原发性肝细胞癌相关HBV G588C突变的功能研究

A study of the functions of G588C mutation associated with HCC

目的探索乙型肝炎病毒(hepatitis B virus,HBV)G588C突变在原发性肝细胞癌(HCC)组织HBV cccDNA中的存在情况及其对HBV复制的影响。方法通过滚环扩增(rolling cycle amplification,RCA)和PCR扩增测序,寻找G588C突变在18对HCC患者癌与癌旁组织中的存在情况。在HBV 1.2×质粒(HBV C基因型)的基础上构建G588C突变位点,将G588C突变质粒与野生型质粒分别转染HepG2和Huh7肝癌细胞系细胞,检测上清和细胞中HBsAg含量及上清中HBV DNA水平。结果在18对癌与癌旁组织中G588C突变只存在于3例癌组织中;G588C突变组上清中HBsAg 3、4d含量分别为HepG2细胞5.605±1.182,8.270±2.241,Huh细胞2.714±0.371,10.148±2.793,细胞内HBsAg 3、4d含量分别为HepG2细胞4.852±1.024,7.736±1.762,Huh细胞18.349±3.040,34.110±2.129;野生型组上清中HBsAg 3、4d含量分别为HepG2细胞8.083±1.428,13.170±0.938,Huh细胞6.231±0.373,23.971±1.573,细胞内HBsAg 3、4d含量分别为HepG2细胞2.937±0.876,4.270±1.659,Huh细胞13.498±3.06,21.010±3.488。G588C突变组上清中HBsAg含量显著低于野生型组(F=44.88,P<0.01),G588C突变组细胞内HBsAg含量显著高于野生型组(F=34.65,P<0.01);G588C突变组上清和细胞中HBsAg总量与HBV 1.2×野生型组比较差异无统计学意义(F=7.69,P>0.05);G588C突变组上清中HBsAg与细胞内HBsAg的比值显著低于HBV 1.2×野生型组(F=23.59,P<0.05),HBV DNA水平与两组间差异无统计学意义(F=6.23,P>0.05)。结论 G588C突变不影响HBV的复制,但影响HBsAg由细胞内向细胞外的分泌,这可能与HCC的发生发展有关。

Objectives To ascertain whether the G588C mutation of hepatitis B virus（HBV）is present in HBV cccDNA from liver tumor tissues and to analyze the functions of the G588C mutation. Methods Rolling cycle amplification（RCA）and PCR direct sequencing were used to detect the G588C mutation in tumor tissues and non-tumor tissues from18 patients with HCC.A G588C mutation plasmid was constructed based on the HBV 1.2× plasmid.The G588C mutation plasmid was transfected into the Huh7 liver cancer cell line,and the HBV 1.2× plasmid was transfected into the HepG2 liver cancer cell line.The level of HBsAg in supernatant and cell lysate was detected using a time-resolved fluoroimmunoassay,and the level of HBV DNA in the supernatant was detected using real-time PCR. Results The G588C mutation was only found in tumor tissues from 3 patients.In HepG2 cells,the level of HBsAg in the supernatant from tissue infected with a virus bearing the G588C mutation was 5.605±1.182 on day 3 and 8.270±2.241 on day 4.In Huh7 cells,the level of HBsAg was 2.714±0.371 on day 3 and 10.148±2.793 on day 4.In HepG2 cells,the level of HBsAg in cell lysates from tissue infected with a virus bearing the G588C mutation was 4.852±1.024 on day 3 and 7.736±1.762 on day 4.In Huh7 cells,the level of HBsAg was 18.349±3.040 on day 3 and 34.110±2.129 on day 4.In HepG2 cells,the level of HBsAg in the supernatant from tissue infected with the wild-type virus was 8.083±1.428 on day 3 and13.170±0.938 on day 4.In Huh7 cells,the level of HBsAg was 6.231±0.373 on day 3 and 23.971±1.573 on day 4.In HepG2 cells,the level of HBsAg in cell lysates from tissue infected with a virus bearing the G588C mutation was2.937±0.876 on day 3 and 4.270±1.659 on day 4.In Huh7 cells,the level of HBsAg was 13.498±3.06 on day 3 and21.010±3.488 on day 4.The level of HBsAg in cell lysates from tissue infected with a virus bearing the G588C mutation was higher than that from tissue infected with the wild-type virus（F=44.88,P〈0.01）,while the level of HBsAg in the supernatant from tissue infected with a virus bearing the G588 C mutation was lower than that from tissue infected with the wild-type virus（F=34.65,P〈0.01）.The total level of HBsAg in the supernatant and cell lysates from tissue infected with a virus bearing the G588C mutation did not differ from the total level from tissue infected with the wild-type virus（F=7.69,P〉0.05）,but the proportion of HBsAg in the supernatant and cell lysates was smaller than that in tissue infected with the wild-type virus（F=23.59,P〈0.05）.Results also indicated that the level of HBV DNA in the supernatant from tissue infected with a virus bearing the G588 C mutation did not differ from the level from tissue infected with the wild-type virus（F=6.23,P〉0.05）. Conclusion The G588C mutation had no effect on the replication capability of HBV,but it can affect the secretion of HBsAg,which might be associated with the development of HCC.