应用微阵列芯片技术分析先天性巨结肠微小RNAs表达谱差异

microRNAs expression profiling analysis in Hirschsprung disease by microarray

目的检测先天性巨结肠（HSCR）微小RNA（microRNAs，miRNA）表达谱，并探讨差异表达的miRNAs在HSCR发生中的作用。方法收集27例HSCR患儿的狭窄段及扩张段肠管组织，选取其中6例，应用miRNA芯片技术筛选HSCR的miRNAs表达谱。运用生物信息学软件预测miRNAs靶基因，采用实时荧光定量（qRT）-PCR验证miR-145-3p、miR-4505及miR-1260a在27例HSCR狭窄段和扩张段组织的表达差异。结果与扩张段肠管组织相比，狭窄段肠管表达上调超过2倍的miRNAs有19个，表达下调超过2倍的有7个（P均〈0．05）。利用靶基因预测软件找到了miRNAs的靶基因，如SOX10、RET、L1CAM。qRT—PCR证实不同组间miR-145—3p（狭窄段1．42±0．42比扩张段0．90±0．31）及miR-4505（狭窄段1．30±0．30比扩张段0．76±0．22）表达量差异具有统计学意义（P均〈0．001），且不同分型之间miR-145—3p（长段型1．53±0．46比短段型1．16±0．12）及miR-4505（长段型1．42±0．26比短段型1．00±0．16）的表达量差异均具有统计学意义（P均〈0．001），而miR-1260a（狭窄段1．11±0．25比扩张段0．99±0．21）的表达差异无统计学意义（P=0．064）。结论HSCR病变段及扩张段肠管存在miRNAs表达差异，miR-145—3p和miR-4505可能与HSCR的发病密切相关。

Objective To investigate the microRNAs（miRNAs） expression profiles in the bowels of patients with Hirschsprung disease （HSCR） ,and to explore the role of differentially expressed miRNAs in the pathogenesis of HSCR. Methods Twenty -seven HSCR tissues, including spastic segments and distending segments, were obtained from patients with HSCR during operation. Then miRNA microarrays were used to investigate the miRNA expression profiles in 6 HSCR specimens. Bioinformatics software was used to predict target genes of miRNA. Three miRNAs （ miR - 145 - 3p, miR - 4505 and miR - 1260a） were chosen and quantificational real - time （qRT） - PCR was per- formed to verify the different expression of those three miRNAs in 27 HSCR tissues. Results The expression of 25 miRNAs in an aganglionic colon segment was found to be more than two fold greater than that in ganglionic segment tis- sues, including 19 up - regulated miRNAs and 7 down - regulated miRNAs in patients with HSCR （ all P 〈 0.05 ）. Target genes of miRNAs were found, such as SOXIO, RET, L1 CAM. qRT - PCR showed the expression of miR - 145 -3p （ 1.42 ± 0.42, aganglionic segment vs 0. 90 ± 0.31, ganglionic segment） and miR - 4505 （ 1.30 ± 0.30, aganglionic segment vs O. 76 ＋0.22, ganglionic segment） displayed a statistical difference between groups （ all P 〈 0. 001 ）. Be- sides, the expressions of miR - 145 -3p （ 1.53± 0.46,long - segment type vs 1.16 ± 0.12, short - segment type） and miR- 4505 （1.42 ±0.26, long- segment type vs 1.00 ± 0.16, short -segment type） showed a statistical difference be- tween different types （ all P 〈 0. 001 ）, but miR - 1260a （ 1.11 ±0.25, aganglionic segment vs 0. 99± 0.21, ganglionic segment） did not show differential expression between different groups （ P = 0. 064）. Conclusions Abnormal expression of miRNAs was found in HSCR spastic segments, suggesting that miRNAs may be involved in the pathogenesis of HSCR.