本文選題：腎癌 + miR-134　； 參考：《南京醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：MicroRNAs (miRNAs,微小RNA)是一類具有調(diào)控功能的RNA,已經(jīng)被發(fā)現(xiàn)在多種惡性腫瘤組織中異常表達(dá)。研究發(fā)現(xiàn)在腎透明細(xì)胞癌(簡(jiǎn)稱腎癌,renal cell carcinoma, RCC)中miR-134呈低表達(dá),但其在腎癌中的作用和調(diào)控機(jī)制不明。本課題旨在研究miR-134在腎癌組織中的表達(dá)情況,探索miR-134在腎癌發(fā)生、發(fā)展中可能的調(diào)控機(jī)制。 材料和方法：采用TaqMan實(shí)時(shí)熒光定量PCR (RT-PCR)方法檢測(cè)4株腎癌細(xì)胞株以及24對(duì)腎癌組織和癌旁無瘤組織樣本中miR-134的表達(dá)水平。運(yùn)用流式細(xì)胞檢測(cè)(周期和凋亡)、遷移實(shí)驗(yàn)、侵襲實(shí)驗(yàn)檢測(cè)評(píng)估m(xù)iR-134變化對(duì)腎癌細(xì)胞系786-0和caki-1細(xì)胞增殖、遷移和侵襲的影響。雙熒光素酶報(bào)告基因?qū)嶒?yàn)確定miR-134是否通過KRAS (Kirsten rat sarcoma viral oncogene homolog)的3'-UTR區(qū)域直接調(diào)控KRAS。miR-134模擬物轉(zhuǎn)染腎癌細(xì)胞后,Western Blot檢測(cè)和分析KRAS.增殖相關(guān)蛋白以及與腫瘤轉(zhuǎn)移相關(guān)的上皮間質(zhì)轉(zhuǎn)化過程(epithelial-mesenchymal transition, EMT)表型蛋白的變化。 結(jié)果：與癌旁無瘤組織相比,miR-134在腎癌組織中的表達(dá)水平顯著下調(diào)(P0.05)：與正常腎小管上皮細(xì)胞相比,4個(gè)腎癌細(xì)胞系(786-0. caki-1、769-P、 ACHN)中miR-134呈低表達(dá)(P0.05)。體外實(shí)驗(yàn)中上調(diào)miR-134的表達(dá)水平可以使腎癌細(xì)胞G0/G1期阻滯(P0.05),顯著降低腎癌細(xì)胞的增殖能力(P0.05)；過表達(dá)的miR-134還可以通過抑制EMT降低腎癌細(xì)胞遷移(P0.05)和侵襲(P0.05)的能力。雙熒光素酶報(bào)告實(shí)驗(yàn)證明KRAS為miR-134直接調(diào)控的靶基因。轉(zhuǎn)染miR-134mimics后,細(xì)胞內(nèi)KRAS蛋白表達(dá)水平顯著降低,并且伴隨著KRAS相關(guān)MAPK/ERK通路的激活以及EMT表型標(biāo)志蛋白E鈣黏蛋白(E-cadherin)、波形蛋白(Vimentin)水平降低(P0.05)及N鈣黏蛋白(N-cadherin)水平的升高(P0.05)。 結(jié)論：在腎癌中,miR-134可作為一種抑瘤因子,可能通過靶向調(diào)控KRAS的表達(dá)抑制腎癌細(xì)胞的增殖和上皮間質(zhì)轉(zhuǎn)化過程。 目的：近年來大規(guī)模的基因組和轉(zhuǎn)錄組分析已經(jīng)發(fā)現(xiàn)了大量的轉(zhuǎn)錄本,這其中就包括長(zhǎng)鏈非編碼RNA (long non-coding RNA, lncRNA), lncRNA被發(fā)現(xiàn)在各種疾病尤其是癌癥中表達(dá)異常。然而,在腎細(xì)胞癌(renal cell carcinoma, RCC)中是否具有特異性異常表達(dá)的lncRNA尚不明確。因此,我們選取了5名腎癌患者的樣本,包括腫瘤組織(T)及其配對(duì)的癌旁無瘤組織(N),通過高通量基因芯片分析來研究腎癌中l(wèi)ncRNA的表達(dá)譜。 材料和方法：我們利用帶有33045個(gè)lncRNA和30215個(gè)mRNA的探針的高通量基因芯片對(duì)5名腎細(xì)胞癌患者的樣本進(jìn)行檢測(cè)分析,篩選差異表達(dá)的lncRNA和mRNA。隨后,我們又根據(jù)芯片結(jié)果選取了兩個(gè)lncRNA (AK096725和ENST00000453068)在70名腎細(xì)胞癌患者的樣本中進(jìn)行了驗(yàn)證,利用定量反轉(zhuǎn)錄聚合酶連鎖反應(yīng)(RT-PCR)檢測(cè)AK096725和ENST00000453068的表達(dá)水平。 結(jié)果：LncRNA芯片在腎細(xì)胞癌組織中檢測(cè)到了27279個(gè)lncRNA有效信號(hào),與癌旁無瘤組織相比其中有480個(gè)lncRNAs表達(dá)上調(diào)(P0.05；T/N1.5),417個(gè)lncRNAs表達(dá)下調(diào)(P0.05；N/T1.5)。另外,19995個(gè)mRNA有效信號(hào)被探針檢測(cè)到,其中458個(gè)mRNAs在腎細(xì)胞癌樣本中表達(dá)升高(P0.05；T/N1.5),413個(gè)表達(dá)降低(P0.05; N/T1.5)。RT-PCR驗(yàn)證顯示AK096725(P=0.043)和ENST00000453068(P＜0.001)在70對(duì)樣本中表達(dá)水平的改變與基因芯片結(jié)果一致。 結(jié)論：本課題利用5對(duì)腎細(xì)胞癌患者的組織樣本,揭示了腎細(xì)胞癌中l(wèi)ncRNA的表達(dá)譜,同時(shí)鑒定出了在腫瘤組織中的異常表達(dá)lncRNA和mRNA。在后期驗(yàn)證試驗(yàn)中,我們發(fā)現(xiàn)AK096725的表達(dá)水平與腎細(xì)胞癌病理類型之間存在一定的相關(guān)性,這一發(fā)現(xiàn)值得關(guān)注。本研究結(jié)果可能對(duì)于尋找腎細(xì)胞癌新的生物學(xué)標(biāo)志物有所幫助。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.11

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