Function And Mechanism Of MYCT1 In Diffuse Large B-cell Lymphoma

Introduction: Lymphoma is a malignant tumor caused by clonal proliferation of lymphoid derived cells.According to histopathological classification,lymphoma is divided into two types,Hodgkin Lymphoma(HL)and Non-Hodgkin lymphoma(NHL).Diffuse large B-cell lymphoma(DLBCL)is the most common subtype of NHL.DLBCL is invasive and often infiltrates bone marrow,indicating that it has a high degree of malignancy,rapid progress and poor prognosis.In recent years,the survival rate of the patients has risen greatly,but 40% of the patients will deteriorate to recurrent and refractory DLBCL(R/R),leading to the mortality rate up to 23.3%,seriously threatening human’s health.DLBCL involves a large number of gene mutations,copy number variations and chromosomal karyotype abnormalities.Study on the mechanism of MYCT1 in the occurrence and development of DLBCL at the levels of cytogenetics and molecular genetics is helpful to find molecular targets for the diagnosis,treatment and prognostic prediction of DLBCL.Chromosomal instability(CIN)is an important trait of tumor cells.It is characterized by abnormal number and structure.CIN is commonly found in a variety of solid tumors and malignant hematological diseases.Studies have shown that specific marker chromosomes have become pivotal targets for the diagnosis,treatment and prognositic prediction of chronic myeloid leukemia and other tumors.MYCT1(MYC Target 1),located on chromosome 6q25,was first discovered and cloned by our research group in laryngeal cancer,which was once named C-Myc target from laryngeal cancer cell(MTLC).Studies have shown that MYCT1 plays different roles in different tumors,suggesting that it has tissue specificity.However,the role and mechanism of MYCT1 in lymphoma including DLBCL have not been reported.In the previous study,we found several differentially expressed genes related to the occurrence and development of lymphogenic malignant diseases in laryngeal cancer cells stably transformed with MYCT1,including RUNX1(runt related protein 1).RUNX1 is an important transcription factor.Similar to MYCT1,RUNX1 shows two-sided roles in different tumors.Even in lymphoma,its role is controversy based on the study from different groups.Moreover,we also found that MYCT1 and RUNX1 were downregulated and upregulated in lymphoma,respectively.Meanwhile,there was deletion of MYCT1 locus and amplification of RUNX1 locus in lymphoma cell karyotypes,suggesting that MYCT1 and RUNX1 have opposite roles in lymphoma,respectively.Previously,we discoveredthat MYCT1 interacted with MAX(myc associated factor X,MAX)in laryngeal carcinoma.Bioinformatics prediction revealed that there was a potential binding site of MAX in the RUNX1 promoter.Since MYCT1 and MAX are widely expressed in a variety of normal tissues,we speculate that MYCT1 may regulate the expression of RUNX1 at the transcriptional level through interaction with MAX,participating in the occurrence and development of lymphoma.In this study,we intend to study the effects of MYCT1 on the karyotype,proliferation and apoptosis of lymphoma cells by using cytogenetic and molecular genetic techniques,and explore the related molecular mechanisms of MYCT1 in lymphoma,so as to provide important clues for the in-depth study of MYCT1-related pathways in the diagnosis,treatment and prognostic prediction of lymphoma.Materials and methods:1.Materials: lymphoma bone marrow specimens with bone marrow infiltration,paraffin-embedded lymphoma tissues,human diffuse large B-cell lymphoma cell lines DB and SU-DHL4.2.Methods: cell culture,gene transfection,chromosome karyotype analysis,fluorescence in situ hybridization(FISH),real-time quantitative PCR(qPCR),Western blot,flow cytometry,CCK-8 assay co-Immunoprecipitation(Co-IP),immunohistochemistry,chromatin immunoprecipitation(Ch IP),dual-luciferase reporter assay.Results:1.Karyotype analysis results(1)Among 209 lymphoma patients with the infiltration of bone marrow,78(37.3%)cases showed abnormal karyotype.The average overall survival(overall survival,OS)and disease-free survival(progression free survival,PFS)in the abnormal karyotype group were significantly lower than those in the normal karyotype group(P < 0.001),and the three-year mortality was significantly higher than that in the normal karyotype group(P < 0.001).(2)Among the 78 patients with abnormal karyotype,68(87.2%)cases showed complex karyotype;and a total of 589 chromosome aberrations.The number aberrations accounted for 53.8%(317/589),and trisomy and haplotype were commonly found.The most affected chromosomes are 3,6 and 11 with rates 7.65%(45/589),7.31%(43/589)and 6.97%(41/589)respectively.On the other hand,the structural abnormality accounted for 46.2%(272/589),mainly involving the chromosome 6(34/272,12.5%),11(28/272,10.3%),14(23/272,8.5%),and the smallest overlapping regions(SOR)were 14q32 qter(18/272,6.6%),6q21-25(16/272,5.9%)and 11q23 qter(13/272,4.8%).2.FISH results of the 78 patients with abnormal karyotypes showed chromosomal deletion of MYCT1 locus in 20 patients and amplification of RUNX1 locus in 16 patients,of which both MYCT1 deletion and RUNX1 amplification were found in 5patients in the same time.3.qPCR and Western blot showed that MYCT1 m RNA and protein levels were significantly lower than those in the control group(P < 0.05),wheras RUNX1 m RNA and protein levels were significantly higher than those in the control group(P < 0.05).4.The karyotype analysis of DB cells stably transfected with MYCT1 overexpression vector showed a translocation between the short arm of chromosome 2 and the long arm of chromosome 8,and a isochromosome of long arm of chromosome 7.SU-DHL4 cells stably transfected with MYCT1 showed the insertions of the short arms of chromosome 9,18,22,anda deletion of the short arm of chromosome 7.5.CCK8 detection showed that the proliferation abilities of the cells stably tranfected with MYCT1 after 48 hours decreased significantly(P < 0.05 in DB group and P <0.01 in SU-DHL4 group)compared to the controls,and the decreasing trend was more obvious after 72 hours.6.Flow cytometry detection showed that the apoptotic DB and SU-DHL4 cells stably tranfected with MYCT1 increased significantly compared to the controls(P < 0.01).7.Flow cytometry detection showed that the number of DB and SU-DHL4 cells stably transformed with MYCT1 increased significantly in G0/G1 phase(P < 0.05),but there was no significant changes in S and G2/M phase.8.qPCR and Western blot results showed that the RUNX1 m RNA and protein levels in DB and SU-DHL4 cells stably transfected with MYCT1 overexpression vector were significantly lower than those in the control group(P < 0.05).Knockdown of MAX could significantly rescue the effect of MYCT1 on RUNX1 expression(P <0.05).9.Co-IP results showed that MYCT1 interacted with MAX in the DLBCL cells.10.Bioinformatics prediction displayed that there was a potential MAX binding site in the RUNX1 promoter.The dual luciferase reporter gene activity detection showed that the luciferase activity of DLBCL cells with MAX knockdown was significantly lower than that of the control group(P < 0.05).Chromatin immunoprecipitation detection confirmed that MAX bound to the RUNX1 promoter region,and MYCT1 significantly inhibit the recruitment of MAX to the RUNX1 promoter(P < 0.001).Conclusions:1.The overall survival and disease-free survival of the lymphoma patients with abnormal karyotypes are shortened,and the 3-year mortality is elongated,suggesting that abnormal karyotype is one of the important factors related to poor prognosis of lymphoma.2.There are three SOR in lymphoma,which are 14q32 qter,6q21-25 and 11q23 qter.Importantly,MYCT1 is located in 6q21-25,suggesting the potential role of myct1 in lymphoma.3.MYCT1 and RUNX1 are down-regulated up-regulated in lymphoma,respectively,suggesting that MYCT1 and RUNX1 play tumor suppressive and oncogenic roles in lymphoma,respectively.4.MYCT1 affects the karyotypes of DLBCL cells,implying the role of MYCT1 in maintaining the chromosome stability of DLBCL,the mechanism of which is required to be explored in the future.5.MYCT1 inhibits the proliferation and promots apoptosis of DB and SU-DHL4 cells,and the cells were arrested in G0 / G1 phase.6.MYCT1 interacts with MAX protein in DLBCL.7.MAX directly binds the RUNX1 promoter region,suggesting that MAX is a transcription factor of RUNX1.8.MYCT1 inhibits the RUNX1 gene expression via MAX.