miR-134通过调控KRAS抑制肾透明细胞癌细胞的增殖和上皮间质转化过程

发布时间：2018-04-24 23:28

本文选题：肾癌 + miR-134　；参考：《南京医科大学》2014年硕士论文

【摘要】：目的：MicroRNAs (miRNAs,微小RNA)是一类具有调控功能的RNA,已经被发现在多种恶性肿瘤组织中异常表达。研究发现在肾透明细胞癌(简称肾癌,renal cell carcinoma, RCC)中miR-134呈低表达,但其在肾癌中的作用和调控机制不明。本课题旨在研究miR-134在肾癌组织中的表达情况,探索miR-134在肾癌发生、发展中可能的调控机制。材料和方法：采用TaqMan实时荧光定量PCR (RT-PCR)方法检测4株肾癌细胞株以及24对肾癌组织和癌旁无瘤组织样本中miR-134的表达水平。运用流式细胞检测(周期和凋亡)、迁移实验、侵袭实验检测评估miR-134变化对肾癌细胞系786-0和caki-1细胞增殖、迁移和侵袭的影响。双荧光素酶报告基因实验确定miR-134是否通过KRAS (Kirsten rat sarcoma viral oncogene homolog)的3'-UTR区域直接调控KRAS。miR-134模拟物转染肾癌细胞后,Western Blot检测和分析KRAS.增殖相关蛋白以及与肿瘤转移相关的上皮间质转化过程(epithelial-mesenchymal transition, EMT)表型蛋白的变化。结果：与癌旁无瘤组织相比,miR-134在肾癌组织中的表达水平显著下调(P0.05)：与正常肾小管上皮细胞相比,4个肾癌细胞系(786-0. caki-1、769-P、 ACHN)中miR-134呈低表达(P0.05)。体外实验中上调miR-134的表达水平可以使肾癌细胞G0/G1期阻滞(P0.05),显著降低肾癌细胞的增殖能力(P0.05)；过表达的miR-134还可以通过抑制EMT降低肾癌细胞迁移(P0.05)和侵袭(P0.05)的能力。双荧光素酶报告实验证明KRAS为miR-134直接调控的靶基因。转染miR-134mimics后,细胞内KRAS蛋白表达水平显著降低,并且伴随着KRAS相关MAPK/ERK通路的激活以及EMT表型标志蛋白E钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)水平降低(P0.05)及N钙黏蛋白(N-cadherin)水平的升高(P0.05)。结论：在肾癌中,miR-134可作为一种抑瘤因子,可能通过靶向调控KRAS的表达抑制肾癌细胞的增殖和上皮间质转化过程。目的：近年来大规模的基因组和转录组分析已经发现了大量的转录本,这其中就包括长链非编码RNA (long non-coding RNA, lncRNA), lncRNA被发现在各种疾病尤其是癌症中表达异常。然而,在肾细胞癌(renal cell carcinoma, RCC)中是否具有特异性异常表达的lncRNA尚不明确。因此,我们选取了5名肾癌患者的样本,包括肿瘤组织(T)及其配对的癌旁无瘤组织(N),通过高通量基因芯片分析来研究肾癌中lncRNA的表达谱。材料和方法：我们利用带有33045个lncRNA和30215个mRNA的探针的高通量基因芯片对5名肾细胞癌患者的样本进行检测分析,筛选差异表达的lncRNA和mRNA。随后,我们又根据芯片结果选取了两个lncRNA (AK096725和ENST00000453068)在70名肾细胞癌患者的样本中进行了验证,利用定量反转录聚合酶连锁反应(RT-PCR)检测AK096725和ENST00000453068的表达水平。结果：LncRNA芯片在肾细胞癌组织中检测到了27279个lncRNA有效信号,与癌旁无瘤组织相比其中有480个lncRNAs表达上调(P0.05；T/N1.5),417个lncRNAs表达下调(P0.05；N/T1.5)。另外,19995个mRNA有效信号被探针检测到,其中458个mRNAs在肾细胞癌样本中表达升高(P0.05；T/N1.5),413个表达降低(P0.05; N/T1.5)。RT-PCR验证显示AK096725(P=0.043)和ENST00000453068(P＜0.001)在70对样本中表达水平的改变与基因芯片结果一致。结论：本课题利用5对肾细胞癌患者的组织样本,揭示了肾细胞癌中lncRNA的表达谱,同时鉴定出了在肿瘤组织中的异常表达lncRNA和mRNA。在后期验证试验中,我们发现AK096725的表达水平与肾细胞癌病理类型之间存在一定的相关性,这一发现值得关注。本研究结果可能对于寻找肾细胞癌新的生物学标志物有所帮助。
[Abstract]:Objective: MicroRNAs (miRNAs, small RNA) is a kind of RNA with regulatory function. It has been found abnormal expression in a variety of malignant tumor tissues. The study found that the expression of miR-134 in renal clear cell carcinoma (abbreviated as renal carcinoma, renal cell carcinoma, RCC) is low, but its role and regulation mechanism in renal cancer is unknown. This subject is aimed at studying miR-. 134 to investigate the possible regulatory mechanism of miR-134 in the occurrence and development of renal cell carcinoma.
Materials and methods: TaqMan real-time fluorescence quantitative PCR (RT-PCR) method was used to detect the expression level of miR-134 in 4 renal cell carcinoma cell lines and 24 pairs of renal cancer tissues and non tumor tissue samples. Flow cytometry (cycle and apoptosis), migration experiment, and invasion test were used to evaluate the change of miR-134 to 786-0 and Caki-1 cells in renal cell line. The effects of proliferation, migration, and invasion. The double luciferase reporter gene experiment determines whether miR-134 is directly regulated by the 3'-UTR region of the KRAS (Kirsten rat sarcoma viral oncogene homolog) to direct the KRAS.miR-134 analogue to renal cancer cells. Western Blot detection and analysis of proliferation related proteins and the epithelial cells associated with tumor metastasis Changes in epithelial-mesenchymal transition (EMT) phenotypic proteins.
Results: the expression level of miR-134 in renal carcinoma tissue was significantly lower than that in non tumor tissues (P0.05). Compared with normal tubular epithelial cells, the miR-134 expression in 4 renal cell lines (786-0. caki-1769-P, ACHN) was low (P0.05). In vitro, up regulation of miR-134 expression level could block the G0/G1 phase of renal cell carcinoma (P0.05). The proliferation capacity of renal cell carcinoma cells (P0.05) was significantly reduced, and the overexpressed miR-134 could also reduce the capacity of renal cell carcinoma cell migration (P0.05) and invasion (P0.05) by inhibiting EMT. The double luciferase reporter experiment proved that KRAS was the target gene directly regulated by miR-134. After transfection of miR-134mimics, the expression level of KRAS protein was significantly reduced and accompanied by miR-134mimics. With the activation of the KRAS related MAPK/ERK pathway and the EMT phenotypic marker protein E calcium mucin (E-cadherin), the level of vimentin (Vimentin) decreased (P0.05) and N calcium mucin (N-cadherin) level increased (P0.05).
Conclusion: miR-134 can be used as a tumor suppressor factor in renal cell carcinoma. It may inhibit the proliferation and epithelial mesenchymal transition of renal cell carcinoma by regulating the expression of KRAS.
Objective: a large number of transcriptional transcripts have been found in large-scale genome and transcriptional analysis in recent years, including long chain non coded RNA (long non-coding RNA, lncRNA), and lncRNA has been found to be abnormal in various diseases, especially in cancer. However, the specific abnormalities in renal cell carcinoma (renal cell carcinoma, RCC) are specific. The expression of lncRNA is not yet clear. Therefore, we selected 5 patients with renal cancer, including tumor tissue (T) and its paired cancerous tissue (N), and study the expression of lncRNA in renal cancer by high throughput gene chip analysis.
Materials and methods: we detected and analyzed samples of 5 renal cell carcinoma patients with high throughput gene chips with 33045 lncRNA and 30215 mRNA probes, screened differentially expressed lncRNA and mRNA., and then we selected two lncRNA (AK096725 and ENST00000453068) in 70 renal cell carcinoma patients according to the results of the chip. The samples were verified by quantitative reverse transcription polymerase chain reaction (RT-PCR) to detect the expression levels of AK096725 and ENST00000453068.
Results: the LncRNA chip detected 27279 lncRNA effective signals in the renal cell carcinoma tissue, 480 of which were up regulated (P0.05; T/N1.5) and 417 lncRNAs expressions (P0.05; N/T1.5) compared with the cancerous tissues near the cancer. In addition, 19995 mRNA effective signals were detected by the needle, 458 of which were in the renal cell carcinoma samples. The level of expression of AK096725 (P=0.043) and ENST00000453068 (P < 0.001) in 70 pairs of samples was consistent with the gene chip results in the 413 expression reduction (P0.05; N/T1.5).RT-PCR verification. The results showed that the expression of AK096725 (P=0.043) and ENST00000453068 (P < 0.001) were in the 70 pairs of samples.
Conclusion: the expression profiles of lncRNA in renal cell carcinoma were revealed in 5 cases of renal cell carcinoma, and the abnormal expression of lncRNA and mRNA. in the tumor tissues was identified in the later verification test. We found that there was a certain correlation between the expression level of AK096725 and the type of renal cell carcinomatosis. This study may be helpful for finding new biomarkers of renal cell carcinoma.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

【相似文献】