Effect Of HPV38E6/E7on The Autophagy Of Keratinocytes And Its Mechanism

Human papillomaviruses (HPV) are small, double-stranded, circular DNA virusesinfecting the epithelium, of which more than100subtypes have been identified so far.HPV infection contributes to a variety of benign and malignant diseases in human beings.It has been demonstrated that prolonged infection of high-risk α-HPV is associated withcervical cancer; plenty of clinical data and research found that β-HPV infection relatesclosely to nonmelanoma skin cancer (NMSC). although features less metastasis andmortality when compared with melanoma, it still imposes heavy medical bills and mentalstress on the affected and the society as a whole due to its increasing incidenceworldwide and localization at sun-exposed regions. Therefore, its pathogenesis hasdrawn intense attention from researchers. Prior study found that contributors to NMSCdo not only include UV radiation, ethnicity, age, geography and immune suppression, butalso involved β-HPV infection.Autophagy is a protective mechanism ubiquitous in eukaryotic cells. It sweeps awaydysfunctioning, degenerated or damaged organelles, macromolecular substances andproteins by producing autolysosomes, therefore is implicated in a multitude of human diseases, including aging, neural degeneration, malignancy and pathogenicmicroorganism infection. Its correlation to tumorigenesis remains a controversial topicdue to its dual effects of both hindering and promoting tumor development. A commonunderstanding on this point is that autophagy plays different roles in different tumors ordifferent stages of the same tumor. In the stage of tumorigenesis, autophagy plays ananti-tumor effect by preventing it from malignant changes; while in the stage of diseaseprogress, autophagy promotes tumor viability by providing energy. Such dual effects arealso found in viral infection. On one hand, upon viral invasion, autophagy plays ananti-virus role by consolidating inherent and adaptive immunity; on the other hand, as theinfection continues, autophagy is took advantage by some viruses by providing venueand energy to viral duplication and transcription, and thereby prolong the infection.However, the precise pathogenesis of β-HPV and existence of high-risk subtypes arenot clearly understood. The effect of β-HPV and autophagy in NMSC pathogenesis hasnot been reported. For this purpose, the author’s research group conducted prior work onexploring high-risk β-HPV subtypes closely related to NMSC in China and theirmolecular mechanism in NMSC pathogenesis. Our results revealed that HPV38was thesubtype most relevant to actinic keratosis (AK) and SCC. We found that autophagy waselevated in SCC tissue using transmission electron microscope (TEM) andimmunohistochemistry (IHC), indicating an important role of autophagy in NMSC. AsHPV38plays an anti-tumor role by potentially regulating autophagy, there raise somequestions to be explored in the present study: how does HPV38affect autophagy on acellular level, and what is its underlying molecular mechanism?MethodsThe present study was conducted on human immortalized HaCaT cells. Firstly,stably transfected cell lines over-expressing HPV38E6/E7were established as thecellular model. Secondly, the effect of HPV38E6/E7on biological functions of cells andeffect of autophagy to cellular proliferation and apoptosis were detected using CellCounting Kit-8(CCK-8), colony formation assay and flow cytometry (FCM).and thelevel of autophagy was observed using Western blot and TEM; Finally, the molecular mechanism of HPV38E6/E7induction to autophagy was explored.Major results1. HPV38E6/E7Plasmids were transformed, amplified, extracted and identified byXbaⅠ/Bam HI double digestion that HPV38E6/E7target fragments were found inthe purchased plasmids. The plasmid was transfected into the human immortalizedcell line HaCaT using lipofectin reagent so as to establish cell lines stably expressingHaCaT-pGCMV and HaCaT-HPV38E6/E7.2. CCK-8and colony formation assay showed that cell proliferation was significantlyincreased after transfection of HPV38E6/E7; repeated determination of apoptosisvia Annexin V/PI method found that apoptosis was significantly higher inHaCaT-pGCMV than in HaCaT-HPV38E6/E7cells (P<0.05); examination of cellcycle by FCM revealed that the portion of S-phase cells were lower inHaCaT-pGCMV than in HaCaT-HPV38E6/E7(P<0.05).3. The expression of autophagy-specific proteins determined by Western blot revealedthat the expression of Beclin1and LC3were elevated while P62were reduced inHaCaT-HPV38E6/E7compared with HaCaT-pGCMV cells; TEM showed thatautophagosome were significantly higher in HaCaT-HPV38E6/E7than inHaCaT-pGCMV cells (P<0.05).4. Western blot showed that autophagy activity of HaCaT-HPV E6/E7was significantlyreduced after the cells were treated with autophagy inhibitor3-methyladenine (MA).CCK-8assay showed that growth of HaCaT-HPV E6/E7cells were inhibited afterthe cells were treated with3-MA, which was not significantly different from thecontrol group (P>0.05). Determination of apoptosis by FCM found that HaCaT-HPVE6/E7reduced cell apoptosis and the apoptosis was significantly improved when theautophagy was inhibited, compared with the control group (P<0.05).5. Western blot showed that the protein expression of p-PKRThr451and p-eIF2αSer51weresignificantly higher in HaCaT-HPV38E6/E7than in HaCaT-pGCMV, p-eIF2αSer51phosphorylation was reduced after the cells were added PKR phosphorylationinhibitor2-aminopurine (2-AP) and that the expression of Beclin1and LC3were significantly reduced, especially when the2-AP was at a concentration of5mmol/L.Major conclusions1. HPV38E6/E7changes cell cycle checkpoint, increases the number of S-phase cells,induces noticeable proliferation or even tumor formation and inhibits apoptosis.2. HPV38E6/E7is able to induce significant increase of autophagy and provide energyand material for high proliferation as well, which may serve as one of its biologicalfunction. When the autophagy is inhibited, the proliferative activity is reduced andapoptosis is increased, therefore HPV38E6/E7might influence cell proliferation andapoptosis via regulating autophagy.3. HPV38E6/E7plays its biological function by inducing autophagy via PKR/eIF2asignaling pathway.