【摘要】：背景:胰腺癌是最具侵襲性的癌癥之一,經(jīng)過大量的努力,胰腺癌的早期診斷有了很大的提高,但總體診斷率還是很低,胰腺癌的預(yù)后仍未得到明顯的改善,五年生存率僅有8%。因此,對(duì)其腫瘤分子生物學(xué)的研究有助于進(jìn)一步揭示其發(fā)生、發(fā)展機(jī)制,有助于早期診斷和治療。miRNAs(MicroRNA)是近來腫瘤分子生物學(xué)研究的一個(gè)熱點(diǎn),雖然很多胰腺癌的研究提示miR-23a是一種致癌調(diào)節(jié)因子,但其潛在的分子機(jī)制依舊不明。PLK-1(Polo樣激酶1)在多種細(xì)胞內(nèi)有絲分裂進(jìn)程中起到至關(guān)重要的作用,許多miRNAs抑制PLK-1的表達(dá),從而抑制腫瘤的發(fā)展。前期的預(yù)實(shí)驗(yàn)研究結(jié)果顯示miR-23a抑制胰腺癌細(xì)胞MIA-PaCa-2的增殖,靶基因預(yù)測(cè)軟件推測(cè)miR-23a與PLK-1可能存在靶向關(guān)系�？紤]到miRNAs和PLK-1的重要作用,有必要對(duì)其進(jìn)一步的研究。目的:1、研究miR-23a和PLK1在人胰腺癌組織和細(xì)胞株中表達(dá)水平,并探討兩者的相關(guān)性。2、研究miR-23a對(duì)胰腺癌細(xì)胞增殖、遷移和侵襲、以及凋亡的影響。3、研究miR-23a靶向PLK-1抑制胰腺癌細(xì)胞增殖的相關(guān)機(jī)制。方法:1、熒光定量PCR檢測(cè)miR-23a和PLK-1在對(duì)胰腺癌與癌旁組織和4種胰腺癌細(xì)胞株中的表達(dá)情況。構(gòu)建miR-23a和PLK-1的過表達(dá)/干擾質(zhì)粒,熒光素酶報(bào)告基因方法證明PLK-1是miR-23a直接調(diào)控的靶基因。2、選取人胰腺癌細(xì)胞株MIA-PaCa-2和Panc-1,CCK8法檢測(cè)轉(zhuǎn)染后細(xì)胞增殖能力的變化;Transwell實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后細(xì)胞侵襲能力的變化;流式細(xì)胞儀檢測(cè)轉(zhuǎn)染后細(xì)胞凋亡的變化。建立MIA-PaCa-2細(xì)胞的裸鼠皮下異種成瘤模型,驗(yàn)證miR-23a對(duì)胰腺癌細(xì)胞成瘤能力的影響。3、選取人胰腺癌細(xì)胞株MIA-PaCa-2,轉(zhuǎn)染miR-23a、PLK-1以及共轉(zhuǎn)染miR-23a和PLK-1,Transwell實(shí)驗(yàn)檢測(cè)后細(xì)胞侵襲能力的變化;流式細(xì)胞儀檢測(cè)轉(zhuǎn)染后細(xì)胞凋亡的變化。熒光定量PCR檢測(cè)體內(nèi)成瘤試驗(yàn)中miR-23a對(duì)PLK-1不表達(dá)的影響。最后,Western Blot檢測(cè)轉(zhuǎn)染后細(xì)胞中PLK-1以及其下游信號(hào)因子Bax、Bcl2、cyclinB1,E-cadherin、Vimentin 蛋白表達(dá)變化。結(jié)果:1、20對(duì)組織中,胰腺癌組織中miR-23a表達(dá)高于相應(yīng)癌旁組織的有19對(duì),PLK-1表達(dá)高于相應(yīng)癌旁組織的有11對(duì)。在人胰腺癌細(xì)胞株中,miR-23a和PLK-1表達(dá)水平均明顯升高,兩者的表達(dá)水平呈負(fù)相關(guān)。雙熒光素酶實(shí)驗(yàn)證明PLK-1是miR-23a直接調(diào)控的靶基因。2、與對(duì)照組相比,MIA-PaCa-2細(xì)胞的miR-23a的過表達(dá)組細(xì)胞增殖能力減弱,細(xì)胞遷移和侵襲能力減弱,細(xì)胞凋亡明顯增強(qiáng),而Panc-1細(xì)胞結(jié)果與MIA-PaCa-2細(xì)胞相反。動(dòng)物實(shí)驗(yàn)結(jié)果表明MIA-PaCa-2細(xì)胞的miR-23a的過表達(dá)組的腫瘤體積和瘤重明顯減小,抑瘤率明顯增加。3、MIA-PaCa-2細(xì)胞過表達(dá)PLK-1組與對(duì)照組相比,細(xì)胞遷移和侵襲能力增強(qiáng),細(xì)胞凋亡明顯減弱;共轉(zhuǎn)染miR-23a和PLK-1組結(jié)果與對(duì)照組相似。裸鼠成瘤實(shí)驗(yàn)MIA-PaCa-2細(xì)胞轉(zhuǎn)染miR-23a后,導(dǎo)致PLK-1表達(dá)的下調(diào),進(jìn)而導(dǎo)致其下游信號(hào)通路因子BCL-2,CyclinB1 and Vimentin表達(dá)的下調(diào),以及Bax and E-cadherin表達(dá)的上調(diào)。而轉(zhuǎn)染過表達(dá)PLK-1載體后,結(jié)果卻相反。體內(nèi)實(shí)驗(yàn)中,當(dāng)miR-23a多表達(dá)時(shí),PLK-1的表達(dá)明顯抑制。結(jié)論:1、miR-23a在胰腺癌組織和胰腺癌細(xì)胞中高表達(dá),在胰腺癌細(xì)胞株中,miR-23a與PLK-1的表達(dá)呈負(fù)相關(guān)性,PLK-1是miR-23a直接調(diào)控的靶基因。2、miR-23a抑制胰腺癌細(xì)胞MIA-PaCa-2增殖、遷移和侵襲,促進(jìn)細(xì)胞凋亡,而Panc-1細(xì)胞卻相反。3、miR-23a通過靶向PLK-1抑制胰腺癌細(xì)胞增殖、遷移和侵襲,促進(jìn)細(xì)胞凋亡。
[Abstract]:Background: Pancreatic cancer is one of the most invasive cancers. After a great deal of effort, the early diagnosis of pancreatic cancer has improved greatly, but the overall diagnostic rate is still low, the prognosis of pancreatic cancer is still not significantly improved, and the five-year survival rate is only 8%. Therefore, the study of molecular biology of tumor helps to further reveal its genesis and development mechanism, which can be helpful for early diagnosis and treatment. MicroRNA is a hot spot in recent tumor molecular biology. Although many pancreatic cancer studies suggest that miR-23a is an oncogenic regulatory factor, its potential molecular mechanism remains unknown. PLK-1 (Polo-like kinase 1) plays an important role in the mitosis of various cells, and many miRNAs inhibit the expression of PLK-1, thus inhibiting the development of tumor. Pre-experimental results showed that miR-23a inhibited the proliferation of MIA-PaCa-2 in pancreatic cancer cells. The target gene prediction software estimated that miR-23a and PLK-1 might have targeted relationship. Considering the important role of miRNAs and PLK-1, it is necessary to further study it. Objective: To study the expression level of miR-23a and PLK1 in human pancreatic cancer tissues and cell lines and to investigate the correlation of miR-23a to pancreatic cancer cell proliferation, migration and invasion and apoptosis. Methods: 1. Fluorescent quantitative PCR was used to detect the expression of miR-23a and PLK-1 in pancreatic cancer and 4 pancreatic cancer cell lines. The expression/ interference plasmids of miR-23a and PLK-1 were constructed, and the luciferase reporter gene method proved that PLK-1 was a target gene directly regulated by miR-23a. Flow cytometry was used to detect the changes of apoptosis after transfection. A nude mouse subcutaneous xenograft model of MIA-PaCa-2 cells was established to verify the effect of miR-23a on the tumorigenicity of pancreatic cancer cells. Flow cytometry was used to detect the changes of apoptosis after transfection. The effect of miR-23a on the expression of PLK-1 in vivo was detected by fluorescence quantitative PCR. Finally, the expression of PLK-1 and its downstream signal factors Bax, Bcl2, cyclin B1, E-cadherin and Vimentin in transfected cells were detected by Western blot. Results: The expression of miR-23a in pancreatic cancer tissues was higher than that of the adjacent tissues, and the expression of PLK-1 was higher than that of the adjacent tissues. In human pancreatic cancer cell line, the expression level of miR-23a and PLK-1 was significantly increased, and the expression level of miR-232a and PLK-1 was negatively correlated. The results of double luciferase assay showed that PLK-1 was the target gene directly regulated by miR-23a. Compared with the control group, the expression of miR-23a in MIA-PaCa-2 cells weakened, cell migration and invasion ability weakened, cell apoptosis was enhanced, and the results of Panc-1 cells were opposite to MIA-PaCa-2 cells. The results of animal experiment showed that the tumor volume and tumor weight of miR-23a in MIA-PaCa-2 cells decreased significantly, and the tumor suppressor rate was increased. The results of miR-23a and PLK-1 group were similar to those in the control group. After Mia-PaCa-2 cells transfected with miR-23a in nude mice, the down-regulation of the expression of PLK-1 resulted in down-regulation of the downstream signal pathway factor BCL-2, CyclinB1 and Vimentin and up-regulation of Bax and E-cadherin expression. However, after the expression of PLK-1 vector was transfected, the results were reversed. In vivo experiments, the expression of PLK-1 was significantly inhibited when miR-23a was expressed. Conclusion: 1. miR-23a is highly expressed in pancreatic cancer tissues and pancreatic cancer cells. In pancreatic cancer cell lines, the expression of miR-23a is negatively correlated with the expression of PLK-1, and PLK-1 is a target gene directly regulated by miR-23a. The miR-23a inhibits the proliferation, migration and invasion of human pancreatic cancer cells MIA-PaCa-2, and promotes apoptosis. and the Panc-1 cell is opposite. 3, miR-23a inhibits proliferation, migration and invasion of pancreatic cancer cells by targeting PLK-1, and promotes apoptosis.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 Milena Ilic;Irena Ilic;;Epidemiology of pancreatic cancer[J];World Journal of Gastroenterology;2016年44期

2 周燕;唐運(yùn)蓮;甘潤(rùn)良;黃s，

本文編號(hào)：2299356