本文選題：Mir-675-5p + ZEB1�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：研究目的:分析mir-675-5p在不同人胰腺癌細(xì)胞中的表達(dá),及其在癌組織中的表達(dá)量與患者生存預(yù)后的關(guān)系,并探究mir-675-5p對胰腺癌細(xì)胞增殖、凋亡、遷移、侵襲等方面的影響和可能的作用機(jī)制。研究方法:1.運(yùn)用SPSS Statistics 20軟件及Graphpad Prism5軟件對從TCGA數(shù)據(jù)庫中獲得的mir-675相關(guān)胰腺癌臨床樣本數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析,獲得mir-675與胰腺癌患者生存時間、最大腫瘤直徑及腫瘤TNM分期的關(guān)系。2.運(yùn)用qRT-PCR檢測不同胰腺癌細(xì)胞中mir-675-5p的表達(dá)水平。利用RNA干擾技術(shù)將mir-675-5p mimics轉(zhuǎn)入相對低表達(dá)mir-675-5p的胰腺癌細(xì)胞株,將mir-675-5p Inhibitor轉(zhuǎn)入相對高表達(dá)mir-675-5p的胰腺癌細(xì)胞株。3.分別運(yùn)用CCK-8法、平板克隆形成試驗、流式細(xì)胞術(shù)、Transwell遷移和侵襲實驗檢測mir-675-5p對胰腺癌細(xì)胞增殖、細(xì)胞克隆形成能力、細(xì)胞凋亡能力及細(xì)胞周期、細(xì)胞遷移和侵襲能力的影響,以探討mir-675-5p對胰腺癌細(xì)胞功能學(xué)方面的影響。4.運(yùn)用qRT-PCR檢測細(xì)胞周期相關(guān)基因RB1及細(xì)胞侵襲相關(guān)基因ZEB1、mir-200家族的表達(dá)量變化,Western blotting檢測細(xì)胞增殖凋亡及侵襲相關(guān)指標(biāo)PCNA、Bcl-2、Bax、cleaved-caspase3、MMP2、MMP9和上皮間質(zhì)轉(zhuǎn)化相關(guān)指標(biāo)E-cadherin、N-cadherin、ZEB1、Snail、Slug、Vimentin的變化。5.運(yùn)用RNA干擾技術(shù)將UBQLN1 si RNAs轉(zhuǎn)染入胰腺癌細(xì)胞使其低表達(dá)UBQLN1,并運(yùn)用qRT-PCR法和Western Blotting法檢測UBQLN1 si RNAs的干擾效率;將UBQLN1si RNAs和mir-675-5p mimics共轉(zhuǎn)染入Patu8988細(xì)胞,將UBQLN1 si RNAs和mir-675-5p Inhibitor共轉(zhuǎn)染入SW1990細(xì)胞,檢測共轉(zhuǎn)染后ZEB1的m RNA和蛋白水平的變化。研究結(jié)果:1.Mir-675的高表達(dá)可使胰腺癌患者獲得相對長的生存時間及相對小的最大腫瘤直徑,而Mir-675的表達(dá)與胰腺癌患者腫瘤TNM分期無相關(guān)關(guān)系。2.Mir-675-5p差異性表達(dá)于4種胰腺癌細(xì)胞株(Patu8988Panc-1Bxpc-3SW1990),在Patu8988細(xì)胞株中表達(dá)量最低,在SW1990細(xì)胞株中表達(dá)量最高。3.上調(diào)mir-675-5p在胰腺癌細(xì)胞株P(guān)atu8988中的表達(dá)能夠抑制細(xì)胞增殖,降低細(xì)胞克隆形成能力,促進(jìn)細(xì)胞凋亡,引起細(xì)胞G1/S期阻滯。降低PCNA蛋白水平,同時能誘導(dǎo)活化的Caspase3表達(dá),降低Bcl-2/Bax比值。上調(diào)mir-675-5p能夠抑制細(xì)胞侵襲轉(zhuǎn)移,下調(diào)侵襲相關(guān)蛋白MMP2及MMP9的蛋白水平,EMT相關(guān)指標(biāo)N-cadherin、ZEB1、Snail、Slug、Vimentin的表達(dá)量減少,E-cadherin的表達(dá)量增加;而下調(diào)mir-675-5p在胰腺癌細(xì)胞株SW1990中的表達(dá)則能夠促進(jìn)細(xì)胞增殖,提高細(xì)胞克隆形成能力,促進(jìn)細(xì)胞侵襲轉(zhuǎn)移。升高PCNA蛋白水平,同時能抑制cleaved-caspase3表達(dá),升高Bcl-2/Bax比值。侵襲相關(guān)蛋白MMP2及MMP9的蛋白水平上升,EMT相關(guān)指標(biāo)N-cadherin、ZEB1、Snail、Slug、Vimentin的表達(dá)量增加,E-cadherin的表達(dá)量下降。4.上調(diào)mir-675-5p在胰腺癌細(xì)胞株P(guān)atu8988中的表達(dá),可以使RB1的m RNA水平升高,ZEB1的m RNA轉(zhuǎn)錄上調(diào),mir-200家族表達(dá)量下降;下調(diào)mir-675-5p在胰腺癌細(xì)胞株SW1990中的表達(dá),可以使RB1的m RNA水平下降,ZEB1的m RNA轉(zhuǎn)錄下調(diào),mir-200家族表達(dá)量上升。5.上調(diào)mir-675-5p在胰腺癌細(xì)胞株P(guān)atu8988中的表達(dá),能夠使泛素化相關(guān)基因UBQLN1的m RNA表達(dá)及蛋白表達(dá)均升高;下調(diào)mir-675-5p在胰腺癌細(xì)胞株SW1990中的表達(dá),能夠使泛素化相關(guān)基因UBQLN1的m RNA轉(zhuǎn)錄及蛋白表達(dá)均下降。6.下調(diào)UBQLN1在已上調(diào)mir-675-5p表達(dá)的Patu8988細(xì)胞株中的表達(dá),可以使ZEB1 m RNA轉(zhuǎn)錄水平上升的趨勢得到逆轉(zhuǎn),使ZEB1蛋白表達(dá)水平下降的趨勢得到逆轉(zhuǎn);下調(diào)UBQLN1在已下調(diào)mir-675-5p表達(dá)的SW1990細(xì)胞株中的表達(dá),可以使ZEB1m RNA表達(dá)水平下降的趨勢得以維持,使ZEB1蛋白表達(dá)水平上升的趨勢得以維持。研究結(jié)論:1.Mir-675的表達(dá)量越高,胰腺癌患者的生存預(yù)后相對越好。2.Mir-675-5p具有抑制胰腺癌細(xì)胞增殖、促進(jìn)凋亡,抑制細(xì)胞遷移及侵襲等生物學(xué)功能。3.Mir-675-5p可引起ZEB1轉(zhuǎn)錄后水平的改變,進(jìn)而引起ZEB1轉(zhuǎn)錄水平的反向改變。4.Mir-675-5p可以促進(jìn)泛素化相關(guān)基因UBQLN1的表達(dá),而UBQLN1可以在蛋白水平下調(diào)ZEB1蛋白的表達(dá),引起ZEB1的m RNA水平反射性升高。Mir-675-5p可通過中間基因ZEB1而引起mir-200家族的變化,提示兩個mi RNA之間也可以互相影響,并且Mir-675-5p可以通過UBQLN1-ZEB1-mir-200環(huán)路影響胰腺癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化的過程。
[Abstract]:Objective: to analyze the expression of mir-675-5p in different human pancreatic cancer cells, the relationship between the expression in the cancer tissues and the survival prognosis of the patients, and to explore the effects and possible mechanisms of mir-675-5p on the proliferation, apoptosis, migration and invasion of pancreatic cancer cells. Research methods: 1. using the SPSS Statistics 20 software and Graphpad Pr Ism5 software was used to analyze the data of mir-675 related pancreatic cancer clinical samples obtained from the TCGA database to obtain the relationship between mir-675 and the survival time of the patients with pancreatic cancer, the maximum tumor diameter and the TNM stage of the tumor..2. was used to detect the expression of mir-675-5p in different pancreatic cancer cells by qRT-PCR. RNA interference technique was used to detect mir-675-5p. Mimics was transferred into the pancreatic cancer cell line with relatively low expression of mir-675-5p, and mir-675-5p Inhibitor was transferred to the pancreatic cancer cell line with relatively high mir-675-5p expression,.3., CCK-8 method, plate clone formation test, flow cytometry, Transwell migration and invasion test to detect the proliferation of pancreatic cancer cells and cell clone formation ability. The effect of apoptosis and cell cycle, cell migration and invasiveness in order to explore the effect of mir-675-5p on the functional aspects of pancreatic cancer cells.4. use qRT-PCR to detect cell cycle related genes RB1 and cell invasion related genes ZEB1, miR-200 family expression changes, Western blotting detection of cell proliferation and apoptosis and invasion correlation Indexes such as PCNA, Bcl-2, Bax, cleaved-caspase3, MMP2, MMP9, and epithelial mesenchymal transition related indicators E-cadherin, N-cadherin, ZEB1, Snail, Slug. Co transfection of UBQLN1si RNAs and mir-675-5p mimics into Patu8988 cells and co transfection of UBQLN1 Si RNAs and mir-675-5p Inhibitor into SW1990 cells. There is no correlation between the expression of Mir-675 and the TNM staging of pancreatic cancer patients.2.Mir-675-5p differentially expressed in 4 kinds of pancreatic cancer cell lines (Patu8988Panc-1Bxpc-3SW1990), and the lowest expression in Patu8988 cell lines. The expression of the highest expression of.3. in the SW1990 cell line can be suppressed by the expression of mir-675-5p in the pancreatic cancer cell strain. Cell proliferation, cell clone formation, cell apoptosis, cell G1/S phase block, PCNA protein level, activated Caspase3 expression and Bcl-2/Bax ratio can be induced. Up regulation of mir-675-5p can inhibit cell invasion and metastasis, decrease the protein level of invasion related protein MMP2 and MMP9, EMT related index N-cadherin The expression of ZEB1, Snail, Slug, Vimentin was reduced and the expression of E-cadherin increased, while the expression of mir-675-5p in the pancreatic cancer cell line SW1990 could promote cell proliferation, improve cell cloning and formation, promote cell invasion and metastasis, increase the level of PCNA protein, and inhibit the expression of cleaved-caspase3 and increase the ratio of Bcl-2/Bax. The protein levels of the related proteins MMP2 and MMP9 increased, and the expression of EMT related indicators N-cadherin, ZEB1, Snail, Slug, Vimentin increased, and the expression of E-cadherin expression decreased.4. up mir-675-5p in the pancreatic cancer cell Patu8988. The expression of mir-675-5p in the pancreatic cancer cell line SW1990 can decrease the m RNA level of RB1, the m RNA transcript of ZEB1, and the miR-200 family expression increase.5. increase in the expression of mir-675-5p in the pancreatic cancer cell Patu8988. The expression in the adenocarcinoma cell line SW1990 can decrease the m RNA transcriptional and protein expression of the ubiquitination related gene UBQLN1, and decrease the expression of.6. down UBQLN1 in the Patu8988 cell line that has increased the mir-675-5p expression. The trend of ZEB1 m RNA transcriptional level is reversed and the downward trend of the ZEB1 protein expression level is reversed. The expression of UBQLN1 in the SW1990 cell lines that have reduced the expression of mir-675-5p can maintain the tendency to decrease the expression level of ZEB1m RNA and maintain the trend of the increase in the expression level of ZEB1 protein. The higher the expression of 1.Mir-675, the better the survival of the patients with pancreatic cancer, the better the better.2.Mir-675-5p has the inhibition of the pancreatic cancer. Cell proliferation, promoting apoptosis, inhibiting cell migration and invasion and other biological functions.3.Mir-675-5p can cause changes in ZEB1 post transcriptional level, and then cause the reverse change of ZEB1 transcriptional level.4.Mir-675-5p to promote the expression of ubiquitination related gene UBQLN1, and UBQLN1 can downregulate the expression of ZEB1 protein at the egg white level, causing m RNA of ZEB1. .Mir-675-5p can cause changes in the miR-200 family through the intermediate gene ZEB1, suggesting that the two mi RNA can also interact with each other, and Mir-675-5p can affect the process of the epithelial mesenchymal transition of pancreatic cancer cells through the UBQLN1-ZEB1-mir-200 loop.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.9

【參考文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 柳長坤;H19衍生的miR-675在膀胱癌中的表達(dá)及其對細(xì)胞生物學(xué)行為影響的研究[D];南京醫(yī)科大學(xué);2016年

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