Mechanism And Intervention Study Of Chromosome Instability And Osteoclasia Induced By CHEK1 In Multiple Myeloma

Multiple Myeloma(MM)is a malignant plasma cell hyperplasia disease and is the second largest malignancy in the blood system.It often happens accompanied by hypercalcemia,osteolytic damage,immunodeficiency and renal damage.MM cells originated from plasma cells in the bone marrow are very hard to kill and clear.Relapse and drug resistance are still the main problems in MM patients.The primary reasons for MM relapse and drug resistance are that MM evolves accompanied by complex genetic changes,such as chromosomal abnormalities and instability,changes in oncogene activation and non-coding RNAs,proliferation and differentiation of osteoclasts in bone marrow microenvironment.Recently,a series of oncogenes related to Chromosomal Instability(CIN),proliferation and drug resistance in MM patients are identified through high-throughput screening.Of which,checkpoint kinase 1(CHEK1)attracts researchers' attention.CHEK1 is a member of serine/threonine protein kinase family and plays an important role in cell cycle regulation and DNA damage response.Through the analysis of plasmocytes gene microarray data obtained from bone marrow of 22 normal patients(NP),44 monoclonal gammopathy of undetermined significance(MGUS)patients and 351 MM newly diagnosed(MM)patients,we find that the expression level of CHEK1 gene in MM patients is significantly higher than that in normal and MGUS patients.Clinical data of patients with MM TT2 treatment regimen show that the expression of CHEK1 gene in MM cells of MM patients with osteolytic lesions was significantly higher than that of patients without osteolytic lesions detected by systemic MRI.In addition,the survival rate of MM patients with high CHEK1 expression was significantly lower than that of patients with low CHEK1 expression.Meanwhile,in the corresponding samples of 88 MM patients before and after recurrence,the expression of CHEK1 gene in the recurrence samples was significantly higher than that in the newly diagnosed samples.We analyzed the sequential samples of 9 patients at the initial diagnosis,before the first bone marrow transplantation,before the second myeloma transplantation and after the second myeloma transplantation,and found that the CHEK1 gene expression level gradually increased as the treatment progressed.In addition,TT2 and APEXS databases showed that the survival cycle of patients with high CHEK1 expression was significantly lower than that of patients with low CHEK1 expression.Subsequently,we constructed CHEK1-overexpression(CHEK1OE)and CHEK1-knockdown(CHEK1KD)MM cell lines.CHEK1 promoting proliferation of MM cells and inducing drug resistance to bortezomib and doxorubicin were confirmed by trypan blue counting and soft agar cloning experiments.Flow cytometry analysis of cell cycle showed that CHEK1 could increase the mitotic phase ratio of MM cells.Methods such as giemsa staining,spindle immunofluorescence staining and exon sequencing confirmed that CHEK1 could induce the deletion and duplication of MM cell chromosome fragments and promote CIN.The results of co-immunoprecipitation technique and immunofluorescence co-localization showed that CHEK1 could bind and phosphorylate centrosomal protein CEP 170,regulate cell division,bind and regulate CIN related protein NEK2,and promote the proliferation and drug resistance of MM cells.Osteoclast differentiation experiment confirmed that CHEK1 can promote the process of osteoclast differentiation in bone marrow microenvironment.CHEK1 binds and regulates differentiation related factor NFATC1,and may be secreted into bone marrow microenvironment through circRNA.LY2603618,one of natural compound monomer modifiers can inhibit the expression of CHEK1.It can enhance the apoptosis of MM cells,and inhibit the expression of centromere related proteins CEP170 and NEK2.It can alleviate the changes in the width of equatorial plate and spindle,and slow down the process of osteoclast differentiation induced by CHEK1.To sum up,CHEK1 gene is closely related to the progress of MM,osteolytic lesions and survival rate of MM patients.It may cause drug resistance by inducing CIN,promoting osteoclast differentiation,aggravating bone destruction,and enhancing the interaction between myeloma cells and the microenvironment matrix.The results provide more theoretical basis for CHEK1 as a molecular target for the diagnosis and treatment of MM.