| Objective: Acute myeloid leukemia(AML)is a kind of hematologic disease with malignant hematopoietic stem cell cloning.Its pathogenesis involves multiple factors and molecular mechanisms.The main pathological features of AML are myeloid cells uncontrolled proliferation,blocked apoptosis,and development arrest.The AML cells accumulate in blood and peripheral tissues,inhibit the proliferation of normal erythroid,granulocyte and megakaryotic cells,cause hematopoietic obstruction of the three lines,and invade other tissues and organs.Some studies have shown that histone epigenetic modification disorder is involved in the development of AML.Luk S-PV is one component of PV-leukocidin secreted by S.aureus.Our previous study found that luk S-PV can exert the anti-leukemia effect in vivo and in vitro through various molecular mechanisms.Besides,Luk S-PV can inhibit the progression of HCC by downregulating the expression of HDAC2,indicating that Luk S-PV can regulating histone epigenetic modification.We further investigate whether Luk S-PV can exert anti-leukemia activity through the epigenetic mechanism and the related molecular mechanism in the present study.Methods:(1)To study whether Luk S-PV has an anti-leukemia effect in vitro,the primary AML blasts were pretreated with different concentrations of Luk S-PV,and the apoptosis was detected by flow cytometry;To determine whether Luk S-PV has an anti-leukemia effect in vivo,the number of AML cells in peripheral blood and spleen was detected by flow cytometry.(2)HL60 cells were pretreated with PBS and Luk S-PV for 24 hours,RNA was extracted and sent to transcriptome sequencing to reveal the transcriptome changes;AML cells were treated with different concentrations of Luk S-PV or different times,and the levels of KMT5 A m RNA and protein were detected by q RT-PCR and Western blot.(3)We detect the expression of KMT5 A in the peripheral blood of normal people and AML patients,and to analyze the effect of KMT5 A expression on the survival of AML patients based on TCGA database;Furthermore,to determine the role of KMT5 A in AML cells,we construct KMT5 A knockdown and overexpression stable cell lines,and to detect the apoptosis and proliferation by flow cytometry.(4)To determine whether Luk S-PV mediates apoptosis and proliferation via KMT5 A,KMT5A KD and KMT5 A OE cells were pretreated with Luk S-PV.Then the apoptosis and proliferation were detected by flow cytometry,and related proteins were detected by Western blot.(5)Chromatin-immunoprecipitation(Ch IP)sequencing and Ch IP-PCR were performed to identify the transcriptional target genes regulated by the KMT5A/H4K20me1;AML cells were pretreated with different concentrations of Luk S-PV,and the expression of PIK3 CB was detected by q RT-PCR and Western blot;The KMT5 A overexpression cell line was pretreated with PIK3 CB inhibitor,and the apoptosis and proliferation were detected by flow cytometry.(6)To determine whether Luk S-PV mediated apoptosis and proliferation via KMT5A/H4K20me1-PIK3 CB pathway in AML cells,western blot was used to detect relative proteins in KMT5 A KD/OE cells and primary AML blasts,which pretreated with PBS and Luk S-PV.Results:(1)Luk S-PV induces apoptosis in a dose-dependent manner in primary AML blasts;The Luk S-PV treatment group’s spleen index was lower than the untreated group.Besides,FCM analysis showed that the percentage of AML cells(CD33+ cells)in peripheral blood and spleen was lower in the treatment group than in the untreated group.(2)RNA-seq results showed that significantly decreased the level of KMT5 A mRNA in HL60 cells pretreated with Luk S-PV;Meanwhile,q RT-PCR and Western blot results showed that Luk S-PV could downregulate KMT5 A in a concentration and time-dependent manner.(3)q RT-PCR and western blot revealed that KMT5 A was significantly upregulated in AML patients compared to the healthy controls,and associated with a poor prognosis.KMT5 A knockdown can dramatically inhibit the proliferation and induce apoptosis of AML cells,while overexpression of KMT5 A can significantly promote the proliferation of AML cells.(4)Luk S-PV can downregulate the level of H4K20me1 in a concentration and time-dependent manner;Overexpression of KMT5 A can resist the apoptosis and proliferation inhibition of AML cells induced by Luk S-PV,indicating that Luk S-PV can promote apoptosis and inhibit AML cells’ proliferation by downregulating KMT5A/H4K20me1.(5)The results of Ch IP-seq and Ch IP-PCR showed that the enrichment of H4K20me1 in the promoter region of PIK3 CB was significantly decreased after Luk S-PV pretreatment,and decreased the m RNA and protein of PIK3 CB in a concentration-dependent manner in HL60 cells,indicating that Luk S-PV inhibited the expression of PIK3 CB via reduce enrichment of H4K20me1 in promoter region by downregulating KMT5A/H4K20me1.Overexpression of PIK3 CB can resist the apoptosis induced by Luk S-PV.Pretreatment with PIK3 CB inhibitor can resist the proliferation promoting effect of KMT5 A overexpression.These results indicate that Luk S-PV-KMT5 A inhibits cell apoptosis and promotes cell proliferation through PIK3 CB.(6)The results of western blot showed that the levels of KMT5 A,H4K20me1,PIK3 CB,p-AKT and Bcl2 was decreased significantly,but the levels of FOXO1,Bak,P21 and P27 was increased in AML cells after Luk S-PV treatment.And this effect was inhibited in KMT5 A overexpress cells,indicating that Luk S-PV mediated AML cell apoptosis and proliferation via KMT5A/H4K20me1-PIK3CB-AKT signal pathway.Conclusion:(1)Luk S-PV induces cell apoptosis in vitro and inhibits cell invasion in vivo;(2)KMT5A is highly expressed in AML and is associated with poor prognosis;Inhibition of KMT5 A could induce apoptosis and inhibit proliferation in AML cells;(3)Luk S-PV exert anti-leukemia effect via KMT5A/H4K20me1-PIK3CB-AKT-FOXO1 signal pathway;(4)KMT5A may be a potential therapeutic target for AML and Luk S‐PV is a targeted drug candidate for the treatment of AML. |
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