【作者】 胡建燃；

【导师】 赫杰；

【作者基本信息】 哈尔滨工业大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 本课题组前期研究发现Utrophin(UTRN)是一个候选的抑癌基因,并在多种肿瘤中表达下调或缺失。鉴于UTRN基因能够编码除了2个全长转录本A和B外,还可能编码包括短转录本Up71和Up140在内的7个变异体,本研究采用半定量RT-PCR方法分析了短转录本Up71和Up140在15种人肺癌细胞系中的表达,结果表明Up71和Up140的表达不影响对UTRN全长转录本的研究。同时,本研究检测全长UTRN(FL-UTRN)在以上15种肺癌细胞系中的表达缺失情况,结果表明FL-UTRN可能在NCI-H69、NCI-H82和NCI-H446细胞中完全表达缺失。另外,本研究采用荧光定量PCR技术检测了FL-UTRN在19种人肺癌细胞系的表达,与正常人胚肺成纤维细胞MRC5和正常成人肺组织比较,结果显示FL-UTRN在NCI-H520、NCI-H661等细胞中表达水平显著增高。此外,本研究分析了FL-UTRN在26例非小细胞肺癌临床病例中的表达,与正常癌旁组织比较,FL-UTRN在19例肺癌组织中表达明显下调。结合已有的细胞定位研究结果,FL-UTRN在其高表达的人肺癌细胞系中存在于细胞核,提示核表达的FL-UTRN在肺癌发生中的可能起重要作用。已有研究证实,在非肿瘤细胞中FL-UTRN表达于细胞膜,但是在前期研究中,同时发现FL-UTRN在NCI-H520等人肺癌细胞系中发生了核转位,在细胞膜上无表达。为了进一步研究FL-UTRN在细胞核中的继发功能,本研究采用RNAi技术沉默NCI-H520细胞中核表达的FL-UTRN,与对照组比较,核FL-UTRN表达沉默的NCI-H520细胞的增殖能力没有显著改变,但是在软琼脂中形成的细胞集落显著增大。另外,作为参照实验,本研究采用RNAi技术同时沉默了在小鼠成纤维细胞NIH3T3中质膜表达的FL-UTRN,在其表达沉默前后,NIH3T3细胞的增殖能力以及黏着斑分子α-actinin、vinculin和paxillin的表达和定位无显著改变。以上研究结果提示,相比质膜表达的FL-UTRN,核FL-UTRN表达沉默可能增强了肺癌细胞的恶性程度。因此,核表达的FL-UTRN可能作为一个候选抑癌基因的产物在细胞核内行使功能,为深入探讨UTRN与肺癌发生的关系提供了新的思路。更多还原

【Abstract】 In previous study, we demonstrated that Utrophin(UTRN) is a candidate tumor suppressor gene,and the low expression or deletion of UTRN occurred in various tumor tissues. In addition, in light of the transcripts of UTRN gene containing full-length transcripts A and B, also seven isoforms including short transcripts Up71 and Up140, we detected the expression of short transcripts Up71 and Up140 in 15 lung cancer cell lines by RT-PCR. The results showed that the expression of Up71 and Up140 did not effect the investigation on the full-length transcripts of UTRN in this study. At the same time, we detected full-length UTRN(FL-UTRN) in the 15 lung cancer cell lines by RT-PCR. The results suggested that the FL-UTRN maybe absent in H69, H82 and H446 cells. Otherwise, we analyzed the expression of full-length transcripts in 19 lung cancer cell lines by real-time PCR. The results showed that the expression of FL-UTRN in several lung cancer cell lines, including NCI-H520 and NCI-H661, much higher than that in MRC5 cells and lung tissue of mormal adults. In addition, the expression of FL-UTRN was detected in 26 paired non-small-cell lung cancer specimens by real-time PCR. The results suggested that the expression level of the FL-UTRN in tumor was significantly lower than in corresponding adjacent normal tissues. Together with the analysis of the cellular location, FL-UTRN was expressed in the nuclears of which lung cancer cell lines the expression of the FL-UTRN were high. The results above implied that the FL-UTRN which expressed in the nuclears maybe play a important role in lung cancer cells.The FL-UTRN distributed at the periphery of the plasma membrane in the non-tumorous cells according to the previous study. However, the FL-UTRN was found nuclear translocation in several lung cancer cell lines including NCI-H520, and non-expression at the periphery of plasma membrane. For further studying the secondum function of the FL-UTRN in nuclears, the FL-UTRN was silenced by RNAi. there was no obvious change of the capability of proliferation of the NCI-H520 cells, which the nuclear FL-UTRN was silenced, except for an increasing tendency of the colons size in the soft agar. Besides, as the controlled experiment, the FL-lUTRN distributed at the periphery of the plasma membrane in NIH3T3 cells was silenced by RNAi. It was found that there was no obvious change of the capability of proliferation and the the anchorage-independent growth, and the expression and subcellular location of the focal adhesion molecules includingα-actinin, vinculin and paxillin. The results above implied that the expression silence of nuclear FL-UTRN maybe enhance the malignant degree of the lung cancer cells according to the FL-UTRN at the periphery of the plasma membrane. Above data demonstrated that nuclear FL-UTRN maybe act as the product of a tumor suppressor gene in the nuclears. Our studies provide new insight into the relation between UTRN and tumorigenesis.更多还原