micro RNA与小儿肝外门静脉梗阻蛋白C减少的相关性研究

Association of micro RNA with a deficiency of protein C in children with extrahepatic portal venous obstruction

目的探讨microRNA(miRNA)与肝外门静脉梗阻患儿蛋白C减少的关系。方法回顾性研究2013年12月至2016年4月于首都儿科研究所普外科接受Rex手术治疗和术后定期随访复查的53例肝外门静脉梗阻患儿(男32例,女21例;术时年龄0.9~12.6岁,平均6.1岁)手术前、后的血液样本。收集同期收治的18例腹股沟斜疝儿童(均为男孩;年龄2~9岁,平均3.6岁)的术前血液样本作为对照组1。采用ELISA法检测两组样本中的蛋白C浓度,并比较其差异。再以Rex术前后蛋白C浓度低于对照组1并排除因门静脉入肝血流减少引发的继发性蛋白C减少和遗传性蛋白C缺乏的肝外门静脉梗阻患儿的全血样本为研究组。另收集相应数量的腹股沟斜疝儿童术前血液样本为对照组2。采用ELISA检测两组(研究组和对照组2)血液中活化蛋白C(activatedProteinC,APC)相对含量,并比较两组差异。进而,采用qRT-PCR检测两组血浆中miR-204-5p、miR-211-5p、miR-218-5p、miR-802、miR-124-3p、miR-506-3p和蛋白CmRNA的相对含量,比较两组间的差异。对研究组的miR-204-5p、miR-211-5p、miR-218-5p、miR-802、miR-124-3p、miR-506-3p和蛋白CmRNA的表达进行Pearson相关性分析。结果6例EHPVO患儿(男4例,女2例;术时年龄3.1~12.6岁,平均5.2岁)Rex术前、后蛋白C浓度均低于对照组1(术前4.30~4.68μg/ml,平均4.50μg/ml;术后4.08~4.97μg/ml,平均4.48μg/ml;对照组15.03~6.22μg/ml,平均5.52μg/ml)。为进一步进行基因研究,将该6例EHPVO患儿Rex术后血液样本作为研究组,新收集的6例腹股沟斜疝儿童(均为男孩;年龄2~5岁,平均4.2岁)术前血液样本作为对照组2。研究组APC含量为(4102.33±154.35)pg/ml显著低于对照组2(5357.42±479.49)pg/ml,组间比较,差异有统计学意义(P<0.01)。研究组的miR-124-3p和miR-506-3p的相对含量分别为17.98±5.32和7.03±5.05显著高于对照组2的1.14±0.61和1.31±0.93,组间比较,差异有统计学意义(P<0.05)。研究组的蛋白CmRNA的相对含量为0.05±0.03显著低于对照组2的2.44±2.63,组间比较,差异有统计学意义(P<0.05)。研究组中miR-211-5p、miR-802、miR-124-3p和miR-506-3p的表达与蛋白CmRNA的表达有显著负相关性(P<0.05)。结论miR-124-3p和miR-506-3p与小儿肝外门静脉梗阻蛋白C减少有显著的相关关系,可能在引发蛋白C减少中有重要作用。

Objective To explore the relationship between micro RNA and protein C deficiency in children with extrahepatic portal venous obstruction. Methods From December 2013 to April 2016, 53 children with extrahepatic portal venous obstruction underwent Rex shunt and received postoperative follow-ups. There were 32 boys and 21 girls with an age range of 0.9-12.6 years. Blood samples were collected pre- and post-operatively. Eighteen blood samples from children with inguinal hernia pre-operatively were used as control group 1. Serum concentration of protein C was detected by enzyme-linked immunosorbent assay (ELISA). As compared with control group 1, children with a lower pre- and post-operative level of protein C, not caused by postoperative decreased hepatopetal blood flow or family factors, were regarded as study group. Furthermore, the same amounts of blood samples from children with inguinal hernia were used as control group 2. Firstly the level of activated protein C (APC) was compared between study group and control group 2. Then the expressions of miR-204-5p, miR-211-5p, miR-218-5p, miR-802, miR-124-3p, miR-506-3p and protein C mRNA were detected by qRT-PCR and compared between two groups. Lastly the correlation between the expressions of protein C mRNA and miRNA was analyzed. Results There was 6 children in study group (4 boys and 2 girls, 3.1-12.6 years old) and 6 children with inguinal hernia (6 boys, 2-5 years old) was used as control group 2. The level of APC was significantly lower in study group than that in control group 2 [(4 102.33±154.35) vs.(5 357.42±479.49) pg/ml, P<0.01]. The expressions of miR-124-3p and miR-506-3p were significantly higher in study group than that in control group 2 (P<0.05) and the expression of protein C mRNA was significantly lower in study group than that in control group 2 (P<0.05). A significant negative correlation existed between the expressions of miR-211-5p, miR-802, miR-124-3p and miR-506-3p and the expression of protein C mRNA in study group (P<0.05). Conclusions The high expressions of miR-124-3p and miR-506-3p were correlated with deficiency of protein C in children with extrahepatic portal venous obstruction. And it may suppress the expression of protein C mRNA.