本文選題：膀胱癌　切入點：血清　出處：《山東大學》2014年碩士論文

【摘要】：目的 通過對非肌層浸潤性膀胱癌患者、肌層浸潤性膀胱癌患者以及健康對照者血清中microRNA(miRNA)表達情況進行檢測,篩選并驗證出適用于膀胱癌患者血清miRNA實時熒光定量PCR法檢測的內(nèi)參基因,為膀胱癌患者血清miRNA類腫瘤標志物的篩選及后續(xù)研究提供可靠的內(nèi)參基因。 方法 1.采用Miseq測序技術(shù),對10例非肌層浸潤性膀胱癌患者、10例肌層浸潤性膀胱癌患者以及10例健康對照者血清中miRNA表達譜進行測序,并篩選出其中表達水平較高且穩(wěn)定性較好的miRNA用于驗證。 2.采用實時熒光定量PCR技術(shù),首先在30例非肌層浸潤性膀胱癌患者、30例肌層浸潤性膀胱癌患者以及35例健康對照者血清中對初步篩選出的miRNA及U6在血清中的表達水平進行驗證,排除部分表達水平較低或者表達穩(wěn)定性較差的內(nèi)參基因分子后,利用geNorm以及NormFinder軟件對剩余候選內(nèi)參基因分子進行計算分析,分別得到最穩(wěn)定內(nèi)參基因以及最佳內(nèi)參基因組合。 3.另選取63例非肌層浸潤性膀胱癌患者、61例肌層浸潤性膀胱癌患者以及67例健康對照者,采用實時熒光定量PCR技術(shù)對軟件所推薦的最穩(wěn)定內(nèi)參基因以及最佳內(nèi)參基因組合表達水平進行檢測。最后選取miR-148b-3p為目的分子,通過采用實時熒光定量PCR技術(shù)檢測其在46例膀胱癌患者(包括26例非肌層浸潤性膀胱癌患者和20例肌層浸潤性膀胱癌患者)以及46例健康對照者中表達差異情況,以驗證所篩選內(nèi)參基因的可靠性及穩(wěn)定性。 4.采用One-way ANOVA檢驗對內(nèi)參基因組間表達差異進行比較。 5.采用Mann-Whitney U檢驗對miR-148b-3p組間表達差異進行比較。 結(jié)果 1. Miseq測序結(jié)果顯示,在非肌層浸潤性膀胱癌患者組、肌層浸潤性膀胱癌患者組以及健康對照者組血清中,分別測得206、259和180個miRNA表達拷貝數(shù)大于10,經(jīng)初步篩選其中共有10個miRNA符合內(nèi)參篩選要求,分別為hsa-miR-193a-5p、hsa-miR-191-5p、hsa-miR-16-5p、hsa-miR-10a-5p、 hsa-miR-345-5p、hsa-miR-143-3p、hsa-miR-140-3p、hsa-miR-502-3p、 hsa-let-7d-3p、hsa-miR-141-3p。U6亦被納入下一步分析。 2.經(jīng)初次實時熒光定量PCR驗證后,hsa-miR-10a-5p和hsa-miR-345-5p因在部分樣本中未表達而被排除,其余9個基因分子在三組之間的表達水平無明顯差異(P0.05),其中4個miRNA(hsa-miR-143-3p、hsa-miR-140-3p、 hsa-miR-502-3p和hsa-miR-141-3p)的表達水平Ct值中位數(shù)大于35被排除。剩余4個miRNA hsa-miR-193a-5p、hsa-miR-16-5p、hsa-miR-191-5p和hsa-let-7d-3p和U6作為候選內(nèi)參基因被納入進一步分析。應用geNorm和NormFinder兩種軟件分別對上述5個候選內(nèi)參基因穩(wěn)定性進行計算分析后,兩種軟件均推薦hsa-miR-193a-5p為最穩(wěn)定內(nèi)參基因,hsa-miR-193a-5p和hsa-miR-16-5p的組合為最佳內(nèi)參基因組合。 3.經(jīng)再次實時熒光定量PCR驗證后表明,hsa-miR-193a-5p、hsa-miR-16-5p及二者的組合(Ct均值)表達水平在3組間均無差異(P0.05)。分別采用hsa-miR-193a-5p、hsa-miR-16-5p及二者組合作為內(nèi)參對miR-148b-3p表達水平進行校正后均發(fā)現(xiàn),其在膀胱癌患者組中的表達顯著高于健康對照組(P0.0001)。以hsa-miR-193a-5p與hsa-miR-16-5p的組合作為內(nèi)參時,miR-148b-3p用于診斷膀胱癌的ROC曲線下面積AUCROC為0.798,以1.0218為cut-off值時,特異度為56.92%,敏感度為89.23%。 結(jié)論 1. hsa-miR-193a-5p和hsa-miR-16-5p在膀胱癌患者及健康對照者血清中均穩(wěn)定表達,采用二者的組合作為實時熒光定量PCR法檢測膀胱癌患者血清miRNA的內(nèi)參基因可使結(jié)果更為準確可靠,并使不同實驗室之間的檢測結(jié)果具有可比性,可進一步推動膀胱癌血清miRNA研究的進展。 2. miR-148b-3p在膀胱癌患者血清中表達水平顯著升高,作為診斷膀胱癌的血清學腫瘤標志物具有良好的應用潛能。
[Abstract]:Purpose

The expression of microRNA ( miRNA ) in patients with non - myocutaneous invasive bladder cancer , muscular layer invasive bladder cancer and healthy controls was detected , screened and verified to find the internal reference gene which was suitable for the detection of serum miRNA real - time fluorescence quantitative PCR method in patients with bladder cancer , and provided a reliable internal reference gene for screening and subsequent research of serum miRNA tumor markers in patients with bladder cancer .

method

1 . The miRNA expression profiles of 10 patients with non - myocutaneous invasive bladder cancer , 10 patients with bladder cancer and 10 healthy controls were sequenced using Miseq sequencing technique .

2 . In the serum of 30 patients with non - myocutaneous invasive bladder cancer , 30 patients with invasive bladder cancer and 35 healthy controls , the expression levels of miRNA and u6 in serum were verified by real - time fluorescence quantitative polymerase chain reaction ( RT - PCR ) .

3 . In 63 patients with non - myocutaneous invasive bladder cancer , 61 patients with myocutaneous invasive bladder cancer and 67 healthy controls , real - time fluorescence quantitative PCR was used to detect the expression of the most stable internal reference gene and the best internal reference gene .

4 . A one - way ANOVA test was used to compare the differences between the genes in the genome of the internal reference .

5 . A Mann - Whitney U test was used to compare the expression differences between miR - 148b - 3 groups .

Results

1 . The results of Miseq sequencing showed that the number of copies of 206 , 259 and 180 miRNA expression copies measured in patients with non - myocutaneous invasive bladder cancer , muscle layer invasive bladder cancer patient group , and healthy control group were greater than 10 , respectively , which were hsa - miR - 193a - 5p , hsa - miR - 191 - 5p , hsa - miR - 16 - 5p , hsa - miR - 145 - 5p , hsa - miR - 145 - 5p , hsa - miR - 50p , hsa - miR - 140 - 3P , hsa - miR - 502 - 3P , hsa - let - 7d - 3P , hsa - miR - 141 - p . u6 were also included in the next analysis .

2 . After initial real - time real - time fluorescence quantitative PCR , hsa - miR - 10a - 5p and hsa - miR - 345 - 5p were excluded because they were not expressed in some samples . There were no significant differences in the expression level of the remaining 9 genes between the three groups ( P0.05 ) . The remaining four miRNA hsa - miR - 193a - 5p , hsa - miR - 16 - 5p , hsa - miR - 191 - 5p and hsa - let - 7d - 3P and u6 were included in the further analysis . Both software recommended that hsa - miR - 193a - 5p be the most stable internal reference gene , and hsa - miR - 193a - 5p and hsa - miR - 16 - 5p are the best internal reference gene combinations .

3 . The expression levels of hsa - miR - 193a - 5p , hsa - miR - 16 - 5p and hsa - miR - 16 - 5p and hsa - miR - 16 - 5p and hsa - miR - 16 - 5p and hsa - miR - 16 - 5p and hsa - miR - 16 - 5p , respectively , showed no difference among the three groups ( P0.05 ) .

Conclusion

1 . hsa - miR - 193a - 5p and hsa - miR - 16 - 5p are stably expressed in serum of patients with bladder cancer and healthy controls .

2 . The expression level of miR - 148b - p in serum of patients with bladder cancer is significantly increased , which has good application potential as a serological tumor marker for the diagnosis of bladder cancer .

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.14

【參考文獻】

相關(guān)期刊論文 前7條

1 薛春曉;;膀胱癌治療方法的選擇[J];臨床醫(yī)學;2011年07期

2 梁宏偉;陳熹;曾科;張辰宇;;循環(huán)microRNA作為腫瘤的非侵入性生物標志物[J];臨床檢驗雜志;2011年07期

3 張薇,項永兵,劉振偉,方茹蓉,阮志賢,孫璐,高立峰,金凡,高玉堂;1973-1999年上海市區(qū)老年人惡性腫瘤發(fā)病趨勢分析[J];中華老年醫(yī)學雜志;2005年09期

4 魏礦榮,陳振雄,梁智恒,何方方,林茂合,歐小文;中山市1970～1999年膀胱癌發(fā)病趨勢分析[J];中國腫瘤;2005年04期

5 韓瑞發(fā);潘建剛;;中國人群膀胱癌發(fā)病危險因素的Meta分析[J];中華泌尿外科雜志;2006年04期

6 高玉堂,鄧杰,項永兵,阮志賢,汪仲賢,胡炳英,郭懋蓉,滕維康,韓家俊,張英史;上海市居民吸煙與癌癥及有關(guān)疾病十年前瞻性研究[J];中華預防醫(yī)學雜志;1999年01期

7 李志剛,張衛(wèi)華,陶明,高海燕,趙亞娥;膀胱癌危險因素交叉配比病例對照研究[J];中國慢性病預防與控制;1996年06期

，

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