细胞与生物信息学鉴定3种circRNA参与调节人类膀胱癌侵袭能力的研究

Appraisal on regulating effects of three types of circRNAs on human bladder cancer invasion ability

目的筛选并鉴定与膀胱癌侵袭性相关的环状RNA(circRNA)。方法通过转录组测序,分析来自9例组织(癌旁、非肌层浸润膀胱癌和肌层浸润膀胱癌组织各3例)的circRNA测序结果。qPCR检测20例临床膀胱癌样本(非肌层浸润和肌层浸润各10例)中候选circRNA表达水平。分析20例临床膀胱癌样本的病理特点与候选circRNA表达水平变化的相关性。qPCR检测4种膀胱癌细胞系中候选circRNA本底表达水平。Transwell侵袭实验检测膀胱癌细胞侵袭能力。结果通过对非肌层浸润膀胱癌与肌层浸润膀胱癌组织测序结果的比对,发现305个具有显著差异的circRNA(210个显著上调,95个显著下调),筛选出其中3个显著上调的候选circRNA( circST6GALNAC6、circPRUNE2、circMYH11)。qPCR检测发现,circST6GALNAC6与circMYH11 在肌层浸润膀胱癌组织中的表达水平显著高于非肌层浸润膀胱癌组织( P <0.01),与circRNA测序数据相符;而circPRUNE2在上述两种膀胱癌组织中的表达差异无统计学意义( P >0.05)。circST6GALNAC6和circMYH11与膀胱肿瘤大小、病理分期和分级存在统计学意义上的相关性,肿瘤恶性程度越高,两者表达水平越高。此外,在膀胱癌细胞系实验中也证实,circST6GALNAC6和circMYH11的表达水平与癌细胞的侵袭性相关。结论本研究初步验证了circST6GALNAC6和circMYH11两种circRNA与膀胱癌侵袭能力的相关性。

Objective The circRNAs correlated to the bladder cancer invasion were screened out, and its regulating effects were also verified in this paper. Methods The RNA-seq was applied to analyze the circRNA sequencing results of 9 cases of bladder tissue (3 cases of adjacent, 3 cases of non-muscle invasive bladder cancer and 3 cases of muscle invasive bladder cancer tissues). The qPCR was used to detect the expression level of candidate circRNAs in 20 clinical bladder cancer samples (10 cases of non-muscle infiltration and 10 cases of muscle invasion). And the correlation between the pathological features of 20 clinical bladder cancers and the changes of candidate circRNA expression levels was analyzed. The qPCR was used to detect the basal expression level of candidate circRNA in four bladder cancer cell lines. The transwell invasion experiments was applied to detect the cell invasive ability. Results Through the comparison on sequencing results between non-muscle invasive bladder cancer and muscle invasive bladder cancer tissues, totally 305 circRNAs with significant difference (210 significant up-regulation, 95 significant down-regulation) were retrieved, and three candidate circRNAs (circST6GALNAC6, circPRUNE2, circMYH11) with significant up-regulation were screened out. Through the qPCR assay carried out among clinical specimens, it is found that the expression levels of circST6GALNAC6 and circMYH11 in muscle invasive bladder cancer tissues were significantly higher than those in non-muscle invasive bladder cancer tissues ( P < 0.01), which is consistent with circRNA sequencing data. However, the expression difference of circPRUNE2 in two types of bladder cancer tissues was of no statistical significance ( P >0.05). Data of circST6GALNAC6 and circMYH11 were statistically significant in aspects of bladder tumor size, pathological stage, and cell grading. The worse the clinical parameters were, the higher the expression level was. In addition, it was also verified in bladder cancer cell line experiments that the expression levels of circST6GALNAC6 and circMYH11 were correlated to the invasion ability of cancer cells. Conclusions The correlation between the two types of circRNAs (circST6GALNAC6 and circMYH11) and the invasion ability of bladder cancer was preliminarily verified.