Mechanism Of Hepatitis B Virus Inhibiting Megakaryocyte Differentiation And Maturation At Different Developmental Stages Leading To Platelet Production Disorder

Objectives:Hepatitis B virus（HBV）infection can not only lead to chronic progressive diseases in liver,but also involve bone marrow and lead to the disorder of platelet production.In this study,the directional differentiation of hematopoietic stem cells into megakaryocytes was used as the research model,and HBV with different viral loads was added to explore the influence and molecular mechanism of HBV on megakaryocytes in different developmental stages,and provide a scientific basis for future clinical targeted therapy.Methods:Part I:The effect of HBV on megakaryocytes in different developmental stages1)Induce directed differentiation of hematopoietic stem cells into megakaryocytes in vitro:take fresh full-term healthy maternal umbilical cord blood,add magnetic beads to sort CD34⁺hematopoietic stem cells,and add megakaryocyte expansion factors（including:IL-6,SCF,IL-9,TPO）,directed differentiation to different developmental stages of megakaryocytes.Different stages of megakaryocytes were determined at different time points,and the surface antigen markers of cells in each stage:pluripotent hematopoietic stem cells are CD34^bright,immature megakaryocytes are CD34^modCD41^modCD42^dim,mature megakaryocytes are CD34^dimCD41^brightCD42^bright,and activated platelets are CD62p^bright.2)Build the HBV-infected megakaryocyte developmental model:extract the supernatant of Hep AD38 cells to prepare HBV virus stock solutions with different viral loads and co-culture with the hematopoietic stem cells extracted in the previous step.At different developmental stages of megakaryocytes,COBAS Taqman HBV DNA analyzer was used to detect the HBVDNA load in the cell supernatant,PCR was used to detect the expression of HBVccc DNA in megakaryocytes at different developmental stages,and HBs Ag in megakaryocytes was detected by immunofluorescence.The distribution of HBV-infected megakaryocytes confirmed that the model of megakaryocyte development was successfully established.3)Effects of HBV on apoptosis,differentiation and platelet function of megakaryocytes in different developmental stages:（1）At the stage of immature megakaryocytes,the proliferation of immature megakaryocytes was detected by CCK-8 method,the apoptosis of cells was detected by Annexin V/PI double staining method,and the proliferation and replication of cells were detected by cell cycle PI;（2）at the stage of mature megakaryocytes,flow cytometry was used to detect the expression of CD34,CD41,and CD42 on the cell surface to evaluate the differentiation and maturation of megakaryocytes;（3）at the stage of thrombopoiesis,the platelet-rich suspension was extracted by stepped centrifugation,and flow cytometry was used to detect the expression of the platelet function protein CD62p to assess the effect of HBV on platelet function.Part II:The molecular mechanism of HBV promoting apoptosis of immature megakaryocytes1)Virus source:The supernatant of Hep AD38 cells was extracted,filtered and precipitated to prepare an HBV virus stock solution(Hep AD38 is a stable cell line derived from Hep G2,which supports tetracycline-induced HBV replication^[1]);2)Source of immature megakaryocytes:The human megakaryoblastic leukemia cell line Meg-01 was used,and thrombopoietin（TPO）was added in vitro^[2],and cultured to the stage of immature megakaryocytes;3)The virus extracted from the above 1)was co-cultured with the cells derived from 2),and at the stage of immature megakaryocytes,the ultrastructure of the cells was observed by transmission electron microscopy,and the red fluorescence distribution of HBs Ag in the cells was detected by immunofluorescence technology.At different time points,COBAS Taqman HBV DNA analyzer was used to detect the load of HBV DNA in the cell supernatant,which confirmed that HBV infected immature megakaryocytes successful;4)The expression of megakaryocyte apoptosis-related factors Bcl2l2 and Bax were detected,and the molecular mechanism of HBV promoting the apoptosis of immature megakaryocytes was analyzed.Part III:The molecular mechanism of HBV inhibiting differentiation of mature megakaryocytes1)Effects of HBV on the polyploidy,cytoskeleton formation and different differentiation stages of mature megakaryocytes.（1）At the stage of mature megakaryocytes,the numbers of DNA ploidy were detected by flow cytometry to determine whether HBV had an inhibitory effect on the polyploid formation;（2）At the same time,immunofluorescence technique was used to detect the expression of cytoskeleton protein phalloidin in cells to evaluate the effect of HBV on cytoskeleton formation;（3）At the early and late stages of mature megakaryocytes,flow cytometry was used to detect the expressions of CD34 and CD42 on the cell surface to evaluate the differentiation and maturation of megakaryocytes;2)Screen the the key proteins that HBV inhibits the differentiation of mature megakaryocytes:At the mature megakaryocyte stage,the key proteins that HBV inhibits the differentiation of cells were screened by Labelfree quantitative proteomics combined with bioinformatics analysis,and the protein and transcription levels were analyzed by Western blot and RT-PCR.Verification;3)Expression of the related proteins in mature megakaryocytes after HBV up-regulating UBE4B.The relative quantitative expressions of p53,p-p53,ERK1/2 and p-ERK1/2 in mature megakaryocytes were detected by combining the protein characteristics of UBE4B and the analysis of regulatory factors during the differentiation of megakaryocytes.4)The molecular mechanism of HBV inhibiting differentiation of mature megakaryocytes by up-regulating UBE4B.After lentivirus transfected megakaryocytes to knock down UBE4B,the maturation of the cells at the above time points were detected to evaluate whether the inhibition of cell differentiation and maturation by HBV were relieved.The relative quantitative expressions of p53,p-p53,ERK1/2 and p-ERK1/2 were detected by Western blot to clarify the molecular mechanism of HBV on megakaryopoiesis.Results:Part I:1)HBV could promote apoptosis of immature megakaryocytes.The experiment was divided into three groups:control group,HBV low viral load infection group(HBV^lowgroup),and HBV high viral load infection group(HBV^high group).Cell proliferation was detected by CCK-8 assay,and the results indicated that there was no difference in the three groups.The cell cycle was detected by flow cytometry,and the results showed that there was no difference in the proportions of G0/G1%,S%,and G2/M%among the three groups.Apoptosis was detected by Annexin V/PI double staining at the same stage,and the results showed that the apoptosis rate（Q2 area+Q3 area）in the HBV^high group was higher than that in the control group,and there was no significant difference between the HBV^low group and the control group.2)HBV could inhibit differentiation of mature megakaryocytes.The results of flow cytometry showed that the expression of CD34 among the three groups was in the control group<HBV^low group<HBV^high group(the control group compared with the HBV^high group,P=0.0090),and there was no significant difference in CD41 among the three groups,CD42 among the three groups was in control group>HBV^low group>HBV^high group(the control group compared with the HBV^high group,P<0.0001).3)HBV could inhibit the activation function of platelets.Flow cytometry detected the expression of platelet activation protein CD62p in the platelet-rich suspensions of the three groups,and the results showed that the control group>HBV^low group>HBV^highgroup(the control group compared with the HBV^high group,P=0.0463).Part II:At the stage of immature megakaryocytes,RT-PCR was used to detect the expression of anti-apoptotic factor Bcl2l2 and pro-apoptotic factor Bax.The results showed that the relative expression of Bcl2l2 in the control group was higher than that in the HBV-infected group（P=0.0002）,and there was no difference in the expression of Bax.Compared with the two groups,the ratio of Bcl2l2/Bax in the control group was higher than that in the HBV-infected group（P=0.0054）.Part III:1)At the stage of mature megakaryocytes,（1）the DNA polyploidy of cells was detected by flow cytometry.The results showed that the proportion of cells with DNA ploidy≤2N in the control group was lower than that in the HBV-infected group（P=0.0238）,and the ratio of≥4N/≤2N was higher than that in the HBV-infected group（P=0.0484）.（2）The expression of cytoskeletal protein phalloidin was detected by immunofluorescence test at the same stage.The distribution and mean fluorescence value of phalloidin in the control group were higher than those in the HBV-infected group（P=0.0022）;（3）At the early stage of mature megakaryocytes,flow cytometry was used to detect the average fluorescence expression intensities of CD34 and CD42in the two groups.The results showed that the expression of CD34 in the control group was lower than that in the HBV-infected group（P=0.015）,and the expression of CD42in the control group was higher than that in the HBV-infected group.At the late stage of mature megakaryocytes,the above surface antigens were also detected,and the results showed that the differences between CD34（P=0.0003）and CD42（P<0.00001）were more obvious,and both had significant statistical significance;2)A total of 3643 proteins were identified in 6 groups of samples from HBV-infected mature megakaryocytes and the control group.Combined with the fold changes of the proteins and the results of the bioinformatics analysis,the difference of UBE4B in mature megakaryocytes before and after HBV infection was statistically significant;3)At the stage of mature megakaryocytes,the protein expressions of the HBV infected group and the control group were compared,and the results showed that the expressions of p53（P=0.031）and p-p53（P=0.0003）in the control group were higher than those in the HBV-infect infected group.The expressions of ERK1/2 and p-ERK1/2（P=0.0015）in the control group were lower than those in the HBV-infected group.Compared with the ratio of p-p53/p53 in the two groups,the phosphorylation level of p53 in the control group was higher than that in the HBV-infected group（P<0.00001）.The phosphorylation level of ERK1/2 in the control group was lower than that in the HBV-infected group（P=0.0009）;4)After knockdown of UBE4B gene in megakaryocytes,the experiment was divided into three groups:control group（NC group）,empty virus vector group（EV group）,and UBE4B gene knockdown group（sh RNA-UBE4B group）.At the early stage of mature megakaryocytes,there were no significant differences in the mean fluorescence expression intensities of CD34,CD41 and CD42 among the three groups.At the late stage of mature megakaryocytes,there were no differences in the expressions of CD34,CD41 and CD42 between the NC group and the EV group.In the sh RNA-UBE4B group,the expression of CD34 was lower than that of the NC group（P<0.00001）,and the expressions of CD41 and CD42 were higher than those of the NC group（P<0.00001）.At the early stage of mature megakaryocytes,the expressions of the related proteins among the three groups were compared.The results indicated that the expressions of p-p53 and p53（P=0.0032,P=0.0041）,p-ERK1/2 and ERK1/2（P=0.0569,P=0.0020）in the NC group were lower than those in the sh RNA-UBE4B group.There was no significant difference of p-p53/p53 among the three groups.However,the ratio of p-ERK1/2 to ERK1/2 in NC group and EV group was higher than that in sh RNA-UBE4B group（P=0.0216）.And there was no statistical difference between the EV group and the NC group;at the late stage of mature megakaryocytes,the protein expressions among the three groups:p-p53 and p53 were lower in the NC group than sh RNA-UBE4B group（P=0.0002,P<0.0001）.The expression of p-p53/p53 was also lower（P=0.0168）.The expressions of p-ERK1/2 and ERK1/2 were both higher（P=0.0028,P=0.0020）,and the ratio of p-ERK1/2 to ERK1/2 was also higher（P=0.0017）,and the differences were more significant.There was no statistical difference between the EV group and the NC groupConclusions:1)HBV can promote the apoptosis of immature megakaryocytes,inhibit the differentiation of mature megakaryocytes,leading to the disorder of platelet production;2)HBV can inhibit platelet activation,leading to abnormal platelet function;3)HBV promotes apoptosis of immature megakaryocytes by down-regulating Bcl2l2/Bax;4)HBV inhibits the polyploidy and skeleton formation of mature megakaryocytes,and has a stronger inhibitory effect on the late stage of differentiation,resulting in the disorder of platelet production;5)HBV inhibits cell differentiation by up-regulating the expression of UBE4B protein in mature megakaryocytes and inhibiting the expression and phosphorylation of p53 in cells;6)HBV inhibits cell differentiation by up-regulating the expression of UBE4B protein in mature megakaryocytes and increasing the expression and phosphorylation of ERK1/2 in cells;In conclusion,HBV can promote the apoptosis of immature megakaryocytes by down-regulating Bcl2l2/Bax,and inhibit the differentiation of mature megakaryocytes by up-regulating the expression of UBE4B protein,inhibiting the expression and phosphorylation of p53,and increasing the expression and phosphorylation of ERK1/2,leading to the disruption of platelet production.