过表达PTENP1 3’UTR对胃癌细胞行为的影响

发布时间：2018-03-30 03:29

本文选题：胃癌　切入点：过表达　出处：《河南大学》2016年硕士论文

【摘要】：背景胃癌是全球最常见的消化道恶性肿瘤之一,中、日、韩等亚洲国家是胃癌的高发区。据统计每年我国胃癌的新发病例约占到世界总发病例数的42%,总数接近40万例。2011年中国城市居民中胃癌的死亡率为19.66/10万,而农村居民胃癌的死亡率为约为22.09/10万,位于恶性肿瘤导致死亡的第2位。由于目前胃癌的外科手术治疗越来越规范化、内科对于胃癌的药物治疗等综合治疗也得到了进一步的发展,许多胃癌患者的生存期及生活质量都得到了很大的提高,尽管如此,胃癌患者的5年生存率仍低于40%。因此从分子水平来全面理解胃癌发生进展的机理,并在此基础上寻求有效的防治方法依然是当前胃癌研究的重点和难点。PTEN基因是一个研究较多的抑癌基因,定位于人染色体l0q23.3,该基因包含有9个外显子和8个内含子,全长约200kb,编码由403个氨基酸残基组成的蛋白质,该蛋白质具有磷酸酶活性,它能特异性地使磷脂酰肌醇-3,4,5-三磷酸(PIP3)去磷酸化,拮抗PI3K/AKT的信号通路,对细胞的生长、增殖、凋亡、存活进行调控,抑制细胞恶性转化。目前PTEN已经被认为癌症诊断和预后的标志物。micro RNA是近年来发现的一类小非编码、内源性的短单链RNA,大约19-22nt,它可以通过与靶基因m RNA 3'UTR区发生互补结合,抑制靶基因功能蛋白质的翻译,在转录水平对基因的表达进行调控。已经得到证明的有胶质瘤、卵巢癌、乳腺癌、膀胱癌等多种肿瘤细胞,在这些研究中mi RNA调控PTEN基因的表达,抑制PTEN蛋白翻译,影响肿瘤细胞增殖的作用。PTENP1是PTEN基因的假基因,位于染色体9p13.3。有研究表明PTENP1 3'非编码区(3'UTR)与PTEN的3'UTR有近95%的相似区域,而其中两基因有相同的mi RNA作用位点。因此,以PTEN为靶基因的mi RNA也会同时作用与PTENP1,与PTEN竞争性的结合mi RNA,使得PTEN的功能性得以保存。本研究克隆出PTEN与PTENP1相同的mi RNA作用片段(本文中用pp表示),构建pc DNA3.1-pp瞬时转染质粒,在细胞水平比较转染重组质粒后的胃癌细胞与未处理和转染空质粒的胃癌细胞的增殖、迁移和侵袭能力的差别,并检测了过表达pp片段后PTEN的表达情况,为肿瘤的治疗提供新的思路。目的构建pc DNA3.1-pp瞬时转染质粒,将重组质粒转染入胃癌细胞后观察对细胞生长、迁移和侵袭能力的影响,研究pp片段对PTEN表达的影响,为进一步的机制研究打下基础。方法1.采用PCR、胶回收、质粒提取的方法获取目的片段,并将其插入真核表达载体pc DNA3.1内,构建重组质粒pc DNA3.1-pp,通过酶切电泳分析及测序的方法对重组质粒进行鉴定。2.应用脂质体法将重组质粒pc DNA3.1-pp及pc DNA3.1空载体转染入BGC823细胞中,然后用Western Blotting的方法从蛋白质水平检测PTEN基因的表达。3.通过细胞计数、划痕实验、细胞侵袭实验对转染前后细胞的生长、迁移和侵袭能力进行比较观察、比较和分析。结果1.Western Blotting检测PTEN基因的蛋白表达结果显示,重组质粒pc DNA3.1-pp转染BGC823细胞株后PTEN的表达明显高于空质粒转染组细胞及未处理组细胞,而空质粒组与未处理组细胞间PTEN的表达量比较无统计学差异。2.2.用自动计数仪记录三组细胞每天的细胞数,共记录7天。用Graph Pad prism处理数据可以看出,重组质粒pc DNA3.1-pp组的细胞数明显低于空质粒组及未处理组细胞,差异具有统计学意义,而空质粒组与未处理组细胞间细胞数的差异无统计学意义。3.划痕实验检测细胞的水平迁移能力,重组质粒pc DNA3.1-pp组的细胞迁移率明显低于空质粒组及未处理组细胞,差异具有统计学意义,而空质粒组与未处理组之间细胞迁移率的差异无统计学意义。4.Transwell实验检测细胞的侵袭能力,与空质粒组与未处理组相比较,重组质粒pc DNA3.1-pp组穿膜细胞数显著增多,差异具有统计学意义,而前两者之间的穿膜细胞数的差异无统计学意义。结论成功构建瞬时转染pc DNA3.1-pp重组质粒的BGC823细胞,过表达pp片段可以增加PTEN的表达,胃癌细胞的生长、迁移及侵袭能力下降。
[Abstract]:Background gastric cancer is one of the world's most common malignant tumor of digestive tract in Japan, South Korea and other Asian countries is the high incidence of gastric cancer. According to statistics, every year new cases of gastric cancer in China accounted for about 42% of the world total number of the cases, a total of nearly 400 thousand cases of gastric cancer.2011 China city residents of the annual mortality rate was 19.66/10 million while rural residents, the mortality rate of gastric cancer is about 22.09/10 million, in malignant tumors led to the deaths of second. Due to the surgical treatment of gastric cancer is more and more standardized, medicine for gastric cancer drug therapy combined therapy has also been further developed, many patients with gastric cancer survival and quality of life have been greatly improve, nevertheless, gastric cancer patients 5 years survival rate is less than 40%. from the molecular level to fully understand the mechanism of progression of gastric cancer, and on this basis for effective prevention and treatment is still The focal point of.PTEN gene in gastric cancer research is a study of many tumor suppressor gene, located on human chromosome l0q23.3. This gene contains 9 exons and 8 introns, approximately 200KB in length, encoding a 403 amino acid protein, the protein with phosphatase activity, it can. Specifically the phosphatidylinositol -3,4,5- three phosphate (PIP3) phosphorylation, signal pathway antagonist PI3K/AKT, on cell growth, proliferation, apoptosis, survival regulation, inhibition of cell malignant transformation. At present, PTEN has been considered markers of.Micro RNA cancer diagnosis and prognosis is a kind of non encoding discovered in recent years. A short, single stranded RNA, endogenous about 19-22nt, it can occur through complementary binding with target gene m RNA 3'UTR region, inhibiting target gene function of protein translation at the transcription level of gene expression regulation has been demonstrated. The glioma, ovarian cancer, breast cancer, bladder cancer and other tumor cells, the expression of MI RNA in the study of gene regulation of PTEN, inhibition of PTEN protein translation,.PTENP1 influences the proliferation of tumor cells is a pseudogene PTEN gene, located on chromosome 9p13.3. research showed that PTENP1 3'encoding region (3'UTR) a similar area of nearly 95% PTEN and 3'UTR, and two of the MI gene RNA site in the same. Therefore, based on PTEN mi RNA target genes will also function with PTENP1, and with MI RNA PTEN competition, the function of PTEN can be preserved. This study cloned mi RNA. A fragment of PTEN with the same PTENP1 (the PP), to construct the PC transient transfection of DNA3.1-pp plasmid, in comparison with the transfection of gastric cancer cells and untreated and empty plasmid transfected gastric cancer cell cell proliferation, migration and invasion of the poor Don't, and detected the expression of PTEN PP fragment, to provide new ideas for the treatment of cancer. Objective to construct PC transient transfection of DNA3.1-pp plasmid, the recombinant plasmid was transfected into gastric cancer cells after observation of the effect of cell growth, migration and invasion, the effects of PP on the expression of PTEN fragment, lay the foundation for further studies. 1. methods of using PCR, plastic recycling, methods of Plasmid Extraction to obtain fragments and inserted into the eukaryotic expression vector PC to construct recombinant plasmid DNA3.1, PC DNA3.1-pp, through the method of enzyme digestion analysis and sequencing for identification of.2. recombinant plasmid by liposome on the recombinant plasmid PC DNA3.1-pp the PC and DNA3.1 vector transfected into BGC823 cells, then use Western Blotting to detect the expression of.3. PTEN gene from protein level by cell counting, scratch test, cell invasion assay in turn After dyeing cell growth, migration and invasion were observed, compared and analyzed. The results of detection of PTEN gene 1.Western Blotting protein expression showed that the expression of recombinant plasmid PC DNA3.1-pp in BGC823 cells transfected with PTEN was significantly higher than that of empty plasmid transfected cells and untreated cells, while the expression of empty plasmid group and the untreated group the amount of PTEN cells had no significant difference in.2.2. with automatic counting the number of cells recorded in three groups of cells were recorded every day, for 7 days. Using Graph Pad prism data can be seen, the number of cells of recombinant plasmid PC in DNA3.1-pp group was significantly lower than the empty plasmid group and untreated cells, the difference was statistically significant, while the differences the empty plasmid group and untreated cells between the cell number was not statistically significant.3. scratch assay the level of migration, recombinant plasmid PC group DNA3.1-pp cell migration rate Significantly lower than the empty plasmid group and untreated cells, the difference was statistically significant, while the empty plasmid group and untreated cell migration rate differences between groups was not statistically significant.4.Transwell assay, cell invasion ability, with the empty plasmid group compared with the untreated group, the recombinant plasmid PC DNA3.1-pp cells increased significantly. The difference was statistically significant, there was no significant difference between the former two transmembrane cell number. Conclusion the successful construction of the recombinant plasmid DNA3.1-pp was transfected into PC BGC823 cells, overexpression of PP fragment can increase the expression of PTEN and gastric cancer cell proliferation, migration and invasion ability decreased.

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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