宏基因组测序技术分析原发性肝癌患者肠道菌群特征

Metagenomic analysis on characteristics of intestinal flora of patients with primary liver cancer

目的宏基因组测序技术分析原发性肝癌患者肠道菌群的分布特征。方法收集2019年3月~6月于首都医科大学附属北京佑安医院就诊的10例原发性肝癌患者(原发性肝癌组)和10例健康体检(健康对照组)者的粪便标本并记录一般资料。行宏基因组测序并根据测序获得的数据利用主成分分析和多样性分析方法分析两组菌群的结构并比较物种丰度差异。采用Spearman相关分析法分析副血链球菌、唾液链球菌、变形链球菌、嗜热链球菌、副流感嗜血杆菌、韦荣球菌、殊异韦荣菌、艾克曼菌、嗜黏蛋白艾克曼菌、普雷沃菌、另枝菌的丰度与血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、谷氨酰转肽酶(GGT)、总蛋白、总胆红素、甲胎蛋白(AFP)水平间的相关性。结果基于bray、jsd距离衡量的Beta多样性分析显示,原发性肝癌组患者粪便菌群多样性较健康对照组显著降低,差异有统计学意义(t=5.402、P<0.001,t=5.248、P<0.001)。属水平方面丰度最高为拟杆菌属,其次为普雷沃菌属,相对丰度分别为[34.94(11.76,56.02)]%和[11.99(1.29,27.82)]%;物种水平方面丰度最高的分别为普通拟杆菌和普拉梭菌,相对丰度分别为[1.55(0.63,3.90)]%和[1.54(0.53,2.84)]%。在细菌分类学的种水平进行丰度比较,137种细菌在健康对照组和原发性肝癌组中的分布差异有统计学意义(P均<0.05),原发性肝癌组患者副血链球菌、唾液链球菌、变形链球菌、嗜热链球菌、副流感嗜血杆菌、韦荣球菌、殊异韦荣菌丰度显著增加(P均<0.05),而艾克曼菌、嗜黏蛋白艾克曼菌、普雷沃菌、另枝菌丰度显著降低(P均<0.05)。相关性分析发现唾液链球菌(r=0.733、P=0.020)、嗜热链球菌(r=0.867、P=0.002)、副流感嗜血杆菌(r=0.721、P=0.023)与血清ALT水平呈显著正相关;嗜黏蛋白艾克曼菌与AST(r=0.646、P=0.049)、GGT(r=0.762、P=0.037)、总蛋白(r=-0.788、P=0.010)有显著相关性。结论原发性肝癌患者的肠道菌群多样性显著降低,物种丰度发生改变,存在显著的肠道菌群失衡,肠道菌群中特定差异的细菌种类可能作为一种生物标志物用于原发性肝癌的早期诊断。

Objective To investigate the distribution characteristics of intestinal flora of patients with primary liver cancer by metagenome sequencing.Methods Fecal samples of 10 patients with primary liver cancer(primary liver cancer group)and 10 healthy people(healty control group)were collected from March to June 2019 in Beijing Youan Hospital,Capital Medical University and the general clinical information was recorded.According to the metagenome sequencing data,principal component analysis and diversity analysis were used to analyze the flora structure and species differences of the two groups of bacteria were also compared.Spearman correlation analysis was used to analyze the correlation between the abundance of Streptococcus parahaemolyticus,Streptococcus salivarius,Streptococcus mutans,Streptococcus_thermophilus,Haemophilus_parainfluenzae,Veillonella_sp._DORA_A_3_16_22,Veillonella_dispar,Akkermansia_sp._CAG.344,Akkermansia_muciniphila_CAG.154,Prevotella_sp._CAG.924 and Alistipes_sp._CAG.268 and serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),glutamyl transpeptidase(GGT),total protein,total bilirubin and alpha fetoprotein(AFP).Results The analysis of beta diversity based on bray and jsd distance showed that the intestinal flora diversity of the two groups of subjects was statistically significant.The diversities of fecal flora in patients of primary liver cancer group were significantly lower than those of healthy control group,with significant differences(t=5.402,P<0.001;t=5.248,P<0.001).At genus level,the highest abundance was Bacteroides,followed by Prevotella,and the relative abundance were[34.94(11.76,56.02]%and[11.99(1.29,27.82]%,respectively;at species level,the highest abundance was Bacteroides vulgatus and Faecalibacterium prausnitzii,and the relative abundance were[1.55(0.63,3.90]%and[1.54(0.53,2.84]%,respectively.The abundance comparison at the species level of bacterial taxonomy showed that the distribution of 137 species in the healthy control group and the primary liver cancer group was statistically significant(all P<0.05).In the primary liver cancer group,the abundance of Streptococcus_parasanguinis,Streptococcus_salivarius,Streptococcus_mutans,Streptococcus_thermophilus,Haemophilus_parainfluenzae,Veillonella_sp._DORA_A_3_16_22 and Veillonella_dispar were significantly increased,while the abundance of Akkermansia_sp._CAG.344,Akkermansia_muciniphila_CAG.154,Prevotella_sp._CAG.924 and Alistipes_sp._CAG.268 were significantly decreased.Correlation analysis found that Streptococcus_salivarius(r=0.733,P=0.020),Streptococcus_thermophilus(r=0.867,P=0.002)and Haemophilus_parainfluenzae(r=0.721,P=0.023)were significantly and positively correlated with serum ALT level.Akkermansia muciniphila was significantly correlated with serum AST(r=0.646,P=0.049),GGT(r=0.762,P=0.037)and total protein(r=-0.788,P=0.010).Conclusions The diversity of intestinal flora in patients with primary liver cancer was significantly decreased,and the species abundance changed.There was a significant imbalance of intestinal flora.Specific differences in intestinal flora may be used as biomarkers for the early diagnosis of primary liver cancer.