The Mechanism Of Long Non-coding RNA Promoting Clonal Proliferation In Paroxysmal Nocturnal Hemoglobinuria

Objective:The long chain non-coding RNA(LncRNA)and mRNA in CD59~-and CD59~+granulocytes and monocytes cells of PNH patients were detected by RNA sequencing,and were screened and analyzed,so as to preliminarily explore the possible mechanism of the proliferation of PNH clones.In order to investigate the relationship between immune status and paroxysmal nocturnal hemoglobinuria(PNH)clonal evolution,the clinical data of 102 severe aplastic anemia(SAA)patients who received anti-human thymocyte globulin(ATG)treatment were collected and retrospectively analyzed.Methods:1.CD59~-and CD59~+granulocytes and monocytes cells from 5 PNH patients were sorted and LncRNAs and mRNAs were detected by RNA sequencing.Function clustering analysis and KEGG signal pathway enrichment analysis of mRNA were analyzed,meanwhile their expression level rate and function were analyzed.The LncRNAs were screened by co-expression.Then the expressions of differential LncRNAs and mRNAs were verified by q RT-PCR in 30 PNH patients,and the correlation were analyzed with clinical indexes,including hemoglobin(Hb),white blood cells(WBC),platelet(PLT),reticulocyte(Ret),lactate dehydrogenase(LDH),total bilirubin(TBIL),free hemoglobin,haptoglobin and PNH clone.2.The Cas9 and sg RNA overexpressed lentivirus were constructed to infect THP-1 cells,and then the GFP(Cas9 vector)and m Cherry(sg RNA vector)positive cells were screened by flow cytometry.The monoclonal cells were selected and amplified after identification,and finally the PIGA gene knock-down monoclonal cell line were obtained,which was verified by Flow cytometry(FLAER and CD59)and Snapgene sequencing validation.Lentivirus transfection was used to knock down LncRNAFAM157C,then detected transfection rate,knock down rate,cell proliferation and apoptosis.3.The clinical data of 102 SAA patients who received ATG plus Cs A from January 2006 to January 2016 were collected and retrospectively analyzed.The evolution characteristics and appearances of PNH clones were summarized.The remission rate(CR+PR%),remission time,hematopoietic and immune status(CD3~+CD4~+/CD3~+,CD3~+CD8~+/CD3~+,CD3~+CD4~+/CD3~+CD8~+,Th1/Th2,m DC/p DC,CD8~+HLADR~+/CD8~+)of every groups at different time points(0,3,6,12,24,36months)were compared.Malignant clones were also observed in the follow-up.Results:1.LncRNA and mRNA detection of PNH clone cells(1)LncRNAs and mRNAs gene sequencing was performed in 5 PNH patients.Transcription analysis revealed742 up-regulated LncRNAs and 1376 down-regulated LncRNAs,3276 up-regulated mRNAs and 213 down-regulated mRNAs in CD59~-granulated and monocytes cells,compared with CD59~+cells(2)The 173 upregulated mRNAs which FPKM>5 and over 3 patients were screened.Then we focused on 30 mRNAs(CXCR2,CXCL8,MCL1,CXCL1,LYN,PTGS2,etc)related to proliferation,apoptosis and thrombosis,etc.The 7 upregulated LncRNAs(MALAT1,RP13-20l14.10,RP11-22N19.2,LUCAT1,FAM157C,CTD-2530H12.2,RP4-669L17.10)which FPKM>5 and over 3 patients were screened by co-expression method.(3)By KEGG pathway enrichment analysis,several pathways were screened,including cell proliferation,autophagy,endocytosis,platelet activation etc.Cell proliferation pathway include TNF,NF-?B,PI3K-AKT signaling pathway and so on.We focused on the proliferation-related NF-?B pathway.The mRNAs which FPKM>10 and over 3 patients were selected to search out the upstream regulation LncRNAs.A total of 8 mRNAs(TAB2,TLR4,LYN,CFLAR,TNFAIP3,PTGS2,TRIM25,CXCL8)and 6 LncRNAs(MALAT1,LINC01002,FAM157C,CTD-2530H12.2,XLOC-064331,XLOC-106677)were verifed.Only MALAT1,LINC01002,and FAM157C were designed with appropriate primers.(4)The expressions of LncRNAs and mRNAs were detected by q RT-PCR in 30PNH patients.As the results of q RT-PCR showed,LncRNA MALAT1(3.070±2.503)and FAM157C(11.530±6.628)expressions in CD59~-granulated and monocytes cells were consistently higher than those in CD59~+granulated and monocytes cells(1.281±1.246,5.482±6.785)(p=0.0004,p=0.0055)from 30 PNH patients.LncRNA LINC01002 expression in CD59~-granulated and monocytes cells(6.139±11.15)was no significant difference with CD59~+granulated and monocytes cells(5.602±10.180)(p=0.8690).While there were no significant differences in mRNAs.(5)The correlation between the expression of LncRNA MALAT1 and FAM157C and the clinical indexes were analyzed.The high expression of MALAT1 and FAM157C were positively correlated with LDH level(r=0.6244,p=0.0307;r=0.4120,p=0.0407)and CD59~-granulated and monocytes cells ratio(r=0.5188,p=0.0049;r=0.4393,p=0.0280).(6)The expression of mRNA CXCR2 which was coexpression with LncRNA MALAT1 was consistently higher in CD59~-granulated and monocytes cells than that in CD59~+granulated and monocytes cells(3.230±2.336 vs 1.049±1.307,p=0.0008)from 30 PNH patients.The high expression of CXCR2 was positively correlated with LDH level(r=0.4941,p=0.0166)and CD59~-granulated and monocytes cells ratio(r=0.4447,p=0.0335).2.The function detection of the gene FAM157C:(1)The PIGA knock out THP-1cells was successfully constructed,and FAM157C was knocked down by lentivirus with a transfection rate of 70%and a knock-down rate of 90%.(2)The cell proliferation assay showed that the cell viability(%)of the control group,empty virus group and FAM157C knock-down group were(100±0),(95.20±3.178)and(91.93±5.423)after 24h transfection respectively.There was no statistical difference between the three groups(p=0.1807).The cell viability of the control group,empty virus group and FAM157C knock-down group were(100±0),(93.75±5.995)and(77.49±6.597)after 48h transfection respectively.The cell viability of the FAM157C knock-down group was significant lower than the control group and empty virus group(p=0.0275,0.0388).There was no statistical difference between the empty virus group and control group(p=0.1630).The cell viability of the control group,empty virus group and FAM157C knock-down group were(100±0),(92.795±5.802),and(60.47±2.059)after 72h transfection respectively.The cell viability of the FAM157C knock-down group was significant lower than the control group and empty virus group(p=0.0009,0.0052).There was no statistical difference between the empty virus group and control group(p=0.1420).(3)The cell apoptosis experiment showed that apoptosis rate increased after transfection of lentivirus FAM157C.The apoptosis rate of control group,empty virus group and FAM157C knock-down group were(2.483±0.3083)%,(2.926±0.5517)%,and(6.256±0.5453)%after 24h transfection respectively.The apoptosis rate of the FAM157C knock-down group was significant higher than the control group and empty virus group(p=0.0138,0.0066).There was no statistical difference between empty virus group and control group(p=0.0899).The apoptosis rate of control group,empty virus group and FAM157C knock-down group were(5.593±0.6400)%,(6.723±0.3256)%,and(11.30±1.075)%after 48h transfection respectively.The apoptosis rate of the FAM157C knock-down group was significant higher than the control group and empty virus group(p=0.0137,0.0217).There was no statistical difference between empty virus group and control group(p=0.0757).The apoptosis rate of control group,empty virus group and FAM157C knock-down group were(9.797±0.3235)%,(10.21±0.3005)%,and(18.81±0.5363)%after 72h transfection respectively.The apoptosis rate of the FAM157C knock-down group was significant higher than the control group and empty virus group(p=0.0012,0.00087).There was no statistical difference between empty virus group and control group(p=0.0633).(4)The results of cell cycle test showed that the G0/G1 phase of the control group,empty virus group and FAM157C knockdown group were(62.98±1.513)%(65.95±1.174)%and(70.00±0.2404)%,S phase were(3.825±0.7849)%,(5.920±0.9192)%and(13.47±1.039)%,G2 phase were(32.81±1.612)%(27.47±1.160)%and(16.54±0.7990)%after transfection of lentivirus FAM157C.There were statistical differences between the three groups(p=0.0269,0.0198,0.0145).Comparison among groups:there were statistical difference in G0/G1 phase between FAM157C knockdown group and empty virus group,FAM157C knockdown group and control group(p=0.0220,<0.0001),There was no statistical difference between empty virus group and control group(p=0.0596).There were statistical difference in S phase between FAM157C knockdown group and empty virus group,FAM157C knockdown group and control group(p=0.0023,0.0059),There was no statistical difference between empty virus group and control group(p=0.1417).There were statistical difference in G2 phase between FAM157C knockdown group and empty virus group,FAM157C knockdown group and control group(p=0.0053,0.0018),There was no statistical difference between empty virus group and control group(p=0.0697).After FAM157C knockdown,the proportion of cells in G0/G1phase and S phase increased,while the proportion of cells in G2 phase decreased,and the cells were blocked in G0/G1 phase and S phase.3.Summary of clinical data of 102 SAA patients:(1)A total of 32 patients(31%)had PNH clones,in which the median PNH clones size was 10.57%(5.22%-42.38%).Among them,22 cases with PNH clones appeared before treatment,and the median PNH clones size was 10.35%(6.6%-28.60%).18 cases with PNH clones appeared after treatment,among then 4 cases with PNH clones disappeared after treatment.PNH clones appeared in 10 cases after ATG,and the median PNH clones size was 13.55%(5.22%-42.38%),the median appearance time of PNH clones was 15 months after ATG.They were divided into three groups:the group with PNH clones appeared before treatment,the group with PNH clones appeared after treatment,and the group without PNH clones.The three groups before treatment showed no significant statistical significance in white blood cells(WBC,×10~9/L),neutrophils(N%),hemoglobin(Hb,g/L),platelets(PLT,×10~9/L),Ret%,LDH(U/L)and immune indexes(p>0.05).(2)Clinical efficacy comparison:The remission rates of the three groups were statistically significant(68%,90%,56%,p=0.0175),the remission rate of the group with PNH clones appeared after treatment was significantly higher than the group without PNH clones(p=0.0031),but the remission time among these groups were not statistically significant(p=0.1728).Analysing response rates at different points,the response rate to IST at 12 months were statistically significant among these groups(p=0.0349),the response rate of the groups with PNH clones appeared before and after treatment(68%,70%)were significantly higher than the group without PNH clones(40%)(p=0.0287,0.0458).(3)Recovery of hematopoietic function:At 6 months after ATG,the neutrophil percentage(N%)in three groups were statistically significant(p=0.0067),the N%of the group with PNH clones appeared after treatment and the group without PNH clones were higher than the group with PNH clones appeared before treatment.At 12 months after ATG,the Hb in three groups were statistically significant(p=0.0484),the Hb of the group with PNH clones appeared before treatment was obviously higher than the group without PNH clones.The Ret%in three groups were statistically significant(p=0.0044),the Ret%of the group with PNH clones appeared after treatment was obviously higher than the rest two groups.(4)Recovery of immune state:At 6 months after ATG,the proportion of CD8~+HLA-DR~+/CD8~+in three groups were statistically significant(22.26±4.993,17.04±6.447,27.41±5.446,p=0.0027).The proportion of CD8~+HLA-DR~+/CD8~+in group with PNH clones appeared after treatment was significantly lower than that in group without PNH clones(p?0.01).At 12 months after ATG,the ratio of Th1/Th2 in three groups were statistically significant(2.070±1.361,1.758±1.598,3.210±1.566,p=0.0266),the ratio of Th1/Th2 in group with PNH clones appeared before and after treatment were significantly lower than that in group without PNH clones(p?0.05).The proportion of CD8~+HLA-DR~+/CD8~+in three groups were statistically significant(22.88±17.50,13.72±4.928,29.93±7.335,p=0.0377),the proportion of CD8~+HLA-DR~+/CD8~+in group with PNH clones appeared before treatment was obviously lower than that in group without PNH clones(p?0.05).(5)All 6 patients had monosomy 7 clonal evolutions,and were diagnosed myelodysplastic syndromes(MDS).Among them,2 patients from the group with PNH clones appeared before treatment,2 patients from the group with PNH clones appeared after treatment,and 2patients from the group without PNH clones.Conclusion:1.The LncRNAs and mRNAs were detected in CD59~-and CD59~+granulated and monocytes cells in PNH patients by gene sequencing.LncRNA MALAT1,LINC01002,FAM157C,CTD-2530H12.2,XLOC-064331,XLOC-106677 were screened which were associated with proliferation,apoptosis and thrombosis,indicating that LncRNAs may play an important role in PNH.2.Through KEGG pathway analysis,the mRNAs and LncRNAs in the proliferation-related NF-?B pathway were verified and analyzed.LncRNA-MALAT1and FAM157C were found to be significantly increased in PNH clone cells,and positively correlated with CD59~-granulated and monocytes cells ratio and LDH level,which indicating that MALAT1 and FAM157C may play an important role in PNH clone.3.The expression of CXCR2 mRNA which was co-exprssion with LncRNA MALAT1 was consistently higher in PNH clone.The high expression of CXCR2 was positively correlated with LDH level and CD59~-granulated and monocytes cells ratio.It is suggested that MALAT1 maybe play a role in PNH proliferation by regulating CXCR2.4.After knockdown of FAM157C gene,it was found that the cells were blocked in G0/G1 phase and S phase,the apoptosis rate increased,the proliferation ability decreased.It suggested that FAM157C gene promote PNH clone proliferation.5.In SAA patients with PNH clones,the cytotoxic T cell function and Th1 cell number recovered more quickly and had better response to IST.During the follow-up,a small number of SAA patients with or without PNH clones developed MDS malignant clones.