Reaserch About Characteristics And Differently Expressed Genes Of Neuronal Cells Infected By Kaposi's Sarcoma-associated Herpesvirus

Objective: To detect whether Kaposi's Sarcoma-associated Herpesvirus(KSHV)can infect neuronal cells SH-SY5 Y in vitro or not.And detect the proliferation and migration characteristics of neuronal cells after infection.Screen the differential expressed genes from the infected and uninfected cells.Methods:(1)islk.219 cell was induced by doxycycline and sodium butyrate to release KSHV virus in the supernatant,and the KSHV virus was extracted by virus concentration method.The infected SH-SY5 Y cells were observed by fluorescence microscopy.(2)q RT-PCR was used to detect the expression levels of KSHV latent gene latent-associated nuclear antigen(LANA)and cleavage genes RTA,ORF26 and K8.1A.(3)The expression level of KSHV latent gene LANA and cleavage state genes RTA and K8.1A were detected by IHC.(4)The effects of KSHV on cell proliferation and migration were detected by MTT and Transwell experiments,and flow cytometry was used to detect the cell cycle of infected and uninfected SH-SY5 Y cells.Western Blot was used to detect the expression levels of CDK4,CDK6,Cyclin D1 and P27.In SH-SY5 Y cells,after transfection of LANA plasmid,the expression levels of CDK4,CDK6,Cyclin D1 and P27 were further compared.(5)Whole transcriptome sequencing was used to screen differential expressed genes after KSHV infection.The sequencing results were analyzed by bioinformatics techniques such as Gene Ontology and KEGG,and the Notch signaling pathway were verified.Results:(1)Fluorescence microscopy found green-fluorescent in neuronal cells SH-SY5 Y after KSHV infection which confirmed the infection.The cell morphology changed from spindle-shaped agglomeration to cell enlargement,and the morphology became round and scattered.(2)The m RNA and protein expression level of the latent gene LANA and the cleavage genes RTA,ORF26 and K8.1A of KSHV infection cells were significantly increased(P<0.05).(3)The cell proliferation ability after KSHV infection was improved,but the migration ability was down-regulated.Cell cycle results showed that KSHV infected SH-SY5 Y cells can promote the G0/G1 phase.The expression of CDK 4,CDK 6 and Cyclin D 1 were up-regulated,but the expression of P27 was reduced.After transfection of LANA plasmid,the expression level of Cyclin D 1 and CDK 4 were increased.(4)The transcriptome sequencing detected that 11258 genes were up-regulated and 1967 genes were down-regulated after KSHV infection,and the differential genes were enriched in the Notch signaling pathway.The expression levels of Notch 1,NICD protein and its downstream genes RBP-JK and Hes 1 were increased.Conclusion:(1)KSHV can successfully infect neuronal cell SH-SY5 Y in vitro and both latent and cleavagerelated genes can be expressed.(2)KSHV can promote cell proliferation,but reduces the ability of cell migration.(3)KSHV infection can up-regulated the expression level of Notch signaling pathway-related proteins,which may be related to improve cell proliferation.