Long non-coding RNA SNHG3 accelerates progression in glioma by modulating miR-384/HDGF axis

Abstract Glioma is a malignant primary brain tumor that occurs in the central nervous system and has threatened the well-being of millions of patients. It is well acknowledged that long non-coding RNA (lncRNA) SNHG3 participates in the regulation of proliferation, inflation, differentiation, and metastasis in many cancers. However, the regulatory effect of SNHG3 on glioma progression is still controversial. The expression of SNHG3 and HDGF was upregulated, whereas miR-384 was downregulated in glioma tissues, compared with the normal tissues. Interestingly, high SNHG3 contributed to low survival rate while low SNHG3 showed the opposite result. Moreover, SNHG3 or HDGF knockdown significantly suppressed proliferation, migration, and invasion and induced apoptosis in glioma. Meanwhile, restoration of HDGF abrogated the inhibition of SNHG3 silencing on glioma cell progression. Besides, miR-384 inhibitor attenuated SNHG3 silencing induced inhibition on HDGF mRNA and protein expression in A172 and SHG44 cells. LncRNA SNHG3 promotes cell proliferation, migration, and invasion in glioma by enhancing HDGF expression via miR-384 sponging, representing the promising targets for the development of novel therapeutic strategies.


Introduction
Glioma, a malignant primary brain tumor derived from the central nervous system, has a high mortality rate and has threatened the well-being of millions of patients [1,2]. Currently, various advanced intervention strategies, such as surgical resection, chemotherapy, and local irradiation, have been achieved; however, 5 year survival rate of glioma is still less than 5% [3][4][5]. Therefore, investigation of the biological mechanism and development of novel therapeutic strategies are needed urgently.
Long non-coding RNAs (lncRNAs) are a subtype of RNAs comprising more than 200 nucleotides. They are essential modulators in cell infiltration, metabolism, differentiation, and metastasis by targeting the microRNAs (miRNAs) and participating in epigenetic transcriptional regulation [6][7][8].
Many lncRNAs have been identified as diagnostic or prognostic biomarkers in multiple cancers, such as nasopharyngeal carcinoma, hepatocellular carcinoma, and glioma [9][10][11]. Small nucleolar RNA host gene 3 (SNHG3), which is located on 1q35.3, is a novel lncRNA and closely related to tumor development [12]. For example, overexpression of SNHG3 promoted the invasive and migratory potentials by targeting miRNA-151a-3p to enhance RAB22A expression in osteosarcoma [13]. Likewise, SNHG3 served as an oncogene to enhance glioma malignant progression by KLF2 and p21 silencing [14]. Liu et al. discovered that SNHG3 was upregulated in lung adenocarcinoma tissues and abundance of SNHG3 promoted cell cycle and proliferation and inhibited apoptosis of lung adenocarcinoma [15]. However, the molecular mechanism in glioma requires an in-depth investigation.
MiRNAs are small chain RNAs with limited protein coding ability and they typically participate in many cellular physiological and pathological processes by altering gene expression at the post-transcription level, leading to mRNA degradation and protein translation suppression [16][17][18]. Aberrant expression of miRNAs is one of the leading causes of tumorigenesis and metastasis [19]. Upregulation of miR-384 suppressed tumor development, while deficiency of miR-384 expedited growth and metastasis by targeting SLBP in osteosarcoma MG63 cells [20]. Similarly, miR-384 induced cell apoptosis and autophagy through downregulation of collagen α-1(X) chain gene in non-small cell lung cancer [21]. Besides, miR-384 was found to repress proliferation, metastasis, and lipogenesis by targeting pleiotrophin in hepatitis B virus induced hepatocellular carcinoma [22]. However, whether miR-384 is involved in glioma development is largely obscure.
In the present study, we suggested that SNHG3 exerts oncogenic function by modulating miR-384/hepatomaderived growth factor (HDGF) axis. The expression of SNHG3 and HDGF was upregulated while miR-384 was downregulated in tumor tissues, compared with normal tissues. Luciferase reporter system further identified the interaction between miR-384 and SNHG3 or HDGF. Moreover, miR-384 inhibitor inversed SNHG3 silencing mediated inhibition on HDGF mRNA and protein expression in A172 and SHG44 cells, implicating that SNHG3 could regulate HDGF expression by competing with miR-384.

Patient tissues
A total of 42 glioma patients were recruited from Zhangzhou Affiliated Hospital of Fujian Medical University. All the patients had not received chemotherapy, radiotherapy, or other combined treatments prior to surgery. The tumor tissues and the corresponding normal tissues were obtained from the recruited volunteers by surgical resection and stored at −80°C immediately.
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration and has been approved by the Ethics Committee of Zhangzhou Affiliated Hospital of Fujian Medical University.

Cell transfection
Glioma cell lines A172 and SHG44 and human astrocyte cell line NHA were purchased from iCell Bioscience Inc.

Luciferase reporter assay
StarBase v2.0 and TargetScan predicted the potential binding sites between miR-384 and SNHG3 or HDGF. SNHG3 sequence or HDGF 3′-UTR sequence containing wild-type or mutant-type putative binding sites to miR-384 was cloned into pmirGLO vector (Promega, Madison, Wisconsin, USA), termed as SNHG3 WT or SNHG3 MUT, and HDGF 3′-UTR WT or HDGF 3′-UTR MUT. DNA sequencing was used to detect the reporters. Then, 400 ng of the constructed plasmids, 50 ng of Renilla luciferase reporter plasmid (pRL-TK), and 50 nM of miR-384 mimics or miR-NC were transfected into A172 and SHG44 cells using Lipofectamine 2000 (Invitrogen). After incubation for 48 h, a microplate reader (Biotek Instruments) and dual luciferase reporter assay kit (Promega) were used to detect the relative luciferase activity. Renilla luciferase activity was used to normalize the firefly luciferase activity.

CCK8 assay
For the CCK8 assay, A172 and SHG44 cells were seeded on a 96-well plate at a density of 5,000 cells/well and continuously cultured for 24, 48, and 72 h. After reacting with 10 µL of CCK8 reagent (Beyotime, Shanghai, China) for 2 h, the optical density value at 450 nm was measured by a microplate reader (Bio-Rad, Hercules, CA, USA).

Apoptosis assay
The fluorescein isothiocyanate (FITC) Annexin V-propidium iodide (PI) Kit (Beyotime) was used to detect cell apoptosis. A172 and SHG44 cells were harvested and costained with Annexin V-FITC/PI for 30 min at 48 h posttransfection. A172 and SHG44 cells were harvested and washed twice using ice-cold phosphate-buffered saline. Cells were resuspended using Annexin V binding buffer for the concentration of 0.25-1 × 10 7 cells/mL. Then, cells were incubated with 5 µL of FITC-Annexin V and 5 µL of PI for 15 min at room temperature in the dark. Stained cells were added with 400 µL of Annexin V binding buffer in each tube, and flow cytometry was used to analyze apoptosis.

Transwell assay
A172 and SHG44 cells (2 × 10 4 cells per well) were plated into a 6-well plate and starved for 24 h. For transwell assay, A172 and SHG44 cells resuspended in serum-free DMEM (Gibco) were seeded on the upper chamber precoated with Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) for invasion assay (without Matrigel coating for migration assay). Meanwhile, the lower chamber containing the medium supplemented with 10% FBS (Gibco) was regarded as a chemoattractant. After incubation for 48 h, the non-invasive and non-migrated cells were scraped, while the migrated and invaded cells at the lower chamber were fixed with methanol and stained with 0.1% crystal violet (Sigma, St. Louis, MO, USA) for 10 min and counted using a microscope.

Statistical analysis
Data were presented as mean ± standard deviation.
Statistical analysis was carried out by SPSS 13.0 software (Chicago, IL, USA) and GraphPad Prism 7 (GraphPad Inc., San Diego, CA, USA). The correlation of miR-384 and SNHG3 or HDGF was determined by Pearson's correlation coefficient analysis. A P-value of <0.05 was considered statistically significant.

Upregulation of SNHG3 in glioma
The expression of SNHG3 in 42 pairs of glioma tumors and normal tissues was determined using qRT-PCR to elucidate the role of SNHG3 in glioma development. As illustrated in Figure 1a, an obvious upregulation of SNHG3 was observed in tumors, compared with the corresponding normal tissues. Consistently, SNHG3 expression was upregulated dramatically in glioma cell lines A172 and SHG44 in comparison with human astrocyte cell line NHA (Figure 1c). Noticeably, high level of SNHG3 resulted in low survival rate, whereas low level of SNHG3 resulted in high survival rate within 60 days in glioma patients ( Figure 1b). Collectively, we suggested that SNHG3 plays an oncogenic role in glioma.

SNHG3 knockdown suppresses cell progression and induces apoptosis in glioma
Identification of the function of SNHG3 in glioma cell progression was evaluated by CCK8, flow cytometry, and transwell assay. Initially, A172 and SHG44 cells were transfected with si-SNHG3 and si-control for the following biological investigation. We noticed that cell viability was repressed significantly in A172 and SHG44 cells after SNHG3 silencing, compared with the si-control group (Figure 2a and b). Consistent with CCK8 results, transwell assay exhibited that migration and invasion ability were suppressed in glioma cells transfected with si-SNHG3 while remaining unchanged in the si-control group (Figure 2e and f). As expected, SNHG3 knockdown induced apoptosis largely in A172 and SHG44 cells (Figure 2c and d). Those findings represented that SNHG3 could accelerate progression and suppress apoptosis in glioma.

HDGF knockdown inhibits cell progression and enhances apoptosis in glioma
Subsequently, we transfected si-HDGF and si-control in A172 and SHG44 cells to explore the influence of HDGF on glioma cell proliferation, apoptosis, migration, and invasion. As displayed in Figure 3a and b, distinct improvement of HDGF mRNA and protein expression in A172 and SHG44 cells was discovered after si-HDGF transfection in comparison with si-control transfection, representing that the transfection efficiency is extremely   high. CCK8 results indicated that downregulation of HDGF remarkably inhibited proliferation ability of A172 and SHG44 cells (Figure 3c and d). Likewise, the number of migration and invasion cells was reduced in glioma cells after HDGF silencing (Figure 3g and h). On the contrary, the apoptotic rate was elevated in A172 and SHG44 cells transfected with si-HDGF, compared with sicontrol (Figure 3e and f). Therefore, we concluded that HDGF functions as an oncogene to accelerate cell growth as well as to suppress apoptosis in glioma.

Restoration of HDGF abrogates the inhibition of SNHG3 silencing on proliferation, migration, and invasion in glioma
To clarify the regulatory effects of SNHG3/HDGF axis on glioma cell progression, A172 and SHG44 cells were transfected with si-SNHG3, si-SNHG3 + pcDNA-HDGF, si-SNHG3 + pcDNA-control, and si-control. Through western blot analysis, we discovered that HDGF protein expression was decreased distinctly in A172 and SHG44 cells after SNHG3 knockdown, indicating that SNHG3 might modulate HDGF positively (Figure 4a and b). Moreover, rescue experiments implicated that restoration of HDGF abrogated SNHG3 silencing induced suppressive effect on glioma cell proliferation (Figure 4c and d). Similarly, HDGF deficiency reduced the number of migratory and invasive cells; however, the abundance of HDGF showed the opposite trend (Figure 4g and h). Interestingly, HDGF rescued SNHG3 silencing mediated promotion on apoptosis in glioma (Figure 4e and f). Altogether, SNHG3 promotes glioma cell development by regulating HDGF.

SNHG3 is a sponger of miR-384
Increasing evidence has identified that lncRNA acts as a competing endogenous RNA (ceRNA) to modulate cell behavior by competing with its miRNA; thus, we searched the online database StarBase v2.0 and found that miR-384 has the potential to bind to SNHG3 (Figure 5a). To certify the prediction, wild-type SNHG3 (SNHG3 WT) and mutanttype (SNHG3 MUT) vectors were constructed and subsequently co-transfected with miR-384 mimics and miR-NC in A172 and SHG44 cells. Reduction of luciferase activity had appeared in both A172 and SHG44 cells co-transfected with SNHG3 WT and miR-384 mimics, confirming the interaction between SNHG3 and miR-384 (Figure 5b and c).

Discussion
Accumulating evidence has elucidated that lncRNA could modulate cell growth and gene expression by interacting with miRNA as a ceRNA [23]. SNHG3 has been regarded as a prognostic factor in multiple cancer types since its expression level is associated with portal vein tumor thrombus, tumor size, and recurrence [24,25]. For instance, upregulation of SNHG3 indicates poor survival since SNHG3 could accelerate cell growth by sponging miR-196a-5p in osteosarcoma patients [26]. In addition, alteration of SNHG3 expression is associated with the onset of many cancers, for example, SNHG3 functioned as ceRNA by sponging miR-182-5p to release c-Myc and promote malignant development in colorectal cancer [27]. Similarly, SNHG3 repressed the degradation of CD151 by competing with miR-128, contributing to the epithelial-mesenchymal transition (EMT) and sorafenib resistance in hepatocellular carcinoma [28]. Therefore, it is of great significance to illuminate the role of SNHG3 in glioma. Numerous research studies have demonstrated that miRNAs regulate the downstream gene and protein expression by base-pairing of 3′-UTR of mRNA to control cell behavior in cancers [29][30][31]. Differentially, expression of miR-384 was diagnosed in multiple cancers, for instance, overexpression of miR-384 inhibited gastric cancer cell growth, migration, and invasion by interacting with metadherin [32,33]. In addition, miR-84 suppressed cell proliferation, migration, and metastasis in renal cell carcinoma and colorectal cancer by targeting and interacting with RAB23 and KRAS/CDC42 axis, respectively [34,35]. Promotion of miR-384 was indicated to inhibit cell growth and EMT by sponging lncRNA TUG1 in nasopharyngeal carcinoma [36]. Therefore, identification of the potential functional miRNA might present novel targets for glioma diagnosis.
In our study, we attempted to illuminate the biological mechanism of SNHG3 on glioma tumorigenesis, proliferation, migration, invasion, and apoptosis processes. It is apparent that SNHG3 and HDGF expression was upregulated while miR-384 was downregulated in tumor tissues. In addition, SNHG3 or HDGF knockdown significantly attenuated glioma cell progression; however, restoration of HDGF expression remarkably enhanced proliferation, migration, and invasion in glioma. The subsequent luciferase activity detection confirmed the interaction between miR-384 and SNHG3 or HDGF. Besides, Pearson's correlation coefficient analysis revealed that miR-384 was correlated with SNHG3 or HDGF inversely. Essentially, miR-384 inhibitor abolished the suppression of SNHG3 silencing on HDGF mRNA and protein expression, validating that SNHG3 accelerates cell progression and induces apoptosis by upregulating HDGF expression via sponging miR-384 in glioma.

Conflict of interest:
The authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.