The P15-piRNAs Induce DNA And Histones Methylation Modifications Of The P15^INK4b Gene In Leukemia Cells

Objective: To exploerthe regulation mechanismof DNA methylation modificationand/or heterochromatin formation of p15^INK4bgene mediated by the piRNAs which isone of non-coding RNAs in leukemia cells, may provide a new direction for studythepathological mechanisms of leukemia. Method: Firstly, the relative expressionlevel of the p15^INK4bgene in patients with acute leukemiahad been detected throughquantitative PCR and Western Blotting, the methylation levels of CpG islands inp15^INK4bgene promoter had been detected through gene methylation detectiontechnique, and the heterochromatin in p15^INK4bgene promoter region had beendetected through chromatin immunoprecipitation precipitation technology. Secondly,the relative expression level of p15-piRNAs had been detected through quantitativePCR, and the level of p15-piRNAs combining to Piwi subfamily protein AGO3hadbeen detected through co-immunoprecipitation, and identified piRNA-Piwi pathwaycomponents and their composition by immunoprecipitation. Finally, the U937cell linehad been induced to overexpress p15-piRNAs. Then the expression level of p15^INK4bgene, DNA methylation and heterochromatin had been detected. Results:（1） Theexpression level of p15^INK4bgene in acute lymphoblastic leukemia （ALL） patients wassignificantly lower than those in normal and acute myeloid leukemia （AML） patients（RQ=0.08±0.06, p=0.00<0.05）.The methylation levels of CpG islands in p15^INK4bgene promoter in ALL patients detected through methylation-specific PCR （MSP） was100%and those in AML patients was42.9%,which the difference was statisticallysignificant （P=0.016<0.05）. ALL the45CG sites of CpG islands in p15^INK4bgenepromoter had been modified with methylation in Molt-4cell line. However there hadno methylation in U937cell line. Histone H3of p15^INK4bgene promoter region hadbeen modified with monomethylation ang dimethylation in the ninth lysine residue inMolt-4cell line detected by chromatinimmunoprecipitation （ChIP） and qualitativePCR（P≤0.05）. But histone H3K9and H3K27of p15^INK4bgene promoter region had been not modified with methylation in U937cell line （P>0.05）.（2）Hsa_piR₀14637and hsa_piR₀11186differentially expressed in Molt-4and U937cell line,and the expression levels of the latter in Molt-4cell line were significantlyhigher than those in U937cell line （p=0.003<0.05）.The combination levels of the twopiRNAs with protein AGO3in Molt-4cell line were also significantly higher thanthose in U937cell line，especially hsa_piR₀11186molecules （p≤0.05）. There hadexisted the p15-piRNA-AGO3complexesin Molt-4and U937cell lines, whosecomponents were differences. In Molt-4cell line the p15-piRNA-AGO3complexesnot only had contained histone methylaseSuv39H1and EZH2, but also contained DNAmethylation transferase DNMT3a and of DNMT3b without DNMT1. However inU937cell line the p15-piRNA-AGO3complexeshad contained DNMT1withoutDNMT3a and of DNMT3b, and also contained Suv39H1and EZH2whose relativecontent were lower than those in Molt-4cell line.（3） U937cell line as amodel,hsa_piR₀14637and hsa_piR₀11186molecules were induced toover-expressiong through RNA interference, and their expression levels werenotdifferent （p=0.80>0.05）. There also existed different combination levels with AGO3protein （p=0.002<0.05）.The combination level with AGO3protein ofhsa_piR₀11186molecules is quite higher than other. After transfection the p15^INK4bgene expression was significantly down in transfected U937cells, especially inhsa_piR₀11186transfected group （p=0.00<0.05）.The DNAof p15^INK4bgene promoterregion had occurred methylated modification in the hsa_piR₀11186transfectedgroup, while the other groups were no methylation.Compared to the negative controlgroup, the histone H3in p15^INK4bgene promoter region also had occurredmethylatedmodification in the transfected cells, but their methylation level were differentaccording to piRNAs, especially in hsa_piR₀11186transfected group （p≤0.05）. Inhsa_piR₀11186transfected group,the histone H3in p15^INK4bgene promoter regionwas mainly modified with dimethylation in the twenty-seventh lysine residue（p≤0.05）.Conclusion: Theexpressiong ofp15-piRNAs in leukemia cells Molt-4and U937weredifferent. There had existed the p15-piRNA-AGO3complexes in Molt-4andU937cell lines which thecomponentscontaineddifferentepigeneticregulatory molecules. ThepiRNAsmediated methylation of DNA and/or histones H3inpromoter region throughtheirsequencecomplementary which guided thepiRNAs-Piwicomplexesspecificallyto combinep15^INK4bgenome, whichplaytheroleofgene expression regulation.