Long non‐coding RNA NEAT1 serves as a novel biomarker for treatment response and survival profiles via microRNA‐125a in multiple myeloma

Abstract Background The present study aimed to explore the association of long non‐coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) with multiple myeloma (MM) risk and further investigate its correlation with clinical features, treatment response, survival profiles, and its interaction with microRNA‐125a (miR‐125a) in MM patients. Methods Totally, 114 de novo symptomatic MM patients and 30 healthy donors (as controls) were recruited. Their bone marrow samples were collected before treatment (MM patients) and at enrollment (healthy donors), respectively. Subsequently, plasma cells were isolated from bone marrow for detection of lncRNA NEAT1 and miR‐125a expression via reverse transcription quantitative polymerase chain reaction. Results lncRNA NEAT1 was upregulated in MM patients compared with healthy donors and presented with excellent value in distinguishing MM patients from healthy donors. In MM patients, lncRNA NEAT1 positively associated with International Staging System (ISS) stage, beta‐2 microglobulin (β2‐MG), and lactate dehydrogenase (LDH), but not correlated with core cytogenetics and other clinical features. Furthermore, lncRNA NEAT1 negatively associated with complete remission (CR), overall remission rate (ORR), progression‐free survival (PFS), and overall survival (OS). Moreover, lncRNA NEAT1 negatively associated with miR‐125a in MM patients. MiR‐125a was downregulated in MM patients compared with healthy donors, and it negatively associated with ISS stage, β2‐MG, and LDH, but positively correlated with CR, ORR, PFS, and OS in MM patients. Conclusion lncRNA NEAT1 might interact with miR‐125a, and serves as a novel biomarker for treatment response and survival profiles in MM, indicating its clinical value for MM management.


| INTRODUC TI ON
Multiple myeloma (MM) is a systemic malignant disease of the blood characterized by the production of nonfunctional intact immunoglobulins or immunoglobulin chains as well as the uncontrolled proliferation of monoclonal plasma cells in the bone marrow. 1 According to the global statistics report, MM is the third most commonly occurred hematological malignancy, accounting for approximately 1% of all cancer cases, and its incidence has undergone an increment in recent decades. 2 The common treatment approaches for MM consist of chemotherapy, autologous stem cell transplants, targeted therapy, and immunotherapy; however, the treatment efficacy is limited and the drug resistance is increasing due to the various chromosomal abnormalities, contributing to poor treatment response and undesirable survival profiles in MM patients. [3][4][5][6] Therefore, discovering novel prognostic biomarker is a necessity for MM management, which can help to estimate treatment response and predict prognosis in MM patients.
Long non-coding RNA (lncRNA) is one type of RNA longer than 200 nucleotides in length with limited protein-coding ability, and accumulating evidence has been reported that lncRNA is implicated in the tumorigenesis of various cancers. 7 Among the discovered carcinogenic lncRNAs, lncRNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) is upregulated and serve as an oncogene during the onset and progression of various hematopoietic malignancies, such as myeloid leukemia and lymphoblastic leukemia. [8][9][10][11] As for in MM, mechanically, one cellular experiment demonstrates that lncRNA NEAT1 knockdown inhibits cell proliferation, but promotes cell-cycle arrest and apoptosis via regulating PI3K/AKT pathway. 12 Another one indicates that lncRNA NEAT1 promotes M2 macrophage polarization, hence accelerating MM progression. 13 In addition, based on miRanda database analysis and previous studies, one of lncRNA NEAT1 target microRNAs is microRNA-125a (miRNA-125a), and miRNA-125a is shown to suppress MM progression. [14][15][16] According to these evidences and the result of our preliminary study with small sample size, which indicated that ln-cRNA NEAT1 was upregulated in MM patients compared with healthy controls, the hypothesis was raised that lncRNA NEAT1 was involved in MM development and prognosis via interaction with miR-125a; however, the related research is limited. Therefore, we performed the present study to explore the association of lncRNA NEAT1 with MM risk and further investigate its correlation with clinical features, treatment response, survival profiles, and its interaction with miR-125a in MM patients.

| Clinical data collection
Patients' age, gender, immunoglobulin subtype, bone lesion status, renal impairment status, Durie-Salmon stage, International Staging System (ISS) stage, biochemical indexes, and cytogenetics status were recorded post-diagnostic examinations. The Durie-Salmon stage was assessed according to the criteria of Durie-Salmon stage system for MM. 18 The ISS stage was evaluated referring to the criteria of ISS for MM. 19

| Sample collection and determination
Before therapy, bone marrow samples of patients were collected; also, the bone marrow samples were collected from healthy donors after recruitment. For the isolation of plasma cells, all bone marrow samples were treated by density-gradient centrifugation and purified by CD138-coated magnetic beads (Miltenyi Biotec). Subsequently, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect the relative expressions of lncRNA NEAT1 and miR-125a in the plasma cells. Total RNA was extracted from plasma cells using TRIzol™ Reagent (Thermo Fisher Scientific) and then reversely transcribed using PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara). Following that, qPCR was performed using SYBR ® Premix DimerEraser™ (Takara) to quantify lncRNA NEAT1 and miR-125a expressions. In addition, the expressions of lncRNA NEAT1 and miR-125a were calculated using 2 −ΔΔCt method with GAPDH and U6 as internal references, respectively. Primers were listed in the Table S1.

| Response and survival evaluation
Patients' therapy in this study was not intervened, which was decided by attending physician based on patients' clinical conditions in accordance with clinical practice guidelines. 20 The patients' response to the induc- V4), which included complete response (CR), very good partial response (VGPR), and partial response (PR). Overall response rate (ORR) was defined as CR + VGPR + PR. Patients were followed up by telephone or clinical visit until 2019/6/30, during which, patients' survival status was documented for the assessment of progression-free survival (PFS) and overall survival (OS). The PFS was defined as the duration from initial treatment to disease progression or death, and the OS was defined as the duration from initial treatment to death. The patients not known whether the disease had progressed or whether they had died at the last follow-up date were censored on the date of last visit or the date last known to be alive. were used to evaluate the value of variables in differentiating different subjects. PFS and OS were presented using Kaplan-Meier curves, and the difference of PFS and OS between two groups was determined by the log-rank test. P value < .05 was considered as statistically significant.   presented with excellent value in distinguishing MM patients from healthy donors (AUC: 0.939, 95% CI: 0.901-0.977) ( Figure 1B). These data suggested that high lncRNA NEAT1 expression was associated with increased MM risk.

| Correlation of lncRNA NEAT1 with immunoglobulin subtype and stages in MM patients
According to the median of lncRNA NEAT1 expression in MM patients, all MM patients were divided into the low lncRNA NEAT1 patients (n = 57) and high lncRNA NEAT1 patients (n = 57  Figure 2C).

| Correlation of lncRNA NEAT1 with PFS and OS in MM patients
PFS was decreased in high lncRNA NEAT1 patients compared with low lncRNA NEAT1 patients (P = .030) ( Figure 5A). Similarly, OS was also reduced in high lncRNA NEAT1 patients compared with low lncRNA NEAT1 patients (P = .014) ( Figure 5B).

| D ISCUSS I ON
In the present study, we found that (a) lncRNA NEAT1 was upregu- The previous data implied the clinical significance of lncRNA NEAT1/ miR-125a complex in MM management.
However, there still exist some limitations in our present study.
(a) Considering that this was a study with a small sample population, further studies with a larger sample size from multiple regions were needed for validation. (b) Our study did not explore the underlying mechanism of interaction between lncRNA NEAT1 and miR-125a in MM, which needed to be investigated via cellular experiments. (c) As we excluded secondary or mixed MM, further studies were needed to investigate the association of lncRNA NEAT1 and miR-125a with clinical indexes and prognosis in these patients.
In conclusion, lncRNA NEAT1 might interact with miR-125a and serves as a novel biomarker for treatment response and survival profiles in MM, indicating its clinical value for MM management.

ACK N OWLED G M ENTS
This study was supported by the Natural Science Foundation of Zhejiang Province (LY19C080001).

CO N FLI C T O F I NTE R E S T
The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.