靶向PIK3CA候选miRNAs在胃癌细胞与血清中表达的分析

Forecasting of candidate miRNAs targeting PIK3CA and their expression in gastric cancer cells and serum

目的:预测和筛选靶向PIK3CA的候选miRNAs并检测其在胃癌细胞和血清中的表达。方法:采用生物信息学并结合既往文献和前期研究,预测和筛选出靶向PIK3CA的候选miRNAs(miR-203和miR-506)。常规提取胃癌细胞和血清总RNA,并用DNaseⅠ进行消化。以miRNAs特异性茎-环引物引导反转录,通过实时荧光定量PCR对miR-203、miR-506和内参U6进行检测,并采用琼脂糖凝胶电泳检测定量PCR产物。结果:电泳检测细胞和血清中RNA纯度较好;胃癌细胞和血清中miR-203、miR-506和内参U6均能实现特异性扩增。miR-203在胃癌细胞SGC-7901及MGC-803中表达分别为1.360±0.102和1.241±0.110,差异无统计学意义,t=3.125,P=0.09;miR-203在胃癌患者血清中表达为1.420±0.122,明显低于健康人的3.450±0.206,t=2.521,P=0.02。miR-506在胃癌细胞SGC-7901和MGC-803及胃癌患者和健康人血清中表达水平分别为1.256±0.113、1.158±0.109、1.735±0.220和1.592±0.134,差异无统计学意义,P>0.05。结论:miR-203可能是调控PIK3CA基因的miRNAs之一。PIK3CA在胃癌中的高表达可能与miR-203的异常低表达有关,而与miR-506无关。胃癌患者血清中miR-203的表达有望成为预测胃癌发生或发展的一个重要的分子标志。

OBJECTIVE：To predict and screen candidate miRNAs targeting PIK3CA gene,and detect their expres- sion in gastric cancer cells and serum. METHODS： Candidate miRNAs （miR-203 and miR-506） targeting PIK3CA were screened through bioinformatics combined with the previous literature and our previous study. Total RNA in gastric cancer cells and serum was extracted routinely and treated with DNase I . miR-203, miR 506 and internal control U6 in total RNA preparation were reverse transcribed by specific stem-loop primers. Real-time fluorescence quantitative polymerase chain reaction （rt-fqPCR） was adopted to quantify target miRNAs and U6. Quantitative PCR product was detected by aga- rose gel electrophoresis. RESULTS： RNA quality isolated from cells and serum was confirmed to be good by electrophore- sis. U6 ,miR-16 and miR-224 were amplified and quantified specifically in gastric cancer cells and serum, miR-203 expres sion levels between gastric cancer cell line SGC-7901 （1. 36010. 102） and MGC-803 （1. 241±0. 110） had no significant difference （t= 3. 125, P=0.09）. But, it was significantly lower in the serum of patients with gastric cancer （1. 420 ± 0. 122） than that in healthy people （3. 450±0. 206）,t=2. 521 ,P=0.02. Expression level of rniR-506 showed no signifi- cant difference between gastric cancer cell line SGC-7901 and MGC-803 as well as in the serum of gastric cancer patients and healthy people,Which were 1. 256±0. 113,1. 158±0. 109,1. 735±0. 220,1. 592±0. 134, respectively （P〉0.05）. CONCLUSIONS： miR-203 may be one of the miRNAs regulating PIK3CA gene. The high expression level of PIK3CA in gastric cancer may be associated with abnormally low expression of miR-203 ,while it has nothing to do with miR-506. The expression of miR-203 in the serum of patients with gastric cancer,is expected to become an important molecular marker for predicting gastric cancer development or progression.