Study On HPV16 E6 Gene Polymorphism And Function In Cervical Cancer Of Uygur And Han Of Women In Xinjiang

Objective: To investigate the status and genotype distribution of human papillomavirus infection of the cervical human papillomavirus(HPV)in Uygur and Han women in Xinjiang.To study the variation of the HPV16 E6 gene sequence in the cervix of Uygur and Han women in Xinjiang.To analyze HPV16 E6 gene polymorphism site and related with the development of cervical cancer.Methods:(1)2879 cases of Uygur and Han women cervical mucus of exfoliated cells samples were collected for epidemiological analysis of HPV.(2)Genomic DNA was extracted from the cervical HPV16 positive tissues of 110 Uygur and Han women,and E6 was amplified by PCR,and this PCR product was directly sequenced.The HPV16 E6 sequence was analyzed with the European standard as the prototype,and the evolutionary tree analysis was carried out.(3)HPV16 E6 T295/T350-GV230,HPV16 E6 G295/G350-GV230 and HPV16 E6 T295/G350-GV230 were stable transfected in human cervical cancer C33 A cells.HPV16 E6 protein expression was certified by immunofluorescence assay.CCK8 assay and clonogenic assay were used for analyzing C33 A cells proliferation.Transwell assay were used for studying C33 A migration and invasion.C33 A cells apoptosis was analzed by FACS experiment.(4)SPSS17.0 statistical software was used for statistical data processing.P<0.05 was considered statistically significant.Results:(1)The total infection rate of HPV was 26.390%(760/2879),among Uygur infection rate was 22.87%(196/857)and Han infection rate was 27.89%(564/2022)(P <0.05).The high-risk types of HPV with higher detection rates in Uygur were HPV16,HPV 52 and HPV 53 respectively,whereas the high-risk HPV with higher detection rate in Han were HPV16,HPV52 and HPV58.Uygur and Han of women infected HPV are common with single infection.(2)A total of 14 mutation sites were found in HPV16 E6 gene with gene sequencing in 110 HPV16-positive samples including 8 missense mutations and 6 synonymous mutations.Among them,65 cases of E6 gene were mutated at nucleotide 350(T350G)with amino acid changing in leucine to valine(L83V)and the mutation rate was 59.09%.In addition,there were 7 cases of E6 gene mutation at nucleotide 295(T295G)with the corresponding amino acid changing aspartic to glutamic(D64E)and the mutation rate was 6.36%.It is noteworthy that these 7 cases of HPV16 E6 T295 G mutations were accompanied by the E6 T350 G mutation.Phylogenetic analysis showed that there were HPV16 European(Ep),European variant(E),Asian(As)in Uygur and Han of women and no African(Af)and Asian American(AA)were found.(3)HPV16 E6 T295/T350,G295/G350 and T295/G350 can promote the proliferation,migration and invasion of cervical cancer C33 A cells.The T295/G350 was stronger than G295/G350,and the G295/G350 was stronger than T295/T350,the difference was statistically significant(P <0.05).All three polymorphisms could inhibit the apoptosis of C33 A cells,but the difference was not statistically significant(P>0.05).Conclusion:(1)The positive rates of HPV infection were different between the Uygur and Han ethnic groups in Xinjiang,and the genotype distribution of HPV infection was different.(2)HPV16 infection is mainly European(E)and Asian(As)between the Uygur and Han in Xinjiang.(3)HPV16 E6-T295/G350 expression promoted the proliferation,migration and invasion of cervical cancer cells stronger than HPV16 E6-G295/G350,while HPV16 E6-G295/G350 was stronger than HPV16 E6-T295/T350.