本文選題：miR-199b-5p + 乳腺癌�。� 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]本研究通過(guò)檢測(cè)miR-199b-5p在乳腺癌患者組織(包括癌、癌旁及正常組織)及血漿外泌體中的表達(dá)情況,探討其臨床意義及相關(guān)性;同時(shí),我們選用三陰性乳腺癌細(xì)胞株HCC1937(ER、PR及HER-2均陰性)及MCF-7 (激素ER陽(yáng)性、PR及HER-2陰性)作為實(shí)驗(yàn)對(duì)象,通過(guò)mimic在這兩株細(xì)胞中過(guò)表達(dá)miR-199b-5p,觀察 miR-199b-5p 對(duì)乳腺癌 HCC1937、MCF-7 細(xì)胞增殖、遷移、侵襲及凋亡的影響;最后,探討miR-199b-5p與DDR1的相互關(guān)系,為臨床治療乳腺癌提供新的標(biāo)志和潛在重要靶點(diǎn)奠定基礎(chǔ)。[方法]1、以miR-199b-5p為作用靶點(diǎn)合成miRNAmimic并用脂質(zhì)體法轉(zhuǎn)染HCC1937、MCF-7細(xì)胞,同時(shí)設(shè)NC作為陰性對(duì)照組。2、通過(guò)熒光及qRT-PCR篩選穩(wěn)定細(xì)胞轉(zhuǎn)染株。3、用細(xì)胞劃痕的方法檢測(cè)過(guò)表達(dá)miR-199b-5p對(duì)乳腺癌細(xì)胞HCC1937、MCF-7遷移能力的影響。4、用Transwell小室檢測(cè)過(guò)表達(dá)miR-199b-5p對(duì)HCC1937、MCF-7細(xì)胞遷移及侵襲能力的影響。5、用CCK8法檢測(cè)過(guò)表達(dá)miR-199b-5p對(duì)HCC1937、MCF-7細(xì)胞增殖能力的影響。6、用流式細(xì)胞術(shù)分析過(guò)表達(dá)miR-199b-5p對(duì)HCC1937、MCF-7細(xì)胞凋亡率的影響。7、用 western blotting 檢測(cè)過(guò)表達(dá) miR-199b-5p 后對(duì) HCC1937、MCF-7 細(xì)胞中DDR1蛋白表達(dá)的影響。8、用試劑盒提取組織及外周血外泌體中的miRNA,并用qRT-PCR方法檢測(cè)miR-199b-5p在乳腺癌組織中及外周血外泌體中的表達(dá)情況。[結(jié)果]1、 qRT-PCR 檢測(cè)顯示 miR-199b-5p mimic 能明顯提高 HCC1937、MCF-7乳腺癌細(xì)胞株中miR-199b-5p表達(dá)水平(p0.05);2、 CCK8 檢測(cè)顯示 miR-199b-5pmimic 轉(zhuǎn)染 HCC1937、MCF-7 細(xì)胞后細(xì)胞增殖受到顯著抑制(p0.05);3、 細(xì)胞劃痕實(shí)驗(yàn)顯示經(jīng)miR-199b-5p mimic轉(zhuǎn)染HCC1937細(xì)胞后遷移速度明顯減慢(p 0.05);4、 流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示miR-199b-5p mimic轉(zhuǎn)染HCC1937、MCF-7細(xì)胞后凋亡率顯著增加(p 0.05);5、 通過(guò)qRT-PCR檢測(cè)乳腺癌患者癌組織、癌旁組織及正常組織發(fā)現(xiàn),相對(duì)于正常組織,乳腺癌組織中miR-199b-5p表達(dá)顯著下調(diào)(p 0.05)6、 Western blotting 結(jié)果顯示:經(jīng) miR-199b-5p mimic 轉(zhuǎn)染后,DDR1 蛋白表達(dá)均降低(p0.05)。[結(jié)論]1、過(guò)表達(dá)miR-199b-5p可明顯抑制乳腺癌細(xì)胞株HCC1937、MCF-7的增殖,減低其遷移速度,促進(jìn)乳腺癌細(xì)胞HCC1937、MCF-7的凋亡。2、在HCC1937、MCF-7乳腺癌細(xì)胞株中過(guò)表達(dá)miR-199b-5p使DDR1表達(dá)明顯降低。3、相對(duì)于正常組織,乳腺癌組織中miR-199b-5p表達(dá)顯著下調(diào)。4、乳腺良惡性腫瘤患者外周血外泌體中miR-199b-5p表達(dá)無(wú)明顯差異。
[Abstract]:[objective] to investigate the clinical significance and correlation of miR-199b-5p expression in breast cancer tissues (including cancer, cancer and normal tissues) and plasma exocrine. Three negative breast cancer cell lines HCC1937 (ER-PR and HER-2 negative) and MCF-7 (hormone ER positive PR and HER-2 negative) were selected as the experimental subjects. The expression of miR-199b-5pin HCC1937 and HER-2 negative breast cancer cell lines was observed by mimic, and the proliferation and migration of MCF-7 cells were observed by miR-199b-5p. Finally, to explore the relationship between miR-199b-5p and DDR1, to provide a new marker and potential important target for clinical treatment of breast cancer. [methods] 1. Using miR-199b-5p as target, miRNAmimic was synthesized and transfected into HCC1937 MCF-7 cells by liposome method. At the same time, NC was used as negative control group. The stable cell transfection cell line. 3 was screened by fluorescence and qRT-PCR. The effect of overexpression of miR-199b-5p on the migration ability of HCC1937 MCF-7 was detected by cell scratch method. The expression of miR-199b-5p in HCC1937 MCF-7 was detected by Transwell chamber. Effect of cell migration and invasion. The effect of overexpression of miR-199b-5p on the proliferation of HCC1937 MCF-7 cells was detected by CCK8 method. Flow cytometry was used to analyze the effect of overexpression of miR-199b-5p on apoptosis rate of HCC1937 MCF-7 cells. Western blotting was used to detect the expression of miR-199b-5p on the apoptosis rate of MCF-7 cells. Effects of DDR1 protein expression on HCC1937 / MCF-7 cells. MiRNAs were extracted from tissues and peripheral blood secretory bodies with kit, and the expression of miR-199b-5p in breast cancer tissue and peripheral blood exocrine was detected by qRT-PCR. [results] 1. The results of qRT-PCR showed that miR-199b-5p mimic could significantly increase the expression level of miR-199b-5p in HCC1937 MCF-7 breast cancer cell line (p0.05). CCK8 assay showed that miR-199b-5pmimic transfection of HCC1977 MCF-7 cells was significantly inhibited (p0.05), and the cell scratch assay showed that miR-199b-5p mimic was transfected into HCC1937 MCF-7 cell line. The migration rate of HCC1937 cells decreased significantly (p0.05). The results of flow cytometry showed that the apoptosis rate of HCC1937 MCF-7 cells transfected with miR-199b-5p mimic was significantly increased (p0.05). The cancer tissues of breast cancer patients were detected by qRT-PCR. The expression of miR-199b-5p was significantly down-regulated (p0.05) in paracancerous tissues and normal tissues. Western blotting showed that the expression of miR-199b-5p mimic was decreased after transfection (p0.05). [conclusion] 1. Overexpression of miR-199b-5p could significantly inhibit the proliferation of MCF-7 cell line HCC1937, decrease its migration speed, and promote apoptosis of MCF-7 cell line HCC1937. Overexpression of miR-199b-5p in HCC1937 / MCF-7 breast cancer cell line significantly decreased DDR1 expression. The expression of miR-199b-5p was significantly down-regulated in breast cancer, but there was no significant difference in the expression of miR-199b-5p in peripheral blood of patients with benign and malignant breast cancer.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

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