膀胱癌患者血清microRNA实时荧光定量PCR法检测中内参基因的筛选及验证

发布时间：2018-04-04 09:53

本文选题：膀胱癌　切入点：血清　出处：《山东大学》2014年硕士论文

【摘要】：目的通过对非肌层浸润性膀胱癌患者、肌层浸润性膀胱癌患者以及健康对照者血清中microRNA(miRNA)表达情况进行检测,筛选并验证出适用于膀胱癌患者血清miRNA实时荧光定量PCR法检测的内参基因,为膀胱癌患者血清miRNA类肿瘤标志物的筛选及后续研究提供可靠的内参基因。方法 1.采用Miseq测序技术,对10例非肌层浸润性膀胱癌患者、10例肌层浸润性膀胱癌患者以及10例健康对照者血清中miRNA表达谱进行测序,并筛选出其中表达水平较高且稳定性较好的miRNA用于验证。 2.采用实时荧光定量PCR技术,首先在30例非肌层浸润性膀胱癌患者、30例肌层浸润性膀胱癌患者以及35例健康对照者血清中对初步筛选出的miRNA及U6在血清中的表达水平进行验证,排除部分表达水平较低或者表达稳定性较差的内参基因分子后,利用geNorm以及NormFinder软件对剩余候选内参基因分子进行计算分析,分别得到最稳定内参基因以及最佳内参基因组合。 3.另选取63例非肌层浸润性膀胱癌患者、61例肌层浸润性膀胱癌患者以及67例健康对照者,采用实时荧光定量PCR技术对软件所推荐的最稳定内参基因以及最佳内参基因组合表达水平进行检测。最后选取miR-148b-3p为目的分子,通过采用实时荧光定量PCR技术检测其在46例膀胱癌患者(包括26例非肌层浸润性膀胱癌患者和20例肌层浸润性膀胱癌患者)以及46例健康对照者中表达差异情况,以验证所筛选内参基因的可靠性及稳定性。 4.采用One-way ANOVA检验对内参基因组间表达差异进行比较。 5.采用Mann-Whitney U检验对miR-148b-3p组间表达差异进行比较。结果 1. Miseq测序结果显示,在非肌层浸润性膀胱癌患者组、肌层浸润性膀胱癌患者组以及健康对照者组血清中,分别测得206、259和180个miRNA表达拷贝数大于10,经初步筛选其中共有10个miRNA符合内参筛选要求,分别为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亦被纳入下一步分析。 2.经初次实时荧光定量PCR验证后,hsa-miR-10a-5p和hsa-miR-345-5p因在部分样本中未表达而被排除,其余9个基因分子在三组之间的表达水平无明显差异(P0.05),其中4个miRNA(hsa-miR-143-3p、hsa-miR-140-3p、 hsa-miR-502-3p和hsa-miR-141-3p)的表达水平Ct值中位数大于35被排除。剩余4个miRNA hsa-miR-193a-5p、hsa-miR-16-5p、hsa-miR-191-5p和hsa-let-7d-3p和U6作为候选内参基因被纳入进一步分析。应用geNorm和NormFinder两种软件分别对上述5个候选内参基因稳定性进行计算分析后,两种软件均推荐hsa-miR-193a-5p为最稳定内参基因,hsa-miR-193a-5p和hsa-miR-16-5p的组合为最佳内参基因组合。 3.经再次实时荧光定量PCR验证后表明,hsa-miR-193a-5p、hsa-miR-16-5p及二者的组合(Ct均值)表达水平在3组间均无差异(P0.05)。分别采用hsa-miR-193a-5p、hsa-miR-16-5p及二者组合作为内参对miR-148b-3p表达水平进行校正后均发现,其在膀胱癌患者组中的表达显著高于健康对照组(P0.0001)。以hsa-miR-193a-5p与hsa-miR-16-5p的组合作为内参时,miR-148b-3p用于诊断膀胱癌的ROC曲线下面积AUCROC为0.798,以1.0218为cut-off值时,特异度为56.92%,敏感度为89.23%。结论 1. hsa-miR-193a-5p和hsa-miR-16-5p在膀胱癌患者及健康对照者血清中均稳定表达,采用二者的组合作为实时荧光定量PCR法检测膀胱癌患者血清miRNA的内参基因可使结果更为准确可靠,并使不同实验室之间的检测结果具有可比性,可进一步推动膀胱癌血清miRNA研究的进展。 2. miR-148b-3p在膀胱癌患者血清中表达水平显著升高,作为诊断膀胱癌的血清学肿瘤标志物具有良好的应用潜能。
[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 .

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

【参考文献】