MALAT1/miR-582-5p/GALNT1/MUC1 Axis Modulates Progression Of Leukemia Stem Cells By Regulating JAK2/STAT3 Cascade

Acute myeloid leukemia(AML)is a malignant disease of the blood system that seriously threatens human life and health.Its leukemia cells are usually derived from stem cells or progenitor cells,and are the most common type of leukemia in adults.At present,although some progress has been made in the treatment of acute myeloid leukemia,its recurrence rate is still high.Glycans is closely related to the occurrence and development of tumor.This study found that N-acetylgalactosaminyltransferase 1(GALNT1)was highly expressed in leukemia stem cells and affected the O-glycosylation modification process of transmembrane protein MUC1.Metastasis-associated lung adenocarcinoma transcript 1(MALAT1),as a long-chain non-coding RNA,indirectly regulated the expression of GALNT1 and affected the biological behavior of leukemia stem cells through sponge miR-582-5p by the mechanism of competing endogenous RNA(ce RNA).JAK2/STAT3 is a key carcinogenic signal pathway,and its abnormality will affect the occurrence and development of tumor.And STAT3 acted as a transcription factor to mediate the transcription of MALAT1 and MUC1.Objective:To explore the molecular mechanism underlying the activation of JAK2/STAT3 pathway by MALAT1/miR-582-5p/GALNT1/MUC1 crosstalk,and to provide potential biomarkers for the diagnosis and treatment of leukemia.Method:1.Leukemia stem cells(CD34+CD38-AML)were extracted from acute myeloid leukemia cells KG-1a and MOLM13 by using the CD34+CD38-cell isolation kit,and the surface markers of the extracted leukemic stem cells were detected by flow cytometry.The expression of GALNT gene family in leukemic stem cells(LSCs)and non-LSCs was detected by real-time quantitative polymerase chain reaction(q RT-PCR),and GALNT1 was taken as the research object.2.The LSCs-KG-1a and LSCs-MOLM13 cell lines that downregulated the expression of GALNT1 were constructed,which were verified by q RT-PCR and Western Blot.The O-glycosylation level measured by VVA in LSCs-KG-1a and LSCs-MOLM13 after down-regulating GALNT1 was analyzed by flow cytometry.The effects of down-regulation of GALNT1 on the biological behavior of leukemic stem cells were verified in vitro and in vivo.In vitro experiment:(1)Analyzed the proliferation ability of cells through Cell Counting Kit-8(CCK-8),methylcellulose colony formation assay and immunofluorescence;(2)Detected the apoptosis level of cells through flow cytometry(Annexin-V/PI staining)and Western Blot experiment.In vivo experiment: The xenografted tumor model was used to analyze the tumorigenicity of nude mice after down-regulating GALNT1,and the levels of GALNT1 and Ki67 in tumor were further detected by immunohistochemistry staining.3.Through the prediction of micro RNAs that can participate in the regulation of GALNT1 through the Bioinformatics prediction website,the obtained data set was screened and miR-582-5p was determined as the research object.The potential sites were verified by dual-luciferase reporter gene experiment,q RT-PCR and Western Blot test.Continued to predict the Lnc RNA that can combine with miR-582-5p through the Bioinformatics prediction website,and found that there was a great possibility of combining MALAT1 with miR-582-5p.The potential sites were further verified by dual-luciferase reporter gene experiment and q RT-PCR experiment.Co-regulated the expression level of MALAT1 and miR-582-5p in LSCs,and detected the change of GALNT1 expression level in LSCs after regulating MALAT1 and miR-582-5p by q RT-PCR and Western Blot.4.The O-glycosylation level of MUC1 was detected by lectin pull-down test and Western Blot test.The Western Blot experiments were used to analyze the effects on JAK2/STAT3 pathway protein expression levels by regulating GALNT1,MUC1 respectively and coregulating MALAT1 and miR-582-5p.5.The possible binding sites between STAT3 and the promoters of MALAT1 and MUC1 were predicted through the Bioinformatics prediction website,and selected the experiments including chromatin immunoprecipitation assay(Ch IP),q RT-PCR,and Western Blot to verified further.Result:1.Leukemia stem cells were successfully isolated and extracted.Compared with non-LSCs,GALNT1 showed a higher expression level in LSCs-KG-1a and LSCs-MOLM13.2.After down-regulating GALNT1 in LSCs-KG-1a and LSCs-MOLM13,the O-glycosylation level of LSCs decreased significantly.In vitro experiments confirmed that their proliferation ability decreased,the apoptosis rate increased and the expression level of apoptotic protein increased.In vivo experiments showed that after knocking down GALNT1,the tumorigenic ability in nude mice was reduced,the levels of GALNT1 and Ki67 showed relatively low.3.The expression levels of miR-582-5p in LSCs-KG-1a and LSCs-MOLM13 were lower than that of in non-LSCs.miR-582-5p can bind to GALNT1 was confirmed by dual-luciferase reporter gene experiment,q RT-PCR and Western Blot.Meanwhile,Lnc RNA MALAT1 can bind to miR-582-5p was confirmed by dual-luciferase reporter gene experiment and q RT-PCR either.Mi R-582-5p inhibitor could partially reverse the inhibitory effect of si-MALAT1 on GALNT1 when si-MALAT1,si-NC,miR-582-5p inhibitor as well as inhibitor-NC were cotransfected to LSCs-KG-1a and LSCs-MOLM13.4.Both the lectin pull-down assay and the Western Blot assay results showed that knockdown of GALNT1 reduced the content of MUC1 bound to VVA and that the O-glycosylation also changed.Blocking MUC1 and VVA or knocking down GALNT1 would inhibit JAK2/STAT3 pathway.In addition,co-transfection of miR-582-5p inhibitor and si-MALAT1 showed that si-MALAT1 could partially alleviate the promotion of miR-582-5p inhibitor on the phosphorylation of LSCs.5.The Ch IP experiment verified that STAT3 could combine with MALAT1 and MUC1 promoters.The results of q RT-PCR and Western Blot showed that STAT3 could effectively mediate the transcription of MALAT1 and MUC1 in LSCs.Conclusion:1.GALNT1 was highly expressed in LSCs-KG-1a and LSCs-MOLM13.GALNT1 participated in regulating the proliferation,apoptosis and subcutaneous tumorigenesis of LSCs-KG-1a and LSCs-MOLM13.2.MALAT1 bound competitively to miR-582-5p and regulated the expression level of GALNT1.3.MALAT1/miR-582-5p/GALNT1 crosstalk mediated O-glycosylation of MUC1 and triggered JAK2/STAT3 pathway.4.STAT3 acted as a transcription factor of MALAT1 and MUC1,and regulated their expression.