Minerva Mata-Rocha1, Ruth Monserrat Rodríguez-Hernández2, Pedro Chávez-Olmos3, Efraín Garrido3, Conrado Robles-Vázquez4, Sergio Aguilar-Ruiz4, Honorio Torres-Aguilar2, Carolina González-Torres5, Javier Gaytan-Cervantes5, Juan Manuel Mejía-Aranguré6, María de Los Angeles Romero-Tlalolini7. 1. CONACyT-Unidad de Investigación Médica en Epidemiologia Clínica, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, IMSS, Mexico City, Mexico. 2. Facultad de Ciencias Químicas, Universidad Autónoma Benito Juárez de Oaxaca, Oaxaca, Mexico. 3. Departamento de Genética y Biología Molecular, CINVESTAV-IPN, Ciudad de México, Mexico. 4. Facultad de Medicina y Cirugía, Universidad Autónoma Benito Juárez de Oaxaca, Oaxaca, Mexico. 5. Laboratorio de secuenciación, Division de Desarrollo de la Investigacion, Centro Médico Nacional Siglo XXI, IMSS, Ciudad de México, Mexico. 6. Coordinación de Investigacion en Salud, IMSS, Torre Academia Nacional de Medicina, Ciudad de México, Mexico. 7. CONACYT-UABJO, Facultad de Medicina y Cirugía, Oaxaca, Mexico. Electronic address: mdlaromerotl@conacyt.mx.
Abstract
INTRODUCTION: The main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODS: We evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTS: We detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSION: HPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell.
INTRODUCTION: The main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODS: We evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTS: We detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSION:HPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell.
Keywords:
DNA del VPH en exosomas; DNA del VPH episomal en exosomas; DNA-seq exosomal; Episomal HPV DNA in exosomes; Exosomal DNA-seq; Exosomal HPV DNA; Exosomas de exudado cervical; Exosomes of cervical exudate
Authors: Leandra B Jones; Sanjay Kumar; Aliyah J Curry; Jayde S Price; Alexandre Krendelchtchikov; Brennetta J Crenshaw; Courtnee' R Bell; Sparkle D Williams; Tambre A Tolliver; Sabita N Saldanha; Brian Sims; Qiana L Matthews Journal: Biomedicines Date: 2019-09-30
Authors: Víctor Acevedo-Sánchez; Ruth M Rodríguez-Hernández; Sergio R Aguilar-Ruíz; Honorio Torres-Aguilar; María de Los A Romero-Tlalolini Journal: Membranes (Basel) Date: 2021-06-20