Although HPV infection is associated with the occurrence of a variety of tumors, it is particularly associated with cervical cancer . Multiple studies have confirmed that persistent infection with HPV is the definitive cause of cervical cancer, making HPV screening critically important. Our results show that the positive rate of total HPV infection in females in Yili, the westernmost part in Xinjiang of China, is 26.39%, the positive rate of Han infection is 27.89%, and the positive rate of Uyghur infection is 22.87%. Thus, there was an observed difference in the positive rate of infection between the two ethnic groups. Among the permanent residents of Yili, the Uygur population accounts for approximately 47%, and the Han nationality accounts for about 36%. However, the Uyghur women in the test population accounted for only 20%, which may be related to health awareness and the socioeconomic status. Studies have shown that  among HPV-infected women, probability of cervical cancer in multiple parturients is significantly higher than in non-fertile women. In contrast, while the Uygur lifestyle is typically associated with early marriage, early childbearing, multiple births, and intensive births. This study shows that HPV16, HPV52, and HPV53 represented the high-risk HPV strains with a high detection rate in this region, especially in Uygur. The HPV16 infection rate in Uyghur was 25.19%, which was significantly higher than that of the women of Han nationality, at 18.04%. HPV16 was identified to be the most carcinogenic of the high-risk HPV strains, which may be related to the high prevalence of cervical cancer in Uygur. There are also regional differences in the positive rate of HPV infection. Li et al.  reported that the positive rate of HPV infection among the Beijing population was 19.1%, of which the most common genotypes were HPV52, HPV16, and HPV58. In addition, Zhang et al.  reported a positive rate of infection in the Shenzhen population of 15.9%. The most common genotypes were HPV52, HPV16, and HPV53. Mijit et al.  reported that the positive rate of HPV infection was 13% in Kashgar, Xinjiang, China, and the most common high-risk types were HPV16, HPV58, and HPV39. The high-risk HPV infection rate in Uruguay was 20.8%, and the most common high-risk types were HPV16, HPV31, HPV51, HPV56, and HPV39 . Although some differences were observed between the positive infection rate and the most common subtypes, HPV16 was the most common genotype in each study. In the present study, the Han infection types were HPV16, 52, 58, 53, 81, 51 42, 33, 31, 56, 66, 68, 43, 6, 18, 59, 14, 11, 35, 45, 73, 82, and 83. The Uyghur infection types were HPV16, 52, 53, 42, 66, 31, 59, 81, 58, 43, 35, 51, 73, 18, 6, 39, 45, 56, 82, 33, 68, 11, and 83. In terms of multiple infections, single infections were most common in both ethnic groups, followed by a double infection, with triple and higher infections being the most rare. Most women are infected with HPV as a transient infection that can be readily cleared by the immune system. Only about 10% of persistent infections are converted into high-grade CIN by low-grade cervical intraepithelial neoplasia (CIN). This type of infection can eventually evolve into cervical cancer, a process that usually takes 5–10 years. It is important to note that while the total infection rate of Uyghur HPV is lower than that of the Han nationality, the infection rate of HPV16 is higher than that of the Han nationality. Thus, Uyghur women may have a decreased ability to clear an HPV16 infection and continue to remain infected with HPV for longer periods of time.
This observed difference may also be related to changes in the body’s hormone levels or HPV susceptibility in Uyghur women. In addition, the prolonged duration of HPV infection may reduce the function of the immune system [27, 28]. At the molecular level, HPV is a double-stranded DNA virus that encodes six early genes (E1, E2, E4, E5, E6 and E7) and two late genes (L1 and L2). Although both E1 and E2 are involved in genomic DNA replication and transcriptional regulation of the virus, the effective tumorigenicity of HPV is primarily caused by its early genes E5, E6, and E7 [29,30,31,32]. Among the HPV subtypes, HPV16 is the most common in cervical cancer and its genome is polymorphic.
Evolutionary analysis shows that the worldwide occurrence of HPV16 genomic polymorphisms have evolved over the past 200,000 years, with the following six phylogenetic branches: European (EUR), Asian (As), Asian-American (AA), African 1 (AF-1), Africa type 2 (Af-2), and North American type (NA). Each branch can be further subdivided into endogenous single nucleotide polymorphisms that can detect different variants in persistently infected precancerous lesions or cancer hosts . Epidemiological investigations have shown that different mutants of the same HPV type have different biological characteristics and may be associated with different risks of disease . Changes in E6 coding can induce strong mechanical and functional changes, resulting in large differences in the cancer risk caused by HPV16 variants . Several studies have reported a high degree of similarity in the distribution of HPV16 subtypes among Chinese Han women, mainly the HPV16 Asian and European strains [36, 37]. Our study observed the genetic diversity and phylogeny of 110 samples by sequencing analysis. Compared with the HPV16 European standard strain, there was a total of 27 European standard strains. A total of 14 mutation sites were identified, including six synonymous mutations and eight missense mutations. In addition, there were 17 cases of E6 gene mutations at 178th nucleotides (T178G) belonging to the Asian standard strain. There were 65 cases of E6 gene mutations at 350 nucleotides (T350G) with a mutation frequency of 63.64%, and corresponding amino acid change of leucine → valine (L83V). This belonged to the European strain mutant, 40 of which were Uygur cervical cancer cases. We believe that HPV16 in Han and Uyghur women in Xinjiang is dominated by the Asian and European strains, and there are no Asian and African strains. These results suggest that the infection and development of invasive cervical cancer in Xinjiang Uygur women may be related to the G mutation in the 350th point of the HPV16 European strain. Studies have shown that HPV16 E6-T350G mutations in Moroccan women are prevalent in high-grade cervical lesions and are closely related to the progression of cervical cancer . In addition, the HPV16 E6-G350 (T295/G350) mutant in HPV16 persistently infected cervical diseases and women with malignant cervical cancer is more common than the HPV16 European original strain, HPV16 E6-T350 (T295/T350) [39, 40]. Our study found seven HPV16 E6 T295G and T350G nucleotide co-mutations, which may serve as a variant of the HPV16 European mutant strain. To further study the function of these mutations, three plasmids of the European strain, HPV16 E6 prototype (T295/T350), HPV16 E6-G295/G350 mutation, and HPV16 E6-G350 mutation (T295/G350) were constructed, and the corresponding cytology was designed to verify their function.
In cytology studies, HPV-negative cervical cancer C33A cells were selected for transfection, and a series of cytological methods were used to analyze the effects of different mutants of the HPV16 E6 oncoprotein gene. C33A cells are cervical cancer cells with mutations in the pRB and p53 genes, which does not have the carcinogenic effects of the high-risk HPV oncoprotein, E6 . Therefore, the effect of the HPV16 E6 gene on this cell line can be studied in this cell model. Each mutation expresses a specific gene, and most of these genes are involved in adhesion, proliferation, apoptosis, migration, and invasion. Compared to the HPV16 E6 European strain prototype (T295/T350) and two mutant types (HPV16 E6-T295/G350 mutation and HPV16 E6-G295/G350 mutation stably transfected C33A cells), the present results showed that the HPV16 E6 prototype, HPV16 E6-G295/G350 mutation, and HPV16 E6-T295/G350 mutation promoted cell proliferation, migration, and invasion, and inhibited apoptosis. The HPV16 E6-T295/G350 mutation had the strongest effect on cell proliferation, migration, and invasion, followed by the HPV16 E6-G295/G350 mutation, and HPV16 E6 prototype with the weakest effect. Thus, our results may partially explain the carcinogenic potential of this mutation.
Some experimental studies have shown that the natural variability of the HPV16 E6 gene mutant is sufficient to alter the cell functional activity induced by E6, including resistance to serum/calcium differentiation, prolongation of the primary human keratinocyte lifespan, and P53 and Bax in human immortalized cells. Decreased expression levels, apoptosis, transformation, and immortalization of human keratinocytes have been demonstrated; however, the mechanisms responsible for these changes and the involvement of genes in their regulation remain unclear [42, 43]. One limitation of this study is that there was no further study of the gene expression regulated by the HPV16 E6-T295/G350 mutation and HPV16 E6-G295/G350 mutation. Thus, such mechanisms will be investigated in future studies. While HPV vaccines are currently used in some developed countries, in underdeveloped countries and regions, including China, the high cost of these vaccines remains an obstacle for their wide-spread use. Therefore, some scholars have proposed a semi-compulsory HPV vaccination program in China .