In the present study, we identified gC1qR as a downstream target for p38 MAPK/JNK signalling in HPV 16 E2-induced cervical squamous carcinoma cell apoptosis. Our analysis provided experimental evidence that silencing the gC1qR gene or inhibiting p38 MAPK/JNK signalling is essential for the in vitro growth and migration properties of cervical squamous carcinoma cells in response to HPV 16 E2 treatment.
The C33a cell line was the primary focus of this experiment because C33a cells are negative for HPV DNA and RNA , and they represent a convenient model to study the effects of HPV 16 E2 on cellular gene expression without the involvement of other HPV types. Traditionally, the effects observed in regulating cellular genes when E2 protein is expressed in cervical carcinoma-derived cell lines result from repressed expression of the viral oncogenes E6 and E7; however, in this work, we demonstrated that HPV16 E2 changes cellular gene expression independently of viral oncoprotein E6 and E7 regulation.
HPV type 16 is the most prevalent type of HPV (84.7%), which is in agreement with other studies, while the frequency of HPV type 18 (3.38%) is very low compared with other ethnic populations . Low-grade dysplasias with HPV 16 infection demonstrated an increased rate of malignancy progression . HPV-16 E6/E7 oncoproteins have been demonstrated to cause immortalisation of primary human keratinocytes and are expressed in malignant cancers . Many studies have previously reported the ability of the HPV-16 E6/E7 oncoproteins to disrupt the normal process of differentiation of human foreskin keratinocytes  by targeting key tumour suppressors, such as p53  and pRb , resulting in increased levels of cell survival proteins, such as Akt , and disruption of the cell cycle . The HPV E2 protein functions as a repressor or an activator of early gene transcription, which regulates viral transcription and genome replication . Disruption of the viral E2-gene, which controls the transcription of oncogenes E6 and E7 that manipulate the cell cycle and the ability of apoptosis, has been associated with poor outcomes. Conversely, the HPV 16 E2 gene acted via mitochondrial-dependent pathways to control cellular apoptosis and fate . Among mitochondrial matrix proteins, gC1qR controls diverse cellular processes, such as cell growth, differentiation and apoptosis . The present study provides an essential framework for assessing the role of gC1qR protein in HPV 16 E2-transfected cervical squamous carcinoma cell apoptosis. gC1qR is a multi-compartmental and multi-functional cellular protein that is distributed in several tissues and cell types, including lymphocytes, endothelial cells, dendritic cells and platelets [29, 30]. However, in our experiment, immunohistochemistry demonstrated that gC1qR expression was significantly decreased in human cervical squamous cell carcinoma tissues compared with normal cervical tissues (see Additional file 2: Figure S2). Although gC1qR is not overexpressed in human cervical squamous cell carcinoma tissues, its expression increased significantly in the HPV16 E2-induced cervical squamous carcinoma cell line.
During complement activation, the biological responses mediated by C1q recognise and activate the signal that triggers the classical complement pathway. C1q functions as a potent extracellular signal for a wide range of cells, resulting in inhibition of T cell proliferation or endothelial cell activation . Additionally, the C1q-gC1qR complex not only may be involved in innate and adaptive immunity , but also may be an underlying molecular mechanism in virus infection. Xu et al.  provided evidence that viruses use host gC1qR protein to inhibit antiviral responses and to promote viral proliferation by activating a suppressive pathway to negatively regulate antiviral signalling. When constitutively expressed in a normal murine fibroblast cell line, gC1qR induces growth perturbation, morphological abnormalities and apoptosis . gC1qR has been extensively studied previously as an inducer of apoptosis . Recent cohort studies have shown that gC1qR is a conserved eukaryotic multifunctional protein that primarily localised in the mitochondrial matrix (see Additional file 3: Figure S3) and on the cell surface . Human gC1qR is expressed as a proprotein of 282 amino acids (aa) whose first 73 amino acids, containing a mitochondrial localization signal, are required for localizing the protein to the mitochondria and are subsequently cleaved to generate mature gC1qR. The mature form of gC1qR has been tied to apoptosis and autophagy via inducing mitochondrial dysfunction . In the present study, we determined that silencing the gC1qR gene in cervical squamous carcinoma cells results in decreased cervical squamous carcinoma cell apoptosis rates.
In the present study, our results indicate that gC1qR is a physiological inhibitor of HPV 16-induced cervical squamous carcinoma cell survival. A role for gC1qR in HPV 16 E2 oncogene-mediated apoptosis was also demonstrated. As shown in Figure 3D, flow cytometry analysis revealed that cells in the subG1 region decreased after gC1qR siRNA vector treatment. Interestingly, we observed that the gC1qR gene has an effect on the p38 MAPK/JNK-pathway in HPV 16 E2 expression. Recently, it was reported that the p38 MAPK/JNK-pathway is activated by HPV 16 E6 and E7 viral oncogene expression . However, our observations suggest that HPV 16 E2 also activates this pathway; however, the consequences of this activation may be different from the activation induced by the viral oncogenes because tight regulation and controlled coordination of the p38 MAPK/JNK signalling cascade is required to maintain the balance between apoptosis and differentiation.