本文選題：宮頸癌 + miR-21��； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：背景宮頸癌是全世界范圍內(nèi)女性中最常發(fā)生的惡性腫瘤之一,其目前的發(fā)病率在女性惡性腫瘤中居第二位。盡管近些年來由于HPV篩查和早期診斷技術(shù)的應(yīng)用,宮頸癌發(fā)生率在發(fā)達(dá)國家中呈逐年下降的趨勢(shì),但在發(fā)展中國家由于醫(yī)療條件的限制,宮頸癌任然是導(dǎo)致女性死亡的主要原因之一。在中國,宮頸癌在女性惡性腫瘤發(fā)生率中居第一位。關(guān)于宮頸癌的發(fā)生機(jī)制,前人已進(jìn)行了一定的研究。如在宮頸中持續(xù)注射高危的人類乳頭瘤病毒(Human Papilloma Virus, HPV)會(huì)促進(jìn)宮頸癌的發(fā)生；而癌基因蛋白HPVs E5、E6和E7也能單獨(dú)促進(jìn)宮頸癌細(xì)胞的增殖、遷移和侵襲。另外,有研究顯示血管內(nèi)皮生長(zhǎng)因子VEGF-C、蛋白酪氨酸磷酸酶SHP-2以及CD147-4等一些分子也能通過一定的機(jī)制促進(jìn)宮頸癌細(xì)胞的增殖、遷移和侵襲。相對(duì)的,一些腫瘤抑制分子女Beclinl、HDAC10等也可以通過一定的機(jī)制抑制宮頸癌細(xì)胞的侵襲和遷移。近年來,越來越多的研究顯示microRNAs在宮頸癌的發(fā)生發(fā)展中也發(fā)揮著重要的作用。如miR-135a、miR-10a和miR-205的異常高表達(dá)可以分別通過下調(diào)β-catenin、CHL1和CYR61、CTGF來促進(jìn)宮頸癌細(xì)胞的生長(zhǎng)、遷移和侵襲；而腫瘤抑制microRNAs如miR-218、miR-372和miR-214的表達(dá)缺失也會(huì)促進(jìn)宮頸癌的發(fā)生。另外,大量研究顯示具有廣泛致癌作用的miR-21在非小細(xì)胞肺癌、結(jié)直腸癌、卵巢癌、乳腺癌以及食管癌中異常高表達(dá),可以通過調(diào)控PTEN信號(hào)通路來促進(jìn)腫瘤的發(fā)生,但miR-21在宮頸癌發(fā)生發(fā)展中的作用尚很少有人進(jìn)行過系統(tǒng)研究。因此,本研究中我們檢測(cè)了宮頸癌患者腫瘤組織中miR-21和PTEN的表達(dá)情況,發(fā)現(xiàn)miR-21在宮頸癌中異常高表達(dá),并與PTEN的表達(dá)呈負(fù)相關(guān)；然后我們?cè)趯m頸癌CaSki細(xì)胞和HeLa細(xì)胞中研究了miR-21對(duì)細(xì)胞增殖、遷移和和侵襲能力的影響；最后我們研究了miR-21對(duì)PTEN表達(dá)的調(diào)控機(jī)制。本研究通過上述實(shí)驗(yàn)最終證明miR-21的異常高表達(dá)可以通過抑制PTEN信號(hào)通路促進(jìn)宮頸癌細(xì)胞的增殖和遷移。第一部分宮頸癌組織中miR-21高表達(dá),并與PTEN表達(dá)呈負(fù)相關(guān)目的：檢測(cè)miR-21在臨床宮頸癌患者腫瘤組織和正常人宮頸組織中的表達(dá)水平,分析二者之間是否存在顯著差異,從而闡明miR-21表達(dá)水平與宮頸癌發(fā)生的相關(guān)性。并進(jìn)一步研究上述宮頸癌患者腫瘤組織中miR-21表達(dá)水平與抑癌基因PTEN的表達(dá)是否存在相關(guān)性,以及miR-21表達(dá)水平與患者HPV陽性/陰性是否存在相關(guān)性,為探討miR-21在宮頸癌發(fā)生中的作用奠定基礎(chǔ)。方法：收集了36例宮頸癌患者的腫瘤組織樣本作為實(shí)驗(yàn)組,21例正常人的宮頸組織樣本作為對(duì)照組,用TRIzol破裂組織并提取mRNA。運(yùn)用Superscript First-Strand Synthesis System for the RT-PCR kit,引物用其中的隨機(jī)Uni-12引物,獲得各組織的cDNA。設(shè)計(jì)針對(duì)PTEN的特異性引物及Taqman引物,以各組織cDNA為模板,使用Taqman探針法進(jìn)行實(shí)時(shí)定量PCR (Real-Time PCR)實(shí)驗(yàn),檢測(cè)PTEN在上述各組織中的mRNA表達(dá)水平。以β-actin作為內(nèi)參,標(biāo)定其表達(dá)量。用mirVana miRNA Isolation kit提取microRNA,用mirVana RT-qPCR miRNA Detection kit對(duì)miR-21進(jìn)行定量分析,U6小核RNA作為內(nèi)部參照。對(duì)得出的結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析,考察宮頸癌組織中miR-21和PTEN的表達(dá)水平是否具有相關(guān)性。同時(shí)由于這36例宮頸癌樣本中有28例HPV陽性、8例陰性,通過統(tǒng)計(jì)分析研究miR-21表達(dá)水平與HPV陽性/陰性的相關(guān)性。結(jié)果：將21例正常人宮頸組織中miR-21的平均表達(dá)值設(shè)為1.00±0.09,而36例宮頸癌患者腫瘤組織中miR-21的平均表達(dá)值為2.14±0.19,說明宮頸癌組織中miR-21顯著高表達(dá)(P0.001)。此外,這36例宮頸癌患者腫瘤組織中miR-21高表達(dá)時(shí),PTEN表達(dá)水平顯著較低(P0.001),二者的表達(dá)水平呈顯著負(fù)相關(guān)性(R2=0.2713,P=0.0011)。而HPV陽性/陰性的宮頸癌患者腫瘤組織中miR-21的表達(dá)沒有明顯差異。結(jié)論：宮頸癌患者腫瘤組織中miR-21高表達(dá),并與PTEN的表達(dá)呈負(fù)相關(guān)；miR-21的表達(dá)水平與HPV陽性/陰性無顯著相關(guān)性。第二部分miR-21過表達(dá)對(duì)宮頸癌細(xì)胞體外生物學(xué)行為的影響目的：在宮頸癌CaSki和HeLa細(xì)胞中轉(zhuǎn)染miR-21 mimics、miR-21抑制劑或miRNA對(duì)照,研究miR-21表達(dá)水平改變對(duì)宮頸癌細(xì)胞的增殖、遷移和侵襲能力的影響,從而闡明miR-21在宮頸癌發(fā)生中的作用。方法：用含10%胎牛血清的RPMI-1640或者DMEM,37℃,5%CO2濃度的環(huán)境下培養(yǎng)宮頸癌CaSki細(xì)胞和HeLa細(xì)胞；使用Lipofectamine 2000試劑將miR-21 mimics、miR-21抑制劑和miRNA對(duì)照分別轉(zhuǎn)染進(jìn)入宮頸癌CaSki細(xì)胞和HeLa細(xì)胞中；使用Real-Time PCR檢測(cè)轉(zhuǎn)染效果,并將轉(zhuǎn)染好的上述細(xì)胞分別進(jìn)行細(xì)胞增殖、克隆形成、遷移和侵襲能力的檢測(cè)。首先,將上述轉(zhuǎn)染好的細(xì)胞在CCK-8孵育,在24h時(shí)檢測(cè)細(xì)胞在450nm下的吸光度,檢測(cè)miR-21表達(dá)水平改變對(duì)上述細(xì)胞增殖能力的影響；另外,同樣將上述轉(zhuǎn)染好的細(xì)胞在CCK-8孵育,分別在0、24、36和48h時(shí)檢測(cè)細(xì)胞在450nm下的吸光度,從而研究miR-21表達(dá)水平改變后上述細(xì)胞增殖能力隨時(shí)間的變化情況。其次,將上述轉(zhuǎn)染好的細(xì)胞計(jì)數(shù)300個(gè)細(xì)胞后在 正常培養(yǎng)基中孵育,48h后用0.005%的結(jié)晶紫染色20min, Image J軟件統(tǒng)計(jì)克隆數(shù),研究miR-21表達(dá)水平改變對(duì)宮頸癌細(xì)胞克隆形成能力的影響。再次,將上述轉(zhuǎn)染好的細(xì)胞接種到12孔板中至密度為85%,進(jìn)行劃痕愈合實(shí)驗(yàn),然后分別在0、24、48h觀察劃痕愈合請(qǐng)況,檢測(cè)miR-21表達(dá)水平改變對(duì)宮頸癌細(xì)胞遷移能力的影響。最后,用Matrigel包被好Transwell小室,將上述轉(zhuǎn)染好的細(xì)胞懸浮于無血清培養(yǎng)基中,放于小室上層,小室浸泡在20%血清的培養(yǎng)基中,24h后觀察穿過Matrigel后附著于基底膜的細(xì)胞并計(jì)數(shù),以確定miR-21表達(dá)水平改變對(duì)宮頸癌細(xì)胞侵襲能力的影響。結(jié)果：在CaSki和HeLa細(xì)胞中轉(zhuǎn)染miR-21 mimics可以使miR-21的表達(dá)水平顯著上升,而轉(zhuǎn)染miR-21抑制劑則可以導(dǎo)致miR-21的表達(dá)水平顯著下降。與對(duì)照組相比,轉(zhuǎn)染miR-21 mimics后CaSki和HeLa細(xì)胞的增殖能力顯著增加,形成的克隆數(shù)顯著增多,細(xì)胞劃痕實(shí)驗(yàn)中劃痕愈合速度顯著加快,Transwell實(shí)驗(yàn)中穿。過Matrigel的細(xì)胞數(shù)顯著增多；而轉(zhuǎn)染。miR-21抑制劑后CaSki和HeLa細(xì)胞的增殖能力顯著降低,形成的克隆數(shù)顯著減少,細(xì)胞劃痕實(shí)驗(yàn)。中劃痕愈合速度顯著變慢,Transwell實(shí)驗(yàn)中穿過Matrigel的細(xì)胞。數(shù)顯著減少。結(jié)論：miR-21過表達(dá)可以顯著提高宮頸癌CaSki細(xì)胞和HeLa細(xì)胞的增殖、克隆形成、遷移和侵襲能力,說明miR-21是宮頸癌的一個(gè)癌基因,miR-21高表達(dá)對(duì) 宮頸癌的發(fā)生和發(fā)展具有顯著促進(jìn)作用。第三部分miR-21通過抑制PTEN調(diào)控宮頸癌細(xì)胞的增殖目的：前期工作發(fā)�，F(xiàn)宮頸癌患者腫瘤組織 中miR-21高表達(dá),PTEN低表達(dá),兩者表達(dá)水平呈。顯著負(fù)相關(guān)。為了明確miR-21和PTE N的上下游關(guān)系,研究。miR-21發(fā)揮作用的分子機(jī)制進(jìn)行了本部分的研究。方法：在宮頸癌CaSki細(xì)胞和HeLa細(xì)胞中分別轉(zhuǎn)染miR-21 mimics和miRNA對(duì)照,通過Real-Time PCR和Western blot分別在mRNA和蛋白水平上檢測(cè)PTEN的變化,以確定PTEN是否作用在miR-21下游。通過分子克隆手段構(gòu)建PTEN-pcDNA3.1質(zhì)粒,將其穩(wěn)定轉(zhuǎn)染到宮頸。癌CaSki細(xì)胞中使其過表達(dá)PTE N基因,通過Real-Time PCR和Western blot分別在mRNA和蛋白水平上檢測(cè)PTEN的表達(dá)水平,以確定是否成功過表達(dá)PTEN基因；然后通過CCK-8實(shí)驗(yàn)檢測(cè)過表達(dá)PTEN對(duì)細(xì)胞增殖 能力的影響,通過克隆形成實(shí)驗(yàn)檢測(cè) 過表達(dá)PTEN對(duì)細(xì)胞克隆形成能力的影響,從而最終確定miR-21是否通過PTEN影響細(xì)胞的增殖能力。結(jié)果：與對(duì)照組相比,在宮頸癌CaSki細(xì)胞和Hela細(xì)胞中轉(zhuǎn)染miR-21 mimics后,PTEN基因 在mRNA和蛋白水平上的 表達(dá)量均有顯著降低。構(gòu)建的PTEN-pcDNA3.1質(zhì)粒能夠在CaSki細(xì)胞成功過表達(dá)PTEN基因,而過表達(dá)PTEN基因后,CaSki細(xì)胞的增殖能力和克隆形成能力顯著下降。結(jié)論：宮頸癌細(xì)胞中轉(zhuǎn)染miR-21 mimics能明顯降低PTEN的表達(dá)水平,PTEN是miR-21的下游基因,而miR-21影響細(xì)胞 的增殖能力也是通過抑制PTEN來實(shí)現(xiàn)的。
[Abstract]:Cervical cancer is one of the most frequently occurring malignant tumors in the world . The incidence of cervical cancer is the second in the malignant tumor of women . Although the incidence of cervical cancer has declined year by year in developed countries due to the application of HPV screening and early diagnosis technology , cervical cancer is the first cause of female death in developing countries . In China , cervical cancer is the first leading cause of female death . In China , cervical cancer has been studied in a certain way .
In addition , some molecules such as VEGF - C , protein tyrosine phosphatase SHP - 2 and CD147 - 4 can promote the proliferation , migration and invasion of cervical cancer cells .
In addition , the expression of miR - 21 and PTEN in non - small cell lung cancer , colorectal cancer , ovarian cancer , breast cancer and esophageal cancer can be promoted by controlling PTEN signal pathway .
Then , we studied the effects of miR - 21 on cell proliferation , migration and invasion ability in cervical cancer cells and HeLa cells .
The expression level of miR - 21 in cervical cancer patients and the expression level of miR - 21 in human cervical tissues were analyzed by using the method of Superscript First - Strand Synthesis System for the RT - PCR kit . Conclusion : The expression of miR - 21 in cervical cancer patients was negatively correlated with the expression of PTEN . Conclusion : The expression of miR - 21 in cervical cancer patients was negatively correlated with the expression of PTEN .
The expression level of miR - 21 was not significantly correlated with HPV positive / negative . The effect of miR - 21 overexpression on the biological behavior of cervical cancer cells was investigated . The effects of miR - 21 expression level on the proliferation , migration and invasion ability of cervical cancer cells were investigated .
The miR - 21 inhibitor , the miR - 21 inhibitor and the miRNA control are respectively transfected into cervical cancer cells and HeLa cells by using the Lipofectamine 2000 reagent ;
firstly , the transfected cells are incubated in CCK - 8 , the absorbance of the cells at 450 nm is detected at 24h , and the influence of the miR - 21 expression level change on the cell proliferation ability is detected ;
The effect of miR - 21 expression level on the invasion ability of cervical cancer cells was investigated . After 24 hours , the transfected cells were cultured in a medium containing 20 % serum , then the transfected cells were incubated with 0.005 % crystal violet for 20 minutes . The results showed that the expression level of miR - 21 was significantly increased after 24 h .
Conclusion : miR - 21 overexpression can significantly improve the proliferation , clonal formation , migration and invasion ability of cervical cancer cells .
The results showed that the expression level of PTEN gene was significantly lower than that of control group , and the expression level of PTEN gene was significantly lower than that of control group . Conclusion : The expression level of PTEN gene was significantly lower than that of control group .
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.33

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本文編號(hào)：2069392