Chromosomes are mainly composed of highly compacted spiral DNA, which is the carrier of genes. Chromosomal instability (CIN) is caused by incorrect separation of somatic cells during mitosis, which can manifest as numerical and structural aberrations and is a notable feature of tumors [17]. The change in chromosome number is known as aneuploidy, that is, the loss or acquisition of the whole chromosome. Chromosomal abnormalities are characteristics of human tumors and are found in almost all solid tumors and malignant hematological diseases. Changes in chromosome structure are mainly the loss of chromosome heterozygosity, chromosome translocation, insertion translocation, inversion and amplification caused by chromosome breakage and error repair. Since Vogelstein [18] and colleagues found the role of CIN in human tumors, an increasing number of researchers have been paying attention to the mechanism of CIN and its relationship with human malignant tumors.
The replication of DNA is a highly regulated process. If the cell cycle is blocked, genetic material will replicate and separate abnormally [19]. In the whole process of the cell cycle, exogenous and endogenous stress responses can lead to abnormal DNA synthesis and damage repair [20]. However, epigenetic abnormalities, such as telomeres and centromeres, can lead to abnormalities in the repetitive sequence of chromosome secondary structure, chromatin conformation, origin and distribution, and replication time, resulting in replication-related DNA double-strand breaks, increased chromosome instability, and thus karyotype abnormalities [21].
Lymphoma is a highly heterogeneous disease. Ninety percent of lymphoma patients have clonal chromosomal abnormalities, with a large number of gene mutations, chromosome number changes and structural abnormalities [22]. DLBCL tumor cells usually have random and complex chromosomal abnormalities and sometimes show more than two kinds of chromosomal abnormal variations, which indicates that DLBCL patients have karyotype genetic instability and may undergo additional genetic changes [23].
In 1972, Manolov [24] first found that 14q + was closely related to Burkitt lymphoma. Later, researchers turned their research focus to lymphoma cytogenetics. Studies have shown that chromosomal reproducibility and clonal abnormalities have greatly affected the classification and subtype diagnosis of NHLs. Specific marker chromosomes of various lymphomas have been found successively, such as t (14; 18) in follicular lymphoma, t (8; 14) in Burkitt lymphoma, t (3; 14) in diffuse large B-cell lymphoma, t (11; 14) in mantle cell lymphoma, and t (2; 5) in anaplastic large cell lymphoma and their derived abnormalities [25, 26].
In this study, we found that the overall abnormal rate of karyotypes in lymphoma patients with bone marrow infiltration can reach 37.3%, in which the abnormal rate of complex karyotypes can be as high as 87.2%, which is consistent with the research results of scholars such as Mitelman F [27]. In addition, we found that the platelet value of patients with abnormal karyotypes was relatively low at the initial diagnosis; the complete remission rate, OS, and PFS were significantly reduced; and the three-year mortality was significantly increased. These findings are consistent with Greenwell [28], but the regulatory mechanism between complex karyotype abnormalities and thrombocytopenia has not been reported.
The smallest overlapping regions of chromosome deletion are important evidence for the discovery of tumor suppressor genes [29]. In this study, we found the three smallest overlapping regions in lymphoma patients with bone marrow infiltration, namely, 14q32-qter, 6q21-25, and 11q23-qter. Patients with abnormalities in these three key chromosomal regions have a significantly shortened trend in OS and PFS. It can be seen that patients with abnormalities in these smallest overlapping regions are prone to rapid disease progression and die in the early stage; thus, the disease is more dangerous in these patients. These results suggest that there may be tumor suppressor genes related to the occurrence, development and prognosis of lymphoma in these regions.
Some scholars have also studied these three smallest overlap regions. In a study of the correlation between the pathogenesis of lymphoma and disease subtypes, Lossos IS [30] and others found that the rearrangement of the IGH gene at the 14q32 site, such as t (14; 18), t (11; 14), t (8; 14) and other initial genetic changes, is related to the occurrence of lymphoma. 11q23.1 is an unstable region of B-cell lymphoma [31]. The Foxr1 gene and MLL in the 11q23 segment are related to the occurrence of B-cell lymphoma [32, 33]. 6q21-25 is also an important chromosomal region associated with lymphoma. Abnormalities at different sites of 6q can affect the malignancy of lymphoma; for example, 6q24-6q27 is related to intermediate lymphoma, 6q21 is related to high-grade lymphoma, and 6q23 is related to low-grade lymphoma [34]. Some scholars have found in T-cell lymphoma that the deletion of the 6q25 chromosome segment is related to the prognosis of lymphoma [35], but these studies involve large chromosome segments and many genes. To date, relevant research reports on specific genes in this segment are rare.
Previously, we showed that MYCT1, the first tumor suppressor gene cloned in our laboratory, plays an important role in the occurrence and development of many kinds of tumors. Moreover, MYCT1 is located in the region of chromosome 6q25. At present, there has been no report on whether MYCT1 can affect the stability of chromosome karyotypes or its relationship with the occurrence and development of lymphoma.
The study of clinical specimens has suggested that MYCT1 expression is reduced in lymphoma and thus may play a role as a tumor suppressor gene in lymphoma. We selected two DLBCL cell lines for culture and karyotype analysis. The DB cell line has a super triploid karyotype, and the SU-DHL4 cell line has a near-diploid karyotype, both of which are complex karyotypes. The analysis showed that both cell primordial karyotypes contained the t(14;18) translocation and involved multiple chromosomal structural and numerical abnormalities. When the MYCT1 stable cell line was successfully constructed, we analyzed the karyotypes of the two cell lines again and found that both cell lines had karyotype evolution and structural abnormalities. In the DB-MYCT1 group, the t (2;8) translocation occurred, and an abnormal chromosome with a long arm of chromosome 7 was added; the SU-DHL4-MYCT1 group showed deletion of the long arm of chromosome 7 and abnormalities of the short arm of chromosomes 9, 18 and 22. MYCT1 has an impact on the stability of DLBCL chromosomes, resulting in the increase or deletion of large segments of chromosomes.
In this study, the proliferation, cycle changes and other biological functions of DLBCL cells stably transformed with MYCT1 were studied. MYCT1 overexpression reduced the proliferation of DLBCL cells and blocked the cells in G0/G1, which played a significant negative regulatory role in DLBCL cells, once again confirming the role of MYCT1 as a tumor suppressor gene in lymphoma.
Studies have found that when some tumor suppressor genes are silenced in cells with stable chromosomes, replication stress increases the number of structural chromosomal aberrations [36]. The two-way interaction between replication stress and chromosomal error segregation has changed chromosomal instability, providing an evolutionary mechanism for cancer cells [37]. Other scholars have found that tumor cells with abnormal karyotypes have evolved a mechanism to escape the immune system, and changing CIN can regulate tumor activity and immunogenicity [38]. In summary, we consider that after MYCT1 overexpression, it may interact with some transcription factors or proteins, causing cell cycle arrest, inactivating oncogenes or activating tumor suppressor genes of the DLBCL cell line itself, and losing the previously stable immune escape function, which will lead to increased apoptosis and inhibit the proliferation of lymphoma cells.
The coding product of the MAX gene is MYC-associated factor X (MAX), which is a highly conserved transcription factor highly homologous to the primary structure of c-myc. MAX can regulate the transcription of target genes and regulate cell proliferation, apoptosis and differentiation [39]. Moreover, MAX is the core component of the C-MYC regulatory transcription complex and is a necessary factor for C-MYC to bind DNA and activate transcription [40].
RUNX1 is a key regulator in hematopoiesis, a common target of multiple chromosome translocations in human leukemia, and it plays an important role in hematopoiesis regulation and the occurrence and development of hematological malignancies [41, 42]. Some scholars have found that combined transgenic mice, with T cells or B cells overexpressing MYC and RUNX1 genes, are easily accessible to lymphoma, suggesting that RUNX1 can accelerate MYC-induced lymphoma [43]. The RUNX1 gene can also be used as a target for mouse leukemia virus (MLV) insertion mutation and lymphoma transcription activation [44]. In contrast, the cells of RUNX1-deficient chimeric mice can also develop T-cell lymphoma after treatment with ENU, suggesting that the loss of RUNX1 activity may also lead to lymphoid malignancies [45]. Gillian [46] also found that RUNX1 deficiency can cause lymphoma and proposed that RUNX1 can be used as a therapeutic target in p53 wild-type or mutant lymphoma. An increasing number of research results have suggested that the combination of positive or negative regulators and RUNX1 may be related to their functions in tumors [47].
In a study of RUNX1 regulating cell proliferation and apoptosis, scholars found that RUNX1 can upregulate centromere-associated protein E (CENPE), lead to the early expression of genes involved in the cell cycle and repeated application, and promote the growth of AML cells through cell proliferation [48]. Catherine [49] found that RUNX1 enhances downstream inositol phosphate 3-kinase Akt signal transduction by upregulating the expression of type 1 insulin-like growth factor receptor (IGF1R), thereby inhibiting T-ALL cell apoptosis and promoting proliferation. Natalia [50] found in a study of cell cycle regulation that RUNX1 mutation in AML can activate the transcription of ccdn2 together with AP-1 and then block the cell in G1 phase. In lymphoma, the mechanism of RUNX1 and cell cycle regulation has not been reported.
In this study, we found that 1) MYCT1 is located in one of the three smallest overlapping regions of diffuse large B-cell lymphoma; 2) MYCT1 alters the chromosomal instability of diffuse large B-cell lymphoma cells; 3) MYCT1 is negatively correlated with RUNX1 in lymphoma patients and MYCT1 represses RUNX1 transcription by binding MAX in diffuse large B-cell lymphoma cells; and 4) MYCT1 inhibits proliferation in diffuse large B-cell lymphoma probably by suppressing RUNX1 transcription.
In conclusion, MYCT1 overexpression can inhibit the positive regulation of RUNX1 by MAX, resulting in the downregulation of RUNX1 expression. Through a series of experiments, we proved the regulation of the MYCT1-MAX-RUNX1 signaling pathway in DLBCL cells and confirmed that MYCT1 plays the role of its tumor suppressor gene in lymphoma. However, the mechanism of RUNX1 in lymphoma needs further study. This experiment is the first to study the function and mechanism of MYCT1 in lymphoma, which provides a new target for further study of the pathogenesis and early diagnosis and treatment of lymphoma.