应用外显子测序对肝癌不同转移潜能的研究

The application of whole exome sequencing in different metastatic potential of heptaocellular carcinoma

目的应用全基因组外显子测序技术对不同转移潜能的肝细胞癌组织及细胞株进行DNA分析,探讨与肝癌转移潜能相关的突变基因及位点.方法提取不同转移潜能的12例肝细胞癌及其癌旁组织和6株不同转移潜能的肝癌细胞(HCC-LM3、MHCC-97L、MHCC-97H、HepG2、SMCC-7721、PLC-RFP-5)的DNA,行全基因组外显子测序,所得结果进行生物信息学比对分析、筛选出与肝细胞癌转移相关的突变基因及其位点.结果在高、低转移潜能肝癌组织中,高转移潜能组中(≥3例)有UIMC1 (rs3733876C/T)、SEMA3C(rs1527482G/A)、SAMD9(rs10279499G/T)、RECK(rs16932912G/A)、PDLIM4(rs4877 G/T)、LIMK2(rs3747154A/G)、CRIM1 (rs3821169G/T)基因突变与肝癌转移相关.仅存在于高转移潜能肝癌细胞(HCC-LM3、MHCC-97L和MHCC-907H)中与转移相关的有CAPZB(rs79308175C/T)、CSF1R(rs10079250A/G)、HSPA5(rs143920039A/T)、SORBS3(rs2449331T/C,rs3758036C/T)等突变基因及位点;仅在3种低转移潜能细胞中(HepG2、SMCC-7721、PLC-RFP-5)与转移相关的有RASAL3(rs146624357 G/A,rs142945276 C/T)等突变位点;对3株同源的高转移性肝癌细胞(HCC-LM3、MHCC-97L和MHCC-97H)的DNA对比分析后发现,仅出现在HCC-LM3中为EHBP1(rs77403174T/C),仅出现在MHCC-97L和MHCC-97H中的为FAM189B(rs150296362C/T)、SERPINB13(rs144903442G/A)、ARHGEF1(rs2303797C/T)等突变位点.结论通过全基因组外显子测序发现UIMC1(rs3733876C/T)、SEMA3C(rs1527482G/A)、SAMD9 (rs10279499G/T)等突变基因及位点与肝癌转移潜在相关.

Objective Genome-wide exome sequencing was used to analyze DNA of hepatocellular carcinoma (HCC) tissues and cell lines with different metastatic potentials to explore the mutation genes and sites related to the HCC metastasis. Methods Genomic DNA extracted from twelve HCC tumors and its adjacent tissues and six HCC cells (HCC-LM3, MHCC-97L, MHCC-97H, HepG2, SMCC-7721 and PLC-RFP-5) with different metastatic potentials were analyzed by whole exome sequencing. Bioinformatics analysis and screening of mutations was used to investigated mutation genes and sites potentially associated with HCC metastasis. Results Comparing high and low metastatic potential HCC, high metastatic potential HCC group had more than three gene mutations including UIMC1 (rs3733876 C/T), SEMA3C (rs1527482 G/A), SAMD9 (rs10279499 G/T), RECK (rs16932912 G/A), PDLIM4 (rs4877 G/T, LIMK2 (rs3747154 A/G), CRIM1 (rs3821169 G/T) which was associated with HCC metastasis. Mutate genes and its sites such as CAPZB (rs79308175C/T), CSF1R (rs10079250A/G), HSPA5 (rs143920039A/T), SORBS3 (rs2449331T/C, rs3758036C/T) only occured in high metastatic potential HCC cells (HCC-LM3, MHCC-97L and MHCC-97H), while mutation sites RASAL3 (rs146624357G/A, rs142945276 C/T) only appeared in three low metastatic potential cells (HepG2, SMCC-7721 and PLC-RFP-5). The DNA comparative analysis of three homologous high metastatic potential HCC cell lines (HCC-LM3, MHCC-97Land MHCC-97H) showed that only EHBP1 (rs77403174T/C) was presented in HCC-LM3, while FAM189B (rs150296362C/T), SERPINB13 (rs144903442G/A) and ARHGEF1 (rs2303797C/T) only presented in MHCC-97L and MHCC-97H. Conclusion Mutations and its sites such as UIMC1 (rs3733876C/T), SEMA3C (rs1527482G/A), SAMD9 (rs10279499G/T) was detected to potentially related to the HCC metastasis by genome-wide exome sequencing.