發(fā)布時間：2018-03-16 06:08

  本文選題：miR-375　切入點：miR-217　出處：《廣東藥科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:通過高通量測序分析,篩選出人乳腺癌耐阿霉素細(xì)胞與野生型的乳腺癌細(xì)胞中差異表達(dá)的miRNAs,研究乳腺癌耐阿霉素細(xì)胞中低表達(dá)的miRNA-375和高表達(dá)的miRNA-217對人乳腺癌耐阿霉素細(xì)胞增殖以及侵襲、遷移能力的影響。方法:通過WST-1實驗先驗證得到的乳腺癌耐藥細(xì)胞是否為穩(wěn)定的耐藥細(xì)胞。通過對野生型乳腺癌細(xì)胞和耐阿霉素的乳腺癌細(xì)胞的樣品進(jìn)行Small RNA高通量測序分析,獲取正常乳腺癌細(xì)胞和耐阿霉素的乳腺癌細(xì)胞差異表達(dá)miRNA信息;選擇一些差異表達(dá)的miRNA,用Real-time RT-PCR實驗對篩選出差異表達(dá)顯著的mi RNA進(jìn)行驗證。將miR-217 inhibitor和inhibitor NC、miR-375模擬物(miR-375 mimics)和mimics NC分別轉(zhuǎn)染MCF-7/Adr;阿霉素處理后用WST-1法檢測耐阿霉素細(xì)胞的增殖情況;流式細(xì)胞儀檢測MCF-7/Adr細(xì)胞周期和凋亡;細(xì)胞劃痕和transwell遷移、侵襲實驗檢測MCF-7/Adr細(xì)胞的遷移和侵襲能力。結(jié)果:1、通過WST-1實驗驗證得到的乳腺癌耐藥細(xì)胞為穩(wěn)定的耐藥細(xì)胞,對不同藥物濃度的阿霉素,耐藥的乳腺癌細(xì)胞比野生型的乳腺癌細(xì)胞有更明顯的耐受性。2、高通量測序結(jié)果顯示:與配對的野生型的乳腺癌細(xì)胞相比,有多個miRNAs在耐阿霉素的乳腺癌細(xì)胞中表達(dá)異常。3、Real-time RT-PCR結(jié)果顯示:miR-375在耐阿霉素的乳腺癌細(xì)胞中表達(dá)顯著降低,miR-217在耐阿霉素的乳腺癌細(xì)胞中表達(dá)顯著提高。4、WST-1實驗顯示在乳腺癌耐藥(MCF-7/adr)細(xì)胞中分別轉(zhuǎn)染高表達(dá)的miR-217、陰性對照組inhibitor NC和低表達(dá)的miR-375、模擬物對照組mimics NC,發(fā)現(xiàn)表達(dá)72h后,與對照組相比轉(zhuǎn)染小分子的乳腺癌耐藥細(xì)胞的生長速度均明顯減慢。5、流式細(xì)胞儀測細(xì)胞周期、凋亡結(jié)果顯示:上調(diào)miR-375和下調(diào)mi R-217的表達(dá)后,可以提高M(jìn)CF-7/Adr細(xì)胞對阿霉素的敏感性并促進(jìn)其凋亡;可以將MCF-7/Adr細(xì)胞周期阻滯在G1期。6、Tranwell遷移實驗結(jié)果顯示:上調(diào)miR-375和下調(diào)miR-217的表達(dá)均可以使乳腺癌耐阿霉素細(xì)胞的遷移能力降低。7、Tranwell侵襲實驗結(jié)果顯示:上調(diào)miR-375和下調(diào)miR-217的表達(dá)均可以使乳腺癌耐阿霉素細(xì)胞的侵襲能力降低。8、細(xì)胞劃痕結(jié)果顯示:上調(diào)miR-375和下調(diào)miR-217的表達(dá)會使MCF-7/Adr細(xì)胞的遷移能力降低。結(jié)論:1、miR-217在乳腺癌耐阿霉素細(xì)胞中表達(dá)水平顯著高于野生型的乳腺癌細(xì)胞;miR-375在乳腺癌耐阿霉素細(xì)胞中表達(dá)水平顯著低于野生型的乳腺癌細(xì)胞。2、上調(diào)乳腺癌耐阿霉素細(xì)胞中miR-375的表達(dá),下調(diào)乳腺癌耐阿霉素細(xì)胞中miR-217的表達(dá)水平,均可以增加MCF-7/Adr細(xì)胞對阿霉素的敏感性。
[Abstract]:Objective: to analyze by high throughput sequencing. The differentially expressed miRNAss in human breast cancer doxorubicin resistant cells and wild type breast cancer cells were screened. The effects of low expression miRNA-375 and high expression miRNA-217 on the proliferation and invasion of human breast cancer doxorubicin resistant cells were studied. Methods: WST-1 assay was used to determine whether the breast cancer cells were stable drug resistant cells. Small RNA high throughput sequencing was performed on the samples of wild type breast cancer cells and adriamycin resistant breast cancer cells. The differentially expressed miRNA information was obtained between normal breast cancer cells and adriamycin resistant breast cancer cells. Some differentially expressed miRNAs were selected, and the differentially expressed miRNA was screened by Real-time RT-PCR assay. The miR-217 inhibitor and inhibitor NCncfmiR-375 mimicswere transfected into MCF-7 / Adr.After treated with adriamycin, the proliferation of adriamycin-resistant cells was detected by WST-1 method. Flow cytometry was used to detect the cell cycle and apoptosis of MCF-7/Adr cells, cell scratch and transwell migration, invasion assay to detect the migration and invasion ability of MCF-7/Adr cells. For different concentrations of adriamycin, drug-resistant breast cancer cells were significantly more tolerant than wild type breast cancer cells. High-throughput sequencing results showed that: compared with matched wild type breast cancer cells, Abnormal expression of miRNAs in adriamycin-resistant breast cancer cells. 3 Real-time RT-PCR results showed that the expression of 1 miR-375 in adriamycin resistant breast cancer cells decreased significantly, and the expression of mmiR-217 in adriamycin-resistant breast cancer cells increased significantly. Breast cancer MCF-7 / adr cells were transfected with high expression miR-217, negative control group inhibitor NC and low expression miR-375, and mimic control group mimics NC2 h. Compared with the control group, the growth rate of drug-resistant breast cancer cells transfected with small molecules was significantly slower than that of the control group. Flow cytometry was used to measure the cell cycle and apoptosis showed that the expression of miR-375 was up-regulated and the expression of miR-217 was down-regulated. It can increase the sensitivity of MCF-7/Adr cells to adriamycin and promote its apoptosis. The results of tranwell migration assay showed that up-regulation of miR-375 and down-regulation of miR-217 could decrease the migration ability of adriamycin-resistant breast cancer cells. The results showed that both up-regulation of miR-375 and down-regulation of miR-217 were observed. The results of cell scratch showed that up-regulation of miR-375 and down-regulation of miR-217 expression could decrease the migration ability of MCF-7/Adr cells. Conclusion\% 1 + miR-217 can reduce the migration ability of MCF-7/Adr cells in adriamycin-resistant breast cancer cells. The expression level of miR-375 was significantly higher than that of wild type breast cancer cells, and the expression level of miR-375 was significantly lower than that of wild type breast cancer cells. The expression of miR-375 was up-regulated in adriamycin resistant breast cancer cells. Down-regulation of miR-217 expression in adriamycin resistant breast cancer cells increased the sensitivity of MCF-7/Adr cells to adriamycin.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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相關(guān)期刊論文 前1條

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本文編號：1618638