Multiple myeloma (MM), accounting for approximately 13% of hematological malignancies, is featured with the aberrant growth of malignant plasma cells ( 1 ). Previous research achievements have greatly increased life expectancy and quality of life. However, MM remains a virtually incurable hematologic malignancy ( 2 ). MM pathogenesis is involved in a series of biological processes, such as gene mutations, chromosomal abnormalities, epigenetic modifications, cell proliferation, tumor-microenvironment, and evolution of drug-resistant tumor cells ( 3 , 4 ). Identifying essential genes in disease progression is of the most significant importance for identifying new therapeutic approaches and prolonging the prognosis in patients ( 5 , 6 ).
MicroRNAs (miRNAs), 18–24 nucleotides in length, are a kind of noncoding RNA. miRNAs regulate posttranscription gene expression by degrading or repressing target mRNAs ( 7 , 8 ). miRNAs mediate various biological functions such as differentiation, proliferation, apoptosis, and migration ( 9 , 10 ). A series of cancer-associated miRNAs have recently been profiled in MM using microarray analysis ( 10 ). Previous findings demonstrated that miR-19a-3p acts as a novel poor prognostic indicator in MM ( 11 ). However, the mechanism of miR-19a-3p regulating cellular function in myeloma cells has not been elucidated.
FBXO32 (atrogin-1) belongs to the F-box protein family ( 12 ). Recent findings have reported that FBXO32 is down-regulated in cancers and may function as a tumor suppressor ( 13 – 17 ). Besides, emerging studies suggest that FBXO32 is a novel apoptosis regulator ( 18 ). However, the upstream regulatory factor of FBXO32 in tumorigenesis remains unclear.
This study showed that miR-19a-3p is significantly overexpressed and significantly suppressed cell apoptosis in MM cells. FBXO32 was negatively regulated by miR-19a-3p. Besides, our results demonstrated that FBXO32 was a miR-19a-3p target gene in MM cells. FBXO32 rescued the function of miR-19a-3p inhibiting cell apoptosis in MM cells. These results suggested that miR-19a-3p promoted MM cells development by regulating FBXO32 expression.
MATERIALS AND METHODS
Cell lines and clinical specimens
Cells (MM.1S, U266, RPMI8266, IM9, H929, and W63) were purchased from the American Type Culture Collection (MD, USA). Bone marrow (BM) specimens were provided by 8 healthy donors (5 males, 3 females; age range=25–55 years) and 8 patients with MM (4 males, 4 females; age range=30–56 years) from (for blind review). Clinical samples were purified with CD138 MicroBeads according to the Miltenyi Biotec protocol. Ethical approval was obtained from Jinshan Hospital of Fudan University. Cells were grown in the Roswell Park Memorial Institute 1640 medium, containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL).
Quantitative real-time polymerase chain reaction
We extracted total RNAs (cells and BM specimens) using TRizol reagent (Invitrogen, MD, USA) following the manufacturer’s instructions. The SYBR Green primers are listed in Table 1 . Reverse transcription of complementary DNA and quantitative real-time polymerase chain reaction (RT-qPCR) was performed using Takara reagent (Takara, Shiga, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were conducted as the standard for normalization. The 2 −ΔΔCT method was used to determine mRNA expression levels.
Transfection
Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA) was responsible for synthesizing the miR-19a-3p mimics (cat. no. MIMAT0000073) and inhibitors (cat. no. MIMAT0021837). FBXO32 overexpression vector was synthesized by Sangon Biotech (Shanghai, China). Vectors were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
Cell counting Kit-8 assays
Cell Counting Kit-8 (CCK-8) (DOJINDO, Japan) reagent was used to test cell viability. U266 and H929 cells (5×10 3 per well) were seeded on plates and incubated for 24 hours, 48 hours, or 72 hours. The medium was replaced with 10 μL CCK-8 reagent and 100 μL fresh medium. After 1 hour at 37°C, the wavelength of optical density values was 450 nm.
Flow cytometry
The FITC/PI reagent (KeyGen Biotech Co, Nanjing, China) was used to detect cell apoptosis. FlowJo_V10 (Becton-Dickinson, New Jersey, USA) software was used to analyze the results. The upper right quadrant represented late apoptotic cells. The right lower quadrant represented viable apoptotic cell.
Bioinformatics analyses
The biological function of miR-19a-3p and FBXO32 in cancer was explored in the Cancer Hallmarks Analytics Tool (CHAT) ( 19 ). Targets of miR-19a-3p were analyzed by TargetScan ( 20 ), miRDB ( 21 ), and DIANA-microT ( 22 ) database. Online Venn tools ( http://bioinformatics.psb.ugent.be/webtools/Venn/ ) visualized the overlapping target genes of these databases. Enrichment analysis (gene ontology and pathway) was performed using the Enrichr analysis tool ( 23 ). The protein interaction network of miR-19a-3p target genes was analyzed using the STRING analysis tool ( https://string-db.org ) ( 24 ) and visualized by Cytoscape software. The Cytoscape MCODE algorithm further extracted the core subnetwork.
Dual-luciferase reporter assays
The estimated binding site of miR-19a-3p or mutant 3′-UTR sequences were cloned in the XhoI and Notl (Promega Corporation, Madison, WI, USA) restriction sites to the psiCHECK2 vector. The cells were cotransfected with the vectors of wild-type or mutated FBXO32 3′-UTR and the miR-19a-3p NC/mimics/inhibitors. Luciferase assay reagent (Promega Corporation, Madison, WI, USA) was conducted to detect the luciferase activity after 48 hours.
Western blotting
Cleaved caspase 3 and B-cell lymphoma-2 BCL-2 (Abcam, San Francisco, CA, USA) expression levels were detected by western blotting using following the manufacturer’s instructions. Cells were lysed with lysis buffer (Cell Signaling Technology, Inc, Danvers, MA, USA). The supernatant protein was collected and normalized, and equivalent protein (30 μg) was electrophoresed and then transferred onto the polyvinylidene difluoride (PVDF) membranes. A blocking buffer was used to block proteins on the PVDF membranes at 4°C overnight. Then, the membranes incubated with the specific antibody and were washed in washing buffer (0.1% Tween 20 in phosphate-buffered saline). Proteins were then visualized by electrochemiluminescence reagents (KeyGen Biotech Co, Nanjing, China). GAPDH (ImmunoWay Biotechnology Company, Plano, TX, USA) expression levels were used as the standard for normalization.
Statistical analysis
We analyzed the results using the Statistical Package for the Social Sciences version 21.0 software (IBM SPSS Corp, Armonk, NY, USA). Normality distribution of variables was tested using the Shapiro Wilk test. Mean±standard deviation was used as descriptive statistics for normal distributed data. Median (min-max) was used as descriptive statistics for non-normal distributed data. The t test and 1-way analysis of variance tested normally distributed results. Multiple comparisons were made using least significant difference and Student-Newman-Keuls tests. Either χ 2 tests or Fisher exact tests were used to analyze non-normal distributed data. All experiments presented here were conducted at least 3 independent times. p<0.05 was considered statistically significant.
RESULTS
Basic expression levels of miR-19a-3p in multiple myeloma cells and patients
Using CHAT, we revealed that miR-19a-3p is associated with inducing angiogenesis and sustaining proliferative signaling ( Figure 1a ). RT-qPCR assays revealed that miR-19a-3p was increased in MM cells and patients (p<0.01) ( Figure 1. b, c ).
miR-19a-3p regulated cell viability and apoptosis in multiple myeloma cells
To further investigate the biological function of miR-19a-3p in MM, we overexpressed or knocked down miR-19a-3p in U266 and H929 cells (p<0.01) ( Figure 1. d, e ). CCK-8 assays revealed that overexpressed miR-19a-3p significantly promotes cell viability (p<0.05). Besides, miR-19a-3p knockdown suppressed cell viability (p<0.05) ( Figure 2. a, b ). Flow cytometry was conducted to detect cell apoptosis in U266 and H929 cells, which showed that miR-19a-3p overexpression significantly inhibits cell apoptosis and miR-19a-3p knockdown increased cell apoptosis (p<0.01) ( Figure 2c ).
Predicting miR-19a-3p target genes
miR-19a-3p target genes were estimated through 3 bioinformatics databases, including TargetScan, miRDB, and DIANA-microT. A total of 692 genes were overlapped in 3 bioinformatics databases ( Figure 3a ). The Enrichr analysis tool was used to analyze the correlated biological function and pathway of these overlapping target genes, the results of which revealed that these target genes are correlated with multiple biological function and pathway, such as negative regulation of cytoplasmic translation, protein kinase activity, and FoxO signaling pathway (p<0.05) ( Figure 3. b–e ). Besides, we analyzed the protein interaction network of these overlapping genes through the STRING analysis tool ( Figure S1 ) and extract core subnetworks using the Cytoscape MCODE analysis tool. Finally, we obtained 5 significant core subnetworks and found that FBXO32 is a core gene in the core subnetworks (p<0.05) ( Figure 3f ).
FBXO32 was a target gene of miR-19a-3p in multiple myeloma cells
In CHAT, FBXO32 is also associated with sustaining proliferative signaling ( Figure 4a ). Then, we found that FBXO32 3′-UTR sequences matched the “seed sequence” of miR-19a-3p, which indicated FBXO32 is related to the regulation of MM ( Figure 4b ). Luciferase report system revealed that miR-19a-3p mimics significantly inhibits the activity of FBXO32-wt 3′UTR among H929 and U266 cells (p<0.05). miR-19a-3p inhibitors significantly promoted the activity of FBXO32-wt 3′UTR among H929 and U266 cells (p<0.01) ( Figure 4. c, d ). miR-19a-3p does not regulate the activity of FBXO32-mt 3′UTR among H929 and U266 cells ( Figure 4. c, d ). Besides, RT-qPCR assays revealed that FBXO32 is significantly down-regulated in patients with MM (p<0.001) ( Figure 4e ) and cells (p<0.05) ( Figure 4f ). miR-19a-3p mimics significantly inhibited FBXO32 mRNA expression (p<0.05) ( Figure 4g ). miR-19a-3p inhibitors significantly promoted FBXO32 mRNA expression in U266 and H929 cells (p<0.001) ( Figure 4h ).
miR-19a-3p regulated cell viability and apoptosis through FBXO32 in multiple myeloma cells
FBXO32 was significantly overexpressed in U266 and H929 cells (p<0.01) ( Figure 5a ). CCK-8 assays revealed that FBXO32 significantly inhibits cell viability (p<0.05) ( Figure 5b ). Flow cytometry showed that FBOX32 significantly promotes cell apoptosis (p<0.01) ( Figure 5. c, d ). Furthermore, we found that FBXO32 significantly reverses the function of miR-19a-3p promoting cell viability (p<0.05) ( Figure 5e ). In addition, FBXO32 significantly reversed the function of miR-19a-3p inhibiting cell apoptosis (p<0.05) ( Figure 5. f, g ). WB revealed that miR-19a-3p significantly inhibits cleaved caspase 3 expression and increases Bcl-2 expression, which was also reversed by FBXO32 overexpression (p<0.05) ( Figure 5h ).
DISCUSSION
MM remains an incurable hematological malignancy given the clinical use of chemotherapeutics, glucocorticoids, and novel treatments ( 25 ). Hence, it is essential to identify new biomarkers in tumorigenesis and progression, which contribute to realizing the pathogenesis and find novel treatment for MM. Recently, studies have demonstrated aberrant miRNA expression in carcinoma tissues and cells, which may indicate significant molecular and clinical implications of miRNA in tumorigenesis and progression ( 26 ).
Previous studies reported that miR-19a-3p was increased in many cancers (including MM, hepatocellular carcinoma, and breast cancer) ( 5 , 27 , 28 ). Currently, our results presented that miR-19a-3p was overexpressed in MM cells and patients with MM ( Figure 1. b, Cc ). Interestingly, the significance level in patients with MM (p<0.001) was higher than in MM cells (p<0.01). Although the significance level does not account for the difference in expression, it is an interesting situation to consider. On the one hand, it may be caused by the lack of clinical samples. The miRNA expression levels of different individuals were significantly different. In contrast, these cell lines are derived from patients with MM, but screened. Cell lines are not equivalent to primary cells. Further studies revealed that miR-19a-3p suppressed cell apoptosis and promoted cell viability in U266 and H929 cells ( Figure 2. a, c ). These results showed that miR-19a-3p is an oncogene in MM cells. miRNAs alter gene expression by degrading or repressing target mRNAs. Subsequently, we predicted miR-19a-3p target genes in MM cells. Bioinformatics analysis ultimately extracted 5 core subnetworks ( Figure 3f ), which provides the research direction. Our previous studies screened these potential regulatory molecules. Eventually, we found that FBXO32 may be regulated by miR-19a-3p.
FBXO32 is a novel E3 ligase, which is one of the 4 subunits of the ligase complex of ubiquitin proteins ( 13 ). Recent evidence revealed the function of FBXO32 in tumorigenesis ( 29 ). FBXO32 was reported to be decreased in cancers, and FBXO32 induced apoptosis and increased cisplatin chemosensitivity ( 14 - 16 , 29 ). Our results demonstrated that FBXO32 is down-regulated in MM cells and tissues ( Figure 4. e, f ). Besides, miR-19a-3p negatively regulated FBXO32 mRNA expression ( Figure 4. g, h ) in U266 and H929 cells. Luciferase assay verified that miR-19a-3p regulates gene expression by degrading FBXO32 mRNA ( Figure 4. c, d ).
Next, FBXO32 was overexpressed in U266 and H929 cells, and the results revealed that FBXO32 significantly suppressed cell viability and increased cell apoptosis ( Figure 5. b, c ). To verify that miR-19a-3p actually regulates a variety of biological functions by regulating FBXO32 expression in MM, both mimics and FBXO32 overexpression plasmid were transfected in U266 cells, so as to detect cell viability, cell apoptosis, and apoptosis-associated protein expression. Our findings demonstrated that FBXO32 overexpression reverses the functions of miR-19a-3p increasing cell viability and suppressing apoptosis ( Figure 5. e, f ). In addition, FBXO32 overexpression ( Figure 5h ) abolishes the influences of miR-19a-3p suppressing cleaved caspase 3 and increasing BCL-2.
Based on the abovementioned results, we preliminarily revealed that miR-19a-3p directly regulates FBXO32. It would be significant to discover detailed mechanism in MM in the future. More studies are still necessary to confirm these findings, which will provide strong evidence supporting the function of miR-19a-3p and FBXO32 in MM occurrence and development.
Our current work confirmed the expression levels of miR-19a-3p in MM cells and patients with MM. miR-19a-3p significantly promoted cell viability and suppressed cell apoptosis. Moreover, FBXO32 was a target gene of miR-19a-3p. FBXO32 was down-regulated and significantly suppressed cell viability and promoted cell apoptosis in MM cells. Rescue assay further suggested that miR-19a-3p plays a role of oncogene by regulating FBXO32 expression in MM cells.
Supplementary Information
Protein interaction network of miR-19a-3p target genes was analyzed at STRING analysis tools and visualized by Cytoscape software.
Ethics Committee Approval: Ethics committee approval was received for this study from the Ethics Committee of the Jinshan Hospital of Fudan University (JIEC-2018-04).
Patient Consent for Publication: Written informed consent was obtained from the all patients.
Author Contributions: Design - D.Z., Y.L.; Analysis and/or Interpretation - Y.Ma, Y.Meng.; Writing Manuscript - Y.L.; S.G.
Data-sharing Statement: N/A.
Conflict of Interest: The authors have no conflicts of interest to declare.
Funding: This work was funded by the Youth Project of Jinshan District Health Committee (JSK-KTQN-2019-02).
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