In the present study, Oct3/4A, Oct3/4B and Oct3/4B1 mRNA expression was investigated in non-ILs, LSILs, HSILs and CCs. In addition, we used a cell line that expresses the HPV16 E7 oncoprotein [C-33 A cells] to evaluate the effect of the HPV16 E7 oncoprotein on the mRNA expression of Oct3/4A, Oct3/4B and Oct3/4B1. Our results suggest that the HPV16 E7 oncoprotein is involved in the overexpression of Oct3/4A mRNA in CC.
The E7 oncoprotein is an important factor in cervical carcinogenesis, and it is also involved in the alteration of a large number of cellular genes [26] and increases the expression of Oct3/4 and stemness-related genes [14]. The human Oct3/4 gene can generate three isoforms by alternative mRNA splicing, Oct3/4A, Oct3/4B and Oct3/4B1 [11, 12]. Oct3/4 plays an important role in maintaining the totipotentiality and pluripotentiality of human embryonic cells [12]. Previous studies have shown that Oct3/4 is overexpressed in CC [8, 9, 27]. Moreover, Oct3/4 can promote carcinogenesis and the development of tumors, while the loss of its expression leads to the loss of self-renewal and proliferation capabilities [28, 29] .
On the other hand, it has been reported that the HPV E7 oncoprotein binds to Oct3/4 protein, forming a complex both in vitro and in vivo and that Oct3/4 is active and expressed in HR-HPV-transformed cells [30, 31], However, it is unknown which isoform is deregulated by the HPV16 E7 oncoprotein. It has also been observed that the HPV16 E7 oncoprotein positively regulates the expression of Oct3/4 at the mRNA and protein levels both in vivo and in vitro, so the HPV16 E7 oncoprotein is proposed as a critical factor for cell self-renewal that facilitates the overexpression of factors related to maintenance of stem features [14, 30]. In 2015, Li et al. observed the expression of Oct3/4 (A and B) isoforms in CC and reported that Oct3/4A is responsible for cell self-renewal, which has also been attributed to the onset of CC, while Oct3/4B increases proliferation and tumor formation due to antiapoptotic activity [8]. Therefore, it is suggested that the expression level of each Oct3/4 isoform could be different in CC due to the presence of oncoproteins, such as the HPV16 E7 oncoprotein. To meet the stated objective, the PEGFP-N1 vector and PEGFP-N1-E7 construct were transiently transfected into HPV-negative C-33 A epithelial cells derived from CC.
In this study, the detection of Oct3/4A, Oct3/4B and Oct3/4B1 mRNA expression was performed to identify which of the Oct3/4 isoforms are expressed in samples of patients with LSILs, HSILs and CC positive for HPV16 infection. Oct3/4A and Oct3/4B1 mRNA were mostly expressed in samples from patients with HSILs and CC. Our results are similar to those of Li et al., 2015 and Assadi et al., 2011, who reported overexpression of Oct3/4A mRNA in samples from CC patients [8] and Oct3/4B1 mRNA in samples from gastric cancer patients [23], which could be due to the presence of the E7 oncoprotein of HPV16. Oct3/4 mRNA overexpression is closely related to HPV-AR infection, and the HPV16 E7 oncoprotein plays an important role in Oct3/4 overexpression [8, 9, 14, 30].
On the other hand, our results showed higher expression of Oct4A mRNA in C-33 A cells in the presence of the HPV16 E7 oncoprotein than in C-33 A cells that were not transfected or in cells transfected with the vector PEGFP-N1. A similar behavior was observed for the expression of Oct3/4B and Oct4B1 mRNA since their expression was very similar between the study groups, so the HPV16 E7 oncoprotein seemed to have no effect on the expression levels of Oct4B and Oct4B1 mRNA. Our data are consistent with those reported by Wang et al., who showed basal expression of Oct3/4 in C-33 A cells, which agrees with our expression data of the different isoforms in C-33 A cells without transfection [9]. There is also similarity in our results and those reported by Liu et al., who showed that the expression of Oct3/4 was higher in HR-HPV-positive cells, such as CasKi (HPV16-positive) and HeLa (HPV18-positive) cells, compared to C-33 A cells (HPV-negative) [13]. Taken together, these data indicate that the HPV16 E7 oncoprotein is directly or indirectly involved in the upregulation of Oct3/4A, which supports the idea that the transformation of cells mediated by the HPV16 E7 oncoprotein is closely related to the overexpression of Oct3/4A.
On the other hand, it has been reported that Oct3/4 can act as an oncogene and is associated with the triggering of cancerous stem cells [8, 32]. Likewise, during HPV-AR infection, viral DNA can be integrated into host DNA and initiate the synthesis of the E6 and E7 oncoproteins, promoting proliferation by inactivating the p53 and pRB tumor suppressors, respectively [33]. Moreover, it has been shown that the increase in cell proliferation leads to high expression of Oct4 in a pRB-dependent manner [34], while the degradation of p53 leads to the upregulation of Nanog [35]. Nanog is a positive regulator of Oct3/4 expression [36]. In addition, it has been observed that the HPV16 E7 oncoprotein also binds to the Oct3/4 protein [30] and that expression of the HPV16 E7 oncoprotein stimulates the Oct3/4 promoter [31]. In addition, it was found that the Oct3/4 gene is autoregulated by the Oct3/4 protein [37]. These data could help to understand why the high levels of Oct3/4 expression in patient samples could be due to the degradation of p53 by the HPV-AR E6 oncoprotein, leading to overexpression of Nanog, which could lead to an increase in the expression of Oct3/4 in the presence of HPV16 infections. Likewise, pRB degradation by the HPV-AR E7 oncoprotein could also lead to overexpression of Oct3/4. In addition, the interaction between the Oct3/4 protein and HPV16 E7 oncoprotein could modify the transcription of the Oct3/4 gene by directly binding to its promoter region, suggesting that the HPV16 E7 oncoprotein promotes self-renewal through the positive regulation of the Oct3/4 gene, thus maintaining the population of CC stem cells (Figure 5).
The data shown here suggest that Oct3/4A mRNA expression is positively regulated by the HPV16 E7 oncoprotein. However, future studies needed to clarify the effect of the HPV16 E7 oncoprotein on the Oct3/4 isoforms and to reveal new perspectives on the potential roles of the HPV16 E7 oncoprotein in Oct3/4 protein regulation during CC development. In conclusion, we showed that the presence of the E7 oncoprotein of HPV16 increases the expression of Oct4A mRNA.