Regulation of human Cripto-1 gene expression by TGF-beta1 and BMP-4 in embryonal and colon cancer cells

Human Cripto-1 (CR-1) is a cell membrane protein that is overexpressed in several different types of human carcinomas. In the present study we investigated the mechanisms that regulate the expression of CR-1 gene in cancer cells. We cloned a 2,481 bp 5'-flanking region of the human CR-1 gene into a luciferase reporter vector and transfected NTERA-2 human embryonal carcinoma cells and LS174-T colon cancer cells to test for promoter activity. Activity of CR-1 promoter in both cell lines was modulated by two TGF-beta family members, TGF-beta1 and BMP-4. In particular, TGF-beta1 significantly up-regulated CR-1 promoter activity, whereas a dramatic reduction in CR-1 promoter activity was observed with BMP-4 in NTERA-2 and LS174-T cells. Changes in the CR-1 promoter activity following TGF-beta1 and BMP-4 treatments correlated with changes in CR-1 mRNA and protein expression in NTERA-2 and LS174-T cells. We also identified three Smad binding elements (SBEs) within the CR-1 promoter and point mutation of SBE1 (-2,197/-2,189) significantly reduced response of the CR-1 promoter to both TGF-beta1 and BMP-4 in NTERA-2 and LS174-T cells. Chromatin immunoprecipitation assay also demonstrated binding of Smad-4 to a CR-1 promoter DNA sequence containing SBE1 in LS174-T cells. Finally, BMP-4 inhibited migration of LS174-T cells and F9 mouse embryonal carcinoma cells by downregulation of CR-1 protein. In conclusion, these results suggest a differential modulation of CR-1 gene expression in embryonal and colon cancer cells by two different members of the TGF-beta family.     

Up-regulated miR155 Reverses the Epithelial-mesenchymal Transition Induced by EGF and Increases Chemo-sensitivity to Cisplatin in Human Caski Cervical Cancer Cells

The epithelial-mesenchymal transition (EMT) induced by EGF promotes cervical cancer progression; however, the mechanisms underlying the EGF-induced EMT remain unclear. In this study, we reported that miR155 overexpression suppressed EGF-induced EMT, decreased migration/invasion capacities, inhibited cell proliferation and increased the chemo-sensitivity to DDP in human Caski cervical cancer cells. Further, the overexpression of miR155 increased TP53 expression but reduced SMAD2, and CCND1 expression levels. These data suggest that miR155 negatively regulates EGF-induced EMT. We conclude that miR155 does not act as an oncogene but as a tumour suppressor in Caski cells.     

用pLNCX2携带人MCPH1基因shRNA重组逆转录病毒载体的构建及鉴定

目的建立逆转录病毒介导的MCPH1基因RNA干扰表达体系并观察其在宫颈癌Caski细胞中对MCPH1表达的影响。方法将人MCPH1基因RNA干扰双链DNA片段重组到逆转录病毒质粒pLNCX2中,构建携带人MCPHI基因RNA干扰的逆转录病毒载体pLNCX2-shRNA—MCPH1,经PT67细胞包装后,产生的重组逆转录病毒感染宫颈癌细胞株Caski细胞,并用G418筛选产生稳定的细胞克隆,用RT—PCR和Western印迹检测细胞中MCPH1mRNA和蛋白表达的变化。结果重组逆转录病毒质粒经测序鉴定正确,逆转录病毒感染Caski细胞后用G418筛选出稳定的细胞克隆,RT—PCR和Western印迹检测人MCPH1mRNA和蛋白表达水平明显低于阴性对照组和未干扰组。结论携带人MCPHI基因RNA干扰双链DNA片段的逆转录病毒感染Caski细胞后能明显抑制MCPHImRNA和蛋白表达,为进一步研究MCPH1在宫颈癌中的作用奠定了基础。     

长链非编码RNA Dleu2 对宫颈癌细胞增殖、迁移及侵袭的影响

目的:研究长链非编码RNA(long non-coding RNA,lnc RNA)Dleu2对宫颈癌细胞增殖、迁移和侵袭能力的影响。方法:利用高通量Lnc RNA芯片技术检测10例宫颈癌组织及对应的癌旁组织,筛选得到一批表达水平具有显著差异的Lnc RNA,进一步针对可能具有生物学功能的Lnc RNA-Dleu2,利用q-PCR验证其在癌组织样本中的相对低表达。再通过在细胞内过表达Lnc RNA-Dleu2研究其对宫颈癌Hela和Caski细胞系增殖、迁移和侵袭的影响。结果:q-PCR结果验证了Lnc RNA芯片筛选的结果,即相较于癌旁组织和正常宫颈上皮细胞系,Lnc RNA-Dleu2在宫颈癌组织和细胞中均低表达。CCK8和克隆形成实验结果显示,过表达Lnc RNA-Dleu2能显著抑制Hela和Caski细胞增殖能力(P0.01);细胞划痕实验结果显示,过表达Lnc RNA-Dleu2能显著抑制Hela和Caski细胞增殖和迁移能力;Matrigel细胞侵袭实验结果显示,过表达Lnc RNA-Dleu2能显著抑制Hela和Caski细胞的侵袭能力(P0.01)。结论:Lnc RNA-Dleu2在宫颈癌中相对低表达,提高Dleu2表达水平能够抑制宫颈癌细胞系Hela和Caski的增殖、迁移和侵袭能力。     

Cripto-1 overexpression leads to enhanced invasiveness and resistance to anoikis in human MCF-7 breast cancer cells

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-CFC protein that has been shown to signal through nodal/Alk-4, PI3K/Akt, and/or ras/raf/MEK/MAPK pathways in mammalian cells, and that is frequently expressed in human primary breast carcinomas. In the present study, the human estrogen receptor positive, MCF-7 breast cancer cell line, that expresses low levels of endogenous CR-1, was transfected with a CR-1 expression vector. MCF-7 CR-1 cells expressed high levels of a 25 kDa recombinant CR-1 protein that was not detected in MCF-7 cells transfected with a control vector (MCF-7 neo). Overexpression of CR-1 did not induce an estrogen independent phenotype in MCF-7 cells. In fact, MCF-7 CR-1 cells showed a response to exogenous estrogens that was similar to MCF-7 neo cells, and failed to grow in immunosuppressed mice in absence of estrogen stimulation. However, MCF-7 CR-1 cells showed a rate of proliferation in serum free conditions, and an ability to form colonies in soft-agar that were higher as compared with MCF-7 neo cells. More importantly, overexpression of CR-1 enhanced the resistance to anoikis and the invasion ability of MCF-7 cells. MCF-7 CR-1 cells showed levels of activation of both Akt and Smad-2 that were significantly higher as compared with MCF-7 neo. These findings suggest that CR-1 overexpression might be associated with the progression towards a more aggressive phenotype in breast carcinoma, through the activation of both Akt and Smad-2 signalling pathways.     

BRCA1 regulates follistatin function in ovarian cancer and human ovarian surface epithelial cells

Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells.     

miR-107 Activates ATR/Chk1 Pathway and Suppress Cervical Cancer Invasion by Targeting MCL1

MicroRNAs (miRNAs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRNAs may function as oncogenes or tumor suppressors. Here we showed that miR-107 directly targeted MCL1 and activated ATR/Chk1 pathway to inhibit proliferation, migration and invasiveness of cervical cancer cells. Moreover, we found that MCL1 was frequently up-regulated in cervical cancer, and knockdown of MCL1 markedly inhibited cancer cell proliferation, migration and invasion, whereas ectopic expression of MCL1 significantly enhances these properties. The restoration of MCL1 expression can counteract the effect of miR-107 on the cancer cells. Together, miR-107 is a new regulator of MCL1, and both miR-107 and MCL1 play important roles in the pathogenesis of cervical cancer. We have therefore identified a mechanism for ATR/Chk1 pathway which involves an increase in miR-107 leading to a decrease in MCL1. Correspondingly, our results revealed that miR-107 affected ATR/Chk1 signalling and gene expression, and implicated miR-107 as a therapeutic target in human cervical cancer. We also demonstrated that taxol attenuated migration and invasion in cervical cancer cells by activating the miR-107, in which miR-107 play an important role in regulating the expression of MCL1. Elucidation of this discovered MCL1 was directly regulated by miR-107 will greatly enhance our understanding of the mechanisms responsible for cervical cancer and will provide an additional arm for the development of anticancer therapies.     

The papillomavirus E7 oncoprotein is ubiquitinated by UbcH7 and Cullin 1- and Skp2-containing E3 ligase

Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2(-/-) mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.     

Fibrodysplasia Ossificans Progressiva-related Activated Activin-like Kinase Signaling Enhances Osteoclast Formation during Heterotopic Ossification in Muscle Tissues

Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues.     

Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor subtypes to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells.

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e. Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [(3)H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca(2+)](i) increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca(2+) responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1-100 nM) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca(2+) system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.     

Antitumor effects of IL-6 on murine liver tumor cells in vivo

IL-6 is a pleiotropic cytokine that is capable of modulating the diverse functions of hepatocytes such as acute phase responses and inflammation in the liver. To learn its antitumor effects in vivo, the cDNA of IL-6 was transfected into murine liver cells, TIB cells. IL-6-transfected TIB cells (TIB73-IL-6 or TIB75-IL-6) produced much higher levels of IL-6 compared with vector-transfected TIB cells (TIB73-vec or TIB75-vec). To investigate the effects of IL-6 on TIB tumor growth in vivo, IL-6-transfected TIB cells or vector-transfected TIB cells were injected subcutaneously into syngeneic mice. Vector-transfected TIB cells grew rapidly 3 weeks after injection, but IL-6-transfected TIB cells did not grow at all for up to 6 weeks. Pathologically, IL-6-transfected TIB cells demonstrated a severe necrosis and apoptotic pattern. Taken together, these results indicate that IL-6 functions as a growth inhibiting factor in vivo, and another biological role of IL-6 in the liver is suggested.     

MicroRNA-30e Functions as a Tumor Suppressor in Cervical Carcinoma Cells through Targeting GALNT7

Cervical cancer is the third most common cancer in women worldwide. However, the underlying mechanism of occurrence and development of cervical cancer is obscure. In this study, we observed that miR-30e was downregulated in clinical cervical cancer tissues and cervical cancer cells. Next, overexpression of miR-30e reduced the cervical cancer cell growth through MTT, colony formation, EdU, and Transwell assay in SiHa and Caski cells. Subsequently, UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) was identified as a potential miR-30e target by bioinformatics analysis. Moreover, we showed that miR-30e was able to bind to the 3′UTR of GALNT7 by luciferase reporter assay. In addition, the mRNA and protein levels of GALNT7 in cervical cancer cells were downregulated by miR-30e. And we validated that downregulation of GALNT7 repressed the proliferation of SiHa and Caski cells by MTT, colony formation, and Transwell assay. We identified that the restoration of GALNT7 expression was able to counteract the effect of miR-30e on cell proliferation of cervical cancer cells. Furthermore, we found that the expression levels of GALNT7 were frequently upregulated and negatively correlative to those of miR-30e in cervical cancer tissues. In addition, we validated that restoration of GALNT7 rescued the miR-30e–suppressed growth of cervical cancer xenografts in vivo. In conclusion, the current results suggest that miR-30e may function as tumor suppressors in cervical cancer through downregulation of GALNT7. Both miR-30e and its novel target, GALNT7, may play an important role in the process of cervical cancer.     

Overexpression of LINC00037 represses cervical cancer progression by activating mTOR signaling pathway

Long non-coding RNAs have been reported to play crucial roles in tumorigenesis including cervical cancer. LINC00037 has been identified as a significant regulator in several cancers. Our study was aimed to investigate the function of LINC00037 in cervical cancer progression. LINC00037 was significantly downregulated in human cervical cancer cells (HeLa, HCC94, HT-3, Caski, and SiHa cells) compared with the ectocervical epithelial cells (End1/E6E7 cells). Overexpression of LINC00037 was able to inhibit cervical cancer cell proliferation, induce cell apoptosis, and restrain the cell migration/invasion capacity. Reversely, knockdown of LINC00037 exhibited an opposite process in vitro. mTOR has been recognized as an atypical serine/threonine kinase that is involved in regulating significant cellular functions. In our present study, we observed that the mTOR signaling pathway was strongly activated in human cervical cancer cells. Meanwhile, upregulation of LINC00037 contributed to the inactivation of mTOR signaling whereas downregulation of LINC00037 activated the pathway. Subsequently, in vivo animal models using SiHa cells were established and we proved that LINC00037 repressed cervical cancer progression via targeting the mTOR signaling pathway. All these findings implied that LINC00037 could regulate cervical cancer pathogenesis via mTOR signaling. In conclusion, a novel role of LINC00037 was manifested in cervical cancer progression.