Gene Polymorphisms And Regulatory Mechanisms Of Human Endogenous Retrovirus HERV-K(HML-2) Family Long Terminal Repeats

Human endogenous retroviruses(HERVs),which stem from exogenous retrovirus infection during the evolution of primates and subsequent integration into the human genome,account for approximately 8%.HERVs are involved in a variety of human physiological functions,and their abnormal expression is closely related to the occurrence and development of various diseases.Due to insertions and deletions,most of the HERVs have evolved into fragmentary remnants,with only the HERV-K family remaining relatively intact.As a result,this family has become a hot area for HERVs research.Based on phylogenetic analyses,the LTR sequences of HERV-K(HML-2)are classified into three subgroups: LTR5 A,LTR5B,and LTR5 Hs.The LTRs of HERV-K(HML-2)contain promoters,enhancers and transcription factor binding sites,which play roles in regulating HERV-K(HML-2)itself and adjacent host genes after binding to specific transcription factors.Currently,many studies have suggested that abnormal transcription of HERV-K(HML-2)is closely related to the occurrence of lung adenocarcinoma.However,there is still a lack of comprehensive understanding of the role and mechanism of HERV-K(HML-2)elements in the development of lung adenocarcinoma.In this paper,we studied the difference in transcriptional activity between LTR5 A,LTR5B and LTR5 Hs of HERV-K(HML-2)and explored the key transcription factor binding sites by luciferase reporting system.The specific contents of the study include: 1.A dual-luciferase reporter assay was carried out based on the representative sequences of three subtypes of HERV-K(HML-2)LTR.The results showed that in the HEK293 T cell line,LTR5 Hs transcriptional activity was about 1.5 and 5 times higher than that of LTR5 A and LTR5 B,respectively.In He La,Beas-2B,L02,A549,and Hep G2 cell lines,LTR5 Hs has the highest transcriptional activity,more than 5 times higher than LTR5 A and LTR5 B.2.LTR5 Hs was divided into four fragments to explore the reasons for the difference in transcriptional activity.The results showed that only the third fragment of LTR5Hs(-263 to 0 bp)significantly enhanced the transcriptional activity of LTR5 B by more than 3 times compared with other regions.3.The key sites on the third segment of LTR5 Hs were explored.The results showed that the TP53-1(GGGCTGG)and TP53-2(GGGCAGC)binding sites on LTR5Hs3-5B specifically decreased the promoter activity of LTR5 Hs.4.After interference with TP53 m RNA,the transcriptional activities of LTR5Hs3-5B and LTR5 Hs decreased significantly,which confirmed that p53 protein regulated the transcriptional activities of LTR5 Hs.5.The Ch IP-q PCR assay and CUT&Tag assay proved the direct binding of the p53 protein with LTR5 Hs sequences on-263 to 0 bp region.Subsequently,we used CRISPR-dCas9 technology to activate and interfere with the transcriptional activity of TP53-1 binding sites,and analyzed its effects on cell phenotypes and changes in gene expression levels.The specific contents of the study include: 1.A sg RNA targeting the TP53-1 binding site of LTR5 Hs sequence was designed and transferred into activation/interference cell lines.Three experimental groups and one control group were screened in each system.Colony formation experiments in the CRISPRa system demonstrated that the rate of colony formation was lower in all three experimental groups than in the control group.Colony formation experiments in the CRISPRi system showed that the rate of colony formation was greater in all three experimental groups than in the control group.The results showed that the enhancement of transcriptional activity of TP53-1 binding site on LTR5 Hs could reverse the phenotypic changes of lung adenocarcinoma cells and had the potential effect of inhibiting the occurrence and progression of lung adenocarcinoma.2.In order to further study the regulatory role of LTR5 Hs,we performed strandspecific transcriptomics sequencing of the activated and interfering cell lines,analyzed the genes that were commonly expressed differently in the three experimental groups of CRISPRa system or CRISPRi system.The results showed that most of these genes are involved in biological processes related to tumor malignant phenotypes,and involved in pathways closely related to tumor formation,growth and metastasis.3.Based on transcriptome analysis results,TCGA database,phenotypic experimental results,the distance between the differential genes and the LTR5 Hs element(less than 100 kb),the differential genes including RNF24,SUSD2,GPX2 in CRISPRa system and KCNK3,DOCK2,ATG9 B in CRISPRi system were selected to identified.The sg RNAs were designed and knocked out on the adjacent LTR5 Hs sequence,the results showed that the expression of SUSD2,KCNK3,and ATG9 B genes were significantly reduced after knocking out the adjacent LTR5 Hs.It suggests that the LTR5 Hs sequences were involved in the regulation of these genes.This study revealed the transcriptional activity and differences of the three subtypes of LTR elements of HERV-K(HML-2),and found that the TP53-1transcription factor binding site on LTR5 Hs has the effect of inhibiting the development of lung adenocarcinoma.This study provides novel strategies for the treatment of lung cancer and novel objectives for the exploration of related medications,which is of great importance.