靶向沉默NOB1基因对乳腺癌细胞MCF-7生长和周期分布的影响

背景与目的:NOB1(NIN1/RPN12 binding protein 1 homolog)是2005年新克隆的一个基因,属于RNA结合蛋白,该类蛋白的功能与恶性肿瘤的发生密切相关。本研究旨在观察利用慢病毒介导的RNAi沉默NOB1基因对人乳腺癌MCF-7细胞增殖及细胞周期的影响。方法:包装表达NOB1短发夹RNA(shRNA)的慢病毒,感染MCF-7细胞,实时定量PCR和蛋白质印迹法(Western blot)验证NOB1的抑制效率;MTT和克隆形成实验检测NOB1对细胞增殖能力的影响;流式细胞术检测细胞周期的变化。结果:NOB1-shRNA慢病毒感染3 d后,可显著下调MCF-7细胞NOB1 mRNA和蛋白的表达水平,显著抑制细胞增殖和体外成瘤能力,并导致细胞周期分布紊乱,G0/G1期及G2/M期细胞增加,S期细胞减少。结论:NOB1基因通过调节细胞周期分布促进乳腺癌细胞恶性增殖,可能是乳腺癌基因治疗的分子靶点。     

Gene amplification CCNE1 is related to poor survival and potential therapeutic target in ovarian cancer

BACKGROUND:

This study examined the clinical significance of CCNE1 (Cyclin E1) amplification and assessed whether CCNE1 is a potential therapeutic target in ovarian cancer.

METHODS:

CCNE1 expression and amplification in ovarian cancer was assessed by immunohistochemistry, fluorescence in situ hybridization and clinical data collected by retrospective chart review. CCNE1 gene knockdown using silencing RNA and a CCNE1 gene transfection system were used to asses CCNE1 function in tissue samples of ovarian cancer.

RESULTS:

Gene amplification was identified in 18 (20.4%) of 88 ovarian carcinomas. CCNE1 copy number significantly correlated with CCNE1 protein expression (r = 0.522, P < .0001). CCNE1 amplification significantly correlated with shorter disease‐free survival and overall survival (P < .001). There were nonsignificant trends between high protein expression and poor disease‐free survival (P = .2865) and overall survival (P = .1248). Multivariate analysis showed gene amplification was an independent prognostic factor for disease‐free survival and overall survival after standard platinum‐taxane chemotherapy (P = .0274, P = .0023). Profound growth inhibition and apoptosis were observed in silencing RNA‐treated cancer cells with gene amplification compared with results in cancer cells with CCNE1 moderate expression without gene amplification or with low CCNE1 expression. CCNE1 overexpression stimulated proliferation in ovarian cancer cell lines ES2 and TOV‐21G, which have lower endogenous CCNE1 expression.

CONCLUSIONS:

These findings indicate that CCNE1 overexpression is critical to growth and survival of ovarian cancer tumors with CCNE1 gene amplification. Furthermore, they suggest that CCNE1 silencing RNA‐induced phenotypes depend on amplification status of ovarian cancers. Therefore, CCNE1‐targeted therapy may benefit ovarian cancer patients with CCNE1 amplification. Cancer 2010. © 2010 American Cancer Society.     

17β‐estradiol upregulates GREB1 and accelerates ovarian tumor progression in vivo

Exogenous 17β‐estradiol (E2) accelerates the progression of ovarian cancer in the transgenic tgCAG‐LS‐TAg mouse model of the disease. We hypothesized that E2 has direct effects on ovarian cancer cells and this study was designed to determine the molecular mechanisms by which E2 accelerates ovarian tumor progression. Mouse ovarian cancer ascites (MAS) cell lines were derived from tgCAG‐LS‐TAg mice. Following intraperitoneal engraftment of two MAS cell lines, MASC1 and MASE2, into SCID mice, exogenous E2 significantly decreased the survival time and increased the tumor burden. Microarray analysis performed on MASE2‐derived tumors treated with E2 or placebo showed that E2 treatment caused the upregulation of 197 genes and the downregulation of 55 genes. The expression of gene regulated by estrogen in breast cancer 1 (Greb1) was upregulated in mouse tumors treated with E2 and was overexpressed in human ovarian cancers relative to human ovarian surface epithelium, suggesting a role for GREB1 in human ovarian tumor progression. RNA interference‐mediated knockdown of GREB1 in MASE2 cells decreased their proliferation rate in vitro and increased survival time in mice engrafted with the cells. These results emphasize the importance of E2 in ovarian tumor progression and identify Greb1 as a novel gene target for therapeutic intervention.     

Lost expression of <Emphasis Type="Italic">DCC</Emphasis> gene in ovarian cancer and its inhibition in ovarian cancer cells

Ovarian cancer is a leading cause of cancer-related women mortality in China. In recent years, the molecular mechanisms involved in ovarian carcinoma development and/or progression have been intensely studied, and several genes have been identified. Deleted in Colorectal Carcinoma (DCC), is an important tumor suppressor gene, which is inactivated in many kinds of tumors, and its function(s) is not clarified. Even though the lost expression of DCC occurred in later stages of multistep colorectal carcinogenesis, its contribution to the onset or progression of ovarian cancer is not fully understood. To investigate DCC expression in ovarian cancer, we studied 254 clinical samples by RT–PCR. Our results revealed that 52% malignant ovarian cancer did not express DCC gene. By contrast, DCC expression was observed in all normal ovary tissues and 80% benign ovarian tumors. Obviously, there was a significant correlation between DCC expression and ovarian cancer, especially in the epithelial ovarian cancer. The present study also suggested that the loss expression of DCC occurred more frequently in the cases of later clinical stage, higher pathological grade, and poorer prognosis. In the other part of this study, we further explored DCC expression after transfection in two kinds of ovarian caner cell lines, namely SKOV3 cell and HO-8910 cell, using RT–PCR and immunocytochemistry. The results indicated that DCC expressed in SKOV3-DCC and HO-8910-DCC cells, and ultrastructural analysis showed the appearance of apoptotic features in them. Furthermore, cell growth was markedly down-regulated in above groups of cells, indicating that transfection with the DCC constructs can suppress the growth of tumor cells. In conclusion, our results suggest an association of lost expression of DCC with the ovarian cancer, and DCC gene may inhibit the growth of ovarian carcinoma cells. However, this result needs further trials with a larger sample.     

NOB1 in Non-small-cell Lung Cancer: Expression Profile and Clinical Significance

Nin one binding (NOB1) gene has been reported up-regulated in several types of cancer. The aim of this study was to investigate the expression profile of NOB1 in non-small-cell lung cancer (NSCLC) and assess the clinical significance. qRT-PCR was used in the detection of NOB1 mRNA expression both in NSCLC tissue and in adjacent normal lung tissue. Western blot analysis and immunohistochemistry were used in the detection of NOB1 protein expression. The clinicopathological implications of NOB1 were analyzed statistically. It was confirmed by RT-qPCR that expression of NOB1 mRNA in NSCLC cells was higher than in human lung cells (P?<?0.05), and NOB1 mRNA was also over-expressed in NSCLC tissue when compared with adjacent tissue and normal lung tissue (P?<?0.05). Western blot analysis showed that NOB1 protein was significant increased in NSCLC cell lines compared with human lung cell line. Western blot analysis and immunohistochemistry showed that NOB1 protein was significant increased in NSCLC tissue compared with adjacent tissue and normal lung tissue (P?<?0.05). There were significant associations between NOB1 expression and TNM stage, lymph node metastasis, and histopathological grade (P?<?0.05), but not gender, age, smoke, or tumor diameter (P?>?0.05). Our results suggest that enhanced expression of NOB1 gene plays an important role in the occurrence and development of NSCLC. NOB1 may be a potential therapeutic target in NSCLC.     

siRNA mediated silencing of NIN1/RPN12 binding protein 1 homolog inhibits proliferation and growth of breast cancer cells

The gene encoding the Nin one binding (NOB1) protein which plays an essential role in protein degradation has been investigated for possible tumor promoting functions. The present study was focused on NOB1 as a possible therapeutic target for breast cancer treatment. Lentivirus mediated NOB1 siRNA transfection was used to silence the NOB1 gene in two established breast cancer cell lines, MCF-7 and MDA-MB-231, successful transfection being confirmed by fluorescence imaging. NOB1 deletion caused significant decline in cell proliferation was observed in both cell lines as investigated by MTT assay. Furthermore the number and size of the colonies formed were also significantly reduced in the absence of NOB1. Moreover NOB1 gene knockdown arrested the cell cycle and inhibited cell cycle related protein expression. Collectively these results indicate that NOB1 plays an essential role in breast cancer cell proliferation and its gene expression could be a therapeutic target.     

RNA干扰沉默基质金属蛋白酶2基因对卵巢癌OVCAR-3细胞诱导体外血管形成能力的影响

 目的　探讨沉默基质金属蛋白酶2（MMP-2）基因对卵巢癌OVCAR-3细胞血管内皮生长因子（VEGF）表达的影响,以及对细胞诱导的体外血管形成能力的影响。方法　合成特异性靶向基因的小干扰RNA （siRNA）并转染卵巢癌OVCAR-3细胞（MMP-2沉默组）,以非特异性序列转染细胞作为阴性对照组,以培养液代替转染siRNA的试剂作为空白对照组;采用实时荧光定量PCR和Western blot法分别检测转染后48 h MMP-2及VEGF的mRNA和蛋白表达水平,通过体外血管形成实验检测卵巢癌OVCAR-3细胞诱导的体外血管形成能力。结果　与阴性对照组相比,MMP-2沉默组转染48 h,OVCAR-3细胞MMP-2和VEGF mRNA表达量分别下降了78.8 %和75.5 %（P＜0.05）,蛋白表达量分别下降了81.2 %和78.3 % （P＜0.05）;体外血管形成实验显示,沉默MMP-2基因后卵巢癌OVCAR-3细胞诱导的体外血管形成能力明显降低（P＜0.05）。结论　沉默MMP-2基因可抑制卵巢癌细胞VEGF表达,并能够抑制卵巢癌细胞诱导的体外血管形成能力,抑制MMP-2基因有助于抗血管形成,可能成为卵巢癌基因治疗的新靶点。     

Retrovirus-Mediated CXCR4-siRNA Can Inhibit the Invasion of Prostate Carcinoma In Vitro

Objective： CXCR4 is a potential target for cancer gene therapy. In this study, an RNA interference retrovirus vector targeting CXCR4 gene was constructed, and its influence on the invasion of prostate cancer cells by depressing CXCR4 gene expression was analyzed. Methods： the CXCR4-specific siRNA gene was cloned by PCR and inserted into the Pgensil-1 plasmid eonstaining U6 promoter and EGFP, and then the recombinant fragment was sub-cloned into PLXSN and tested by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected into prostate cancer cells. The expression of CXCR4 mRNA was detected by RT-PCR. The ability of invasion of prostate cancer cells in vitro was estimated by Transwell experiment. Results： it was showed that with the recombinant PLXSN transfection, the expression of CXCR4 mRNA in prostate cancer cells PC-3m and LNCaP was reduced at rates of （84.26±10.20）% and （88.17±11.23）%, respectively. The results of Transwell experiment also showed that the number of cells under micro-membrane were 14.7±3.1 and 18.9±4.2 in the treated group of PC-3m and LNCaP, respectively, compared with 46.9±5.3 and 66.7±6.0 in the control group （P〈0.05）. Conclusion： PLXSN/EGFP-U6-siCXCR4 can significantly depress the expression f CXCR4, by which the invasiveness of prostate cancer cells in vitro was inhibited as well. This recombinant fragment would be helpful in the treatment of prostate cancer.     

Aberrant methylation of breast and ovarian cancer susceptibility gene 1 in chemosensitive human ovarian cancer cells does not involve the phosphatidylinositol 3′‐kinase–Akt pathway

Methylation is an important silencing mechanism of breast and ovarian cancer susceptibility gene 1 (BRCA1) expression in sporadic ovarian cancer. However, the role of BRCA1 methylation in chemotherapy in sporadic ovarian cancer and the related pathways have not been understood completely. This study has determined the roles of BRCA1 hypermethylation in chemotherapy of sporadic ovarian cancer and its related signaling pathways. We used bisulfite sequencing, real‐time polymerase chain reaction, and western blotting to check the methylation state and expression levels of BRCA1 of the following cell lines: platinum‐sensitive human ovarian cancer cell line COC1, platinum‐resistant cell line COC1/DDP, SKOV‐3, and 5‐Aza‐dC treated COC1. The cisplatin sensitivity of ovarian cancer cells was examined by MTS (methyl‐thiazol tetrazolium) assay. Tumorigenicity in vivo and DDP‐based chemosensitivity were compared among the above cells. Phosphatidylinositol 3′‐kinase (PI3K)–Akt pathway activation in ovarian cancer cells was studied by western blotting. The frequency of BRCA1 methylation in the COC1 cell line was higher than in COC1/DDP and SKOV‐3 cell lines, whereas the mRNA and protein expression of BRCA1 were lower than in the COC1/DDP and SKOV‐3 cell lines. DNA demethylation decreased the chemosensitivity of COC1 cells and partially increased the expression levels of BRCA1. The activation of the PI3K‐Akt pathway was low in ovarian cancer cells. Our results indicate that hypermethylation of BRCA1 might play an important role in the chemosensitivity of ovarian cancer, and that the PI3K–Akt pathway is not involved in this response. (Cancer Sci 2010)     

Fem1b,a proapoptotic protein,mediates proteasome inhibitor‐induced apoptosis of human colon cancer cells

In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization‐1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis‐inducing proteins Fas, tumor necrosis factor receptor‐1 (TNFR1), and apoptotic protease activating factor‐1 (Apaf‐1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization‐1 (FEM‐1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT‐116, and DLD‐1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT‐116, and DLD‐1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor‐induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor‐induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer. © 2009 Wiley‐Liss, Inc.     

Nucleoside analog inhibits microRNA‐214 through targeting heat‐shock factor 1 in human epithelial ovarian cancer

The important functions of heat shock factor 1 (HSF1) in certain malignant cancers have granted it to be an appealing target for developing novel strategy for cancer therapy. Here, we report that higher HSF1 expression is associated with more aggressive malignization in epithelial ovarian tumors, indicating that targeting HSF1 is also a promising strategy against ovarian cancer. We found that a nucleoside analog (Ly101‐4B) elicits efficient inhibition on HSF1 expression and potent anticancer activity on epithelial ovarian cancer both in vitro and in vivo. Moreover, by targeting HSF1, Ly101‐4B inhibits the biogenesis of microRNA‐214, which has been revealed to be overexpressed and to promote cell survival in human ovarian epithelial tumors. These findings demonstrate that Ly101‐4B is a promising candidate for ovarian cancer therapy, and expand our understanding of HSF1, by revealing that it can regulate microRNA biogenesis in addition to its canonical function of regulating protein‐coding RNAs.     

Influence of the prodrugs 5‐fluorocytosine and CPT‐11 on ovarian cancer cells using genetically engineered stem cells: tumor‐tropic potential and inhibition of ovarian cancer cell growth

Recent studies have shown that genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non‐toxic prodrugs to toxic metabolites selectively migrate toward tumor sites and reduce tumor growth. In the present study, we evaluated whether these GESTECs were capable of migrating to human ovarian cancer cells and examined the potential therapeutic efficacy of the gene‐directed enzyme prodrug therapy against ovarian cancer cells in vitro. The expression of cytosine deaminase (CD) or carboxyl esterase (CE) mRNA of GESTECs was confirmed by RT‐PCR. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to ovarian cancer cells. GESTECs (HB1.F3.CD or HB1.F3.CE cells) engineered to express a suicide gene (CD or CE) selectively migrated toward ovarian cancer cells. A [³H] thymidine incorporation assay was conducted to measure the proliferative index. Treatment of human epithelial ovarian cancer cell line (SKOV‐3, an ovarian adenocarcinoma derived from the ascites of an ovarian cancer patient) with the prodrugs 5‐fluorocytosine (5‐FC) or camptothecin‐11 (CPT‐11) in the presence of HB1.F3.CD or HB1.F3.CE cells resulted in the inhibition of ovarian cancer cell growth. Based on the data presented herein, we suggest that GESTECs expressing CD/CE may have a potent advantage to selectively treat ovarian cancers. (Cancer Sci 2010; 101: 955–962)     

<Emphasis Type="Italic">Parkin</Emphasis> Gene Alterations in Ovarian Carcinoma from Northern Indian Population

Parkin, a tumor suppressor gene located on chromosome 6q25-27, has been identified as a target for mutation in many human malignancies like breast, ovaries, cervical and lungs etc. After a preliminary report on the loss of heterozygosity and altered Parkin expression in breast and ovarian tumors, we aimed to study loss of heterozygosity in the Parkin gene associated microsatellite markers and its expression in human ovarian cancer patients from Indian population. We examined 102 paired normal and ovarian cancer samples for allelic loss in Parkin gene locus using Parkin gene associated microsatellite markers through loss of heterozygosity and changes in its expression through semiquantitative RT-PCR. Loss of heterozygosity identified common region of loss in Parkin locus with highest frequency for the intragenic marker D6S1599 (53%) whereas, 49 of 102 (48%) specimens showed decreased or no expression of Parkin in ovarian tumors. The study revealed that presence of loss of heterozygosity was significantly higher in both the intragenic markers (D6S1599 and D6S305) as compared with the locus of flanking region (D6S1008) with their p value 0.000001 and 0.00008, respectively. It also revealed that Parkin inactivation is probably a combination of loss of heterozygosity coupled with downregulation of Parkin gene through an alternative means like epigenetic mechanism. These data strongly supports the previous study and argue that Parkin is a tumor suppressor gene whose inactivation may play an important role in ovarian carcinoma.     

High Incidence of 4153delA <Emphasis Type="Italic">BRCA1</Emphasis> Gene Mutations in Lithuanian Breast- and Breast-ovarian Cancer Families

Summary BRCA1 and BRCA2 gene mutations confer a high lifetime risk to breast and ovarian cancers. We have screened cancer patients from 13 families with at least three breast and/or ovarian cancers from Lithuania for 5382insC, C61G and 4153delA BRCA1 gene mutations. One of three mutations was found in 9 of the 13 studied families (69%). 4153delA was the most frequently detected and accounted for 56% of all identified mutation. 5382insC and C61G accounted for 33% and 11% of found mutations, respectively. Significantly higher, than in other populations, incidence of 4153delA indicates that this may be founder BRCA1 mutation characteristic for Lithuanians. Our analysis shows that testing of 4153delA, 5382insC, C61G BRCA1 mutations should be extremely effective and inexpensive tool in testing Lithuanian population aimed to identify individuals with high risk of breast and ovarian cancers.