The Clinical Significance Of Serum Microrna, Long Non-coding RNA And CRBN Protein In Multiple Myeloma

Background: Multiple myeloma (MM) is a malignant plasma cell disordercharacterized by the accumulation of clonal malignant plasma cells in the bone marrow and production of a monoclonal immunoglobulin,with median age ofonset60-65years old. Typical clinical and laboratory features in patients withMM include lytic lesions or osteoporosis, anaemia, renal insufficiency, andhypercalcemia.With the increasingly rapid aging of population in China,the incidence of multiple myeloma is on the rising trend,which is a serious threat topeople’s life and health.It causes about1%of neoplastic diseases and13%ofhematological malignancies.The outcome for patients with multiple myeloma ishighly variable. Although the median overall survival time is3to4years, therange is from less than6months to more than10years.MM classification using biomarkers may effectively define risk, which allows for the use of appropriate treatments to acquire a better response and prognosis, then realize individual treatment.But, to date, few satisfactory biomarkers for the early diagnosis,monitoring of response effect and predicting prognosis of multiple myeloma havebeen identified. Recently, a number of groups have investigated that miRNAsand long non-coding RNAs have been associated with various cancers and playimportant roles in a variety of physiological and pathological processes,meanwhile they are stable in the serum or plasma.Cereblon (CRBN) has recently beenidentified as a target for immunomodulatory drugs (IMiDs) including lenalidomide and its expression levels correlated significantly with response to lenalidomide treatment.The downregulation of CRBN has been linked to resistance to IMiDs.These datas indicates that circulating miRNAs,long non-coding RNAs andthe CRBN protein levels are potential as diagnostic or prognostic biomarkers in myeloma.We first used bioinformatics prediction to identify particular miRNAs--miR-223,miR-451,miR-144and long non-coding RNAs--CRNDE-h,GAS5which were differentially expressed in MM patients.Object: To detect the expression levels of the circulating miR-223, miR-451, miR-144,long non-coding RNA CRNDE-h,GAS5and CRBN protein in theMM patients and healthy control subjects,and analysis whether the expression level of target gene and CRBN protein are associated with clinical characteristics,responsive effect,progression free survival and over survival,so that we can fin d the biomarkers of diagnosis and prognosis in MM.Method: We detected miRNA and long non-coding RNA in the serum of98newly diagnosis MM patients before received4courses of PAD treatments and52healthy control subjects by quantitative real-time PCR.The CRBN protein weredetected in the serum of95refractory/relapsed MM patients before receivedlenalidomide plus dexamethasone and69healthy control subjects by ELISA.Differences in target gene or CRBN protein levels were compared with the Student ttest or Mann-Whitney U test.We therefore set cutoff values for each target gene orCRBN protein at the median point,then divided the patients into high and low groups,and analyzed whether the expression levels were associated with clinicalcharacteristics,responsive effect and outcome of MM patients.Results：①we found that levels of miR-451,miR-144were significantly lower inMM patients (MMmedian0.059VS controlmedian0.9716[p＜0.001], MMmedian0.237VScontrolmedian0.920[p＜0.001]), and levels of long non-coding RNA CRNDE-h,GAS5were significantly higher in MM patients(MM0.527±0.802VS control-0.035±0.402for CRNDE-h [p＜0.001], MM0.402±0.940VS control0.088±0.479for GAS5[p＜0.001]) than healthy control subjects.We also used receiver operating characteristic(ROC) curves showing that serum miR-451yielded an AUC (the area under the ROCcurve) of0.818,which indicated it was a good biomarker for discriminating myelomapatients from healthy controls.②The expression level of circulating miR-223andmiR-144were both associated with renal insufficiency (P=0.007);while the proportionof hypercalcemia was higher in the high groups of miR-451and miR-144(p=0.04);theexpression level of circulating CRNDE-h was associated with age（p=0.046）,hypercalcemi（ap=0.014）, and13q14.3deletion（p=0.023）;the expressionlevel of circulating GAS5was associated with hypercalcemia （p=0.04）,and13q14.3deletion（p=0.023）.③We found that the circulating miR-223expression was highlycorrelated with response to2courses of PAD treatments (p=0.021).④The PFS and OSwere significantly higher in patients with the smaller ratio of miR-451/miR-144or thelower circulating CRNDE-h leve,but Cox regression analysis revealed that the ratio ofmiR-451/miR-144and the circulating CRNDE-h are not independent prognosisfactors.⑤CRBN levels were significantly higher in MM patients than the healthycontrols(p＜0001).The responsive rate in the higher CRBN protein group was higherthan the lower one(p=0.004).In responding patients significantly higher CRBNexpression levels compared to non-responding patients were noted（p=0.006）. Conclusion:We found that levels of miR-451,miR-144were significantly lowerin MM patients,while levels of long non-coding RNA CRNDE-h,GAS5weresignificantly higher in MM patients,and circulating miR-451was a good biomarkerfor discriminating myeloma patients from healthy controls,the circulating miR-223expression was highly correlated with response to2courses of PAD treatments.Thesmaller ratio of miR-451/miR-144and the lower circulating CRNDE-h level wereassociated with the longer PFS and OS.CRBN levels were significantly higher in MMpatients than the healthy controls,the responsive rate to lenalidomide in the higherCRBN protein group was higher than the lower one. In responding patientssignificantly higher CRBN expression levels compared to non-responding patientswere noted.It indicates that the serum CRBN protein has a potential to predictlenalidomide effect, is expected to be a non-invasive and convenient predictivebiomarker.