| Objective: To investigate the molecular evolution of cervical cancer caused by HPV16 virus in Xinjiang Uygur and Han women,analyze the HPV16 genome-wide genetic polymorphisms in Xinjiang Uygur and Han women,and analyze its relationship with disease occurrence and development,and analyze the effect of HPV16 E6 and E7 gene polymorphic sites on cervical cancer progression.Methods:(1)DNA was extracted from 188 samples of cervical tissues infected with HPV16 in Xinjiang Uygur and Han women,and the genes were amplified by PCR and sequenced.The HPV16 genome sequence was compared with the European standard sequence,and its evolutionary tree was established.(2)Stable transfection of HPV16 E6 prototype 295 T /350 T,HPV16 E6 mutant 295G/350 G and 295T/350G-GV230 expression vectors in cervical cancer C33 A cells,respectively,HPV16 E7 prototype 647A-GV144,HPV16 E7 mutation 647G-GV144 and HPV16 E6+E7 prototype E6-178T/E7-647A-GV144,HPV16 E6+E7 mutation E6-178G/E7-647G-GV144 recombinant expression vectors.(3)C33A cells stably transfected with recombinant expression vectors for different polymorphic sites of E6 were seeded subcutaneously in the left hind leg of nude mice.Observe and measure the nude mice's weight and tumor size on average 2-3 days.After the nude mice were sacrificed to obtain the tumors,the tumors of each group were weighed.(4)Detect the expression of apoptosis-associated protein Caspase 3,cyclin protein CDK 4,immunoprotein IFN-? and MHC-?by immunohistochemistry experiments and Western Blot experiments,and analyze HPV16 E6 prototype E6-295T/350 T,HPV16 E6 mutant E6-295G/350 G and E6-295T/350 G,HPV16 E7 prototype E7-647 A,mutant E7-647 G and HPV16 E6 + E7 prototype(E6-178T/E7-647A),mutant E6-178G/Effects of four recombinant expression vectors E7-647 G on cell cycle,apoptosis and immune function of cervical cancer C33 A.(5)The experimental data were statistically analyzed using SPSS22.0.P <0.05 considered the difference to be statistically significant.Results:(1)DNA sequencing was performed on 48 HPV16-positive cervical tissues,and the entire gene sequence was compared with the European standard strain sequence.It was found that 48 samples had nucleotide variation,and the variation rate was 100%.The nucleotides at 24 positions on the E1 gene have changed,and the sample with the A978 G mutation is in largest amount,accounting for 52.1%(25/48),causing the amino acid change from isoleucine to methionine(I326M);the nucleotides at 23 positions in the E2 gene sequence were changed.In 42 samples,there was a C2546 T mutation,occupying the highest frequency of 87.5% and the mutation changed proline to serine(P219S);7 sites were found on the E5 gene that had the nucleotide changes,and there were 36 samples with A3115 C mutation at a frequency of 75%.This mutation caused isoleucine to become leucine(I44L);there were 22 nucleotides mutation on the L2 sequence occurred,the highest mutation site was A4362 C,accounting for 62.5%(30/48),the amino acid change was leucine ? phenylalanine(L330F);23 sites of mutations were found on the L1 gene,and the mutation rate that reached the highest 85.4%(41/48)was A5570 G,the amino acid change caused was threonine ? alanine(T266A);16 sites of nucleotide changes were found on E6,and the site with the highest mutation rate is A7220 G,accounting for 45.8%(22/48),the amino acid changes from aspartic acid to glutamic acid(D32E);8 positions of nucleotides were changed in the E7 gene sequence.A7689 G mutation occurred in 25 samples,the mutation rate was 52.1%,and the amino acid changed from asparagine to serine(N29S);a total of 34 mutation sites on LCR sequence was found,the highest mutation site is G6657 A,and the mutation frequency is 93.75%.Phylogenetic tree analysis of the sequence showed that the HPV16 infection type in this group of samples was mainly European type,accounting for 70.8%(34/48),followed by Asian type,accounting for 29.2%(14/48),and no Asian and African types were found.(2)Nude mice were inoculated with C33 A cells stably transfected with the mutant recombinant plasmid 3 days after purchase.Nude mice were killed from the day of inoculation to the nude mice for a total of 30 days.Observation and measurement of nude mice and tumors were performed once every 3 days.According to the experimental analysis of nude mice xenograft tumor models,there was no significant difference in the terminal mean tumor volume between the HPV16 prototype group,the E6-295G/350 G mutation group,and the NC group(P>0.05),while HPV16 E6-295T/350 G mutation group was significantly larger than the NC group and other experimental groups(P<0.05);compared with the NC group,compared with the NC group,the terminal tumor weight of the HPV16 E6-295T/350 G mutation group was significantly increased(P<0.05).The experimental results show that HPV16 E6-295T/350 G mutation can promote tumor growth of cervical cancer more.(3)In cytology experiments,the expression of Caspase 3 was reduced in the HPV16 E6-295T/350 G mutation group compared with the HPV16 E6-295T/350 T prototype group and the HPV16 E6-295G/350 G mutation group(P<0.01);the expression of CDK4 in the HPV16 E6-295T/350 T prototype group was higher than the two mutant groups of HPV16 E6-295G/350 G and HPV16 E6-295T/350 G,and was similar to HPV16 E6-295G/350 G compared with significant difference(P<0.05);HPV16 E6-295T/350 G mutant group IFN-? expression was significantly lower than HPV16 E6-295T/350T(P<0.05)and HPV16 E6-295G/350G(P<0.05),and the results of Western Blot detection showed that the HPV16 E6-295G/350 G mutant group was better than the HPV16 E6-295T/350 T prototype group and HPV16 E6-295T/350 G,IFN-? expression decreased(P<0.01,P <0.001);Western Blot results showed HPV16 E6-295G/350 G mutation group MHC-? expression Significantly lower than HPV16 E6-295T/350 T prototype group and HPV16 E6-295T/350G(P<0.001,P <0.01).The expression of Caspase 3 in the HPV16 E6-178G/E7-647 G mutant group was significantly lower than that in the HPV16 E6-178T/E7-647 A prototype group and the HPV16 E7-647 G single point mutation group(P<0.05);HPV16 E7-647 A,HPV16 E7-647 G,HPV16 E6-178T/E7-647 A,HPV16 E6-178G/E7-647 G,the expression of CDK4 is higher than that of NC;the expression of IFN-? Reduced in HPV16 E7-647 A HPV16 E7-647 G,HPV16 E6-178T/E7-647 A,HPV16 E6-178G/E7-647 G,and HPV16 E7-647 G mutations were lower than HPV16 E7-647 A decreased significantly(P<0.05);only IHC results showed that the expression of MHC-? in the HPV16 E6-178G/E7-647 G mutant group was significantly lower than that of the HPV16 E6-178T/E7-647 A prototype Group(P<0.01).Conclusion:(1)The HPV16 gene sequence in Xinjiang is polymorphic,and the main infection types are European type(Ep)and Asian type(As).(2)HPV16 E6-295T/350 G mutation can further promote the growth of cervical cancer.In vitro experiments have shown that HPV16 E6-295T/350 G is stronger than the prototype and E6-295G/350 G polymorphism site in inhibiting cervical cancer C33 A cell apoptosis and promoting its proliferation.The effect of HPV16 E6-178G/E7-647 G in inhibiting cervical cancer C33 A cell apoptosis and promoting its proliferation is stronger than that of its prototype and E7-647 G and E7-647 A polymorphic sites.(3)HPV16 E6 and E7 can degrade the expression of immune proteins IFN-? and MHC-?,and the mutation group has a stronger effect than the prototype group. |
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