| Objective:In this study,an attempt was made to explore Helicobacter pylori(H.pylori)subgroups related to gastric cancer via an analysis of the cytotoxin associated gene A(cagA),vacuolating cytotoxin A(vacA)and blood group antigen binding adhesin(babA)of H.pylori and a further investigation of the sequence variation and evolutionary characteristics of the carboxyl terminal(cagA 3’)variable region and babA gene of H.pylori.Through this study,the relationship between the difference of H.pylori strains and the risk of gastric cancer was finally elucidated,and the classification system of H.pylori that can effectively distinguish the difference of carcinogenicity was established.Methods:1.A total of 150 clinical isolates of H.pylori were collected from the central laboratory of Qingdao Municipal Hospital from 2012 to 2020,including 91 strains of H.pylori from patients with gastritis and 59 strains of H.pylori from patients with gastric cancer.The H.pylori clinical isolates were cultured for the extraction of genomic DNA.The genotypes of cagA,vacA and babA gene were specifically amplified by polymerase chain reaction(PCR),and a comparison was drawn on the distribution differences of each virulence genotype in the gastritis group and the gastric cancer group.2.The H.pylori cagA 3’variable region and babA gene were specifically amplified for sequencing.The Neighbour-Joining(N-J)phylogenetic tree were based on DNA sequence of the H.pylori cagA 3’variable regions and babA,and constructed by MAGE software.The sequence variation and evolutionary characteristics of cagA 3’variable region and babA gene were analyzed by N-J phylogenetic tree.Results:1.A total of 150 H.pylori strains were included in the study,59 of which were derived from gastric cancer patients.Their average age was 54±11.5 years,involving 66.10%of male patients and 33.90%of female patients.In addition,91 strains were derived from gastritis patients with an average age of 52±12.3 years,and among them,there were 51.65%of male patients and 48.35%of female patients.There was no significant difference in age and sex between gastritis group and gastric cancer group(P>0.05).2.The virulence genotype of 150 H.pylori strains were amplified by PCR,with the results showing that:(1)H.pylori strains with cagA~+,vacA s1/m1,vacA s1/m2 and babA~+genotypes accounted for 100.00%,44.07%,55.93%and 98.31%in gastric cancer group;Those in the gastritis group accounted for 93.41%,48.35%,50.55%and 98.90%,respectively.(2)The proportion of cagA~+/vacA s1/m1,cagA~+/vacA s1/m2,babA~+/vacA s1/m1,babA~+/vacA s1/m2,babA~+/cagA~+and babA~+/cagA~-genotype strains in gastric cancer group was 44.07%,55.93%,43.10%,56.90%,100.00%and 0.00%,respectively;The proportion in gastritis group was 49.41%,50.59%,49.44%,50.56%,93.26%and 6.74%.(3)The proportion of cagA~+/vacA s1/m1/babA~+and cagA~+/vacA s1/m2/babA~+genotype strains in gastric cancer group was 43.10%and 56.90%,that in gastritis group was 50.00%and 50.00%,respectively.There was no significant difference in the above virulence genotypes between gastritis group and gastric cancer group(P>0.05).3.Among the 150 H.pylori strains,the cagA~+genotype was 100.0%in gastric cancer group and 93.41%in gastritis group;The EPIYA motif of cagA 3’variable region of 140 H.pylori strains was typed.92.14%(129/140)of the strains carried the East Asian EPIYA-ABD motif,and only 6 strains carried the EPIYA-C motif,all of which were in the gastritis group.There was a significant difference in the EPIYA-C motif between the two groups(P=0.04),which indicated that H.pylori strain containing EPIYA-C motif may have a lower risk of gastric cancer.Besides,the N-J phylogenetic tree was constructed based on the cagA3’variable region gene sequence of 140 H.pylori strains,with the results showing that in the cagA 3’Cluster I subgroup,the number of H.pylori strains in the gastric cancer group was in a dominant position,accounting for 84.48%,which was significantly higher than that in the gastritis group(29.27%),and the difference was statistically significant(P=1.18E-10).4.The N-J phylogenetic tree was constructed based on babA gene sequences of 132strains.It was also found that the proportion of H.pylori strains in the babA Cluster I subgroup in the gastric cancer group was 80.36%,which was significantly higher than that in the gastritis group(39.47%).The difference was statistically significant(P=3.00E-6)according toχ~2test.Conclusion:1.H.pylori strains with EPIYA-C motif in cagA 3’variable region had a lower risk of gastric cancer.2.H.pylori strains associated with gastric cancer obviously clustered into subgroups in the phylogenetic tree constructed by cagA 3’variable region and babA gene sequence,respectively,which had more similar genetic evolution characteristics.Based on the evolutionary characteristics of cagA 3’variable region and babA sequence,H.pylori subgroups related to gastric cancer can be identified,which would provide a theoretical basis and a new idea for the establishment of H.pylori classification system that can effectively distinguish carcinogenic differences. |
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