurrence and development of many related diseases such as gastric cancer, breast cancer, cervical cancer, colorectal cancer, renal injury, neurodegeneration, neuroinflammatory, human atherosclerotic plaque, hepatocarcinoma, acute kidney injury, amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. In short, PGRN plays a very critical role in injury repair and tumorigenesis, it provides a new direction for succeeding research and serves as a target for clinical diagnosis and treatment, thus warranting further investigation. Here, we discuss the potential therapeutic utility and the effect of PGRN on the relationship between inflammation and cancer.

Alterations in KEAP1/ NF-E2 p45-related factor 2 (NFE2L2/Nrf2) signaling pathway have been reported in 23% lung adenocarcinoma patients, suggesting that deregulation of the pathway is a major cancer driver. Here we report that mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) drives tumor growth and drug resistance by up regulating transcription factor Nrf2. In non-small cell lung cancer (NSCLC) cells and xenografts, MKP-1 knockdown triggered the down-regulation of the metabolic enzymes and cytoprotective proteins, which are the target genes of Nrf2. Consequently, the cell growth was markedly inhibited with decrease of tumor metabolisms and GSH contents. Moreover, MKP-1 silencing sensitized NSCLC cells to cisplatin treatment. Mechanistically, MKP-1 inhibited the ubiquitylation of Nrf2 via a direct interaction with the transcription factor. Nrf2 was hence stabilized and its transcriptional program was activated. Notably, Nrf2 elevated MKP-1 expression at transcriptional level. In human lung adenoma tumor samples, high levels of expression of MKP-1, Nrf2, and its target gene heme oxygenase 1 were closely correlated. Thus, MKP-1 and Nrf2 form a forward feedback loop in lung cancer cells, which stabilizing and activating Nrf2 to promote anabolic metabolism and GSH biosynthesis. This study uncovers a novel role of MKP-1 in the malignant evolution of cancers.

Background CCL22 played critical roles in Tregs recruitment. The upstream regulators modulating CCL22 in hepatocellular carcinoma (HCC) were not clearly understood. Methods MiR‐23a, p‐p65, p65, CCL22, and Foxp3 levels were monitored by RT‐qPCR and western blotting. Immunofluorescence assay was used to perform the costaining of Foxp3 and CD4 on liver tissues. Transwell assay was applied to evaluate the migration ability of Tregs. Dual‐luciferase assay was performed to determine relationship of miR‐23a/CCL22 and p65/miR‐23a. Chromatin immunoprecipitation (ChIP) was applied to detect the direct binding of p65 to miR‐23a promoter. Xenograft tumor models were developed to investigate the functions of p65 and miR‐23a in vivo. Results HBV infection was associated with reduced survival and increased Tregs recruitment in HCC patients. MiR‐23a was decreased, whereas p65, CCL22, and Foxp3 were increased in HBV+ tumors. MiR‐23a was inversely correlated with CCL22 and Foxp3 expression in HCC. MiR‐23a directly targeted CCL22 3’UTR, leading to CCL22 reduction and attenuated Tregs recruitment. Meanwhile, p65 functioned as a transcription repressor of miR‐23a by directly binding to its promoter. Inhibition of p65 induced miR‐23 expression, leading to less CCL22 expression and Tregs recruitment in vitro. CCL22 was the indispensable effector underlying p65/miR‐23a axis and Tregs recruitment. MiR‐23a inhibitor promoted xenografted tumor growth accompanying with upregulation of CCL22, whereas p65 inhibition exerted opposite effects. Conclusion Blockage of p65 disinhibited miR‐23a expression, leading to CCL22 reduction and repress Tregs recruitment. Targeting p65/miR‐23a/CCL22 axis was a novel approach for HBV+ HCC treatment., MiR‐23a expression is significantly decreased in HBV+ hepatocellular carcinoma tissues. MiR‐23a was inversely correlated with CCL22 and Foxp3 in HBV+ hepatocellular carcinoma tissues. MiR‐23a directly targeted CCL22 to attenuate Tregs recruitment.

Enzalutamide, a second-generation androgen receptor (AR) antagonist, has demonstrated clinical benefit in men with prostate cancer. However, it only provides a temporary response and modest increase in survival, indicating a rapid evolution of resistance. Previous studies suggest that enzalutamide may function as a partial transcriptional agonist, but the underlying mechanisms for enzalutamide-induced transcription remain poorly understood. Here, we show that enzalutamide stimulates expression of a novel subset of genes distinct from androgen-responsive genes. Treatment of prostate cancer cells with enzalutamide enhances recruitment of pioneer factor GATA2, AR, Mediator subunits MED1 and MED14, and RNA Pol II to regulatory elements of enzalutamide-responsive genes. Mechanistically, GATA2 globally directs enzalutamide-induced transcription by facilitating AR, Mediator and Pol II loading to enzalutamide-responsive gene loci. Importantly, the GATA2 inhibitor K7174 inhibits enzalutamide-induced transcription by decreasing binding of the GATA2/AR/Mediator/Pol II transcriptional complex, contributing to sensitization of prostate cancer cells to enzalutamide treatment. Our findings provide mechanistic insight into the future combination of GATA2 inhibitors and enzalutamide for improved AR-targeted therapy.

As an important medium of intercellular communication, exosomes play an important role in information transmission between tumor cells and their microenvironment. Tumor metastasis is a serious influencing factor for poor treatment effect and shortened survival. Lung cancer is a major malignant tumor that seriously threatens human health. The study of the underlying mechanisms of exosomes in tumor genesis and development may provide new ideas for early and effective diagnosis and treatment of lung cancer metastasis. Many studies have shown that tumor-derived exosomes promote lung cancer development through a number of processes. By promoting epithelial–mesenchymal transition of tumor cells, they induce angiogenesis, establishment of the pretransfer microenvironment, and immune escape. This understanding enables researchers to better understand the mechanism of lung cancer metastasis and explore new treatments for clinical application. In this article, we systematically review current research progress of tumor-derived exosomes in metastasis of lung cancer. Although positive progress has been made toward understanding the mechanism of exosomes in lung cancer metastasis, systematic basic research and clinical translational research remains lacking and are needed to translate our scientific understanding toward applications in the clinical diagnosis and treatment of lung cancer metastasis in the near future.

DNA double strand breaks (DSBs) are a severe threat to genome integrity and a potential cause of tumorigenesis, which is a multi-stage process and involves many factors including the mutation of oncogenes and tumor suppressors, some of which are transcribed microRNAs (miRNAs). Among more than 2000 known miRNAs, miR-21 is a unique onco-miRNA that is highly expressed in almost all types of human tumors and is associated with tumorigenesis through its multiple targets. However, it remains unclear whether there is any functional link between DSBs and miR-21 expression and, if so, does the link contribute to DSB-induced genomic instability/tumorigenesis. To address this question, we used DNA-PKcs-/- (deficient in non-homologous end-joining (NHEJ)) and Rad54-/- (deficient in homologous recombination repair (HRR)) mouse embryonic fibroblasts (MEFs) since NHEJ and HRR are the major pathways for DSB repair in mammalian cells. Our results indicate that levels of miR-21 are elevated in these DSB repair (DSBR) deficient cells, and ionizing radiation (IR) further increases these levels in both wild-type (WT) and DSBR-deficient cells. Interestingly, IR stimulated growth in soft agar and this effect was greatly reduced by blocking miR-21 expression in both WT and DSBR-deficient cells. Taken together, our results suggest that either IR or DSBR-deficient can lead to an upregulation of miR-21 levels and that miR-21 is associated with IR-induced cell growth in soft agar. These results may help our understanding of DSB-induced tumorigenesis and provide information that could facilitate the development of new strategies to prevent DSB-induced carcinogenesis.

Objective. The primary purpose of this study was to evaluate the reparative efficacy of a novel antimicrobial peptide, Nal-P-113, in shortening the healing time of oral mucosal ulcers by promoting cell proliferation and migration and accelerating the cell cycle. Methods. Cell counting kit-8 (CCK-8) and wound-healing assays were used to evaluate the proliferation and migration of human immortalized oral epithelial cells (HIOECs). The cell cycle distribution of HIOECs was analyzed by flow cytometry. Additionally, the RNA levels of EGF, FGF-2, and TGF-β1 of HIOECs were assessed by real-time PCR. Rats were divided into three groups randomly: (a) blank control group; (b) 20 μg/mL Nal-P-113; and (c) 10 ng/mL rhEGF. An oral mucosal ulcer was induced in every rat by the application of 30% acetic acid. An immunohistochemical assay was used to assess the expression of EGF, FGF-2, and TGF-β1 in the rat oral mucosa. Results. In the CCK-8 assay, the optical density values in the Nal-P-113 and rhEGF groups were found to be significantly higher than that in the blank control group. In addition, the scratch areas in the Nal-P-113 and rhEGF groups were found to be significantly smaller (Pβ1 at 24 h and 48 h. Lastly, the immunohistochemical assay verified that Nal-P-113 changed the expression of the above cytokines in rat mucosal ulcers. Conclusion. Nal-P-113 promoted the repair of oral mucosal ulcers by increasing the EGF and FGF-2 expression and decreasing that of TGF-β1 in HIOECs, accelerating their proliferation and cell cycle progression. The application of Nal-P-113 might serve as an effective therapeutic approach for recurrent aphthous stomatitis.

Despite a number of studies have emphasized the extensive role of microRNA (miRNA) in the development of multiple cancers, the role of miR-30a-5p in the progression of osteosarcoma (OS) and the underlying mechanism are still limited. We detected the expression level of MiR-30a-5p and forkhead box D1 (FOXD1) in Clinical OS specimens and found that miR-30a-5p was significantly decreased while FOXD1 was markedly increased. Dual luciferase assay confirmed that FOXD1 was directly regulated by miR-30a-5p. In vitro assay showed that inhibitior of FOXD1 suppressed cell proliferation, migration and invasion in MG63 and U2OS cells, while overexpression of FOXD1 promoted OS cell proliferation and migration. In vivo assay further showed the inhibition of tumor growth after knockdown of FOXD1. These results suggested that FOXD1 might play key roles in OS development and progression, and was negatively regulated by miR-30a-5p in OS.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Phelps et al., 2016) that described how we intended to replicate selected experiments from the paper ‘Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs’ (Tay et al., 2011). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact PTEN 3’UTR regulation using a reporter, while the original study reported decreased activity when SERINC1, VAPA, and CNOT6L were depleted (Figure 3C; Tay et al., 2011). Using the same reporter, we found decreased activity when ceRNA 3’UTRs were overexpressed, while the original study reported increased activity (Figure 3D; Tay et al., 2011). In HCT116 cells, ceRNA depletion resulted in decreased PTEN protein levels, a result similar to the findings reported in the original study (Figure 3G,H; Tay et al., 2011); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (DicerEx5) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs VAPA or CNOT6L did not statistically impact DU145, wild-type HCT116, or DicerEx5 HCT116 cell proliferation. The original study reported increased DU145 and wild-type HCT116 cell proliferation when these ceRNAs were depleted, which was attenuated in the DicerEx5 HCT116 cells (Figure 5B; Tay et al., 2011). Differences between the original study and this replication attempt, such as variance between biological repeats, are factors that might have influenced the results. Finally, we report meta-analyses for each result.

Minichromosome maintenance protein 5 (MCM5) has been suggested overexpressed in cervical cancer, but the clinical value and biological function of MCM5 in cervical cancer is still unknown. In our study, MCM5 mRNA and protein were significantly overexpressed in cervical cancer tissues and cell lines compared with normal cervical tissues and cell lines, and were obviously increased in cervical adenocarcinoma tissues and cell lines in comparison to cervical squamous cell carcinoma tissues and cell lines. In cervical adenocarcinoma patients, we firstly found that MCM5 expression was closely correlated with clinical stage, lymph node metastasis, distant metastasis and histological grade. Univariate and multivariate analysis showed MCM5 high-expression was an independent unfavorable prognostic factor. In conclusion, MCM5 is associated with the malignant status and poor prognosis in cervical adenocarcinoma patients, and modulates cervical adenocarcinoma cells proliferation.

// Qin Yuan 1, * , Jiayin Song 2, * , Weiwei Yang 3 , Hongyan Wang 1 , Qianyu Huo 1 , Jie Yang 1 , Xiaoxu Yu 3 , Yunde Liu 1 , Chen Xu 4 and Huijing Bao 1 1 School of Laboratory Science, Tianjin Medical University, Tianjin, China 2 The Department of Clinical Laboratory, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China 3 The Department of Laboratory Science, Tianjin Central Hospital of Gynecology Obstetrics, Tianjin, China 4 The Department of Laboratory Science, Tianjin Fourth Central Hospital, Tianjin, China * These authors have contributed equally to this work Correspondence to: Huijing Bao, email: kris_10713@126.com Chen Xu, email: skyalong1028@163.com Yunde Liu, email: yundeliu@163.com Keywords: ovarian cancer, metastasis, CA125, Wnt pathway, cut-off value Received: September 30, 2016 Accepted: May 04, 2017 Published: June 07, 2017 ABSTRACT CA125 has been used extensively to screen for neoplasms, especially in ovarian cancer. The serum CA125 level can be used as a better prognosis evaluation and it may dynamic monitoring the disease progression. We explored the effect of CA125 on ovarian cancer cell migration and its underlying mechanism. Transwell assays showed that exposure to 0.2 μg/ml or 0.4 μg/ml CA125 for 48 h increased migration of A2780 and OVCAR-3 ovarian cancer cells. This effect of CA125 was blocked addition of 200 ng/ml DKK-1, a Wnt pathway inhibitor. Conversely, addition of CA125 reversed the inhibitory effect of Wnt inhibition in A2780 cells pretreated with DKK-1. Examination of CA125 levels in serum from 97 ovarian cancer patients revealed no relationship between a patient’s age or CA125 level currently used clinically for ovarian cancer diagnosis and metastasis. However, using receiver operating characteristic (ROC) curves, we identified a new cut-off value for the serum CA125 concentration (82.9 U/ml) that is predictive of metastasis. The area under the curve is 0.632. This new cut-off value has the potential to serve as a clinically useful indicator of metastasis in ovarian cancer patients.

To investigate the effects of excision repair cross complement-1 (ERCC-1) gene on the proliferation and apoptosis of ovarian cancer SKOV3 cells.Ovarian cancer SKOV3 cell line was cultured and ERCC-1 overexpressing and ERCC-1 silencing cells were constructed via overexpression and silencing of ERCC-1 gene. The expression level of ERCC-1 in cells in each group was detected via quantitative polymerase chain reaction (qPCR). The expressions of apoptosis-related proteins [B-cell lymphoma 2 (Bcl-2), Fas and caspase-3] in cells in each group were detected via Western blotting. In addition, bromodeoxyuridine (BrdU) labeling and direct cell counting methods were performed, and the cell growth curve was drawn. The apoptosis rate in each group was detected via flow cytometry. SPSS 17.0 statistical software was used for analyses. Students' t-test and one-way analysis of variance (ANOVA) were performed. P0.05 suggested statistically significant difference.Overexpression of ERCC-1 could downregulate the expression levels of apoptosis proteins (Fas and caspase-3) and upregulate the expression level of antiapoptotic protein Bcl-2. The apoptosis rate of SKOV3 cells in ERCC-1 overexpressing group was higher than those in ERCC-1 silencing group and control group.ERCC-1 gene was not associated with the proliferation of ovarian cancer cells. ERCC-1 gene can inhibit the apoptosis of ovarian cancer SKOV3 cells.

Annexin A1 (ANXA1) is a member of the annexin superfamily. Previous studies have reported that ANXA1 is highly expressed in various types of malignant tumor; however, its role in the progression of non-small cell lung cancer (NSCLC) remains to be fully clarified. The present study aimed to investigate the oncogenic role of ANXA1 in NSCLC cells in vitro. RNA interference was used to down-regulate ANXA1 expression in A549 and H1299 cells using a small interfering RNA lentiviral vector. Subsequently, cell proliferation and migration were detected using Cell Counting kit-8, clone formation, wound healing and Transwell chamber assays. Successful transfection was confirmed using fluorescence microscopy, which demonstrated that ANXA1 had been efficiently inhibited. ANXA1 knockdown suppressed the proliferation, migration and invasion of NSCLC cells. In conclusion, the present study provided evidence suggesting that ANXA1 may contribute to the growth and invasion of NSCLC cell lines, and ANXA1 may be exploited as an in vitro therapeutic target for the treatment of NSCLC.

Our previous study suggested that minichromosome maintenance protein 5 (MCM5) overexpression was observed in cervical adenocarcinoma and closely associated with advanced clinical stage, more metastatic lymph nodes, present distant metastasis, low histological grade, and poor prognosis. Down-regulation of MCM5 inhibited cervical adenocarcinoma cell proliferation. The purpose of the present study is to search and confirm valuable microRNAs (miRNAs), which target MCM5 to modulate cervical adenocarcinoma cell proliferation. In our results, we found that levels of miR-362-3p expression were reduced in cervical adenocarcinoma tissues and cell lines. Moreover, 3′-UTR of MCM5 had binding site of miR-362-3p through analyzing Targetscan database and miRanda database, and there were an inverse association between miR-362-3p and MCM5 in cervical adenocarcinoma tissues. Furthermore, we verified miR-362-3p directly targeted to 3′-UTR of DCLK1 by luciferase reporter assay, and negatively regulated mRNA and protein expressions of MCM5 by qPCR and Western blot. Then, we conducted gain-of-function study and rescued-function study, and found that miR-362-3p served as a tumor suppressive miRNA to modulate cervical adenocarcinoma cell proliferation through regulating the functional target MCM5. Finally, we analyzed correlations between miR-362-3p expression and clinicopathological characteristics and observed that miR-362-3p low expression was associated with advanced clinical stage and poor prognosis. In conclusion, miR-362-3p is a tumor suppressive miRNA in cervical adenocarcinoma.

Myosin VI has been reported to be associated with the progression of ovarian and prostate cancer. The aim of the present study was to reveal the role of myosin VI in the proliferation of melanoma. Briefly, lentivirus‑mediated short hairpin RNA (shRNA) was designed specifically to silence myosin VI in A375 and A431 cell lines. Expression levels of myosin VI were then analyzed in the two cell lines by quantitative polymerase chain reaction and western blot analyses. Cell viability was assessed using MTT and colony formation assays. In addition, the cell cycle distribution was determined by flow cytometry. The results demonstrated that knockdown of myosin VI significantly suppressed melanoma cell viability and proliferation, and induced cell cycle arrest in G0/G1 phase. To the best of our knowledge, the present study was the first to assess the role of myosin VI in the growth of melanoma. Knowledge of the underlying mechanism of the role myosin VI in skin cancer cells may aid in the development of novel methods of melanoma diagnosis and therapy in the future.

Muscle stem cells (MuSCs, satellite cells) are the major contributor to muscle regeneration. Like most adult stem cells, long-term expansion of MuSCs in vitro is difficult. The in vivo muscle regeneration abilities of MuSCs are quickly lost after culturing in vitro, which prevents the potential applications of MuSCs in cell-based therapies. Here, we establish a system to serially expand MuSCs in vitro for over 20 passages by mimicking the endogenous microenvironment. We identified that the combination of four pro-inflammatory cytokines, IL-1α, IL-13, TNF-α, and IFN-γ, secreted by T cells was able to stimulate MuSC proliferation in vivo upon injury and promote serial expansion of MuSCs in vitro. The expanded MuSCs can replenish the endogenous stem cell pool and are capable of repairing multiple rounds of muscle injuries in vivo after a single transplantation. The establishment of the in vitro system provides us a powerful method to expand functional MuSCs to repair muscle injuries.

MiR-217 can function as an oncogene or a tumour suppressor gene depending on cell type. However, the function of miR-217 in lung cancer remains unclear to date. This study aims to evaluate the function of miR-217 in lung cancer and investigate its effect on the sensitivity of lung cancer cells to cisplatin. The expression of miR-217 was detected in 100 patients by real-time PCR. The effects of miR-217 overexpression on the proliferation, apoptosis, migration and invasion of SPC-A-1 and A549 cells were investigated. The target gene of miR-217 was predicted by Targetscan online software, screened by dual luciferase reporter gene assay and demonstrated by Western blot. Finally, the effects of miR-217 up-regulation on the sensitivity of A549 cells to cisplatin were determined. The expression of miR-217 was significantly lower in lung cancer tissues than in noncancerous tissues (p < 0.001). The overexpression of miR-217 significantly inhibited the proliferation, migration and invasion as well as promoted the apoptosis of lung cancer cells by targeting KRAS. The up-regulation of miR-217 enhanced the sensitivity of SPC-A-1 and A549 cells to cisplatin. In conclusion, miR-217 suppresses tumour development in lung cancer by targeting KRAS and enhances cell sensitivity to cisplatin. Our results encourage researchers to use cisplatin in combination with miR-217 to treat lung cancer. This regime might lead to low-dose cisplatin application and cisplatin side-effect reduction.

Previous studies have shown that cysteine-rich buccal gland protein (CRBGP) from buccal glands of Lampetra japonica could suppress angiogenesis in chick chorioallantoic membrane models. As CRBGP is composed of a pathogenesis-related group 1 (PR-1) domain and a cysteine-rich domain (CRD), which domain accounts for the effects of CRBGP on anti-angiogenesis? In the present study, recombinant PR-1 and CRD (rL-PR-1 and rL-CRD) were obtained. MTT assays showed rL-PR-1 inhibited the proliferation of HUVECs significantly in a dose-dependent manner with an IC50 of 2μM, while rL-CRD had no obviously inhibitory effect on the proliferation of HUVECs, suggested that PR-1 is the main function domain on the anti-angiogenic activity of CRBGP. Similar to CRBGP, rL-PR-1 induced apoptosis in HUVECs in a mitochondrial-dependent pathway by affecting the level of BAX, BCL2 and caspase 3. Also, the cytotoxic property of rL-PR-1 might be one of the factors which suppressed the proliferation of HUVECs. Furthermore, rL-PR-1 blocked the adhesion, migration, invasion and tube formation of HUVECs by disturbing the cytoskeleton arrangement and down-regulating the level of matrix metallo-peptidase 2. In summary, rL-PR-1 has the anti-angiogenic activity which would provide the information on the functions and mechanisms of cysteine-rich secretory protein family members.

Recent epidemiological studies revealed a significant association between oral squamous cell carcinoma (OSCC) and Porphyromonas gingivalis, a major pathogen of periodontal disease. As a keystone pathogen of periodontitis, P. gingivalis is known not only to damage local periodontal tissues, but also to evade the host immune system and eventually affect systemic health. However, its role in OSCC has yet to be defined. To explore the underlying effect of chronic P. gingivalis infection on OSCC and to identify relevant biomarkers as promising targets for therapy and prevention, we established a novel model by exposing human immortalized oral epithelial cells (HIOECs) to P. gingivalis at a low multiplicity of infection (MOI) for 5–23 weeks. The P. gingivalis infected HIOECs were monitored for tumor biological alteration by proliferation, wound healing, transwell invasion, and gelatin zymography assays. Microarray and proteomic analyses were performed on HIOECs infected with P. gingivalis for 15 weeks, and some selected data were validated by quantitative real-time PCR and (or) western blot on cells infected for 15 and 23 weeks. Persistent exposure to P. gingivalis caused cell morphological changes, increased proliferation ability with higher S phase fraction in the cell cycle, and promoted cell migratory and invasive properties. In combining results of bioinformatics analyses and validation assays, tumor-related genes such as NNMT, FLI1, GAS6, lncRNA CCAT1, PDCD1LG2, and CD274 may be considered as the key regulators in tumor-like transformation in response to long-time exposure of P. gingivalis. In addition, some useful clinical biomarkers and novel proteins were also presented. In conclusion, P. gingivalis could promote tumorigenic properties of HIOECs, indicating that chronic P. gingivalis infection may be considered as a potential risk factor for oral cancer. The key regulators detected from the present model might be used in monitoring the development of OSCC with chronic periodontal infection.

Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. Here, we show that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. We show that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or β-tubulin in pins- mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. Our data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells.

The differentially expressed in adenocarcinoma of the lung-1 (DAL-1) protein is a member of the membrane-associated cytoskeleton protein 4.1 family. This protein was previously found to be downregulated or lost in more than half of primary non-small cell lung cancers (NSCLC). In this study, the relationship between DAL-1 expression and NSCLC metastasis was examined. DAL-1 mRNA and protein levels were measured in NSCLC cell lines and in tumor cells isolated from the pleural fluid of NSCLC patients clinically diagnosed with distant metastases to the bone or brain. The results revealed that DAL-1 expression was observed in two (GLC-82 and NCI-H460) out of seven metastatic NSCLC cell lines examined. DAL-1 expression was not observed in the cells isolated from the pleural fluid in nine out of ten patients. Overexpression of DAL-1 in A549 cells, a cell line lacking endogenous DAL-1, inhibited cell migration and invasion by approximately 38 and 48 %, respectively. In contrast, DAL-1 knockdown in NCI-H460 cells enhanced the migration and invasion potential of this cell line 4.6- and 3-fold, respectively. Furthermore, DAL-1 promoter methylation was observed in six of nine pleural fluid NSCLC cell isolates and in two cell lines (A549 and H1299), as evidenced by a lack of endogenous DAL-1. Demethylation in A549 cells successfully restored DAL-1 mRNA and protein expression levels, resulting in a parallel remarkable inhibition of migration and invasion. These results indicated that DAL-1 was pivotal in triggering NSCLC migration and invasion and that loss of DAL-1 expression was due to the epigenetic methylation.

BackgroundAlthough radiotherapy is one of the mainstream approaches for the treatment of head and neck squamous cell carcinoma (HNSCC), this cancer is always associated with resistance to radiation. In this study, the mechanism of action of isoalantolactone as well as its radiosensitizing effect was investigated in UMSCC-10A cells.MethodsThe radiosensitization of UMSCC-10A cells treated with isoalantolactone was analyzed by colony formation assay. The radiosensitization effects of isoalantolactone on cell proliferation, cell cycle and apoptosis regulation were examined by BrdU incorporation assay, DNA content assay and flow cytometry, respectively. Western blotting was performed to determine the effects of isoalantolactone combined with radiation on the protein expression of Mek, extracellular signal-regulated kinase (Erk1/2) as well as phosphorylated Mek and Erk1/2. Erk1/2 knockdown by siRNA was used to confirm that isoalantolactone specifically inhibited the activation of Erk1/2 signaling pathway in UMSCC-10A cells.ResultsIsoalantolactone enhanced the radiosensitivity of UMSCC-10A cells; the sensitivity enhanced ratios (SERs) were 1.44 and 1.63, respectively, for 2.5 and 5 μM. Moreover, isoalantolactone enhanced radiation-induced cell proliferation and apoptosis and cell cycle arrested at G2/M phase. Furthermore, no marked changes were observed in the expression of total Erk1/2 and Mek protein after radiation treatment. However, isoalantolactone was significantly reduced radiation-induced the phosphorylation of Erk1/2, whereas it altered the phosphorylation of Mek to a lesser extent. In addition, the radiosensitivity of UMSCC-10A cells with Erk1/2 knockdown was increased. Isoalantolactone cannot further prevent the proliferation of UMSCC-10A cells with Erk1/2 knockdown which other mechanism regulated cell proliferation.ConclusionOur results suggested that isoalantolactone enhanced radiation-induced apoptosis, cell cycle arrested and reduced the cell proliferation of UMSCC-10A cells via specifically inhibited the phosphorylation of Erk1/2. Thus a low concentration of isoalantolactone may be used to overcome the resistance of UMSCC-10A cells to radiation and may be a promising radiosensitizer in cancer therapy.

We investigated the effects of isoalantolactone on cell growth inhibition and underlying cell death mechanisms in SKOV3 human ovarian cancer cells. The effects of isoalantolactone on cell proliferation and cell cycle were examined by EdU incorporation assay and DNA content assay. Western blotting was performed to determine the protein expression effects of isoalantolactone on cell cycle‑related proteins, autophagic regulators and PEA‑15. Autophagic vacuoles were observed by acridine orange staining. PEA‑15 knockdown by siRNA was used to confirm that PEA‑15 was involved in isoalantolactone‑induced autophagy of SKOV3 cells. Isoalantolactone inhibited the viability and proliferation of SKOV3 cells in a dose‑ and time‑dependent fashion. Isoalantolactone induced cell cycle arrest at G2/M phase and decreased the expression of cell cycle‑related proteins cyclin B1 and CDK1 in SKOV3 cells. Accordingly, isoalantolactone also induced SKOV3 cell autophagy via accumulation of autophagic vacuoles in the cytoplasm, increased Beclin1 protein expression, and increased LC3 cleavage. Furthermore, we observed that isoalantolactone‑induced autophagy was through increased PEA‑15 expression and the phosphorylation of ERK, whereas less change was observed to autophagy on SKOV3 cells through PEA‑15 knockdown by siRNA. Isoalantolactone‑induced autophagic cell death was further confirmed by pretreatment with the autophagy inhibitor 3‑methyladenine (3‑MA). In conclusion, isoalantolactone induced cell cycle arrest and autophagy and inhibited cell proliferation of SKOV3 cells via the upregulated PEA‑15 expression and the phosphorylation of ERK.

The contribution of fiber alignment of scaffold on cellular mechanisms was evaluated by a comparative study of two different cells sourced from cornea. Electrospun scaf-folds with similar composition and comparable fiber size were fabricated into randomly oriented and aligned scaffolds, which bear paralleled degradation of gelatin. Tensile test of wet scaffolds indicated that fiber alignment could influence its mechanical properties. Due to the unidirectional fiber orientation, aligned scaffold exhibited higher tensile modulus, higher break strength, and lower elongation at break than randomly oriented scaffold. The effect of fiber alignment on cells behavior was evaluated by cell morphology, specific protein expression, adhesion, and proliferation. Different corneal cells responded uniquely to fiber alignment of scaffold, keratocytes interacting more favorably on alignment scaffold and corneal epithelial cells more favorably on randomly oriented scaffold. These results confirmed that fiber alignment of scaffold would be benefit for cell proliferation if its contact guidance coincided with the cell shape and cytoskeletal tension. This finding is important to envisage an advanced composite scaffold that incorporates randomly oriented and aligned fibers for the growth and control of different cell types required for the successful development of corneal grafts by tissue engineering. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.

Fission yeast mutants defective in actomyosin ring formation and function exhibit a prolonged G2 delay following cytokinesis failure. This G2 delay depends on the SIN, a signaling network essential for cytokinesis, and the non-essential Cdc14p family phosphatase, Clp1p/Flp1p and has been proposed to signify a cytokinesis checkpoint mechanism. However, the physiological relevance of this proposed Clp1p/Flp1p-dependent checkpoint is unclear because all previous studies were carried out using mutations in essential actomyosin ring components under fully restrictive conditions and thus these cells would have died regardless of the presence of the checkpoint. Here we show that delays in cytokinesis caused by minor perturbations to different components of the cytokinetic machinery, which normally cause only mild defects, become lethal when Clp1p/Flp1p is inactivated. In addition, we show that Clp1p/Flp1p does not function simply to inhibit further rounds of nuclear division, but also allows damaged actomyosin rings to be maintained to facilitate completion of cell division. Ectopic activation of the SIN significantly bypasses the requirement of Clp1p/Flp1p for G2 delay as well as for completion of cytokinesis. We conclude that the Clp1p/Flp1p-dependent cytokinesis checkpoint provides a previously unrecognized cell survival advantage when the cell division apparatus is mildly perturbed.

Skin cancer is the most common malignancy with increasing incidence rates worldwide. The advanced form of skin cancer, melanoma, is resistant to conventional treatment methods, which motivated researchers to identify an alternative effective therapeutic approach. This study was designed to identify the effects of small interfering RNA (si-RNA) mediated silencing of eukaryotic translation initiation factor 3, subunit D (EIF3D) against melanoma cell survival. Briefly, a lentivirus-mediated RNA interference system was employed to knock down EIF3D expression in A375 and A431 melanoma cells. The cell proliferation was analyzed by methylthiazoletetrazolium (MTT) and colony formation assays. The cell cycle progression was investigated using flow cytometry. Results revealed that si-RNA-mediated knockdown of EIF3D significantly reduced the gene and protein expression levels of EIF3D in melanoma cells. Furthermore, knockdown of EIF3D led to a significant reduction in cell proliferation due to G2 /M phase cell cycle arrest. Apparently, the study demonstrated the critical involvement of EIF3D in the survival and progression of melanoma cells and depletion of EIF3D could be developed as a possible therapeutic option in the gene-targeted treatment of melanoma.

We evaluated the in vitro and in vivo inhibition of S180 tumor cell growth of the water extract of Agrimonia pilosa. Inhibitory effect of Agrimonia pilosa on in vitro-cultured S180 cell proliferation was measured by MTT assay; mice model of transplanted tumor was established, after 8 days of continuous administration, the tumors were removed, weighed, and compared with the control group, and the in vivo anti-tumor effect of Agrimonia pilosa on mouse sarcoma S180 was compared using inhibition rate. The in vitro anti-tumor experiment indicated that the inhibition rate gradually increases with the increase of extract concentration and the extension of time, with IC50 175.64, 90.59, and 71.74 µg/ml at 24 h, 4 h, and 72 h respectively. In this study, the in vitro MTT assay was used to determine the inhibitory effect of Agrimony pilosa water extract on S180 tumor cells, the method is simple, reliable, and practical; mice model of in vivo transplanted S180 tumor was established, which allowed direct observation of tumor inhibitory effect, and thus found out that the water extract of Agrimonia pilosa has inhibitory effect on S180 tumor cells.

The development of tumor cell drug resistance is the primary reason for treatment failure in lung cancer chemotherapy. Therefore, overcoming multidrug resistance is currently an urgent issue to be addressed in lung cancer treatment. Sunitinib is a tyrosine kinase inhibitor with confirmed inhibitory effects on tumor growth and metastasis; however, the effects of sunitinib and mechanisms of action in lung cancer multidrug resistance are yet to be determined. The present study was designed to examine the effects of sunitinib and the mechanisms underlying lung cancer multidrug resistance. It was observed that sunitinib was able to improve the sensitivity of A549/DDP lung cancer cells to cisplatin, enhance tumor apoptosis, arrest the cell cycle in G0/G1 phase, upregulate intracellular Rh-123 content, downregulate the expression of P-glycoprotein, multidrug resistance protein 1, multidrug resistance-associated protein 1, lung resistance protein, glutathione-S-transferase, ERCC1, survivin and Bcl-2 in tumor cells, phosphorylation of AKT and extracellular signal-regulated kinase (ERK), glutathione activity, and transcriptional activity of nuclear factor-κB, Twist, Snail and AP-1. The results demonstrated that sunitinib was able to reverse the multidrug resistance of A549/DDP lung cancer cells, which was possibly associated with the downregulation of multidrug resistance-associated gene expression and the inhibition of AKT and ERK phosphorylation.

NOD-like receptors (NLRs) are a family of intracellular proteins that play critical roles in innate immunity against microbial infection. NLRC5, the largest member of the NLR family, has recently attracted much attention. However, in vitro studies have reported inconsistent results about the roles of NLRC5 in host defense and in regulating immune signaling pathways. The in vivo function of NLRC5 remains unknown. Here, we report that NLRC5 is a critical regulator of host defense against intracellular pathogens in vivo. NLRC5 was specifically required for the expression of genes involved in MHC class I antigen presentation. NLRC5-deficient mice showed a profound defect in the expression of MHC class I genes and a concomitant failure to activate L. monocytogenes-specific CD8(+) T cell responses, including activation, proliferation and cytotoxicity, and the mutant mice were more susceptible to the pathogen infection. NLRP3-mediated inflammasome activation was also partially impaired in NLRC5-deficient mice. However, NLRC5 was dispensable for pathogen-induced expression of NF-κB-dependent pro-inflammatory genes as well as type I interferon genes. Thus, NLRC5 critically regulates MHC class I antigen presentation to control intracellular pathogen infection.

It has been reported that metastasis-associated gene 1 (Mta1) is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.

Harnessing the regenerative potential of the central nervous system to repopulate depleted cellular populations from endogenous stem cells would be a novel approach for the treatment of neurological diseases resulting from cell death. Consequently, understanding if and how the central nervous system is capable of such regeneration would determine if such an approach is feasible. In this report, we provide evidence of widespread regenerative response in the spinal cord of amyotrophic lateral sclerosis transgenic mice. However, this regenerative response appears to be largely unproductive. We demonstrate that there is significantly increased gliogenesis, but an absence of convincing neurogenesis. The fact that the neurodegenerative process stimulates a regenerative response suggests that the adult spinal cord has at least limited ability for regeneration. Further studies will determine if this endogenous regenerative process can be enhanced and directed so as to slow or even reverse the natural progression of this devastating disease.

Recent studies using the Drosophila central nervous system as a model have identified key molecules and mechanisms underlying stem cell self-renewal and differentiation. These studies suggest that proteins like Aurora-A, atypical protein kinase C, Prospero and Brain tumor act as key regulators in a tightly coordinated interplay between mitotic spindle orientation and asymmetric protein localization. These data also provide initial evidence that both processes are coupled to cell cycle progression and growth control, thereby regulating a binary switch between proliferative stem self-renewal and differentiative progenitor cell specification. Considering the evolutionary conservation of some of the mechanisms and molecules involved, these data provide a rationale and genetic model for understanding stem cell self-renewal and differentiation in general. The new data gained in Drosophila may therefore lead to conceptual advancements in understanding the aetiology and treatment of human neurological disorders such as brain tumor formation and neurodegenerative diseases.