The Study Of Transcription Level Of Human Endogenous Retrovirus In Patients With Hematological Malignancies

Background:Human Endogenous Retroviruses(HERVs)are remnants of ancient exogenous retrovirus infections in human ancestors that gradually evolved and integrated into the human genome,following Mendelian law and accounting for approximately 8-9% of the human genome.The structure of HERVs includes four coding regions(gag,pro,pol,env)and long terminal repeat(LTR)linked to the 3’ and 5’ ends.HERVs have multiple physiological functions and play important roles in embryonic development,gene transcription and expression.Over millions of years,most of the integrated HERVs have lost their coding abilities due to frameshift,mutations,or sequence deletions and remain silent.However,some HERVs genes still have complete open reading frame(ORF)that encode biologically active proteins or intact pre-viruses.Among them,HERV-K is the latest HERVs to be integrated into the human genome and has the strongest biological activity.In normal cells of healthy individuals,HERV-K is usually in a silenced state,with low transcription levels.However,in malignant tumors such as lung cancer,breast cancer,and melanoma,or other pathological conditions,abnormal transcription and expression of HERV-K m RNA or protein are observed.This phenomenon has also been observed in patients with hematologic malignancies such as leukemia and lymphoma.However,research on the transcription characteristics of HERV-K in hematologic malignancies is currently limited to scattered reports with few cases and inconsistent conclusions.Usually,only the transcription levels of a particular coding region of HERV-K are measured,and there has been no systematic investigation into the transcription levels of HERV-K’s different coding regions and their integration sites.As a result,the features of aberrant HERV-K transcription in hematologic malignancies cannot be fully characterized,and the potential contribution of abnormal HERV-K transcription to the occurrence and development of hematologic malignancies cannot be analyzed comprehensively.Objectives:This study focuses on leukemia,lymphoma,and other hematological malignancies.The transcription levels of HERV-K in peripheral blood mononuclear cells(PBMC)and different cell subsets of patients were detected to analyze the possible impact of HERV-K abnormal transcription on cell function.Further detection of HERV-K integration sites with transcriptional activity is conducted,and the differences in transcriptional levels of these integration sites are analyzed.Additionally,the biological functions of HERV-K integration sites with elevated transcriptional levels are preliminarily explored.Ultimately,the data will be comprehensively analyzed to gain a complete understanding of the characteristics of aberrant transcription of HERV-K in hematological malignancies,to analyze its relationship with tumor occurrence and development,and to provide a basis for a deeper understanding of the pathogenesis of hematological malignancies and the development of novel diagnostic and treatment strategies.Methods:(1)Recruitment of leukemia,lymphoma,multiple myeloma,and myelodysplastic syndrome patients and healthy controls will be conducted to collect peripheral blood samples and background information.The HERV-K gag,pol,and env transcription levels in PBMC will be detected using real-time quantitative PCR methods.The differences in HERV-K transcription levels among different populations will be analyzed,and the correlation between HERV-K transcription levels and patient pathological types or clinical stages will be evaluated.Furthermore,the HERV-K transcription and cytokine expression levels in different PBMC cell subsets will be determined.(2)The HERV-K integration sites with transcriptional activity in leukemia and lymphoma patients will be analyzed.The HERV-K integration sites will be sequenced using next-generation sequencing methods.Bioinformatics analysis tools,such as Hisat2,Samtools,Feature Counts,and Deseq2,will be used to perform differential expression analysis of the HERV-K integration sites.The significantly altered HERV-K integration sites and sequences with transcriptional activity in patients will be screened out,and analyzed the HERV-K integration sites for their biological function through GO analysis and KEGG enrichment.(3)A preliminary identification and analysis of the function of the long non-coding RNA(Lnc RNA)MIR548XHG,which is encoded by the 21q21.1 locus,will be conducted.The leukemia cell line KASUMI-1 will be used as a model,and small interfering RNA(si RNA)targeting the MIR548 XHG m RNA and MIR548 XHG over-expression plasmid will be transfected to explore the regulatory relationship between MIR548 XHG and its downstream mi RNA-548 X.Additionally,the impact of the different transcription and expression levels of MIR548 XHG and mi RNA-548 X on cell proliferation will be investigated.Results:(1)A total of 352 lymphoma patients,263 leukemia patients,20 multiple myeloma patients,and 14 myelodysplastic syndrome patients,as well as 64 healthy controls,were recruited based on diagnostic criteria.The transcription levels of HERV-K gag,pol,and env in PBMCs from all patient groups were significantly higher than those of the healthy controls(P<0.0001).HERV-K transcription levels were higher in patients with acute myeloid leukemia(AML)than in patients with acute lymphoblastic leukemia(ALL).In AML patients,there was a trend towards higher HERV-K transcription levels in patients with worse clinical stage and prognosis.Among the different PBMC cell subpopulations in leukemia and lymphoma patients,only B lymphocytes showed elevated HERV-K transcription levels(P<0.01),which were accompanied by decreased expression levels of B lymphocyte-related cytokines(P<0.01).There were no significant changes in HERV-K transcription levels(P>0.05)or related cytokine expression levels(P>0.05)in T lymphocytes and neutrophils.(2)A total of 82 HERV-K integration sites with transcriptional activity were identified in leukemia and lymphoma patients,including 23 gag,25 pol,and 34 env integration sites.Compared to healthy controls,23 HERV-K integration sites showed altered transcription levels in leukemia patients,with 20 sites displaying increased transcription levels and 3 sites showing decreased transcription levels.Similarly,20HERV-K integration sites exhibited altered transcription levels in lymphoma patients,with 12 sites displaying increased transcription levels and 8 sites showing decreased transcription levels.The up-regulated integration sites could encode Lnc RNAs such as ERVK-28,aspartic and cysteine proteases,while the downregulated integration sites could encode defensins,Lnc RNA 2614,and some integration sites had no annotation in the human genome.Three HERV-K sequences with up-regulated expression levels were found in leukemia and lymphoma patients,respectively.GO analysis and KEGG pathway enrichment analysis indicated that HERV-K is involved in various biological functions in leukemia and lymphoma patients,including regulation of interleukin-4secretion,negative regulation of immune response,and negative regulation of adaptive immunity.(3)In leukemia patients,the HERV-K integration site 21q21.1 encodes a Lnc RNA,MIR548 XHG.After transfection with si RNA targeting MIR548 XHG transcription,both MIR548 XHG transcription and mi RNA-548 X expression levels were decreased,and the proliferation ability of the leukemia cell line KASUMI-1 was also reduced(P<0.05).Conversely,after transfection with the overexpression plasmid of MIR548 XHG,both MIR548 XHG transcription and mi RNA-548 X expression levels were increased,and the proliferation ability of KASUMI-1 cells was enhanced(P<0.05).Conclusion:(1)The PBMCs of patients with various hematological malignancies generally exhibit elevated levels of HERV-K gag,pol,and env transcripts,which are associated with the clinical progression and prognosis of the disease.The increased HERV-K transcription levels in the PBMCs of leukemia and lymphoma patients are originated from B lymphocytes and are accompanied by a decrease in the expression levels of B lymphocyte-associated cytokines and changes in their functions.(2)Transcriptionally active HERV-K integration sites have been identified in patients with leukemia and lymphoma.While the transcription levels of HERV-K in PBMCs of these patients are significantly elevated,not all HERV-K integration sites show an increase in transcription levels.Some HERV-K integration sites exhibit a decrease in transcription levels.HERV-K integration sites in the peripheral blood of patients with leukemia and lymphoma,whose transcription levels have changed,may negatively regulate the host’s immune response,thereby inhibiting the host’s immune function.(3)Three HERV-K sequences with specific expression in leukemia and lymphoma patients have been identified and are expected to become diagnostic markers for leukemia and lymphoma based on HERV-K.(4)The HERV-K integration site with elevated transcription levels encodes a Lnc RNA,MIR548 XHG,which can positively regulate the expression of downstream mi RNA-548 X and thereby influence the proliferation of leukemia cells.