HPV16 E6-178G/E7-647G Promotes Proliferation and Inhibits Apoptosis in Cervical Cancer C33A Cells

Background HPV16 is the main cause of cervical cancer. In our study, we aimed to investigate the role of HPV mutants HPV16 E6-178G/E7-647G in the proliferation and apoptosis of cervical cancer C33A cells. Methods Plasmids encoding the HPV16 E7 prototype (E7-647A)-GV144, E7 mutant (E7-647G)-GV144, HPV16 E6/E7 prototype (E6-178T/E7-647A)-GV144, and E6/E7 mutant (E6-178G/E7-647G)-GV144 were stably transfected into cervical cancer C33A cells. Western blot analysis, CCK8 proliferation assay, cell cloning assay and ow cytometry were used to detect the effects of the different polymorphism sites in HPV16 on cell proliferation and apoptosis.


Introduction
According to GLOBOCAN's latest estimate, there will be 570,000 cases of cervical cancer and 311,000 deaths from this disease worldwide in 2018. Cervical cancer is also the fourth major cause of cancer deaths in women. Its morbidity and mortality have always been high, second only to breast cancer [1].
Human papillomavirus (HPV) vaccination programs have been successful in preventing cervical cancer in some developed countries, but global coverage remains low [2,3]. The incidence and mortality rates of cervical cancer in China are 7.5 and 3.4 per 100,000 women, respectively [4,5]. Therefore, cervical cancer remains an important public health issue in China.
Currently, more than 200 gene sequences have been identi ed from HPV genotype-speci c sequence information. According to the transformation characteristics of HPV, 14 types, including HPV-16, -18 and − 31, were classi ed as high-risk by WHO [6][7][8]. Epidemiological data has con rmed [9] that persistent infection by high-risk HPV can lead to cervical atypical hyperplasia and cancer through the E6 and E7 genes, which are the two major cancer genes which target a variety of tumor suppressor proteins including p53 and pRb.
Evolutionary analysis has shown that globally, the diversity of the HPV16 genome has been evolving for more than 200,000 years [10]. Epidemiological studies have shown that mutations in the HPV16 gene may contribute to persistent viral infection and the development of cervical cancer. For example, Villa et al [11] showed that non-European variants have a general tendency to lead to persistent infection and are associated with cervical lesions. Zhang et al. [12] found that the European variant containing the T350G mutation results in the replacement of valine by leucine in the E6 protein, which is considered to be an additional risk factor for persistent infection and cervical lesions. In addition, the prevalence of polymorphic cervical cancer with E6 T178G mutations was much higher in Asia (65.5% in China, 85.2% in South Korea and 44% in Japan) than in Europe (2%) and North America (3%). However, little is known about the carcinogenic potential of the HPV16 variant in Asian women compared to many studies in European and American populations [13]. Because of the E6 gene encodes the major transforming protein that inhibits cell apoptosis and promotes cell proliferation, and it is associated with cancer invasiveness, most studies of HPV16 variations have focused on the E6 gene [14], while relatively few studies have analyzed gene variations in E7 as the other major oncogene of HPV16. Studies have shown [15] that HPV16 E7 induces upregulation of KDM2A, inhibits mir-132 and promotes proliferation of cervical cancer cells, thus leading to malignant progression of cervical cancer that is related to poor prognosis for cervical cancer patients.

Tissue samples
The study subjects were female patients who were treated at the Friendship Hospital (central Xinjiang,

Cell culture and transfection
The E6 + E7 gene was cloned into GV144 recombinant vector and veri ed by sequencing. In addition, GV144(-) vectors were used as controls. C33A cells were seeded at 1×10 6 per well in a six-well plates 24 h before transfection. Subsequently, 2 µg E6/E7 recombinant vector and the control vector were mixed with 5 µL of FuGENE HD (Roche), respectively. The FuGENE/DNA complexes were added to C33A cells. Transfected C33A cells were then incubated at 37 °C in 5% CO2 for 24 h.

Analysis of cell proliferation
Proliferation of C33A cells transfected by the E6/E7 GV144 construct and GV144 was determined using CCK-8 assays. C33A cells were seeded in 96-well plates at 3000 cells per well in DMEM(Gibco, USA)containing 10% FBS(Biological Industries, Israel). The cells were incubated for 24 h at 37 °C. Then, 10 µL of CCK-8 solution(Dojindo, Japan) was added to each well and incubated for 4 h.
For analysis of clonal formation, C33A stably transfected cells, selected by G418, were inoculated into six-well plates, 1,000 cells per well, and after 14 d, colonies were xed in 2 mL of 4% paraformaldehyde, followed by the addition of 0.4% crystalline purple staining dye. The number of clones with more than 50 cells were counted under a microscope.
Scratch wound healing assay Cells were seeded onto six-well plates (1×10 6 cells/dish). When cells reached 90% con uence, a scratch was made across the cell monolayer. The cells were washed with PBS for three times to remove detached cells and debris, and cultured in fresh medium without serums in an incubator of 5%CO 2 at 37℃. Then, size of wounds were observed and measured at the indicated times and photographed using an inverted tissue culture microscope at 40× magni cation. Assays were performed at least three times, and data are presented as means ± SD.

Cell apoptosis detection by ow cytometry
Apoptosis analysis was assessed with Annexin V-APC/7-AAD Apoptosis Detection kit (Joint Biology, Hangzhou). Cells were plated in a 6-well plate at a density of 1 × 106 per well. After incubation for 48 h post-transfection, cells were harvested. Apoptosis was induced in accordance with the experimental protocol, the cells were washed with precooling PBS for three times to remove detached cells and debris, and incubated with 5 µl Annexin V-APC and 10 µl 7-AAD Staining Solution.

Model of xenotransplanted tumors in nude mice
For xenograft experiments, 7×10 6 C33A cells stably expressing NV-GV144, HPV16 E7 or E6/E7 prototype, E7 mutation or E6/E7 co-mutations from recombinant expression vectors were injected subcutaneously into 5-week-old female nude mice. The tumor volumes were measured using digital calipers every 3 days and calculated using the equation: length (mm) × width 2 (mm) × 0.52. All animals were treated in accordance with institutional guidelines, and the experimental protocol was approved by the Ethics Committee guidelines of the First A liated Hospital, Shihezi University School of Medicine (approval number A2019-038-01).

GenBank accession number
The HPV16 prototype (European prototype, GenBank accession: NC_001526.2) Statistical analysis All data were analyzed using SPSS 22.0 software (IBM, Armonk, NY, USA). The measurement data is represented by means ± SD. The data were normally distributed, displayed homogeneous variance and was subsequently analyzed by ANOVA. A P-value < 0.05 was considered statistically signi cant.

Results
Mutation of the HPV16 E7 gene at nt 647 in cervical cancer and non-cancerous tissues In this study, a total of 66 cervical cancer and non-cancerous tissue samples were collected from women in Xinjiang with HPV16 infections. Among them, the A647G mutation was found in four out of 28 cases (14.3%) with non-cancerous tissues and in 14 out of 38 cases (36.8%) with cervical cancer ( Table 1).
Construction of HPV16 E7 prototype and HPV16 E7 mutant recombinant vectors According to the sequencing results, we found 18 cases with the A647G mutation in the E7 gene and 17 cases with the T178G mutation in the E6 gene. Therefore, the following four recombinant vectors were designed and constructed (Fig. 1 Effect of the HPV16 E7 mutation and E6/E7 co-mutation on proliferation of C33A cells The HPV16 E7 prototype (E7-647A)-GV144 vector, HPV16 E7 mutant (E7-647G)-GV144 vector, HPV16 E6/E7 prototype (E6-178T/E7-647A)-GV144 vector and HPV16 E6/E7 mutant (E6-178G/E7-647G)-GV144 vector were transfected into C33A cells in 96-well plates. Non-transfected C33A cells were used as a blank control group, and the NC-GV144 vector group was used as a negative control group. OD values in the above groups at 450 nm were detected at 0 h, 24 h, 48 h, and 72 h after the addition of CCK-8 to determine the effect of transfection on proliferation of C33A cells. As shown in the Fig. 3A, compared with the NC-GV144 control group, E7 and E6/E7 prototypes signi cantly promoted the proliferation of C33A cells 72 h after transfection (P < 0.05). The HPV16 E7-647G mutant group and the E6-178G/E7-647G co-mutant group showed signi cant cell proliferation 48 h and 72 h after transfection (P < 0.05). The four recombinant expression vectors were then combined and compared (Fig. 3B). At 24 h and 48 h, the HPV16 E6-178G/E7-647G co-mutant group showed signi cantly increased proliferation of C33A cells (P < 0.05). The results suggested that compared to NC-GV144 control group, the recombinant vector in the experimental group had a signi cant effect on the proliferation of C33A cells and that cell proliferation in the HPV16 mutant group was higher than in the HPV16 prototype group. In the mutant group, comutation of HPV16 E6-178G/E7-647G had the greatest impact on the proliferation of C33A cells.
Effects of HPV16 E7 mutation and E6/E7 co-mutation on clonal formation of C33A cells C33A cells stably transfected with NC-GV144, HPV16 E7 prototype (E7-647A)-GV144 vector, HPV16 E6/E7 prototype (E6-178T/E7-647A)-GV144 vector and HPV16 E6/E7 mutant (E6-178G/E7-647G)-GV144 vector were seeded on 6-well plates at a cell density of 1,500 cells per well for 2 weeks and then xed with 4% paraformaldehyde. Colonies were stained with 0.1% Crystal Violet at room temperature and the number of cell colonies were counted at low power under a microscope. The results showed that compared with the control group of NC-GV144, the number of cell colonies in the four recombinant expression vector groups was signi cantly increased (P < 0.05) and the number of cell colonies in the HPV16 mutant group was higher than in the HPV16 prototype group. Among the mutant groups, the co-mutation of HPV16 E6-178G/E7-647G had the greatest effect on clonal formation of C33A cells, indicating that the proliferation of C33A cells was greatest after co-mutation of HPV16 E6-178G/E7-647G (Fig. 3C and 3D).

Discussion
In the present study, we found that the rate of the E7 gene A647G mutation in cervical cancer tissue was higher than in non-cervical cancer tissue (Table 1). In addition, HPV16 E7-A647G and E6-T178G nucleotide co-mutations have been demonstrated in our previous studies [16], consistent with results of Ding et al. [17] The E7 oncoprotein encoded by HPV16 E7 is 11 kDa in size and contains about 100 amino acids. E7 protein can promote the malignant transformation of cervical epithelial cells that are infected with HPV and maintain the malignant phenotype of cervical epithelial cells. The interaction between the E7 gene and the retinoblastoma tumor suppressor Rb is one of the leading causes of cervical cancer. The region where the E7 protein binds to Rb is on amino acids 21-34, which include the 29th amino acid encoded by the nucleotide at position 647. The A647G mutation suppresses the physiological functions of Rb so as to maintain the HPV infection in the host over the long-term. Other studies have found that the incidence of A645C (L28F) can reach 19% in the cervical cancer tissues of Korean women [18], and the mutation is also present in Japan and Italy [19,20]. However, only one mutation at this site was found in our study, further suggesting that HPV mutations have regional characteristics.
In order to study the effect of the HPV16 E7 mutation on malignant progression of cervical cancer, we conducted cytological experiments to analyze and verify its function. Initially, four recombinant expression vectors of GV144, namely, HPV16 E7 prototype (E7-647A), HPV16 E7 mutant (E7-647G), HPV16 E6/E7 prototype (E6-178T/E7-647A) and HPV16 E6/E7 co-mutant (E6-178G/E7-647G) were designed and constructed according to sequencing results. HPV-negative C33A cervical cancer cells were selected for stable transfection, and the expression of HPV16 E7 protein in each group of cells after transfection was detected by western blot. The results suggested that the recombinant vector was successfully transfected (Fig. 2), and further in vitro cytology experiments were conducted to study and analyze the effect of different HPV16 E7 gene mutants on cervical cancer cells. The results of CCK-8, colony formation and proliferation experiments showed that among the four recombinant expression vectors, the co-mutation of HPV16 E6-178G/E7-647G had the strongest effect on proliferation of cervical cancer cells, followed by the mutation of HPV16 E7-647G. Flow cytometry detection showed that both HPV16 E6-178G/E7-647G co-mutation and the E7-647G mutation inhibited apoptosis of cervical cancer cells. Through cell scratch experiments, we also observed that co-mutation of HPV16 E6-178G/E7-647G promoted the migration of cervical cancer cells to the greatest extent, followed by the mutation of E7-647G. These results thus demonstrated that the mutations changed the carcinogenic properties of the virus compared to the HPV16 prototype, and the mutations of these two HPV16 variants in this experiment promoted the malignant progression of cervical cancer cells, especially the co-mutation of HPV16 E6-178G/E7-647G. It has been reported that HPV16 E6 directly induces cervical cancer cell migration by regulating p53 signaling [21]. In order to further study the effect of the HPV16 E7-647G mutation and E6-178G/E7-647G co-mutation on cervical cancer cells, a xenograft tumor model was established in nude mice. Four days after inoculation of C33A cells stably transfected with the four recombinant expression vectors, tumor formation was observed in nude mice at the inoculation site. Tumor growth curve (Fig. 5H) analysis showed that 20 days after inoculation, the tumors of nude mice with the HPV16 E6-178G/E7-647G co-mutation showed signi cant differences from the other experimental groups. As could be seen from the appearance of the mice, the tumor volume was largest in the co-mutation group of HPV16 E6-178G/E7-647G. The tumors were removed and the terminal weight and volume of the tumors were analyzed, which showed that the HPV16 E6-178G/E7-647G polymorphic site promoted the growth of cervical cancer to a greater extent than did HPV16 E7-647G.
To summarize, our in vivo and in vitro experimental studies con rmed that the co-mutation of HPV16 E6-178G/E7-647G promoted the malignant progression of cervical cancer, and we intend to investigate the cancer-causing molecular mechanism of this mutation type.

Conclusions
The genetic differences among HPV16 sub-types may be related to their carcinogenic potential. HPV mutations can differ in biology and etiology, leading to differences in tumor development and behavior. In our research, HPV16 E6-178G/E7-647G can thus promote the proliferation and inhibit the apoptosis of cervical cancer cells.

Declarations
Ethics approval and consent to participate This study was conducted according to the guidelines of the Declaration of Helsinki and all procedures involving human subjects were approved by the First A liated Hospital, Shihezi University School of