Stanniocalcin1 (STC1) Inhibits Cell Proliferation and Invasion of Cervical Cancer Cells

STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-κB p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-κB p65 activation in cervical cancer.     

Identification of NDRG1-regulated genes associated with invasive potential in cervical and ovarian cancer cells

N-myc downstream regulated gene 1 (NDRG1) is an important gene regulating tumor invasion. In this study, shRNA technology was used to suppress NDRG1 expression in CaSki (a cervical cancer cell line) and HO-8910PM (an ovarian cancer cell line). In vitro assays showed that NDRG1 knockdown enhanced tumor cell adhesion, migration and invasion activities without affecting cell proliferation. cDNA microarray analysis revealed 96 deregulated genes with more than 2-fold changes in both cell lines after NDRG1 knockdown. Ten common upregulated genes (LPXN, DDR2, COL6A1, IL6, IL8, FYN, PTP4A3, PAPPA, ETV5 and CYGB) and one common downregulated gene (CLCA2) were considered to enhance tumor cell invasive activity. BisoGenet network analysis indicated that NDRG1 regulated these invasion effector genes/proteins in an indirect manner. Moreover, NDRG1 knockdown also reduced pro-invasion genes expression such as MMP7, TMPRSS4 and CTSK. These results suggest that regulation of invasion and metastasis by NDRG1 is a highly complicated process.     

A major constituent of green tea,EGCG, inhibits the growth of a human cervical cancer cell line,CaSki cells,through apoptosis,G(1) arrest,and regulation of gene expression

A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG) has been known to possess antiproliferative properties. In this study, we investigated the anticancer effects of EGCG in human papillomavirus (HPV)-16 associated cervical cancer cell line, CaSki cells. The growth inhibitory mechanism(s) and regulation of gene expression by EGCG were also evaluated. EGCG showed growth inhibitory effects in CaSki cells in a dose-dependent fashion, with an inhibitory dose (ID)(50) of approximately 35 microM. When CaSki cells were further tested for EGCG-induced apoptosis, apoptotic cells were significantly observed after 24 h at 100 microM EGCG. In contrast, an insignificant induction of apoptotic cells was observed at 35 microM EGCG. However, cell cycles at the G1 phase were arrested at 35 microM EGCG, suggesting that cell cycle arrests might precede apoptosis. When CaSki cells were tested for their gene expression using 384 cDNA microarray, an alteration in the gene expression was observed by EGCG treatment. EGCG downregulated the expression of 16 genes over time more than twofold. In contrast, EGCG upregulated the expression of four genes more than twofold, suggesting a possible gene regulatory role of EGCG. This data supports that EGCG can inhibit cervical cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene expression in vitro. Furthermore, in vivo antitumor effects of EGCG were also observed. Thus, EGCG likely provides an additional option for a new and potential drug approach for cervical cancer patients.     

ARL6IP5 reduces cisplatin-resistance by suppressing DNA repair and promoting apoptosis pathways in ovarian carcinoma

Ovarian carcinoma (OC) is the most lethal gynecological malignancy due to frequent recurrence resulting from cisplatin-resistance. ARL6IP5 is a novel gene implicated to suppress cisplatin-resistance by activating apoptosis and inhibiting DNA repair through XRCC1 and PARP1. We investigated the clinicopathological and prognostic significance of the immunohistochemical ARL6IP5 expression on 79 post-chemotherapy OC patient tissue samples; in vitro, the effect of ARL6IP5 overexpression (OE) and knockdown (KD) on cancer hallmark functions and the effect of ARL6IP5 on the expression of DNA repair and apoptosis-related proteins were observed in OC cells and their cisplatin-resistant (CisR) counterparts. ARL6IP5 expression was significantly associated with chemotherapeutic response and was an independent prognosticator of progression-free and overall survival of high-grade serous OC patients. ARL6IP5-OE decreased cellular proliferation, invasion, migration, adhesion, and increased apoptosis (p < 0.05); the opposite was observed for ARL6IP5-KD. Notably, ARL6IP5-OE reduced cisplatin-resistance of both OC and CisR OC cells, while ARL6IP5-KD increased cisplatin-resistance (p < 0.05). ARL6IP5-OE suppressed the expressions of DNA repair proteins and increased those of pro-apoptotic proteins; the opposite was observed for ARL6IP5-KD. The recombinant ARL6IP5 protein (rARL6IP5) had the greatest apoptotic effect among cisplatin and olaparib, in both OC and CisR OC cells; moreover, rARL6IP5 was the only single agent in CisR OC cells to retain higher apoptotic efficacy compared with control (p < 0.05), indicating that the apoptotic pathway influenced by rARL6IP5 remained effective in CisR OC cells compared to cisplatin and olaparib. In conclusion, we demonstrated that ARL6IP5 is an independent prognosticator of OC patients with cellular functions of a tumor-suppressor, possibly influencing the development of cisplatin-resistance and progression of OC cells through regulation of DNA repair and apoptosis. rARL6IP5 had significantly greater apoptotic efficacy compared to conventional chemotherapeutic agents in both OC and CisR OC cells, suggesting that ARL6IP5 may be a valuable novel chemotherapeutic against CisR OC.Subject terms: Prognostic markers, Ovarian cancer     

Fibronectin promotes cervical cancer tumorigenesis through activating FAK signaling pathway

Cervical cancer is a cancer arising from the cervix, and it is the fourth most common cause of death in women. Overexpression of fibronectin 1 (FN1) was observed in many tumors and associated with the survival and metastasis of cancer cells. However, the mechanism by which FN1 promotes cervical cancer cell viability, migration, adhesion, and invasion, and inhibits cell apoptosis through focal adhesion kinase (FAK) signaling pathway remains to be investigated. Our results demonstrated that FN1 was upregulated in patients with cervical cancer and higher FN1 expression correlated with a poor prognosis for patients with cervical cancer. FN1 knockdown by small interfering RNA (siRNA) inhibited SiHa cell viability, migration, invasion, and adhesion, and promoted cell apoptosis. FN1 overexpression in CaSki cell promoted cell viability, migration, invasion, and adhesion, and inhibited cell apoptosis. Further, phosphorylation of FAK, a main downstream signaling molecule of FN1, and the protein expression of Bcl-2/Bax, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), and N-cadherin was upregulated in CaSki cells with FN1 overexpression, but caspase-3 protein expression was downregulated. The FAK phosphorylation inhibitor PF573228 inhibited FN1 overexpression-induced expression of those proteins in CaSki cells with FN1 overexpression. In vivo experiment demonstrated that FN1 knockdown significantly inhibited FN1 expression, phosphorylation of FAK, and tumor growth in xenograft from the nude mice. These results suggest that FN1 regulates the viability, apoptosis, migration, invasion, and adhesion of cervical cancer cells through the FAK signaling pathway and is a potential therapeutic target in the treatment of cervical cancer.     

The coxsackie adenovirus receptor inhibits cancer cell migration

The coxsackie and adenovirus receptor (CAR) is a key factor in adenoviral cancer gene therapy. Reduced expression of CAR during progression of prostate and bladder cancer has been reported. In embryonic development and tissue differentiation, CAR is also differentially expressed. This study suggests a role of CAR expression in cell adhesion and cell motility of human cancer cells. Stable CAR-expressing clones from E-cadherin-deficient A2780 ovarian and CaSki cervical cancer cells with originally low and high CAR expression levels, respectively, were established. CAR reexpression in otherwise singularly growing A2780 parental cells resulted in formation of cell-cell contacts and aggregation in cell clusters. CAR overexpression in cell adhesion-forming CaSki cells did not result in morphological changes. Migration of the A2780 CAR clones was strongly reduced as characterized by using spread-off assays. Using migration chambers, formation of satellite colonies was reduced by 97% in CAR-expressing A2780 cell clones and by 23% in CAR-expressing CaSki cell clones. Parental A2780 and CaSki cells selected for high migratory ability by using migration chambers expressed endogenous CAR on lower levels associated with lower adenoviral transduction efficiency. Our data suggest CAR as a new inhibitory factor for cancer cell migration.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells

Wogonin is a flavonoid compound extracted from Scutellaria baicalensis and is well known as a benzodiazepine receptor ligand with anxiolytic effects. Many recent studies have demonstrated that wogonin modulates angiogenesis, proliferation, invasion, and tumor progress in various cancer tissues. We further explored the mechanism of action of wogonin on cervical cancer cells that contain or lack human papillomavirus (HPV) DNA. Wogonin was cytotoxic to HPV 16 (+) cervical cancer cells, SiHa and CaSki, but not to HPV-negative cells. We demonstrated that wogonin induced apoptosis by suppressing the expressions of the E6 and E7 viral oncogenes in HPV-infected cervical cancer CaSki and SiHa cells. The modulation of p53 and protein retinoblastoma (pRb) were also triggered by the suppression of E6 and E7 expressions. However, p53 was not altered in HPV-negative cervical cancer C33A cells. Moreover, wogonin modulated the mitochondrial membrane potential and the expression of pro- and anti-apoptotic factors such as Bax and Bcl-2. Wogonin also provoked the cleavage of caspase-3, caspase-9, and poly ADP ribose polymerase. After transfection of siRNAs to target E6 and E7, additional restoration of p53 and pRb was not induced, but processing of caspases and PARP was increased compared with wogonin treatment alone. Together, our findings demonstrated that wogonin effectively promotes apoptosis by downregulating E6 and E7 expressions and promoting intrinsic apoptosis in human cervical cancer cells.     

Inhibition of BAP31 expression inhibits cervical cancer progression by suppressing metastasis and inducing intrinsic and extrinsic apoptosis

Cervical cancer is reported as one of the most lethal types of cancer among female. However, extensive studies of the molecular mechanisms that regulate the progression of cervical cancer are still required. B-cell associated protein (BAP)-31 is a 28-kDa integral membrane protein in the endoplasmic reticulum (ER), playing essential role in modulating various physiological processes. The present study indicated that BAP31 was a novel gene associated with cervical cancer development. Here, we demonstrated that BAP31 was significantly increased in human cervical cancer specimens, which was positively correlated to histological grade of the cancer. BAP31 knockdown suppressed cell proliferation, clonogenic ability and metastasis-associated traits in vitro, as well as carcinogenesis and pulmonary metastasis in vivo. Further studies indicated that the expression levels of transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-2, MMP-9, Rho-associated protein kinase 1 (ROCK1), α-smooth muscle actin (α-SMA), Vimentin and N-cadherin were markedly reduced by BAP31 knockdown in cervical cancer cells. In addition, intrinsic and extrinsic apoptosis was significantly induced in BAP31 knockdown cells, as evidenced by the increased expression of cleaved Caspase-8/-9/-3 and poly (ADP-ribose) polymerases (PARP). Notably, suppressing the activities of Caspase-8/-9 and ?3 obviously diminished BAP31 silence-triggered apoptosis. Together, these findings highlighted an essential role for BAP31 in the modulation of tumorigenesis and metastatic potential of cervical cancer, and demonstrated a promising application of BAP31 in cancer prevention.     

LncRNA DANCR promotes cervical cancer progression by upregulating ROCK1 via sponging miR-335-5p

Emerging evidence highlights the key regulatory roles of long noncoding RNAs (lncRNAs) in the initiation and progression of numerous malignancies. The lncRNA identified as differentiation antagonizing nonprotein coding RNA (DANCR) is a novel lncRNA widely involved in the development of multiple human cancers. However, the function of DANCR and its potential molecular mechanism in cervical cancer remain unclear. In this study, we discovered that DANCR was significantly elevated in cervical cancer tissues and cells, and was closely correlated with poor prognosis of cervical cancer patients. In addition, knockdown of DANCR inhibited proliferation, migration, and invasion of cervical cancer cells in vitro, indicating that DANCR functioned as an oncogene in cervical cancer. Moreover, we verified that DANCR could directly bind to miR-335-5p, isolating miR-335-5p from its target gene Rho-associated coiled-coil containing protein kinase 1 (ROCK1). Functional analysis showed that DANCR regulated ROCK1 expression by competitively binding to miR-335-5p. Further cellular behavioral experiments revealed that miR-335-5p mimics and ROCK1 knockdown reversed the effects of upregulated DANCR on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer cells by rescue assays. In summary, this study demonstrated that DANCR promoted cervical cancer progression by functioning as a competing endogenous RNA (ceRNA) to regulate ROCK1 expression via sponging miR-335-5p, suggesting a novel potential therapeutic target for cervical cancer.     

靶向HPV16-E6的siRNA对宫颈癌CaSki细胞的抑制作用

以RNA干扰技术为手段,HPV编码的癌蛋白E6为靶标．探讨靶向HPV16-E6的siRNA对宫颈癌细胞生物学行为的影响,并试图阐明该实验的临床意义．构建靶向HPV16-E6的siRNA表达载体,应用体外转染试剂转染HPV16-E6阳性的宫颈癌CaSki细胞．以RT—PCR检测CaSki细胞中E6蛋白的mRNA的表达．借助细胞色素c测定来分析细胞凋亡相关分子的表达和活性．从而研究靶向HPV16-E6的siRNA诱导细胞凋亡的分子机制．RT．PCR检测结果表明,将靶向HPV16-E6的siRNA的表达载体瞬时转染到HPV16-E6阳性的CaSki细胞后,其所舍E6蛋白质和mRNA的表达下调;Westernblotting栓出抑凋亡蛋白Bcl．2的表达亦告下调;细胞色素C释放实验结果显示,HPV16-E6siRNA能够诱导细胞色素c从线粒体释放到细胞浆中。从而诱导细胞凋亡．靶向HPV16-E6的siRNA能够有效抑制细胞增殖并诱导细胞凋亡．靶向HPV16-E6的siRNA为研究重要致瘤蛋白HPV16-E的功能开辟了新途径,给HPV16-E6阳性肿瘤的靶向基因治疗提供新的实验依据．并探索了HPV感染及宫颈癌的新基因疗法．     

Role and mechanism of miR-4778-3p and its targets NR2C2 and Med19 in cervical cancer radioresistance

The aim of this study was to investigate the effect of miR-4778-3p on the radiosensitivity of cervical cancer cells and to elucidate the underlying mechanism. Tissue samples were collected from eight patients with cervical cancer prior to chemoradiotherapy. MicroRNA chip analyses, RT-PCR, gene transfection, CCK8, wound healing and Transwell assays, colony-forming assay, western blot, and the Dual-Luciferase Reporter Assay System were used to evaluate the role of miR-4778-3p in cervical cancer radiosensitivity and its relationships with target molecules NR2C2 and Med19. Thirty-two differentially expressed miRNA molecules (fold-change?>?2; p?<?0.05) associated with cervical cancer radioresistance were identified. The expression of miR-4778-3p was significantly lower in recurrent or metastatic patients than in control subjects. In vitro studies using radioresistant HeLa and SiHa cervical cancer cell lines showed that miR-4778-3p upregulation significantly inhibited cell proliferation, invasiveness, and migration after irradiation. There was also a significant increase in apoptosis and a significant decrease in the proportion of cells at the G2/M phase. Further, miR-4778-3p upregulation led to increased expression of apoptosis-related molecules, such as Bax, Caspase-3, Caspase-8, and Caspase-9. Reporter gene assays showed that miR-4778-3p bound specifically to NR2C2 and Med19 and negatively regulated their expression. Thus, miR-4778-3p reduces the vitality, proliferation, and migration of radioresistant cervical cancer cells and may regulate the radiosensitivity of cervical cancer by targeting and regulating NR2C2 and Med19 expression.     

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

UHRF1基因在乳腺癌循环肿瘤细胞中的表达研究

外周血液中乳腺循环癌肿瘤细胞的生物表征与转移性乳腺癌的严重程度密切相关,本研究的目的在于结合体外细胞实验探讨UHRF1基因对乳腺癌进展的意义。多重RNA原位分析乳腺癌循环肿瘤细胞(circulating tumor cells,CTCs)中UHRF1的表达;MTT法检测UHRF1基因转染对正常乳腺细胞增殖的影响;蛋白免疫印迹检测UHRF1基因转染对正常乳腺细胞中Bax蛋白和Bcl-2蛋白的影响;Caspase-3检测试剂盒检测正常乳腺细胞中Caspase-3活性;Transwell侵袭实验和划痕愈合实验检测UHRF1基因转染对正常乳腺细胞侵袭和迁移能力的影响。研究发现,UHRF1 RNA水平在乳腺癌循环肿瘤细胞中高表达;UHRF1基因增加正常乳腺细胞增殖率;UHRF1基因降低正常乳腺细胞中Caspase-3活性;UHRF1基因降低正常乳腺细胞中Bax蛋白的表达,增加Bcl-2蛋白的表达;UHRF1基因增强正常乳腺细胞侵袭和迁移能力。本研究初步说明,UHRF1可促进正常乳腺细胞增殖,抑制正常乳腺细胞凋亡,增强正常乳腺细胞侵袭和迁移能力。     

NGX6 gene inhibits cell proliferation and plays a negative role in EGFR pathway in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is a common cancer in South China but is rare in other parts of the world. A novel NPC-related gene was isolated by location candidate cloning strategy, whose expression was down-regulated in NPC. This gene was designated human NGX6 (Genbank accession AF188239) and encoded a predicted protein of 338 amino acids that harbors an EGF-like domain. The effects of NGX6 on cells from human NPC cell line HNE1 were investigated. The cells transfected with NGX6 had a markedly high expression of NGX6, leading to significant decrease in cell proliferation and the capability to form colonies in soft agar, delaying the G0-G1 cell cycle progression. Flow cytometry assay indicated that the expression of cyclin D1 significantly decreased in NGX6-transfected HNE1 cells as well as cyclin A and E. There was a delay in tumor formation and a dramatic reduction in tumor size when cells transfected with NGX6 were injected into nude mice. In another way, we found NGX6 played a negative role in EGFR Ras/Mek/MAPK pathway. We propose that NGX6, as an EGF-like domain gene, could delay cell cycle G0-G1 progression and thus inhibit cell proliferation by negatively regulating EGFR pathway in NPC cells and down-regulating the expression of cyclin D1 and E.