本文選題：乳腺癌 + Let-7c-5p��； 參考：《浙江理工大學(xué)》2017年碩士論文

【摘要】：乳腺癌是女性最普遍的惡性疾病之一,它的高發(fā)生率及死亡率嚴(yán)重威脅女性身心健康。隨著精準(zhǔn)醫(yī)療的提出,乳腺癌的治療方法將更加多樣化,治療效果也將更加令人滿意。在精準(zhǔn)醫(yī)療中基因工程將發(fā)揮重要作用,所以探究基因在乳腺癌中扮演的角色以及其發(fā)揮作用的分子機(jī)制能夠?yàn)榫珳?zhǔn)醫(yī)療奠定理論基礎(chǔ)。Let-7c-5p是抑癌基因let-7家族中的一員,它在前列腺癌、非小細(xì)胞肺癌以及結(jié)腸癌中都起著一定的抑癌作用,在乳腺癌患者血清中檢測(cè)到其表達(dá)量呈明顯下降趨勢(shì),但是let-7c-5p在乳腺癌中的生物學(xué)功能尚未明了。本研究選取9對(duì)乳腺癌以及相對(duì)應(yīng)癌旁組織的石蠟包埋樣本為實(shí)驗(yàn)對(duì)象,提取其總RNA后,進(jìn)行RT-qPCR檢測(cè)let-7c-5p在乳腺癌及癌旁組織中的表達(dá)情況;同時(shí)在乳腺癌細(xì)胞MCF-7中過表達(dá)let-7c-5p,采用MTT的方法測(cè)試let-7c-5p對(duì)MCF-7細(xì)胞增殖能力的影響,利用流式細(xì)胞儀探測(cè)let-7c-5p對(duì)MCF-7細(xì)胞凋亡的影響;然后利用生物信息學(xué)軟件預(yù)測(cè)let-7c-5p潛在的靶標(biāo)基因,并用雙熒光素酶實(shí)驗(yàn)加以驗(yàn)證,再采用熒光定量PCR和Western blotting實(shí)驗(yàn)深入探索其調(diào)控方式。結(jié)果顯示let-7c-5p在9例乳腺癌組織中的表達(dá)量均低于對(duì)應(yīng)的癌旁組織,其在癌組織中的表達(dá)量約為癌旁組織的0.23。對(duì)乳腺癌細(xì)胞MCF-7轉(zhuǎn)染let-7c-5p mimics后,細(xì)胞中l(wèi)et-7c-5p表達(dá)量較對(duì)照組顯著升高(P0.01),轉(zhuǎn)染后細(xì)胞的增殖能力受到明顯抑制(P0.01),且抑制效果與let-7c-5p mimics終濃度呈正相關(guān);流式細(xì)胞儀檢測(cè)結(jié)果表明過表達(dá)let-7c-5p后細(xì)胞凋亡率明顯上升(P0.01)。生物信息學(xué)軟件預(yù)測(cè)ERCC6為let-7c-5p潛在的靶標(biāo)基因,雙熒光素酶實(shí)驗(yàn)、Western blotting和熒光定量PCR結(jié)果表明let-7c-5p能夠結(jié)合至ERCC6 mRNA的3’UTR,從而抑制ERCC6蛋白的合成,但是ERCC6 mRNA的表達(dá)水平并不受let-7c-5p影響。綜上,本研究結(jié)果表明let-7c-5p在乳腺癌組織中呈低表達(dá),在乳腺癌細(xì)胞中過表達(dá)let-7c-5p能明顯抑制細(xì)胞增殖,并且引發(fā)細(xì)胞凋亡,ERCC6為let-7c-5p的靶基因,let-7c-5p可能通過抑制ERCC6蛋白表達(dá)發(fā)揮其抑癌作用。
[Abstract]:Breast cancer is one of the most common malignant diseases in women. Its high incidence and mortality seriously threaten women's physical and mental health. With the development of precise medicine, the treatment of breast cancer will be more diversified and the therapeutic effect will be more satisfactory. Genetic engineering will play an important role in precision medicine, so exploring the role of genes in breast cancer and its molecular mechanisms could lay the theoretical foundation for precision medicine. Let-7c-5p is a member of the let-7 family of tumor suppressor genes. It plays a certain role in the inhibition of prostate cancer, non-small cell lung cancer and colon cancer. The expression of let-7c-5p in serum of breast cancer patients shows a downward trend, but the biological function of let-7c-5p in breast cancer is not clear. In this study, 9 pairs of paraffin embedded samples of breast cancer and corresponding adjacent tissues were selected as experimental objects. The total RNA was extracted and the expression of let-7c-5p in breast cancer and adjacent tissues was detected by RT-qPCR. At the same time, let-7c-5p was overexpressed in MCF-7 of breast cancer cells. The effect of let-7c-5p on the proliferation of MCF-7 cells was tested by MTT method, the effect of let-7c-5p on MCF-7 cell apoptosis was detected by flow cytometry, and the potential target gene of let-7c-5p was predicted by bioinformatics software. The double luciferase experiment was used to verify it, and then fluorescence quantitative PCR and Western blotting experiments were used to explore its regulation mode. The results showed that the expression of let-7c-5p in 9 breast cancer tissues was lower than that in the corresponding paracancerous tissues, and the expression level in the cancer tissues was about 0.23 of that in the paracancerous tissues. The expression of let-7c-5p in breast cancer cells transfected with let-7c-5p mimics by MCF-7 was significantly higher than that in the control group (P 0.01). The proliferation ability of breast cancer cells was significantly inhibited after transfection, and the inhibitory effect was positively correlated with the final concentration of let-7c-5p mimics. The results of flow cytometry showed that the rate of apoptosis increased significantly after overexpression of let-7c-5p. The bioinformatics software predicted that ERCC6 was a potential target gene for let-7c-5p. The results of double luciferase assay and fluorescent quantitative PCR showed that let-7c-5p could bind to the 3- UTRs of ERCC6 mRNA, thereby inhibiting the synthesis of ERCC6 protein. However, the expression of ERCC6 mRNA was not affected by let-7c-5p. In conclusion, the results showed that the expression of let-7c-5p in breast cancer tissues was low, and overexpression of let-7c-5p in breast cancer cells could significantly inhibit the proliferation of breast cancer cells. ERCC6, a target gene of let-7c-5p, may play an inhibitory role by inhibiting the expression of ERCC6 protein.
【學(xué)位授予單位】：浙江理工大學(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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