發(fā)布時(shí)間：2018-05-18 09:23

  本文選題：子宮頸癌 + miR-195��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景：子宮頸癌是全世界女性最常見(jiàn)的生殖系統(tǒng)惡性腫瘤之一,發(fā)病率僅次于乳腺癌而位居第二位,死亡率為第五位,嚴(yán)重威脅人類(lèi)生命和健康。據(jù)統(tǒng)計(jì),全世界每年大約有50萬(wàn)新發(fā)子宮頸癌病例,占所有癌癥新發(fā)病例的5%；近27萬(wàn)患者死亡。我國(guó)每年新發(fā)病例13-15萬(wàn),大約占全球發(fā)病總數(shù)的三分之一；我國(guó)每年子宮頸癌死亡人數(shù)大約為5萬(wàn)。子宮頸癌的主要病理類(lèi)型有鱗狀上皮癌和腺上皮癌,其進(jìn)展是一個(gè)相當(dāng)復(fù)雜的過(guò)程,與多基因多階段的異常調(diào)控有關(guān),目前我們對(duì)子宮頸癌發(fā)生發(fā)展機(jī)制的了解仍不全面。研究已證實(shí),高危型人乳頭瘤病毒(HPV)持續(xù)感染是子宮頸癌明確的致病原因。然而,感染HPV的女性并不一定發(fā)展成為子宮頸癌；此外,并不是所有處于子宮頸上皮癌變的患者最終均能發(fā)展成為子宮頸浸潤(rùn)癌。這說(shuō)明子宮頸癌的發(fā)生發(fā)展是個(gè)復(fù)雜的過(guò)程,除了人類(lèi)HPV病毒之外,還有其他因素在子宮頸癌的發(fā)生發(fā)展中發(fā)揮重要作用。因此,子宮頸癌發(fā)病和進(jìn)展的分子機(jī)制仍需深入研究。MicroRNAs (miRNAs)是由20-25個(gè)核苷酸組成的RNA,通過(guò)與靶基因的3’非翻譯區(qū)(3’-UTR)結(jié)合進(jìn)而促進(jìn)mRNA降解或者抑制mRNA翻譯而發(fā)揮作用。通過(guò)在轉(zhuǎn)錄后水平調(diào)控基因表達(dá),miRNAs在很多生命活動(dòng)中發(fā)揮其重要功能,包括細(xì)胞增殖、分化、凋亡、發(fā)育等。已有研究報(bào)道,miRNAs表達(dá)異常與許多疾病如糖尿病、心血管疾病、類(lèi)風(fēng)濕關(guān)節(jié)炎等的發(fā)病及進(jìn)展有密切相關(guān)性。近年來(lái)研究表明, miRNAs在人類(lèi)許多癌癥中也發(fā)揮重要作用,參與了腫瘤的發(fā)生、發(fā)展以及轉(zhuǎn)移等多個(gè)過(guò)程。研究發(fā)現(xiàn),有些miRNAs發(fā)揮癌基因作用,另外一些miRNAs發(fā)揮抑癌基因作用。miRNAs與其調(diào)節(jié)的靶基因形成調(diào)控網(wǎng)絡(luò)共同調(diào)節(jié)腫瘤的發(fā)生及發(fā)展。研究還證實(shí),miRNAs在癌癥患者血清及癌組織中表達(dá)異常與腫瘤的發(fā)病、進(jìn)展、轉(zhuǎn)移、侵襲及耐藥有密切的關(guān)系,因此miRNAs可能成為腫瘤診斷的良好靶標(biāo)分子。MicroRNA-195 (miR-195)是一重要的抑癌miRNA,在肺癌、乳腺癌等多種腫瘤中均發(fā)揮抑癌基因的作用。然而,miR-195在子宮頸癌中的作用尚不完全清楚。為明確miR-195在子宮頸癌發(fā)生發(fā)展中的作用,本學(xué)位論文第一部分首先從臨床樣本出發(fā),檢測(cè)miR-195在子宮頸癌組織及癌旁組織中的表達(dá)情況,分析其表達(dá)與子宮頸癌發(fā)生、發(fā)展、轉(zhuǎn)移的相關(guān)關(guān)系。結(jié)果提示miR-195在子宮頸癌中可能發(fā)揮抑癌基因作用。腫瘤細(xì)胞的增殖和轉(zhuǎn)移是子宮頸癌發(fā)生發(fā)展的關(guān)鍵環(huán)節(jié)。腫瘤細(xì)胞的增殖與腫瘤的生長(zhǎng)密切相關(guān),而腫瘤細(xì)胞的侵襲、遷移能力決定了其轉(zhuǎn)移能力。為進(jìn)一步明確miR-195對(duì)子宮頸癌發(fā)病的抑制作用,本學(xué)位論文第二部分在體外培養(yǎng)的子宮頸癌細(xì)胞(HeLa)中表達(dá)miR-195,檢測(cè)miR-195對(duì)細(xì)胞增殖、侵襲、遷移能力的影響。在確定miR-195能抑制HeLa細(xì)胞增殖、侵襲及遷移能力的基礎(chǔ)上,本學(xué)位論文第三部分深入研究了miR-195發(fā)揮作用的分子機(jī)制,篩選并鑒定了細(xì)胞周期蛋白D2 (CyclinD2, CCND2)和轉(zhuǎn)錄因子MYB為miR-195直接調(diào)控的靶基因。細(xì)胞增殖與細(xì)胞周期密切相關(guān),而CCND2是一重要的細(xì)胞周期蛋白。CCND2通過(guò)與周期蛋白依賴(lài)的激酶4(CDK4)及周期蛋白依賴(lài)的激酶6(CDK6)結(jié)合,進(jìn)而調(diào)節(jié)CDK4和CDK6的活性,從而促進(jìn)細(xì)胞從G1期轉(zhuǎn)化至S期。研究表明,CCND2異常表達(dá)與人腦組織膠質(zhì)瘤、胃癌等多種腫瘤的發(fā)生具有密切相關(guān)性。MYB是廣泛存在的轉(zhuǎn)錄因子,它可以通過(guò)調(diào)節(jié)基因表達(dá)從而在細(xì)胞的分化與增殖過(guò)程中起到重要作用。MYB在神經(jīng)母細(xì)胞瘤及乳腺癌等多種腫瘤組織中呈現(xiàn)高表達(dá)狀態(tài),并且其表達(dá)水平與預(yù)后密切相關(guān)。此外還有研究表明,MYB蛋白和腫瘤侵襲及轉(zhuǎn)移密切相關(guān)。可見(jiàn),CCND2和MYB是兩個(gè)重要的促腫瘤蛋白。我們第三部分實(shí)驗(yàn)結(jié)果顯示,miR-195可作用于CCND2及MYB基因的3’-UTR而直接抑制蛋白質(zhì)表達(dá),該結(jié)果初步闡明了miR-195抑制宮頸癌HeLa細(xì)胞增殖、侵襲及遷移的分子機(jī)制。本論文的基本框架和主要研究?jī)?nèi)容如圖1所示,各部分的詳細(xì)研究思路和技術(shù)路線(xiàn)將在論文中作進(jìn)一步的闡述。第一部分：miR-195在子宮頸癌組織中的表達(dá)研究目的：檢測(cè)子宮頸癌組織及癌旁組織中miR-195的表達(dá)情況,并結(jié)合臨床病理資料分析miR-195與子宮頸癌發(fā)生、轉(zhuǎn)移的關(guān)系。方法：在本部分研究中,我們首先收集69對(duì)子宮頸癌的臨床樣本,并提取子宮頸癌組織和對(duì)應(yīng)癌旁組織的RNA,然后利用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)miR-195表達(dá)水平進(jìn)行檢測(cè),明確miR-195在子宮頸癌組織及鄰近癌旁組織中的表達(dá)情況。最后,分析miR-195表達(dá)與子宮頸癌發(fā)病的相關(guān)性；進(jìn)一步結(jié)合臨床病理資料分析miR-195表達(dá)與子宮頸癌轉(zhuǎn)移的相關(guān)性。結(jié)果：1.相對(duì)于癌旁組織,miR-195在子宮頸癌組織中的表達(dá)明顯下調(diào),統(tǒng)計(jì)分析結(jié)果顯示差異具有顯著性。2.與沒(méi)有發(fā)生轉(zhuǎn)移的子宮頸癌樣本(38例)相比,miR-195在發(fā)生轉(zhuǎn)移的子宮頸癌樣本(31例)中表達(dá)顯著下調(diào)(降為0.43倍)。3.在沒(méi)有發(fā)生淋巴結(jié)轉(zhuǎn)移的子宮頸癌病例中,18例(47%)腫瘤組織中miR-195表達(dá)水平較癌旁組織降低,8例(21%)腫瘤組織中miR-195表達(dá)水平較癌旁組織未發(fā)生改變,12例(32%)腫瘤組織中miR-195表達(dá)水平較癌旁組織升高。在發(fā)生淋巴結(jié)轉(zhuǎn)移的病例中,20例(65%)腫瘤組織中miR-195表達(dá)水平較癌旁組織降低,6例(19%)腫瘤組織,中miR-195表達(dá)水平較癌旁組織未發(fā)生改變,5例(16%)腫瘤組織中miR-195表達(dá)水平較癌旁組織升高。結(jié)論：1.相對(duì)于癌旁組織,miR-195在子宮頸癌組織中的表達(dá)明顯下調(diào),說(shuō)明miR-195在子宮頸癌中可能作為抑癌基因發(fā)揮作用。2.相對(duì)于未發(fā)生盆腔淋巴結(jié)轉(zhuǎn)移的子宮頸癌樣本,miR-195在發(fā)生轉(zhuǎn)移的子宮頸癌樣本中表達(dá)顯著下調(diào),說(shuō)明miR-195在子宮頸癌的發(fā)展過(guò)程中可能發(fā)揮了重要的作用。第二部分：miR-195對(duì)子宮頸癌細(xì)胞(HeLa)增殖、遷移及侵襲能力的影響目的：體外細(xì)胞模型研究miR-195對(duì)子宮頸癌細(xì)胞(HeLa)增殖、遷移、侵襲能力的影響。方法：在本部分研究中,我們?cè)贖eLa細(xì)胞中高表達(dá)miR-195,通過(guò)對(duì)細(xì)胞進(jìn)行直接計(jì)數(shù),從而明確miR-195對(duì)HeLa細(xì)胞增殖的影響。利用流式細(xì)胞術(shù)檢測(cè)HeLa細(xì)胞中表達(dá)miR-195后細(xì)胞周期的改變,從而明確miR-195對(duì)HeLa細(xì)胞周期的影響。利用傷口愈合模型來(lái)研究miR-195對(duì)HeLa細(xì)胞遷移能力的影響。利用基于Matrigel的Transwell法研究miR-195對(duì)HeLa細(xì)胞侵襲能力的影響。結(jié)果：1.miR-195對(duì)HeLa細(xì)胞增殖的影響第5天假轉(zhuǎn)染組(Mock)細(xì)胞數(shù)為(14.5±2.1)×105；對(duì)照組細(xì)胞數(shù)為(14.6±0.8)×105；而表達(dá)miR-195組細(xì)胞數(shù)為(10.4±1.0)×105,與對(duì)照組比較差異有顯著性(P0.05)。CCK-8檢測(cè)結(jié)果顯示,與Mock組相比,轉(zhuǎn)染對(duì)照RNA對(duì)活力細(xì)胞數(shù)無(wú)影響(0.97±0.11倍),而高表達(dá)miR-195組細(xì)胞生長(zhǎng)較對(duì)照組比較顯著降低(0.69±0.08倍),兩者比較差異有顯著性(P0.05)。2.miR-195對(duì)HeLa細(xì)胞周期的影響Mock組中處于G0/G1期的細(xì)胞占總細(xì)胞數(shù)的50%,S期細(xì)胞約占39%；G2/M期細(xì)胞約占11%；對(duì)照組中處于G0/G1期的細(xì)胞占總細(xì)胞數(shù)的49%,S期細(xì)胞約占41%；G2/M期細(xì)胞約占10%；高表達(dá)miR-195后,處于G0/G1期的細(xì)胞比例增加(60%)；處于S期的細(xì)胞比例減少(30%)。三次實(shí)驗(yàn)結(jié)果統(tǒng)計(jì)顯示,表達(dá)miR-195組G0/G1期細(xì)胞比例較對(duì)照組顯著增加(P0.05),而S期細(xì)胞比例較對(duì)照組顯著減少(P0.05)。3.miR-195對(duì)子宮頸癌HeLa細(xì)胞傷口愈合能力的影響與對(duì)照組相比,高表達(dá)miR-195的HeLa細(xì)胞傷口愈合能力顯著下降。4.miR-195對(duì)子宮頸癌HeLa細(xì)胞侵襲能力的影響細(xì)胞接種24小時(shí)后,miR-195高表達(dá)組遷移穿過(guò)Matrigel的細(xì)胞數(shù)明顯少于對(duì)照組；結(jié)晶紫染色結(jié)果顯示,miR-195高表達(dá)組遷移穿過(guò)Matrigel的細(xì)胞染色吸光值為對(duì)照組的0.69±0.06倍(P0.05)。結(jié)論：1.miR-195在子宮頸癌中可能作為抑癌基因發(fā)揮作用,可抑制HeLa細(xì)胞生長(zhǎng)、增殖、遷移和侵襲。2.miR-195可作為子宮頸癌治療的潛在靶點(diǎn),為子宮頸癌的治療提供新的思路。第三部分：miR-195功能靶基因CCND2和MYB的鑒定目的：預(yù)測(cè)并驗(yàn)證miR-195在HeLa細(xì)胞中的靶向基因,從而明確miR-195調(diào)控子宮頸癌細(xì)胞行為的作用機(jī)制。方法：本部分研究中,我們選擇較為權(quán)威的生物信息學(xué)工具TargetScan、DIANA microT和PicTar對(duì)miR-195的靶基因進(jìn)行預(yù)測(cè),得到一批共同預(yù)測(cè)到的靶基因。經(jīng)過(guò)功能注釋以及文獻(xiàn)檢索,最終將目標(biāo)鎖定在CCND2和MYB兩個(gè)基因上。進(jìn)一步利用定量PCR、蛋白質(zhì)免疫印跡等方法來(lái)驗(yàn)證miR-195對(duì)CCND2口MYB表達(dá)的調(diào)控作用；并將包含有miR-195結(jié)合區(qū)的CCND2和MYB基因的3’-UTR序列構(gòu)建到熒光素酶報(bào)告質(zhì)粒中,然后將該質(zhì)粒與miR-195共同轉(zhuǎn)染到HeLa細(xì)胞中進(jìn)行雙熒光素酶活性檢測(cè)。最后,在高表達(dá)miR-195的細(xì)胞中回補(bǔ)CCND2和MYB,檢測(cè)細(xì)胞增殖、遷移及侵襲能力。結(jié)果：1.經(jīng)過(guò)生物信息學(xué)預(yù)測(cè)及功能注釋和文獻(xiàn)檢索,最終將目標(biāo)鎖定在CCND2和MYB兩個(gè)基因上。其中CCND2與細(xì)胞周期密切相關(guān),而MYB與腫瘤細(xì)胞轉(zhuǎn)移、侵襲有密切相關(guān)性。2.定量PCR實(shí)驗(yàn)結(jié)果顯示,在HeLa細(xì)胞中高表達(dá)miR-195后,CCND2基因的mRNA水平下降至對(duì)照組的0.85±0.07倍(P0.05),MYB基因的mRNA水平下降至對(duì)照組的0.79±0.06倍(P0.05)。蛋白質(zhì)免疫印跡結(jié)果顯示,高表達(dá)miR-195后CCND2和MYB的蛋白水平也顯著降低。以上結(jié)果表明,miR-195在HeLa細(xì)胞中抑制CCND2和MYB基因的mRNA及蛋白質(zhì)表達(dá)。熒光素酶檢測(cè)結(jié)果顯示,含CCND2基因3’-UTR的報(bào)告系統(tǒng)中,高表達(dá)miR-195組熒光素酶活性降低為對(duì)照組的0.65±0.07倍(P0.05)；含MYB基因3’-UTR的報(bào)告系統(tǒng)中,高表達(dá)miR-195組熒光素酶活性降低為對(duì)照組的0.53±0.08倍(P0.05)。3.在高表達(dá)miR-195的細(xì)胞中回補(bǔ)CCND2可以使細(xì)胞增殖能力回復(fù)到對(duì)照組的0.85±0.07倍,與轉(zhuǎn)染miR-195組(為對(duì)照組的0.65±0.07倍)相比差異有顯著性,回補(bǔ)CCND2對(duì)miR-195抑制的細(xì)胞遷移、侵襲能力無(wú)影響。4.在高表達(dá)miR-195的細(xì)胞中回補(bǔ)MYB可以使細(xì)胞增殖能力回復(fù)到對(duì)照組的0.77±0.05倍,與轉(zhuǎn)染miR-195組(為對(duì)照組的0.65±0.07倍)相比差異有顯著性；回補(bǔ)MYB使miR-195抑制的細(xì)胞遷移得以部分回復(fù)；回補(bǔ)MYB可以將被miR-195抑制的細(xì)胞侵襲能力回復(fù)到對(duì)照組的0.86±0.07倍,與轉(zhuǎn)染miR-195組(為對(duì)照組的0.69±0.05倍)相比差異有顯著性。結(jié)論：1.在子宮頸癌細(xì)胞中,miR-195可以直接與CCND2及MYB基因的3’-UTR區(qū)結(jié)合,從而抑制二者的表達(dá)。2.miR-195可以直接抑制CCND2和MYB基因的表達(dá),從而發(fā)揮其抑制子宮頸癌細(xì)胞增殖、遷移和侵襲的作用。
[Abstract]:Background: cervical cancer is one of the most common female malignant tumors in the world. The incidence of the disease is the second place next to breast cancer, and the mortality rate is fifth. It is a serious threat to human life and health. According to statistics, there are about 50 new cases of cervical cancer in the world, accounting for 5% of all new cases of cancer in the world and nearly 270 thousand. The death of the patients is 13-15 million new cases in China each year, accounting for about 1/3 of the global total. The death toll of cervical cancer in China is about 50 thousand each year. The main pathological types of cervical cancer are squamous and adenocarcinoma. The progress is a very complicated process, which is related to the abnormal regulation of multi gene and multiple stages. Our understanding of the mechanism of the development of cervical cancer is still not comprehensive. Research has proved that persistent infection of high risk human papillomavirus (HPV) is a clear cause of cervical cancer. However, women infected with HPV do not necessarily develop cervical cancer; in addition, not all patients in the cervical epithelial cancer can eventually develop. This suggests that the development of cervical cancer is a complex process, other than human HPV virus, and other factors play an important role in the development of cervical cancer. Therefore, the molecular mechanism of the pathogenesis and progression of cervical cancer still needs to be studied in depth (.MicroRNAs (miRNAs) is composed of 20-25 nucleotides. " RNA, by combining with the 3 'untranslated region of the target gene (3' -UTR) to promote the degradation of mRNA or inhibit the translation of mRNA. By regulating gene expression at the post transcriptional level, miRNAs plays its important functions in many life activities, including cell proliferation, differentiation, withering, and development. Many diseases such as diabetes, cardiovascular disease, and rheumatoid arthritis are closely related. In recent years, studies have shown that miRNAs plays an important role in many human cancers, and participates in many processes, such as the occurrence, development and metastasis of cancer. Some studies have found that some of the miRNAs play the role of oncogene, and some other miRNAs hair. The role of the oncogene.MiRNAs and its regulatory target gene regulation network together regulate the occurrence and development of the tumor. The study also confirmed that the abnormal expression of miRNAs in the serum and cancer tissues of cancer patients is closely related to the incidence, progression, metastasis, invasion and resistance of cancer. Therefore, miRNAs may be a good target for the diagnosis of cancer. Sub.MicroRNA-195 (miR-195) is an important tumor suppressor miRNA, which plays the role of tumor suppressor gene in all kinds of tumor, such as lung cancer and breast cancer. However, the role of miR-195 in cervical cancer is not completely clear. In order to clarify the role of miR-195 in the development of cervical cancer, the first part of this thesis first started from clinical samples to detect M The relationship between the expression of iR-195 in cervical cancer tissue and para cancerous tissue and the correlation between its expression and the occurrence, development and metastasis of cervical cancer. The results suggest that miR-195 may play the role of tumor suppressor gene in cervical cancer. The proliferation and metastasis of tumor cells are the key link in the development of cervical cancer. In order to further clarify the inhibitory effect of miR-195 on the pathogenesis of cervical cancer, the second part of this dissertation expresses miR-195 in the cervical cancer cell (HeLa) cultured in vitro, and detects the effect of miR-195 on cell proliferation, invasion and migration. Based on the ability of miR-195 to inhibit the proliferation, invasion and migration of HeLa cells, the third part of this thesis has studied the molecular mechanism of miR-195, and screened and identified the target gene directly regulated by cell cycle protein D2 (CyclinD2, CCND2) and transcription factor MYB. Cell proliferation is closely related to cell cycle, and CCN D2 is an important cyclin.CCND2 that combines with cyclin dependent kinase 4 (CDK4) and cyclin dependent kinase 6 (CDK6) to regulate the activity of CDK4 and CDK6, thereby promoting the transformation of cells from the G1 phase to S phase. The study shows that the abnormal expression of CCND2 is closely related to the occurrence of a variety of tumors such as glioma and gastric cancer in human brain tissue Related.MYB is a widely existing transcription factor. It can play an important role in the differentiation and proliferation of cells by regulating gene expression..MYB is highly expressed in a variety of tumor tissues such as neuroblastoma and breast cancer, and the expression level is closely related to the prognosis. In addition, the study shows that the MYB protein is also expressed. It is closely related to tumor invasion and metastasis. It is seen that CCND2 and MYB are two important tumor stimulating proteins. Our third experimental results showed that miR-195 could act on the 3 '-UTR of CCND2 and MYB gene and directly inhibit protein expression. The results preliminarily elucidate the molecular mechanism of miR-195 inhibiting the proliferation, invasion and migration of HeLa cells in cervical cancer. The basic framework and main research contents of this paper are illustrated in Figure 1. The detailed research ideas and technical routes of each part will be further elaborated in this paper. Part 1: the purpose of miR-195 expression in cervical cancer tissue: to detect the expression of miR-195 in cervical cancer tissues and adjacent tissues, and to combine with clinical pathology. Data analysis of the relationship between miR-195 and the occurrence and metastasis of cervical cancer. Methods: in this part of the study, we first collected 69 clinical samples of cervical cancer and extracted the cervical cancer tissue and the RNA corresponding to the paracancerous tissue, and then detected the level of miR-195 by real time fluorescence quantitative PCR technique, and made clear the miR-195 in the cervical cancer group. In the end, the correlation between the expression of miR-195 and the incidence of cervical cancer was analyzed. The correlation between the expression of miR-195 and the metastasis of cervical cancer was analyzed by the clinicopathological data. Results: 1. compared with the adjacent tissue, the expression of miR-195 in the cervical cancer group was obviously down down, and the statistical analysis showed that the results were obvious. Compared with non metastatic cervical cancer samples (38 cases), the expression of miR-195 was significantly reduced in 31 cases of metastatic cervical cancer (0.43 times),.3. in cervical cancer cases without lymph node metastasis, and 18 (47%) of the tumor tissues in 18 cases (47%) were lower than those in the paracancerous tissue, 8 cases (2). 1%) the expression level of miR-195 was not changed in tumor tissue, and in 12 cases (32%), the expression level of miR-195 was higher than that of para cancerous tissue. In the cases of lymph node metastasis, the expression level of miR-195 in 20 cases (65%) was lower than that of para cancer tissue, 6 cases (19%), and the expression level of miR-195 was less than that of para cancerous tissue. The expression level of miR-195 in 5 cases (16%) was higher than that of para cancerous tissue. Conclusion: the expression of miR-195 in cervical cancer tissue is obviously down regulated by 1. relative to the paracancerous tissue, indicating that miR-195 may play a role in the tumor suppressor gene in cervical cancer as compared to the cervical cancer samples without pelvic lymph node metastasis, miR- 195 significantly down-regulation in the metastasis of cervical cancer samples, indicating that miR-195 may play an important role in the development of cervical cancer. Second: the effect of miR-195 on the proliferation, migration and invasion of cervical cancer cells (HeLa): in vitro cell model study on the proliferation of cervical cancer cells (HeLa) by miR-195 The effect of migration and invasion ability. Methods: in this part of the study, we expressed miR-195 in HeLa cells, and by direct counting the cells to determine the effect of miR-195 on the proliferation of HeLa cells. Flow cytometry was used to detect the changes in the cell cycle of the HeLa cells after the expression of miR-195, thus defining miR-195 to HeLa cell cycle. The effect of the wound healing model was used to study the effect of miR-195 on the migration ability of HeLa cells. The effect of miR-195 on the invasive ability of HeLa cells was studied using the Transwell method based on Matrigel. Results: the effect of 1.miR-195 on the proliferation of HeLa cells (Mock) was (14.5 + 2.1) x 105, and the number of cells in the control group was (14.6 + 0) (14.6 + 0). .8) x 105, and the number of cells expressed in the miR-195 group was (10.4 + 1) x 105, and there was a significant difference between the control group and the control group (P0.05).CCK-8 detection results showed that compared with the Mock group, the transfected control RNA had no effect on the number of active cells (0.97 + 0.11 times), while the cell length of the high expression miR-195 group was significantly lower than that of the control group (0.69 + 0.08 times), and the differences were poor. The effect of P0.05.2.miR-195 on the cell cycle of HeLa cells was 50% of the total number of cells in the G0/G1 stage, 39% in S, 11% in G2/M, 49% in the total number of cells in the control group, 41% in S, 10% in G2/M, and in G0/G1, in G0/G1, and in G0/G1. The proportion of cells in the period of S was increased (60%), and the proportion of cells in the stage of stage was decreased (30%). The three times of the experimental results showed that the proportion of G0/G1 phase cells in the miR-195 group was significantly higher than that of the control group (P0.05), while the proportion of S stage cells was significantly lower than that of the control group (P0.05) the effect of.3.miR-195 on the healing ability of cervical cancer HeLa cells was compared with the control group. The wound healing ability of HeLa cells with high expression of miR-195 significantly decreased the effect of.4.miR-195 on the invasive ability of HeLa cells in cervical cancer. After 24 hours of cell inoculation, the number of cells migrating through Matrigel in miR-195 high expression group was significantly less than that in the control group; the result of crystal violet staining showed that the miR-195 high expression group migrated through Matrigel cells. The value of light was 0.69 + 0.06 times (P0.05) of the control group. Conclusion: 1.miR-195 may play a role in the tumor suppressor gene in cervical cancer. It can inhibit the growth, proliferation, migration and invasion of HeLa cells as potential targets for the treatment of cervical cancer, and provide new ideas for the treatment of cervical cancer. The third part: miR-195 function target gene CCN. The purpose of D2 and MYB identification: to predict and verify the targeting gene of miR-195 in HeLa cells, and to clarify the mechanism of miR-195 regulating the behavior of cervical cancer cells. Methods: in this part, we choose the more authoritative bioinformatics tool TargetScan, DIANA microT and PicTar to predict the target gene of miR-195, and obtain one of the target genes of miR-195. Through functional annotation and literature retrieval, the target was finally locked on two genes of CCND2 and MYB. Quantitative PCR, protein immunoblotting and other methods were used to verify the regulation of miR-195 on the MYB expression of CCND2 mouth, and the 3 '-UTR sequence containing the CCND2 and MYB genes in the miR-195 binding region. The plasmid was built into the luciferase reporter plasmid, and then the plasmid and miR-195 were transfected into HeLa cells for double luciferase activity detection. Finally, the CCND2 and MYB were supplemented in the cells with high expression of miR-195, and the cell proliferation, migration and invasion ability were detected. Results: 1. through bioinformatics prediction, functional annotation and literature retrieval, the results will eventually be carried out. The target is locked on two genes of CCND2 and MYB, of which CCND2 is closely related to the cell cycle, while MYB and tumor cell metastasis, and the invasion of the.2. quantitative PCR test results show that the mRNA level of the CCND2 gene drops to 0.85 + 0.07 times as much as the control group (P0.05) in the HeLa cell, and the level of the MYB gene drops to the level to the control group. 0.79 + 0.06 times (P0.05) of the control group. Protein immunoblotting showed that the protein levels of CCND2 and MYB were also significantly reduced after high expression of miR-195. The results showed that miR-195 inhibited the mRNA and protein expression of the CCND2 and MYB genes in HeLa cells. The results of luciferase detection showed that the high table in the reporting system containing the CCND2 gene 3 '-UTR. The activity of luciferase in the miR-195 group decreased to 0.65 + 0.07 times (P0.05) of the control group. In the report system containing MYB gene 3 '-UTR, the luciferase activity of the high expression miR-195 group decreased to 0.53 + 0.08 times of the control group (P0.05).3. in the cells with high expression of miR-195, which could restore the cell proliferation ability to 0.85 + 0.07 times as much as the control group. Compared with the transfected miR-195 group (0.65 + 0.07 times as the control group), there was a significant difference between the transfected group and the transfected group (CCND2). The reactivation of miR-195 inhibited cell migration and invasion.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R737.33

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