RNAi技术在乳腺癌基因治疗中的研究进展

乳腺癌是当前发病率较高的女性恶性肿瘤，严重威胁女性的身体健康。传统治疗以手术治疗为主，辅以化疗、放疗。而化疗和放疗对肿瘤非特异性、敏感性差，副作用大。因此乳腺癌的基因治疗受到充分重视。1998年RNA干扰（RNAinterference，RNAi）的发现，特别是在哺乳动物细胞应用小干扰RNA（small interfering，siRNA）成功介导RNAi，为基因功能的研究提供了强大的武器，也为乳腺癌基因治疗增添了新的活力。现对RNAi在乳腺癌基因治疗中的研究现状与进展方面作一综述。

Survivin在乳腺癌中的表达及其与Caspase-3、PCNA表达的关系

目的探讨Survivin在乳腺癌发生、发展中的作用及其与Caspase-3蛋白表达和细胞增殖的相互关系。方法用免疫组化S-P法检测Survivin在正常乳腺组织(10例)、乳腺囊性增生组织(18例)、不典型增生组织(20例)和乳腺癌组织(50例)中的表达以及Caspase-3、PCNA在乳腺癌组织中的表达。结果Survivin蛋白在正常乳腺组织无表达,在乳腺囊性增生组织、不典型增生组织、乳腺癌组织中的阳性率分别为5.6%(1/18)、45.0%(9/20)、72.0%(36/50),差异有显著性(P<0.05)。Survivin蛋白的表达与临床分期和淋巴结转移有关(P<0.05),与年龄、是否绝经、肿瘤大小和组织学分级均无关。Survivin与Caspase-3的表达呈负相关(P<0.01)。Survivin蛋白表达阳性的乳腺癌PCNA的标记指数明显高于Survivin蛋白表达阴性的乳腺癌细胞PCNA的标记指数(P<0.01)。结论Survivin蛋白在乳腺癌中表达上调,提示其通过抑制凋亡在乳腺癌发生、发展中起重要作用,可能成为乳腺癌基因治疗的新靶点。Survivin通过与激活的Caspase-3结合,抑制其活性,从而阻止细胞凋亡。Survivin不仅参与凋亡的调控,还促进了细胞增殖。

尿激酶氨基末端基因抗乳腺癌细胞转移的实验研究

目的 探讨尿激酶氨基末端 (ATF)基因转移对肿瘤转移的抑制作用。方法 构建重组ATF基因真核表达载体pcDNA3 ATF ,用脂质体Lipofectin介导 ,将其导入其证明尿激酶 (uPA)和尿激酶受体 (uPAR)均有表达的人乳腺癌细胞系MCF 7。用Westernblot检测转染细胞ATF基因的表达 ;体外观察并比较了野生型和转基因MCF 7细胞侵袭人工基底膜能力 ;裸鼠皮下接种癌细胞 ,复制自发性肿瘤转移模型 ,观察成瘤性和转移性。结果 uPA/uPAR在MCF 7中有较高水平的表达 ;转ATF基因后 ,可检测到ATF的明显表达 ,体外侵袭穿透人工基底膜的能力明显降低 ,体内原位成瘤性不受影响 ,但自主性肺转移能力有一定程度的下降。结论 ATF基因转移后 ,ATF的表达可能竞争性抑制内源性uPA与uPAR的结合 ,在一定程度上抑制癌细胞的侵袭和转移。

Tet-On调控HSVtk表达的重组腺相关病毒载体的构建和感染活性的检测

构建含有Tet基因调节系统及自杀基因HSVtk的重组腺相关病毒载体pAAV TRE HSVtk Tet_On ,并使用PCR技术和限制性内切酶消化进行鉴定。用构建好的重组质粒分别与辅助质粒pAAV_RC、pHelper以磷酸钙共沉淀法转染HEK2 93细胞,进行病毒包装后得到了AAV TRE HSVtk Tet_On重组腺相关病毒,以氯化铯密度梯度离心对包装好的病毒进行纯化。用纯化后的重组腺相关病毒感染乳腺癌细胞株MCF_7后,斑点杂交检测结果显示,HSVtk基因整合进入MCF_7细胞基因组中。有感染活性的重组腺相关病毒能将目的基因转移到宿主细胞中,在Dox诱导下,GCV对AAV感染的MCF_7细胞具有明显的杀伤作用。

HER2 over-expression and response to different chemotherapy regimens in breast cancer

Purpose: To exam the relationship between HER2 over-expression and different adjuvant chemotherapies in breast cancer. Patients and Methods: A total of 1625 primary breast cancer patients who received post-surgery adjuvant chemotherapy in Tianjin Cancer Hospital,China,from July 2002 to November 2005 were included in the study. Among them,600 patients were given CMF (CTX+MTX+5-Fu) regimen,600 given CEF (CTX+E-ADM+5-Fu) regimen,and 425 given anthracyclines plus taxanes regimen,with mean follow-up time of 42 months. Results: In CMF treatment group,the 3-year disease free survival (DFS) in HER2 over-expressed patients was lower than that of the HER2-negative ones (89.80% vs 91.24%,P=0.0348); in node-positive subgroup,the 3-year DFS was 84.72% in HER2 over-expressed patients,and 90.18% in the HER-2-negative ones (P=0.0271). Compared to CMF regimen,anthracyclines and anthracyclines plus taxanes regimens are more effective (P<0.05) in node-positive HER2 over-expression than those in the node-negative. Conclusion: HER2 over-expression is an independent index for predicting poor prognosis and short DFS for breast cancer patients. HER2 over-expressed patients are resistant to CMF regimen chemo-therapy,but sensitive to anthracyclines-based or anthracyclines plus taxanes regimen. HER2 expression can be taken as a marker for therapies in breast cancer.

The expressions and significance of α-,β-catenins and cyclin D1 in breast cancers

Objective： To study the relationship between expressions of α-, β-catenins and cyclin D1 and the occurrence, infiltration and metastasis of breast cancer. Methods： High sensitive S-P immunohistochemical method was used to detect the protein expressions of α-, β-catenins and cyclin D1 in the 60 cases of breast cancer tissues. Results： Abnormal immunoreactivities of α- and β-catenins were observed in 37 （61.7%） and 42 （70%） cases of breast cancer tissues, respectively. There were 28 cases （46.7%） who showed cyclin D1 overexpression. The abnormal expression rates of α- and β-catenins in infiltrating Iobular carcinoma （ILC） were significantly higher than those in infiltrating ductal carcinoma （IDC） （P 〈 0.05）, but they had no relations to the extent of differentiation and lymphatic metastasis of breast cancer （P 〉 0.05）. The overexpression rate of cyclin D1 was correlated with tumor stage and lymphatic metastasis of breast cancer （P 〈 0.05）, but not with histological type and the extent of differentiation （P 〉 0.05）. Cyclin D1 overexpression was observed in 57.1% （24/42） of these cases that showed abnormal staining of β-catenin, but only observed in 22.2% （4/18） of these cases with normal membranous staining of β-catenin. There was a significantly positive correlation between the abnormal expression of β-catenin and overexpression of cyclin D1 （r, = 0.321, P 〈 0.05）. Conclusion： The abnormal expression of β-catenin may play an important role in the genesis of breast cancer by triggering cyclin D1 overexpression in breast cancer. The abnormal expressions of α- and β-catenins are not a key factor in malignant cell metastasis in breast cancer, but may also involve in the progress.

P53 gene could be a new effective therapeutic target in triple-negative breast cancer: a Meta-analysis

Objective： The aim of this study was to explore the relationship between p53 gene and triple-negative breast cancer （TNBC）, and determine that whether p53 gene could be a new effective therapeutic target. Methods： We identified studies with quantitative data on the relation of p53 gene and TNBC through searching 12 databases online （Oct. 1999-Oct. 2012） and reviewing the references, which were written in English or Chinese. Summary estimates of odds ratio （OR） was calculated using the fixed-effects model or the random-effects model as appropriate. Results： We identified 12 eligible stud- ies with 1532 cases of TNBC patients and 6329 controls of non-TNBC patients. The test for homogeneity resulted in X^2 = 200.16 （P 〈 0.05）, it showed significant heterogeneity so that a random effect model was applied. Our results showed that the expression of p53 gene could be much stronger in TNBC group than that in non-TNBC group [OR = 2.10, 95% confidence interval （CI） = 1.21-3.65]. In ethnicity-subgroup analysis, we found that in Caucasian group, the expression of p53 gene were stronger in TNBC group （OR = 2.60, 95% CI = 1.21-5.57）, but there was no statistical significance in Asian group （OR = 1.69, 95% CI = 0.83-3.45）. Conclusion： P53 gene could be an effective predictor and a good therapeutic target for TNBC patients in the future, especially in Caucasian. Further researches focusing on p53 gene would gain a breakthrough in the treatment of TNBC.

High Expression of the RECK Gene in Breast Cancer Cells is Related to Low Invasive Capacity

OBJECTIVE To investigate the expression of the RECK gene in human breast （cancer） cell lines, and to determine the relationship between RECK gene expression and the invasive capacity of the breast cancer cell lines.METHODS The invasive capacity of breast （cancer） cell lines including HBL-100, MCF-7 and MDA-MB-435S were determined by the Tran- swell method. The protein expression levels of RECK, MMP-2 and MMP- 9 genes in these three cell lines were measured by immunocytochemical methods. The expressions of the RECK gene and protein level were measured hv RT-PCR and Westrn hints in the cell lines respectively.RESULTS The order of the nvasive capacity of the breast （cancer） cell lines was MDA-MB-435S, being the highest, and HBL-100, being the lowest. The invasive capacity difference between any two groups among the three groups was significant （P〈0.01）. The protein expression level of the RECK gene in the HBL-100 cell line was highest, and no expression was detected in MDA-MB-435S cells. Moreover, the expression of the RECK gene was negatively correlated with the expression of the MMP-2 and MMP-9 genes. The mRNA level of the RECK gene in HBL-100 cells was the highest, but no expression was found in the MDA-MB-435S cells （P〈0.001）.CONCLUSION There was a significant negative correlation between the expression level of the RECK gene and invasive capacity in vitro, and the RECK gene expression showed an inverse proportion to that of the MMP-2, MMP-9 genes.

Specific killing effect of diphtheria toxin A fragment under control of DF3 promotor on human breast cancer cells

Objective: To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods: Constructing recombinant expression vector PGL3-DF3-DTA and transfecting it into human breast cancer cells of DF3 positive and negative. By means of RT-PCR to measure the expression of DTA in human breast cancer cells. MTT color-imetry was used to examine the effect of PGL3-DF3-DTA on growth of human breast cancer cells. By experiment on nude mice to observe the killing effect of PGL3-DF3-DTA on human breast cancer cells. Results: Recombinant expression vector PGL3-DF3-DTA was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Conclusion: Recombinant expression vector PGL3-DF3-DTA could produce specific killing effect on human breast cancer cell line of DF3 positive.

针对细胞周期蛋白E的有效小干扰RNA靶向位点的筛选

乳腺癌是威胁女性健康的常见恶性肿瘤，寻找乳腺癌分子靶向治疗的新靶点，对于乳腺癌的基因治疗尤为重要。细胞周期蛋白E（CyclinE）是细胞周期中G1／S期的关键调节蛋白。很多国内外乳腺癌疾病的研究者都注意到了CyclinE同乳腺癌预后之间的密切关系，并考虑可否将其作为一个乳腺癌基因治疗的新靶点。我们设计并合成了针对CyclinE的小干扰RNA（siRNA）序列，采用RNA干扰（RNAi）方法沉默乳腺癌细胞系中CyclinE的表达，为进一步探究CyclinE在乳腺癌的发生、发展及转归中所产生的分子生物学效应提供实验工具，并鉴定其基因沉默的效率，同时探讨影响RNAi效率的因素。

MicroRNAs in breast cancer: oncogene and tumor suppressors with clinical potentia

MicreRNAs （miRs） are small single-stranded RNA molecules, which function as key negative regulators of post-transcriptional modulation in almost all biological processes. Abnormal expression of microRNAs has been ob- sewed in various types of cancer including breast cancer. Great efforts have been made to identify an association between microRNA expression profiles and breast cancer, and to understand the functional role and molecular mechanism of aberrant-expressed microRNAs. As research progressed, ＇oncogenic microRNAs＇ and ＇tumor sup- pressive microRNAs＇ became a focus of interest. The potential of candidate microRNAs from both intercellular （tissue） and extraceUular （serum） sources for clinical diagnosis and prognosis was revealed, and treatments involving microRNA achieved some amazing curative effects in cancer disease models. In this review, advances from the most recent studies of microRNAs in one of the most common cancers, breast cancer, are highlighted, especially the functions of specifically selected microRNAs. We also assess the potential value of these microRNAs as diagnostic and prognostic markers, and discuss the possible development of microRNA-based therapies.