Lower expression of the RASSF10 gene induces myeloma cell proliferation due to hypermethylation of the gene promoter in multiple myeloma

Background: Multiple myeloma (MM) is an incurable malignant neoplasm of plasma cells, in which genetic defects, epigenetic aberrations and bone marrow microenvironment are involved in the pathogenesis. RASSF10 acts as a tumor suppressor gene by methylation in glioma and several other cancers, but its role in MM remains unknown. Methods: In order to explore the role of RASSF10, mRNA expression was detected in MM patients and analyzed with overall survival. Results: Expression of the RASSF10 gene signicantly decreased in newly diagnosed MM patients, and was positively correlated with overall survival. RPMI-8226 and OPM-2 cell lines with lower RASSF10 expression were selected for further study. Overexpression of RASSF10 in these two cell lines inhibited proliferation and induced apoptosis. The RASSF10 gene promoter in MM cell lines was hypermethylated, and downregulated after decitabine treatment. Meanwhile, expression of the RASSF10 gene was upregulated. MM cells with overexpression of RASSF10 were injected into nude mice and exerted anti-MM activity in vivo. Conclusions: Low expression of RASSF10 contributed to the proliferation of myeloma cells by hypermethylation of its promoter.


Background
Multiple myeloma (MM) is a malignant neoplasm of plasma cells that accumulate in the bone marrow (BM) and is characterized by end-organ damage (CRAB) caused by secretion of a monoclonal protein (Mprotein) (1). Over the last 10 years, therapy of MM has improved using novel agents, including proteasome inhibitors and immunomodulatory drugs, in combination with autologous stem cell transplantation, alkylating agents and/or glucocorticoids. Unfortunately, MM remains an incurable disorder and all patients ultimately relapse due to the development or drug resistance. Therefore, further breakthroughs are still needed to improve patient outcome (2). Apart from the central role of genetic defects, an increasing number of studies has shown that epigenetic aberrations are also involved in the pathogenesis of MM. DNA methylation and post-translational histone modi cations are the most common epigenetic mechanisms known to disturb normal gene expression (3)(4)(5)(6).
The RASSF family encodes distinct tumor suppressors, which consists of six classical members (RASSF1-6) and four N-terminal members (RASSF7-10). The former group contains both the RA domain and the SARAH domain, and the latter contains an RA domain within its extreme N termini but lacks the SARAH domain. RASSF functions shown epigenetic silencing, RAS signaling, microtubule stability, cell cycle apoptosis, and immune system and nuclear transport (7,8). RASSF10, as the newest member, has a CpG island of 2254 bp with 209 CpGs and acts as a tumor suppressor gene by methylation of the gene promoter in glioma and several other cancers, such as thyroid cancer, prostate carcinoma, and leukemia (9)(10)(11)(12). Recent research shows that RASSF10 is hypermethylated in B-acute lymphocytic leukemia (B-ALL) and chronic Lymphocytic Leukemia (CLL), and associated with prognostic parameters (13). MM is a Bcell malignant disease, which has been investigated for treatment with DNA methyltransferase (DNMT) inhibitors (decitabine or 5-azacytidine) (14). However, the role of RASSF10 in MM remains unknown.  Table 1. The remission patients who received bortezomib-basic regimens for at least four cycles were de ned as very good partial response, complete response or stringent complete responses. Meanwhile, supportive therapies were given in these patients, including blood transfusion and anti-infective agents. Ten milliliters of bone marrow (BM) was taken from the patients and normal controls. This study was approved by the Ethical Committee of the Tianjin Medical University (IRB2020-WZ-075 and IRB2020-DW-04). Written informed consent was obtained from the patients for publication.

Cell lines
Human MM cell lines RPMI-8226, OPM-2 and U266 were obtained from the Cell Culture Center, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. All cell lines were cultured in 90% RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) at 37°C with 5% CO 2 and 95% humidity.
Magnetic-activated cell sorting (MACS) BM mononuclear cells (BMMCs) were isolated from heparin-anticoagulated BM of MM and normal controls using Ficoll-Hypaque density gradient centrifugation. CD138 + cells were puri ed using the anti-CD138 mAb-conjugated MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Ten million cells were resuspended in 80 ml buffer. Then, 20 ml CD138 MicroBeads (Miltenyi Biotec) were added and incubated at 4°C in the dark for 15 min. After washing with 2 ml buffer, the cells were centrifuged at 300 g for 5 min. The cells were resuspended up in 500 ml buffer. The MS column was placed in the magnetic eld of a suitable MACS separator (Miltenyi Biotec). After preparing the column by rinsing with 3´ 500 ml buffer, the cells were applied to the column. The column was washed with 1 ml buffer and all owthrough containing unlabeled cells was collected. Magnetically labeled cells were immediately ushed out by rmly pushing the plunger into the column and collected. The purity of enriched CD138 + cells isolated was evaluated by ow cytometry and was generally >90%.
DNA isolation and methylation analysis by bisul te sequencing DNA was extracted from MM cell lines and normal controls with a TIANamp Genomic DNA kit (Tiangen), and concentrations of DNA were determined by a micro-ultraviolet spectrophotometer (Bio-Rad). Bisul te sequencing was performed by Genechem (Shanghai, China). The experiment was repeated three times and the values were averaged.

Lentivirus transfection
RASSF10 lentivirus was purchased from Genechem, and lentivirus was transfected into MM cell lines and cells. The MOI=100, the cells were not selected using antibiotics, and Con238 (Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin) was used as a control. The e ciency of transfection was measured by ow cytometry and inverted microscopy.
Cell proliferation MM cell lines and cells at 72 h after transfection were analyzed for proliferation and apoptosis. Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8) (Engreen, Beijing, China). Absorbance at 450 nm was read at 1, 2, 3 and 4 days using a 96-well microplate reader (BioTek, Winooski, VT, USA). The experiment was repeated three times and the values were averaged.

Flow cytometry
The proportion of cells undergoing apoptosis was measured using the Apoptosis Detection kit (BD Bioscience, San Diego, CA, USA). Cells were stained with uorescein isothiocyanate-annexin V and propidium iodide (PI) and analyzed with a ow cytometer (FACScan; BD Biosciences, Mountain View, CA, USA). All assays were conducted in triplicate. The experiment was repeated three times and the values were averaged.

Western blotting
Cellular proteins were extracted in radio-immunoprecipitation assay buffer (Beyotime, Shanghai, China) and protein concentrations were determined using a BCA assay kit (Beyotime). Cell extracts (30 mg) were boiled with equal amounts of loading dye for 10 min and separated by 12% polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Hybond-ECL; Thermo Fisher Scienti c, Shanghai, China). Membranes were blocked in Phosphate buffered saline (PBS) with 0.1% Tween 20 (PBS-T) containing 5% non-fat milk for 1 h, and incubated with primary and secondary antibodies in PBS-T containing 5% non-fat milk. The following primary antibodies were used: RASSF10 (diluted 1:1000) (Abcam, Cambridge, UK), bcl-2 (diluted 1:1000), caspase3 (diluted 1:500), GAPDH (diluted 1:1000) (Cell Signaling Technology, Danvers, MA, USA). Primary antibody incubation was carried out overnight at 4℃. The membranes were washed with wash buffer (1×PBS and 0.01% Tween-20) and incubated with anti-rabbit or anti-mouse secondary antibody. The experiment was repeated three times and the values were averaged.

In vivo tumor growth in nude mice
Female BALB/c-nu nude mice aged 4-5 weeks were purchased from Beijing Hua Fukang Bioscience Company and were housed and monitored in a pathogen-free environment. RPMI-8226 MM cell line and transfected cells (n=10 7 ) were prepared in 100 ml serum-free RPMI-1640 medium and injected subcutaneously into the right dorsal ank of nude mice (n=3 each group). Measurement of tumor volume and tumor quality was performed after 21 days, and tumor volume (V) was calculated using the formula: V=0.5×a×b 2 , where a and b represent the longer and shorter tumor diameters, respectively. At the end of each study, animals were killed and tumors were collected and xed in formalin for hematoxylin and eosin (HE) staining and immunohistochemical staining of anti-CD138 or anti-RASSF10 antibody to assess tumor growth. CD138 and RASFF10 staining was quanti ed by Image J software. All procedures were approved by the Animal Ethics Committee of the Tianjin Medical University General Hospital.

Statistical analysis
Student's t-test was conducted for two-group comparisons. For many-group comparisons, one-way ANOVA (if the data were normally distributed) or Kruskal-Wallis test (if the data were not normally distributed) was used. The data are expressed as the mean ± SEM or median. Kaplan-Meier survival curves were constructed, and difference in survival rates was tested by log-rank test. Statistical analyses were performed using SPSS version 21.0. A value of p<0.05 was considered signi cant.

Results
Expression of the RASSF10 gene signi cantly decreased in newly diagnosed MM patients and MM cell lines, and was positively correlated with overall survival The purity of CD138 + cells sorted from BMMCs was >90%. Expression of RASSF10 mRNA in the newly diagnosed MM (NDMM) group (0.20±0.29) was signi cantly lower than that in the remission group (0.64±0.61) and normal control group (0.62±0.61) (both p<0.01), while there was no signi cant difference between the latter two groups (Fig. 1A). Expression of RASSF10 mRNA in MM cell lines RPMI-8226, OPM-2 and U266 was signi cantly more decreased than NCI-H929 and LP-1 (Fig. 1B). Protein expression of the RASSF10 gene was downregulated in the newly diagnosed MM group and MM cell lines RPMI-8226, OPM-2 and U266 (Fig. 1C).

Overexpressed RASSF10 exerts anti-MM activity in vivo
Nude mice (BALB/c-nu) were injected subcutaneously with RPMI-8226 and OPM-2 cells in the right scapular region. The RPMI-8226 nude mice had subcutaneous tumor formation. The lymph nodes, liver and spleen were signi cantly enlarged, and myeloma cells in ltrated the liver. After the recovery of RASSF10 expression (RPMI-8226/RASSF10 cells), the subcutaneous tumor formation volume was reduced, and myeloma cells did not in ltrate the liver. The tumor volume and mass of the RPMI-8226 control group were 96.97±15.22 mm 3 and 87.57±19.75 mg at 21 days, respectively. The tumor volume and mass of the RPMI-8226/RASSF10 group were signi cantly reduced to 16.56±3.15 mm 3 and 19.90±4.60 mg, respectively (both p<0.001) (Fig. 4A, B).
The tumor tissue formed subcutaneously in nude mice was stained with HE and showed a large number of irregular cells, short spindle or cubic, with irregular nuclei, coarse staining, and obvious nucleoli.

Discussion
RASSF10 acts as a tumor suppressor gene in some cancers, including non-hematological and hematological malignancies, such as ALL and CLL. However, the role of RASSF10 remains unknown in MM. In our study, we found that RASSF10 mRNA expression was signi cantly lower in newly diagnosed MM patients compared with normal controls, and upregulated after remission, which was associated with survival. Furthermore, overexpression of the RASSF10 gene inhibited proliferation of MM cell lines in vitro. After injection of RPMI-8226/RASSF10 cells into mice, we found that myeloma mass decreased. These results indicate that the RASSF10 gene may contribute to the pathogenesis of MM. However, the mechanism of this gene remains unclear in MM.
Wei et al. (16) studied the function of RASSF10 in gastric cancer, and showed that the RASSF10 gene was silenced in 75% of cell lines and totally methylations and partly methylations in promotor at six cell lines by Methylation-Speci c PCR (MSP). Furthermore, that study indicated that the RASSF10 gene inhibited proliferation of cancer cells by Wnt/β-catenin signaling in vivo. Jin et al. (17) showed similar results in hepatocellular carcinoma, in which RASSF10 suppressed hepatocellular carcinoma growth by activating p53 signaling. RASSF10 induced blockage of the G2/M phase and made cancer cells sensitive to docetaxel, which indicates that RASSF10 is a resistance marker. Western blotting showed upregulation of p53 and p21 and downregulation of MDM2 and bcl-2 after overexpression of RASSF10. In our study, we treated MM cell lines with decitabine, a demethylation agent, to observe the level of RASSF10 gene expression. As we expected, methylation of the RASSF10 promoter was downregulated and RASSF10 gene expression upregulated after decitabine treatment.
Epigenetic aberrations play an important role in the mechanism of MM, especially DNA methylation.
Hypermethylation of genes seems to be associated with the progression of monoclonal gammopathy of undetermined signi cance to MM and to plasma cell leukemia (18). Many studies have revealed hypermethylation of speci c loci (19) and some of them are associated with poor prognosis of MM patients, including SPARC, BNIP3, DAPK, RARβ, EGLN3, DCC, TGFβR2, CD9, RASD1 and p16 (20)(21)(22)(23)(24)(25)(26). These ndings stress the importance of hypermethylated genes in MM. Therefore, DNMT inhibitors can target this aberrant DNA methylation in MM, such as 5-azacytidine (AZA) and decitabine. Lavelle et al. revealed that decitabine restored the expression of p16 by DNA demethylation in MM cell lines. In addition, decitabine induced G0/G1-and G2/M-phase arrest linked with p21 or p38, respectively (27). Another study showed that decitabine has potent anti-myeloma activity in vitro by depleting myeloid-derived suppressor cells in BM (28). However, use of AZA or decitabine in MM is limited because of induction of DNA damage. Recent research showed that decitabine enhanced the effect of bortezomib in an MM cell line (29). Decitabine, combined with quisinostat, a histone deacetylase inhibitor, showed increased antimyeloma effects and altered immune cell constitution, such as increased dendritic cells and naive T cells, in a mouse myeloma model (30). Another study indicated that decitabine-mediated apoptosis in MM can be enhanced by combination with histone deacetylase inhibitor (31). In our study, decitabine alone reduced apoptosis by 50% in vitro, which indicates decitabine may be used in combination with other drugs.

Conclusion
The RASSF10 gene is signi cantly downregulated in newly diagnosed MM patients and positively correlated with overall survival. The RASSF10 gene inhibits the proliferation of myeloma cells in vitro and in vivo. We demonstrated the hypermethylation of the promoter of RASSF10, which can be modi ed by decitabine. To our knowledge, this is the rst study to reveal the role of RASSF10 in MM and explore the effect of decitabine on this gene. However, the effect of decitabine alone in MM is not satisfactory, and combination with other types of drugs should be tried in the future.

Declarations
Ethics approval and consent to participate The present study was approved by the Ethics Committee of Tianjin Medical University (IRB2020-WZ-075 and IRB2020-DW-04). Written informed consent was obtained for all patients.