肝癌转移相关的微小RNAs在不同转移潜能肝癌细胞系的定量研究

目的 研究与转移密切相关的微小RNAs(miRNAs)在不同转移潜能肝癌细胞系的表达水平,探讨其在肿瘤转移过程中的生物学功能.方法 提取细胞系MHCC97H、MHCC97L、HepG2、L02的总RNA,通过反转录获得特异miRNA(miR-122a、miR-124a、miR-148a、miR-148b、miR-15a、miR-219、miR-30c、miR-338、miR-34a、Let-7g、miR-9)的cDNA,并应用TaqMan MGB探针法对其进行定量检测.采用AB17500系统软件V1.3.1采集Ct值,并使用miRNA内参基因RNU6B校正,相对定量计算公式RQ=2-△Ct,A Ct=CtmiRNAs-CtRNu6B.数据均经SPSS13.0统计软件包处理,采用t检验或非参数检验. 结果 转移相关的miRNAs(miR-124a除外)在MHCC97H与MHCC97L中表达,差异均有统计学意义.HepG2中miR-30c、miR-338、miR-34a和Let-g的表达水平明显高于L02,分别为miR-30c(8.41±0.40比6.82±0.29),miR-338(3.14±0.29比-2.36±0.32),miR-34a(0.71±0.40比-2.95±0.26),Let-7g(-4.07±0.55比-6.98±0.56),t值依次为2.948,12.656,7.484,3.684,P值均＜0.05,差异有统计学意义.而miR-148b,miR-9的表达则显著低于正常肝细胞,分别为miR-148b(1.96±0.51比3.76±0.28),miR-9(-4.38±0.86比-1.10±0.53),t值依次为-3.073,-3.324,P值均＜0.05,差异有统计学意义.miR-148家族中miR-48b在所测细胞系中的表达(5.46±1.21)均显著强于miR-148a的表达(1.29±0.35),Z=-5.097,P=3×10-7,差异有统计学意义.结论 可以利用肝癌细胞系列细胞平台进一步研究肝癌转移相关miRNAs在肿瘤转移过程中的生物学功能.

Identification of metastasis-related mleroRNAs of hepatocellular carcinoma in hepatocellular carcinoma cell lines by quantitive real time PCR

Objective To identify the metastasis-related miRNAs in hepatocellular carcinoma (HCC)cell lines. Methods AqRT-PCR method was established and optimized. Results All candidate metastasis associated miRNAs except miR-124a were expressed in high metastasis cell line MHCC97H and low metastasis cell line MHCC97L, while some miRNAs were differentially expressed between liver cancer cell line (HepG2) and hepatic cell line (L02) (P ＜ 0.05), these miRNAs include: miR-148b (1.96 ± 0.51 vs 3.76± 0.28), miR-9 (-4.38 ± 0.86 vs -1.10 ± 0.53), miR-30e (8.41 ± 0.40 vs 6.82 ± 0.29), miR-338 (3.14 ±0.29 vs -2.36± 0.32), miR-34a (0.71 ± 0.40 vs -2.95 ± 0.26), Let-7g (-4.07 ± 0.55 vs -6.98 ± 0.56). miR-148b expression was about 4 times higher than miR-148a 5.46 (IQR 4.25-6.67) vs 1.29 (IQR 0.94-1.64)] in all cell line tested (Z = -5.097, P = 3 × 10-7). Conclusion This study may help to understand the biological significance of miRNAs in HCC metastasis.