Study On The Molecular Mechanism Of Human Cytomegalovirus Influencing The Differentiation Of Stem Cells From Human Exfoliated Deciduous Teeth Into Neuronal Cells

Objective: Human cytomegalovirus（HCMV）is one of the most important congenital infectious pathogens that cause neonatal malformations and birth defects.Human cytomegalovirus innate infection causes a variety of diseases related to the neonatal nervous system by affecting the differentiation and development of embryonic nerve cells,including central nervous developmental disorders such as microcephaly,and peripheral nerve absence or injury diseases such as hirscholon and hearing loss.So far,the molecular mechanism of HCMV innate infection affecting neuronal differentiation has not been clarified.Previous studies on the molecular mechanism of HCMV congenital infection affecting neuronal cell differentiation were mainly carried out in neural progenitor/precursor cells（NPCs）and human induced pluripotent stem cells（i PSCs）.The experiment confirmed that HCMV could not infect i PSCs and neurons induced by i PSCs;NPCs are mainly isolated from fetal forebrain tissue,and their sustainable separation and acquisition are very limited.Therefore,establishing an in vitro neural cell differentiation model with sustainable acquisition and HCMV infection permissibility is a key step to study the molecular mechanism of neuronal cell differentiation affected by HCMV congenital infection.In this study,stem cells from human exfoliated deciduous teeth（SHED）were used to establish the research model of HCMV infection affecting neuronal cell differentiation;The SHEDs infection model was used to explore the key pathways and transcriptional regulators of HCMV infection affecting neuronal differentiation,and to clarify the molecular mechanism of HCMV infection affecting congenital infection and neuronal cell differentiation.Methods: 1.Establishment and identification of HCMV infection affecting neuronal differentiation model: HCMV clinical low-passage strain Han BAC infected SHEDs at different multiplicities of infection（MOI）.The culture supernatant and cell lysate were collected at different time points after infection,and the content of virus DNA in supernatant and cells was detected by real-time fluorescence quantitative PCR（q PCR）.The transcription and expression levels of HCMV key genes（UL123,UL32,UL55,UL44,UL83 and UL99）were detected by q PCR and Western blot.At the same time,the collected supernatant was inoculated into human embryonic lung fibroblasts（HELF）,and the expression of green fluorescent protein（GFP）was observed and counted to confirm the infectivity of offspring virus.2.Induced differentiation of SHEDs into neuronal cells in vitro: SHEDs and HCMV infected SHEDs was induced.Immunofluorescence assay（IFA）and Western blot were used to detect the markers related to neuronal differentiation（nestin,β3-tubulin,MAP2 and Neu N）.3.Transcriptome analysis of SHEDs in different treatment groups: the transcriptome highthroughput sequencing of SHEDs in different treatment groups was carried out by using Illumina Hiseq4000 sequencing platform,and the genome comparison and differential expression analysis were carried out by using HISAT2 and DESeq2 software.The differentially expressed genes were enriched and analyzed by Cluster Profiler R software combined with KEGG database.The data of different treatment groups were analyzed for biological function and activity by using GSEA online analysis tool（http://www.broadinstitute.org/gsea/index.jsp）.4.Detection of cell cholesterol: the lysed samples of SHEDs in different treatment groups were collected and Amplex^TMRed Cholesterol Assay Kit was used to detect the contents of total cholesterol,free cholesterol and cholesterol esters.5.Detection of transcription level of key enzymes in cholesterol biosynthesis pathway: SHEDs from different treatment groups were collected to extract total RNA.The transcription levels of key enzymes in cholesterol biosynthesis pathway（HMGCR,FDPS,HSD17B7,MSMO1,FDFT1,NADHL,SQLE,EBP,DHCR7,DHCR2,ACAT1 and ACAT2）were detected and compared by q PCR.6.Detection of sterol regulatory element binding protein（SREBP）: SREBP and SREBP cleavage activating protein（SCAP）in SHEDs of different treatment groups were detected and analyzed by immunofluorescence staining and Western blot.Results: 1.SHEDs are permissive to HCMV infection: SHEDs can be infected with HCMV at both high and low multiplicities of infection（MOI=0.1～10）;The key genes of HCMV at different stages were transcribed and translated in SHEDs;HCMV infected SHEDs can release mature and infectious HCMV virus particles.2.HCMV infection disorderss the differentiation of SHEDs into neuronal cells: there are significant differences in various neuronal markers in induced SHEDs under HCMV infection and non-infection conditions.Compared with non-infected cells,the expression levels of microtubule proteins including nestin,β3-tubulin and MAP2,decreased significantly,while the expression level of Neu N increased significantly.3.HCMV interferes with the neuronal differentiation of SHEDs by influencing the cholesterol synthesis pathway: transcriptome sequencing results suggested significant gene enrichment and activation in the cholesterol synthesis pathway of induced differentiated SHEDs,and this phenomenon disappears in HCMV infected cells.4.HCMV disordered the neuronal differentiation of SHEDs by inhibiting the synthesis of endogenous cholesterol.After HCMV infection,the levels of total cholesterol and free cholesterol in differentiated SHEDs were significantly lower than those in uninfected cells;inhibition of endogenous cholesterol synthesis in SHEDs influences the differentiation of SHEDs into neuronal cells,which is consistent with the effect of HCMV infection.5.HCMV infection decreased the transcription level of key enzymes in the cholesterol synthesis pathway of SHEDs: q PCR results showed that HCMV infection significantly reduced the transcription level of key enzymes in the cholesterol synthesis pathway,including HMGCR,FDPs,HSD17B7,MSMO1,FDFT1,NADHL,SQLE,DHCR7,DHCR24,ACAT1 and ACAT2.It is suggested that HCMV may inhibit endogenous cholesterol synthesis in SHEDs at the level of transcriptional regulation.6.HCMV infection changes the expression and cellular localization of SREBPs and SCAP,the transcription regulators related to cholesterol synthase.HCMV infection increased the expression of mature SREBP1 and clustered around the nucleus;HCMV infection significantly decreased mature SREBP2 at 7 days post infection and showed specific aggregation in the nucleus;HCMV infection increased the expression of SCAP and showed perinuclear aggregation.Conclusion: 1.SHEDs that can be induced into neuronal cells are permissive to HCMV infection and have no effect on the replication of HCMV.It can be utilized as an in vitro study model of HCMV infection influcencing neuronal differentiation.2.HCMV infection interferes with the differentiation of SHEDs into neuronal cells by inhibiting the synthesis of endogenous cholesterol.3.HCMV infection regulates the activity of cholesterol synthesis pathway by regulating the expression and cell localization of transcription regulators SREBPs and SCAP related to cholesterol synthesis.