Knockdown of lncRNA SNHG16 suppresses multiple myeloma cell proliferation by sponging miR-342-3p

Background Aberrant expression of long non-coding RNAs (lncRNAs) is closely associated with development and prognosis of human cancers. LncRNA SNHG16 is reportedly involved in human cancer; however, its roles in multiple myeloma (MM) remain unclear. Methods In this study, we investigated the function and molecular mechanisms of SNHG16 in MM. MM cells were transfected with si-SNHG16 or si-NC. SNHG16 expression levels was measured by qRT-PCR. Cell proliferation was monitored using the MTS. Flow cytometry assay was performed to measure the cell cycle and apoptosis. Luciferase reporter assay were performed to confirm the sponged miRNAs of SNHG16. Results SNHG16 expression was up-regulated in MM tissues. SNHG16 knockdown suppressed cell proliferation, arrested cell cycle transition from G1 to S phase, and promoted the apoptosis of MM cells. Moreover, SNHG16 knockdown promoted cleaved-Caspase-3, cleaved-Caspase-9, Foxa3a, and Bax expression, while markedly inhibiting CCND1, Bcl-2, Cyclin D1, PI3K, and p-AKT expression in MM cells. miR-342-3p was a direct target of SNHG16. SNHG16 knockdown significantly increased miR-342-3p expression in MM cells. Overexpression miR-342-3p markedly suppressed cell proliferation, arrested cell cycle transition from G1 to S phase, and promoted apoptosis of MM cells. Overexpression of miR-342-3p markedly promoted cleaved-Caspase-3/-9, Foxa3a, and Bax expression, and inhibited CCND1, Bcl-2, Cyclin D1, PI3K, and p-AKT expression in MM cells. Additionally, repression of miR-342-3p could rescue the effect of SNHG16 knockdown on MM cell proliferation, cycle arrest, apoptosis, and related protein expression. Conclusion Knockdown of lncRNA SNHG16 suppresses MM cell proliferation by sponging miR-342-3p, implicating SNHG16 as a novel therapeutic target for MM.


Background
Multiple myeloma (MM) is a fatal plasmocyte malignancy [1]. MM is the most common hematological cancer worldwide, and its incidence has continued to rise annually [2]. Despite advances in the diagnosis and treatment for MM, including chemotherapy, autologous/allogeneic stem cell transplantation, and monoclonal antibodies therapy such as daratumumab, elotuzumab, indatuximab, SAR650984, the results of clinical treatment of MM remain unsatisfactory [3,4]. MM pathogenesis is very complex and the detailed underlying mechanisms for the development and progression of MM remain largely unknown.
Increasing evidence indicates that the aberrant expression of long non-coding RNAs (lncRNAs) are closely associated with the development and prognosis of

Open Access
Cancer Cell International *Correspondence: hongliu_xy@sina.com; linzenghua@126.com † Xi Yang and Hongming Huang contributed equally to this work 2 Department of Hematology, Affiliated Hospital of NanTong University, No.20 Xishi Road, Nantong 226001, Jiangsu, People's Republic of China Full list of author information is available at the end of the article various types of cancer, including MM [5]. Liu et al. [6] reported that lncRNA TUG1 were significantly up-regulated in MM samples and cell lines, and that down-regulation of TUG1 markedly inhibited MM cell proliferation and promoted apoptosis. LncRNA Small Nucleolar RNA Host Gene 16 (SNHG16), an SNHG member, is up-regulated and functions as an oncogene in pancreatic cancer [7] and gastric cancer [8]. Although SNHG16 plays important roles in different cancers, its functional role and underlying molecular mechanism in MM tumorigenesis are still largely unclear.
LncRNAs act as competing endogenous RNAs (ceR-NAs) to sponge microRNAs (miRNAs). Aberrant expression of miRNAs play critical roles in multiple biological processes in cancer, including MM [9]. miR-342-3p, which is localized to chromosome 14q32, is a tumor suppressor miRNA involved in non-small cell lung cancer [10] and osteosarcoma [11]. Given the suppressive role of miR-342-3p in cancer, we aimed to determine whether SNHG16 acts as an miR-342-3p sponge to regulate the proliferation and apoptosis of MM cells.
In this investigation, we first evaluated the expression of SNHG16 in MM samples cell lines. Subsequently, we explored the effects of SNHG16 on MM cell proliferation, cycle and apoptosis. Finally, the interaction between SNHG16 and miR-342-3p and the underlying mechanisms of SNHG16 in MM cells were investigated.

Clinical specimen collection
Twenty primary (MM patients 66.30 ± 8.21; male, 15) and 15 marrow healthy samples (control, age, 59.6 ± 11.89; male, 10) were collected from June 2018 to January 2019 at Affiliated Hospital of NanTong University. No treatment was applied before the sample collection. MM specimens were taken from intramedullary regions. This study obtained approval from the Clinical Research Ethics Committee of Affiliated Hospital of NanTong University. Informed written consent for the use of the tissue samples was obtained from all patients and healthy controls. All fresh tissues were frozen in liquid nitrogen immediately and stored at − 80 °C until use.

Cell proliferation, cycle, and apoptosis assay
Cell proliferation was evaluated using a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS assay; Promega, Madison, WI, USA). The absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell Cycle Detection Kit (Keygentec, Nanjing, China) was used to assessed the cell cycle. An Annexin V-FITC Apoptosis Detection Kit (Keygentec, Nanjing, China) was used to assessed cell apoptosis. The percentages of the cell population in different phases and cell apoptosis were assessed with flow cytometry (BD Biosciences, San Jose, CA, USA). All experiments were repeated in independent triplicate.

Luciferase reporter assays
StarBase 3.0 software was used to predict miRNAs that targeted SNHG16. There are two miR-342-3p binding sites in the region of SNHG16. Wild-type SNHG16 (WT-SNHG16) containing putative miR-342-3p binding sites and SNHG16 containing mutated binding sites (MUT-SNHG16) (two miR-342-3p binding sites) were synthesized and then cloned into the luciferase reporter vector psi-CHECK-2 (Promega, Wisconsin, WI, USA). For luciferase reporter assays, HEK293 cells were co-transfected with luciferase reporter plasmids and miR-342-3p mimics, miR-342-3p inhibitor, or a negative control miRNA using Lipofectamine 2000. At 48 h post-transfection, cells were collected and relative luciferase activity was assessed using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. The relative luciferase activity was normalized with Renilla luciferase activity. All experiments were repeated in independent triplicate.

Statistical analysis
Statistical analyses were performed using SPSS 19.0 statistical software (IBM Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation (SD). Differences were analyzed with t-test or one-way ANOVA. A P-value < 0.05 was regarded as statistically significant.

SNHG16 is significantly up-regulated in MM samples and MM cells
First, we found that SNHG16 expression was significantly up-regulated in MM patients compared with that in controls (normal marrow tissue) (Fig. 1a). Additionally, SNHG16 expression was significantly up-regulated in MM cell (RPMI-8226 and NCI-H929) compared with that in PBMC (Fig. 1b). The result suggested that SNHG16 might be involved in the progression of MM.

SNHG16 directly interacts with miR-342-3p
To further investigate the molecular mechanism of SNHG16 on MM cell proliferation and apoptosis, potential target miRNAs were predicted using StarBase 3.0 online bioinformatics software. Two miR-342-3p binding sites in the region of SNHG16 were predicted (Fig. 4a). To confirm this predication, luciferase reporter assay was performed. The result revealed that miR-342-3p mimics markedly decreased the relative luciferase activity of the WT-SNHG16, while miR-342-3p inhibitor markedly increased the relative luciferase activity of the WT-SNHG16, but both had no effect on MUT-SNHG16 (Fig. 4b). In addition, miR-342-3p expression was significantly inhibited in MM tissues and RPMI-8226 and NCI-H929 cells (Fig. 4c, d). SNHG16 knockdown significantly increased miR-342-3p expression both in RPMI-8226 and NCI-H929 cells (Fig. 4e). These data indicated the direct interaction of SNHG16 with miR-342-3p in MM.

Discussion
Abnormal expression of lncRNAs contribute to the tumorigenesis and development of MM, and can act as valuable diagnosis markers and attractive therapeutic targets for MM [13,14]. In the present study, SNHG16 expression was markedly increased in tissue from MM patients. Furthermore, SNHG16 knockdown suppressed SNHG16 is reportedly increased and acts as a tumor promoting role in various cancers. For example, Xie et al. [15] found that SNHG16 was up-regulated in hepatocellular carcinoma tissues and cell lines, and that SNHG16 promoted hepatocellular carcinoma cell proliferation, invasion and tumorigenesis. Liu et al. [7] reported that SNHG16 was increased in pancreatic cancer tissues, SNHG16 knockdown suppressed cell proliferation and metastasis. Presently, SNHG16 expression was markedly up-regulated in samples from MM patients and in cell lines. Function assays showed that SNHG16 knockdown suppressed cell proliferation, arrested cell cycle transition from G1 to S phase, and promoted cell apoptosis in MM cells, similar with the roles of SNHG16 in hepatocellular carcinoma and pancreatic cancer. The PI3K/AKT pathway has been demonstrated to play a central role in cell growth and proliferation, and is associated with various Frist red peak represented G1 phase. Grey peak represented S phase. Second red peak represented G2 phase. Right histograms: the bar graph represents the distribution of G1, S, and G2 phase per group. *P < 0.05, **P < 0.01, ***P < 0.001 cancers [16]. To further investigate the regulatory mechanism of SNHG7 on MM cell proliferation at the protein level, we focused on the effect of SNHG16 knockdown on the expression of relevant proteins. SNHG16 knockdown promoted cleaved-Caspase-3, cleaved-Caspase-9 expression, Foxo3a, and Bax, markedly inhibited CCND1, Bcl-2, Cyclin D1, PI3K, and p-AKT expression in MM cells. Thus, SNHG16 might acted as an oncogene and promote cell proliferation and apoptosis by regulating PI3K/AKT pathway in MM cells.
Recent evidence has indicated that lncRNAs can function as molecular sponges for miRNAs to regulate the The bar graph represents the total apoptotic rate. The total apoptosis includes early apoptotic rate (LR) and late apoptotic rate (UR). ***P < 0.001. c The CCND1 expression was measured by qRT-PCR at 48 h after transfected. d, e The expression of Cleaved-Caspase-3, Cleaved-Caspase-9, Cyclin D1, PI3K, p-AKT, AKT, Foxo3a, Bax, Bcl-2, and total Caspase-3/9 proteins in RPMI-8226 and NCI-H929 cells was measured by western blot at 48 h after transfection with miR-342-3p mimics or miR-NC expression and function of target miRNAs [17]. SNHG16 has been reported to function as a molecular sponge for multiple miRNAs in cancers, such as miR-98-5p [18], miR-135a [8], and miR-373 [19]. We investigated the molecule mechanism of SNHG16 regulation in the progression of MM by using bioinformatics analysis to predict putative binding miRNAs. Researchers have reported miR-342-3p as tumor suppressors in various cancers. For instance, overexpression of miR-342-3p inhibits cell proliferation in hepatocellular carcinoma through the inhibition of insulin-like growth factor 1-mediated Warburg effect [20]. miR-342-3p inhibits non-small cell lung cancer cell growth and migration by targeting Anterior Gradient 2 [10]. Here, we predicted that SNHG16 shares two complementary binding sites for miR-342-3p. Furthermore, SNHG16 functioned as a molecular sponge for miR-342-3p and SNHG16 knockdown significantly increased miR-342-3p expression in MM cells. Importantly, we further demonstrated that the overexpression of miR-342-3p has similar effects with SNHG16 knockdown on MM cell proliferation, cycle, apoptosis, and related protein expression, while repression of Frist red peak represented G1 phase. Grey peak represented S phase. Second red peak represented G2 phase. Right histograms: the bar graph represents the distribution of G1, S, and G2 phase per group. *P < 0.05, **P < 0.01, ***P < 0.001 miR-342-3p could rescue the effect of SNHG16 knockdown on MM cell proliferation, cycle arrest, apoptosis, and related protein expression. Taken together, these findings indicate that SNHG16 has an oncogenic role by sponging miR-342-3p in MM cells. The bar graph represents the total apoptotic rate. The total apoptosis includes early apoptotic rate (LR) and late apoptotic rate (UR). ***P < 0.001. c The CCND1 expression was measured by qRT-PCR at 48 h after transfected. d, e The expression of Cleaved-Caspase-3, Cleaved-Caspase-9, Cyclin D1, PI3K, p-AKT, -AKT, Foxo3a, Bax, Bcl-2, and total Caspase-3/9 proteins in RPMI-8226 and NCI-H929 cells were measured by western blot at 48 h after transfected