Study On Telomere-related Functions And Mechanisms Of NFRKB

Objective:Identifying new telomere-associated proteins and exploring novel regulatory mechanisms for telomeres.We found that NFRKB(Nuclear Factor Related to Kappa B Binding Protein)is a new telomere-associated protein,and preliminarily explored its molecular functions involved in telomere regulation and explored its expression and potential function in cancer.Methods:1.Identification of NFRKB as a telomere-associated protein: Using bioinformatics method,we analyzed Ch IP-Seq data of more than 200 nuclear proteins,according to the relative content of telomere DNA in the sequence,the ability of these proteins to bind telomeres is judged;The association was validated by immunofluorescence fluorescence in situ hybridization(IF-FISH)and DNA pull down.2.Exploring the domains of NFRKB responsible for telomere binding: Construct a series of NFRKB domain mutation recombinant expression plasmids.Exogenous expression of NFRKB domain mutant proteins then related domains of NFRKB and telomere were found by IFFISH experiments.3.Exploring the molecular functions associated with the telomeres:To speculate whether the NFRKB is involved in telomere protection,the changes of the co-localization between the ?-H2AX(the injury markers of DNA)and telomere DNA before and after knocking were observed by IF-FISH.4.Exploring cancer-related functions of NFRKB: WB and q RT-PCR were used to detect the expression of target genes in different cell lines and tissues,and the relationship between the NFRKB expression level and the survival time of hepatocellular carcinoma patients was evaluated by global survival analysis.Immunohistochemistry(IHC)was used to detect the distribution of NFRKB in liver cancer tissues.Results:1.NFRKB antibody can immunoprecipitated(IP)a large portion of telomere tandem repeats,suggesting that NFRKB is a potential telomereassociated protein.2.The co-localization of endogenous and exogenous NFRKB with telomeres was observed in U2 OS cells(ALT cells),DNA pull down results suggested that NFRKB directly bind to telomeres,which further validate the hypothesis that NFRKB is a telomere associated protein.3.IF-FISH showed that co-localization of NFRKB with telomeres decreased when one of the N-terminal and middle regions were removed,suggesting that these regions may be responsible for the NFRKB binding to telomeric DNA.4.NFRKB is involved in telomere protection in ALT cells.Knockdown NFRKB significantly increased the proportion of dysfunctional telomeres in U2 OS cell compared with the control group,indicating that NFRKB may have the function to protect telomere DNA from abnormal DNA damage repair process.5.NFRKB may associate with occurrence and development of Hepatocellular carcinoma.NFRKB is expressed in most human cell lines and was relative highly expressed in normal mouse liver.It was found that the abundance of NFRKB in HCC cancer tissue was significantly different from related paracancerous tissue.Survival analyses suggested that NFRKB is a potential Hepatocellular carcinoma-related gene.Conclusion:1.NFRKB is a novel telomere-associated protein involved in telomere protection in ALT cells.2.NFRKB may be related to the occurrence and development of Hepatocellular carcinoma.