本文選題：肝細(xì)胞肝癌 + miR-105-1　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:越來越多的證據(jù)表明micro RNA通過調(diào)節(jié)各自的靶基因在癌癥的發(fā)生發(fā)展過程中起到重要作用。本研究通過篩選出肝癌可能相關(guān)的miR-105-1及其靶基因,隨后驗(yàn)證在肝癌和正常肝組織中進(jìn)行驗(yàn)證miR-105-1及其靶基因NCOA1的表達(dá)量及兩者的關(guān)系,并分析患者的預(yù)后,研究miR-105-1與肝癌的發(fā)生發(fā)展的可能機(jī)制。方法:我們通過使用基因微陣列和分層聚類分析,篩選肝癌可能相關(guān)的miR-105-1,收集34對人肝癌組織及對應(yīng)的癌旁組織和另外120個(gè)肝癌組織,通過RT-q PCR的方法檢測miR-105-1在肝癌及癌旁正常組織中的表達(dá)量差異加以驗(yàn)證,并進(jìn)一步分析miR-105-1和患者預(yù)后的關(guān)系。通過數(shù)據(jù)庫尋找miR-105-1可能的靶基因NCOA1,并通過RT-q PCR、Western-Blot及肝癌組織的免疫組化來驗(yàn)證miR-105-1和NCOA1的靶向關(guān)系,并聯(lián)合分析miR-105-1和NCOA1與患者預(yù)后的關(guān)系。結(jié)果:我們從GEO和TCGA數(shù)據(jù)庫芯片數(shù)據(jù)分析得出miR-105-1可能為我們新發(fā)現(xiàn)的差異基因,我們檢測了miR-105-1在肝癌組織及癌旁正常組織中均有表達(dá),miR-105-1在34個(gè)配對的HCC與癌旁正常組織中下調(diào)0.59倍(P=0.041),與34個(gè)癌旁正常組織相比,miR-105-1在154個(gè)HCC中下調(diào)0.51倍,(P=0.032)。miR-105-1在154個(gè)HCC中Kaplan-Meier生存分析OS的Log-Rank P=0.031,表明miR-105-1表達(dá)越低的HCC病人生存時(shí)間越短。PFS的Log-Rank P=0.038,表明miR-105-1表達(dá)越低的HCC病人疾病進(jìn)展時(shí)間越短。我們通過GEO數(shù)據(jù)庫及三個(gè)靶基因預(yù)測網(wǎng)站預(yù)測出NCOA1可能為miR-105-1的靶基因,Hela細(xì)胞Realtime RT-PCR檢測miR-105-1 mimics(過表達(dá))和inhibitor(knock-down)對NCOA1 m RNA含量的影響。在驗(yàn)證試驗(yàn)中,Hela細(xì)胞RT-PCR檢測中,與對照組相比,miR-105-1過表達(dá)組,NCOA1表達(dá)量低(P0.001),而miR-105-1敲除組,NCOA1表達(dá)量高(P0.001)。WB檢測miR-105-1過表達(dá)和敲除對NCOA1及已知的下游分子CSF1蛋白水平的影響,與對照組相比,miR-105-1過表達(dá)NCOA1及已知的下游分子CSF1蛋白表達(dá)低,相反,miR-105-1敲除后,NCOA1及已知的下游分子CSF1蛋白表達(dá)高。異位表達(dá)的miR-105-1降低NCOA1的3'-UTR的熒光素酶活性;然而,miR-105-1突變體含有三個(gè)突變的核苷沒有對熒光素酶活性的抑制作用。在癌組織及癌旁組織石蠟切片的組化中,我們發(fā)現(xiàn)NCOA1在正常肝組織中低表達(dá),在肝癌組織中高表達(dá)。NCOA1在154個(gè)HCC中Kaplan-Meier生存分析OS的Log-Rank p=0.011,NCOA1表達(dá)越高的HCC病人生存時(shí)間越短。PFS的Log-Rank p=0.033,NCOA1表達(dá)越高的HCC病人疾病無進(jìn)展時(shí)間越短。聯(lián)合分析miR-105-1和NCOA1在154個(gè)HCC中Kaplan-Meier生存分析OS的Log-Rank p0.001,miR-105-1表達(dá)越低同時(shí)NCOA1表達(dá)越高的HCC病人生存時(shí)間越短。PFS的Log-Rank p=0.002,miR-105-1越低同時(shí)NCOA1表達(dá)越高的HCC病人疾病無進(jìn)展時(shí)間越短。結(jié)論1、miR-105-1在肝癌中的表達(dá)低于正常肝組織。2、miR-105-1可能通過調(diào)控靶基因NCOA1來調(diào)節(jié)肝癌的發(fā)生發(fā)展。3、miR-105-1高表達(dá)的肝癌患者預(yù)后較好,相反,低表達(dá)的患者預(yù)后不良。4、NCOA1高表達(dá)的肝癌患者預(yù)后不良,相反,低表達(dá)的患者預(yù)后較好。5、miR-105-1及NCOA1可能成為判斷肝癌預(yù)后的重要的生物學(xué)指標(biāo)。
[Abstract]:Objective: more and more evidence shows that micro RNA plays an important role in the development of cancer by regulating their target genes. This study screened the possible miR-105-1 and target genes of liver cancer, and then verified the expression of miR-105-1 and its target gene NCOA1 in liver cancer and normal liver tissues and both of them and both. The relationship, and the analysis of the prognosis of patients, the possible mechanism of the development of miR-105-1 and liver cancer. Methods: we use gene microarray and stratified cluster analysis to screen the possible miR-105-1 related to liver cancer, collect 34 human liver cancer tissues and corresponding para cancer tissues and other 120 liver cancer tissues, and detect miR through the RT-q PCR method. The expression of -105-1 in the normal tissues of liver cancer and para cancer was verified, and the relationship between miR-105-1 and patient's prognosis was further analyzed. The possible target gene NCOA1 of miR-105-1 was searched through database, and the targeting relationship between miR-105-1 and NCOA1 was verified by RT-q PCR, Western-Blot and liver cancer tissue, and miR-10 was analyzed jointly. The relationship between 5-1 and NCOA1 and the prognosis of patients. Results: We obtained from GEO and TCGA database chip data analysis that miR-105-1 may be our new differentially identified gene. We detected the expression of miR-105-1 in liver cancer tissues and normal tissues adjacent to cancer, and miR-105-1 was reduced by 0.59 times (P=0.041) in 34 paired HCC and adjacent normal tissues (P=0.041). Compared with 34 adjacent normal tissues, miR-105-1 was down 0.51 times in 154 HCC, and (P=0.032).MiR-105-1 in 154 HCC, Kaplan-Meier survival analysis of OS Log-Rank P=0.031, indicating that the lower the miR-105-1 expression, the shorter the survival time of the HCC patient was, the shorter the disease progression time for the patients with the lower expression. The GEO database and three target gene prediction sites were used to predict that NCOA1 might be the target gene of miR-105-1. Hela cells Realtime RT-PCR detected the influence of miR-105-1 mimics (over expression) and inhibitor (knock-down) on NCOA1 m. P0.001, miR-105-1 knockout group, NCOA1 expression high (P0.001).WB detection of miR-105-1 overexpression and knockout of NCOA1 and known downstream molecules CSF1 protein level, compared with the control group, miR-105-1 overexpression NCOA1 and known downstream molecules CSF1 protein table, on the contrary, miR-105-1 knockout, and the known downstream points The expression of CSF1 protein was high. The miR-105-1 of ectopic expression reduced the luciferase activity of NCOA1 3'-UTR; however, the miR-105-1 mutant contained three mutant nucleosides that did not inhibit the activity of luciferase. In the histochemistry of paraffin sections of cancer tissue and paracancerous tissue, we found that NCOA1 was low expression in normal liver tissue and in liver cancer tissue The higher expression of.NCOA1 in 154 HCC is Log-Rank p=0.011 for the survival analysis of OS, the higher the NCOA1 expression is, the shorter the.PFS Log-Rank p=0.033 of HCC patients, the higher the NCOA1 expression, the shorter the disease progression time. The lower the expression of -105-1 and the higher the expression of NCOA1 in HCC patients, the shorter the.PFS Log-Rank p=0.002, the lower the miR-105-1 and the higher the NCOA1 expression, the shorter the progression free time of the disease. Conclusion 1, the expression of miR-105-1 in the liver cancer is lower than that of the normal liver tissue, and miR-105-1 can regulate the hair of the liver cancer by regulating the target gene. The prognosis of liver cancer patients with high expression of.3 and miR-105-1 is better. On the contrary, the prognosis of patients with low expression is poor.4, and the prognosis of the patients with high expression of NCOA1 is poor. On the contrary, the prognosis of the patients with low expression is better.5. MiR-105-1 and NCOA1 may be the important biological indicators to judge the prognosis of liver cancer.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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本文編號：1984603