| Obj ectives1.To explore the relationship between human papilloma virus(HPV)infection and its subtypes and the occurrence,development and malignant transformation of nasal and sinonasal inverted papilloma(NSIP).2.To study the role of activation of PI3K/Akt/mTOR signaling pathway in HPV-infected nasopharyngeal cells.BackgroudNasal and sinonasal inverted papilloma(NSIP)is a common innocuousness tumor of the otolaryngology,presented locally invasive and characterized by a tubular or finger-like depth of the nasopharynx epithelial tissue and break through the basement.The etiology and pathogenesis of NSIP are unclear,but studies have reported that human papilloma virus(HPV)infection is a major risk factor of NSIP.HPV is a kind of race-specific double-strand closed-loop DNA virus with multiple subtypes.A large number of studies have proved that HPV is closely related to the formation,development and prognosis of various tumors.Since Syrjanen et al.first identified HPV in NSIP cases in 1987,the role of HPV infection in NSIP has been extensively studied.The literatures reported that the positive rate of HPV in NSIP patients was significantly higher than that in normal tissues.However,the reports on the relationship between HPV infection and its subtypes and the development and malignant transformation of NSIP were rare.Studies have reported that HPV infection could interact with host cells through a variety of signaling pathways,resulting in promoting cell proliferation and tumorigenesis.However,the mechanism by which HPV infection induces NSIP and malignant transformation is unclear.A large numbers of evidences indicated that HPV proto-oncogene E6/E7 could effectively activate phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Protein kinase B,PKB,Akt)/mammalian rapamycin target(mTOR)signaling pathway,and activate the MAPK signaling pathway,leading to mediation a variety of cellular and molecular functions by inducing regulation of its target genes expression,which plays a key role in tumorigenesis,development and prognosis.Among them,the activation of PI3K can promote the expression of EGFR and lead to the activation of MAPK/ERK signaling.Akt phosphorylation and its activation play an important role in cancer caused by HPV infection.mTOR is involved in various biological signaling pathways such as growth factor induction for the regulation of the energy and nutritional status.However,the role of the PI3K/Akt/mTOR signaling pathway in HPV-infected NSIP is unclear.Methods1.Study on the relationship between NSIP and HPV infection and its subtypes.The control(n=40)and NSIP patients(n=80)paraffin-embedded tissue samples of pathology department in Shandong Zibo Central Hospital from August 2005 to June 2017 were collected.Logistic regression was performed for the analysis of risk factors for NSIP.Detection of tissue HPV E6/E7 DNA positive rate was completed by specific PCR.Subtypes analysis of HPV was measured by genotypic flow-through hybridization through HybirMax method.H&E staining was performed to observe histopathology of NSIP.Immunohistochemistry was used to detect the expression of pS6 and pAkt protein in tissues.2.The role and mechanism of PI3K/Akt/mTOR signaling pathway in HPV-infected cells.Human normal nasopharyngeal epithelial cell line NP69-SV40T and human nasopharyngeal carcinoma cell line CNE1 were selected for cell experiments.HPV11 E6/E7 plasmid was constructed and transfected into NP69-SV40T and CNE1 cells.CCK-8 assay was performed for measurement of the plasmid transfection cell proliferation activity after 1-5 d with or without the administration of mTOR inhibitor rapamycin.Apoptosis was determined by Annexin V-FITC/PI staining using the flow cytometry.The mRNA expression of PI3K,mTOR,Akt,S6,Caspase-3,cyclin D1,p53 and p21 in NP69-SV40T and CNE1 cells was detected by qRT-PCR at 48 h after HPV11 E6/E7 plasmid transfection.And the PI3K,mTOR,Akt,S6,Caspase-3,cyclin D1,p53,p21 protein and its phosphorylation expression levels in HPV11 E6/E7 plasmid transfection cells were detected by Western Blot analysis.Results1.Study on the relationship between NSIP and HPV infection and its subtypes.The average age of 80 patients with NSIP was(50.9±14.3)years old(%male = 63.7%),and that in control group was(n=40,51.3±13.2)years old(%male = 60.0%).The HPV positive rate in the NSIP group(47/80,58.8%)was significantly higher than that in the control group(3/40,7.5%)(P<0.001).HPV infection is a major risk factor for NSIP(OR=33.138,P<0.001).The positive rate of HPV in patients with moderate to severe dysplasia(78.3%)was significantly higher than that in patients with NSIP alone or low dysplasia(49.1%,6/53)(P<0.05).The HPV positive rate was significantly higher in Krouse ?(75.0%)and Krouse IV(85.7%)stage than that in Krouse ?(37.5%,P<0.05).A total of 12 subtypes were detected in 47 HPV positive samples,and HPV11 was the most detected genotype(37.7%).The high-risk type(33.3%and 50.0%)and the mixed type(22.2%and 66.7%)in NSIP combined with SCC and moderate to severe NSIP were significantly higher than those in NSIP alone or low-grade dysplasia(36.0%and 0.0%)(P<0.05).The high-risk(55.6%and 33.3%)and the mixed type HPV infection rates(22.2%and 50.0%)in Krouse ? and ? patients and were significantly higher than patients with Krouse I(0.0%and 16.7%)(P<0.05).The positive rates of pAkt and pS6 protein expression in tissues in HPV+ NSIP patients(74.5%and 80.9%,respectively)were significantly higher than those in HPV'NSIP patients(51.6%and 58.1%,respectively)and the control group(26.3%and 21.1%,respectively)(P<0.05).2.The role and mechanism of PI3K/Akt/mTOR signaling pathway in HPV-infected cells.The cells proliferation activity of HPV11 E6/E7 plasmid transfection NP69-SV40T and CNE1 was significantly higher than that in the control group in a time dependent manner(1.25 to 1.62 folds,P<0.01),and mTOR inhibitors rapamycin could effectively reverse the increase of the cell proliferation activity induced by HPV 11 E6/E7.HPV11 E6/E7 plasmid transfection significantly inhibited cell apoptosis in a time dependent manner(reduce by 17%to 25.1%,P<0.05).There was no obvious effects of HPV E6/E7 plasmid transfection on the transcription and protein expression of Akt,mTOR,S6,MAPK and p21(P>0.05).However,a significant elevated of the p-PI3K(1.47/1.54 folds),pAkt(1.3/2.42 folds),pmTOR(1.6/2.2 folds),pS6(1.6/1.7 folds)and pMAPK(2.47/2.53 folds)protein expression was observed in HPV11 E6/E7 plasmid transfection cells.Furthermore,HPV11 E6/E7 plasmid transfection could significantly inhibit the transcription and protein expression of p53(reduce by 37.9%/59.3%)and Caspase-3(reduce by 54.3%/61.2%),but increase the expression of Cyclin D1(1.85/2.28 folds)(P<0.01).Conclusion1.The HPV infection rate in NSIP was 58.8%,HPV 11 is the main virus subtype of the HPV infection related NSIP.High-risk type HPV infection prescribe the severe of atypical hyperplasia,malignant transformation and higher clinical stage of NSIP.There was a close relationship of HPV infection and its subtype with the occurrence,development and malignant transformation of NSIP.2.HPV11 E6/E7 can activate the PI3K/Akt/mTOR signaling pathways,subsequently activate MAPK,leading to inhibiting the expression of p53 and Caspase-3,and promoting the Cyclin D1 expression.As a result,apoptosis of HPV infection associated cells in nasopharyngeal is inhibited and the proliferation of cells is increased. |
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