LncRNA SNHG4 promotes osteosarcoma proliferation and migration by sponging miR‐377‐3p

Abstract Background Long non‐coding RNAs (lncRNAs) have been identified as crucial regulatory factors in the occurrence and progression of osteosarcoma. Methods Quantitative real‐time polymerase chain reaction was used for detecting small nucleolar RNA host gene 4 (SNHG4) and miR‐377‐3p in osteosarcoma cells and tissues. Kaplan–Meier method was applied for evaluating the association between SNHG4 expression and the overall survival of osteosarcoma patients. CCK8, EdU, flow cytometry, and transwell assay were performed to examine the cell proliferation, apoptosis, cycle, and migration of osteosarcoma cells. Results In our study, we found that lncRNA SNHG4 was highly expressed in osteosarcoma tissues and cell lines. Additionally, the SNHG4 expression was related to distant metastasis, TNM stage, and survival of osteosarcoma patients. Through SNHG4 knockdown, the proliferation of osteosarcoma cells was considerably restrained and the cell apoptosis was induced in vivo and in vitro. Moreover, downregulated SNHG4 inhibited the cell migration and epithelial‐mesenchymal transition in HOS and MG63 cells. In mechanism, we found that SNHG4 acts as a competing endogenous RNA to sponge miR‐377‐3p, which is downregulated in osteosarcoma. Our results showed that there is a negative correlation between SNHG4 and miR‐377‐3p expression in osteosarcoma patients. Conclusion Taken together, SNHG4 promotes cell proliferation and migration by sponging miR‐377‐3p in osteosarcoma.


R E T R A C T E D
. Different degrees of lung metastasis are observed in many osteosarcoma patients upon preliminary diagnosis (Simpson & Brown, 2018;Zhou et al., 2014). In the case of any lung metastasis in osteosarcoma patients, their 5-year survival rate remains lower than 20% (Chou, Geller, & Gorlick, 2008).. The occurrence of osteosarcoma includes multiple processes and may be induced by epigenetic modification or abnormal gene expression (Biazzo & De Paolis, 2016). In recent several decades, people have strived to investigate the molecular mechanisms by which the origin of osteosarcoma is regulated. Growing evidence have indicated that long non-coding RNA (lncRNA) is pivotal in the occurrence and progression of osteosarcoma (Li, Dou, Liu, & He, 2017;Li, Yu, & Shen, 2016). However, little is known in regard to the roles of a majority of lncRNAs in osteosarcoma.
The cell proliferation of lung cancer was evidenced to be promoted by lncRNA small nucleolar RNA host gene 4 (SNHG4, HGNC: 32964) (Tang, Wu, et al., 2019). In addition, it was proposed that SHNG4 contributed to the osteosarcoma (Xu, Feng, Yu, Liu, & Lao, 2018); however, the specific mechanism remains not entirely clear.
In this study, osteosarcoma tissues showed a dramatic upregulation of SNHG4 in comparison with normal tissues. Moreover, SNHG4 expression was related to tumor severity. SNHG4 knockdown presented with significant inhibition on the proliferation and induction effect on the apoptosis in osteosarcoma cells in vivo and in vitro. In addition, the migration of HOS and MG63 cells was restrained via the downregulation of SNHG4. As for mechanical aspects, the proliferation and migration of osteosarcoma cells were adjusted by SNHG4 as a sponge of miR-377-3p. Collectively, we proved the major roles of the SNHG4/miR-377-3p axis in osteosarcoma, creating a fresh insight into the therapy of osteosarcoma.

| Ethical compliance
This study was approved by the Ethics Committee of Shaoxing Central Hospital.

| Clinical specimens and cell lines
A total of 24 osteosarcoma specimens were sampled from the Affiliated Hospital of Shaoxing Central Hospital, all of which were diagnosed with osteosarcoma by postoperative histopathological examination. Immediately after resection, primary cancer tissues and para-carcinoma tissues were quick-frozen in liquid nitrogen, followed by storage at −80°C before RNA extraction.

| RNA interference
Small interfering RNAs of SNHG4 and their negative control siRNA were acquired from Sigma-Aldrich, while miR-377-3p mimics, inhibitor, and their negative control were commercially purchased from RiboBio. As per the manufacturer, cell transfection was performed using Lipofectamine 2000 (Invitrogen).

| Cell proliferation assay
Cell Counting Kit (7 sea biotech) was applied for the detection of cell proliferation. Cell growth was performed in a 96-well plate at 1 × 10 4 /well, while cell incubation was performed in the atmosphere with 5% CO 2 at 37°C up to 70% cell confluent. After 48 hr of transfection with plasmids, another 24, 48, and 72 hr of incubation was further performed. And then each well was seeded with 10 ml of CCK8 solution. SUNRISE Microplate Reader (Tecan) was used for the measurement of absorbance at 450 nm.
For EDU assay, EDU labeled solutions (KeyGen Biotech) was used. Nuclei staining was carried out using DAPI.
For colony-formation assays, single-cell suspensions were first prepared by the trypsinization of different cell cultures and then seeded into 6-well plates at 150 cells/well for an incubation of 14 days at 37°C. For the calculation of cell number in each colony, the formed visible colonies were subjected to methanol fixation and then 0.5% crystal violet staining. We counted and recorded the colonies with more than 50 cells for statistical analysis.

| Apoptosis and cell cycle assay
On a FACSAria flow cytometer (BD Biosciences), cell apoptosis was determined by flow cytometry with Annexin V-FITC/PI apoptosis detection kit (KeyGen).
PI was used for cell staining. Meanwhile, Flow cytometer (FACScan; BD Biosciences, USA) installed with Cell Quest software (BD Biosciences) was used for analyzing the result of cell cycle.

| In vitro migration assays
For Transwell migration assays (Corning Costar), 24-well insert, and 8 mm pore polycarbonate membrane were used.
The lower chamber was added with 700 ml of media with 20% FBS, while upper insert was added with 1 × 10 5 cells re-suspended in serum-free media post-transfection. After Transwell membranes were fixed for the set time, they were subjected to crystal violet staining. A light microscope (Olympus) was used for counting the cells adhered to the lower membrane surface.
For wound healing assay, following 24 hr of transfection, a micropipette tip was employed for straight scratch in each well center. In each well, cells migrated to the scratch, which was observed to analyze cell migration. Twenty-four hours later, the speed of wound closure was measured and normalized to the length at 0 hr. Each assay was conducted in triplicate.

| Reverse transcription and realtime PCR
As instructed by the IFU, TRIzol reagent (Invitrogen) was used for the extraction of total RNA from cultured cells. Total RNAs were used for synthesizing cDNA using a PrimeScript F I G U R E 1 SNHG4 is highly expressed in osteosarcoma cell lines and tissues. (a) qRT-PCR for relative SNHG4 expression in osteosarcoma tissues and adjacent tissues (n = 24). (b) qRT-PCR for SNHG4 expression in osteosarcoma cell lines. (c) Kaplan-Meier analysis for overall survival of osteosarcoma patients stratified by SNHG4 expression based on our dataset (d) TCGA dataset. All the data are from three individual experiments and shown as mean ± SD. *p < .05, **p < .01, and ***p < .001

R E T R A C T E D
RT Reagent kit (Takara). PrimeScript miRNA cDNA Synthesis Kit (Takara) was used for the reverse transcription of MiRNA from total RNAs. SYBR Green Premix Ex Taq II (Takara) was utilized for reverse transcription and real-time PCR (RT-PCR) on Applied Biosystems Step One Plus Real-Time PCR System (Applied Biosystems). U6 was considered to be nuclear control, whereas GAPDH to be cytoplasmic control.

| In vivo assays
Athymic BALB/c nude mice (4-week old) were raised in a pathogen-free environment. HOS cells harvested were washed using PBS. Subsequently, for tumor formation assays, the ventral sides of all mice were administered with 1 × 10 7 cells by subcutaneous injection. We examined the volume of tumors every seven days and completed the calculation. The Committee on the Ethics of Animal Experiments of Affiliated Hospital of Shaoxing College of Arts and Sciences approved this protocol. Animal experiments took place in SPF Animal Laboratory at Shaoxing College of Arts and Sciences.

| Luciferase reporter assay
Cloning was performed for SNHG4 sequences containing supposed miR-377-3p binding sites which were then inserted into the pmirGLO vector (Promega). Cells were subjected to seeding into 96-well plates and then co-transfection with empty pmirGLO vector or recombinant plasmids, miR-377-3p mimics, inhibitors or negative control. Dual-Luciferase reporter assay system (Promega) was adopted for measuring luciferase activity 24 hr later. Renilla luciferase activity was utilized for the normalization of firefly luciferase activity.

| Western blot detection
Cells were lysed with RIPA buffer. An equal amount of proteins was loaded and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. Afterward, primary and proper secondary antibodies (Beyotime) were utilized to incubate the membranes at 4°C overnight subsequent to washing with TBST. All data were analyzed using ImageJ software (NIH).

| Statistical analysis
All statistical analyses were performed with GraphPad Prism and SPSS 20.0 (IBM, SPSS). For the statistical significance, the analysis of two or more groups was conducted by student's t test and one-way analysis of variance, respectively. Correlations were determined by Pearson correlation coefficient analysis. Kaplan-Meier method and log-rank test were adopted for the calculation and analysis of overall survival curves. In all analyses, for statistically significant differences, the p value was set to <.05.

| SNHG4 is highly expressed in osteosarcoma cell lines and tissues
For the determination of roles of SNHG4 in osteosarcoma tissues, qRT-PCR analysis was first conducted to measure the features of SNHG4 expression in 24 osteosarcoma tissues and 24 para-carcinoma tissues, and revealed considerable upregulation of SNHG4 expression in osteosarcoma tissues (Figure 1a). Likewise, osteosarcoma cell lines presented with increased SNHG4 expression  ( Figure 1b). Additionally, the association between SNHG4 expression and clinicopathological conditions of osteosarcoma patients was analyzed. The results indicated that patients with higher expression of SNHG4 was related to distant metastasis, lager tumor size and advanced pathological stage (Table 1). There was no correlation between SNHG4 expression and age, sex and anatomic location of osteosarcoma patients. Next, these samples were divided into SNHG4-low and SNHG4-high subgroups (the median was used as the cutoff). Kaplan-Meier analysis was used for survival determination. The survival rate was poorer in osteosarcoma patients with higher expression of SNHG4 from the results (Figure 1c). The TCGA database also confirmed this result (Figure 1d).

| SNHG4 knockdown significantly suppresses cell proliferation and induces apoptosis in osteosarcoma in vivo and in vitro
For the exploration of physiological functions of SNHG4 in osteosarcoma, SNHG4 was knocked down in MG63 and HOS cells by transfecting with si-NC and si-SNHG4 (Figure 2a). Subsequently, for impact analysis of SNHG4 knockdown on cell proliferation, CCK8, EdU, and colony-formation assays were carried out and suggested that the proliferation was notably restrained by SNHG4 knockdown (Figure 2b-d). The influence of depleted SNHG4 on cell apoptosis was also evaluated via flow cytometric analysis. Based on these results, the apoptotic ratio in MG63 and HOS cells was markedly promoted by SNHG4 knockdown (Figure 2e). In addition, flow cytometric analysis suggested that there were less MG63 and HOS cells in S phase in the SNHG4 siRNA group (Figure 2f). Tumor xenograft experiments were conducted in nude mice to assess the effects of SNHG4 on tumor growth in vivo. The volume of tumors was measured every seven days and observed that tumor growth was considerably delayed by SNHG4 knockdown in vivo (Figure 2g). Additionally, tumor weights were measured when the experiment was completed. The tumor size was notably reduced by SNHG4 knockdown based on the results (Figure 2h). Subsequently, western blot was utilized for measuring the protein levels of biomarkers associated with cell proliferation and apoptosis with tumors in nude mice. SNHG4 knockdown presented with significant inhibition effects on the expression of proliferation biomarkers (PCNA and Cyclin D1) and promotion effects on  (Figure 2i). Collectively, SNHG4 knockdown represented a significant inhibitor on cell proliferation and induction factor on cell apoptosis in vivo and in vitro.

| SNHG4 regulates osteosarcoma cell migration in vivo and in vitro
Wound healing assay and Transwell migration assays were conducted for further determination of the influences of SNHG4 on tumor migration and revealed that the migration in MG63 and HOS cells was notably damaged by SNHG4 knockdown (Figure 3a,b). Moreover, SNHG4 knockdown presented with repression effects on vimentin expression and enhancement effects on the E-Cadherin expression in MG63 and HOS cells (Figure 3c). This study then detected the protein levels of MMP9, MMP2, E-cadherin, and Snai1 proteins associated with tumor migration in xenograft tumors as indicated in Figure 2g. Tumor tissues from SNHG4depleted cells emerged with higher E-Cadherin expression and lower expression of vimentin, MMP2, Snai1, and MMP9 ( Figure 3d). Collectively, SNHG4 knockdown compromised the cell migration in vivo and in vitro.

| SNHG4 sponged miR-377-3p in osteosarcoma
There are emerging evidence that lncRNA exerts its regulatory roles as competing endogenous RNAs (ceRNA) (Zhang et al., 2016;Zhou et al., 2016). An online tool (http://mirdb. org/miRDB /index.html) was utilized for predication to further explore the molecular mechanism of the roles of SNHG4 in osteosarcoma. Furthermore, miR-377-3p was reported as a target candidate of SNHG4, with a potential binding site in SNHG4 with miR-377-3p (Figure 4a). For its validation, Dual-Luciferase reporter assays were completed and revealed that the luciferase activity in MG63 and HOS cells was notably repressed via transfection with miR-377-3p mimics ( Figure 4b). However, the luciferase activity could not be regulated by miR-377-3p mimics upon the mutation  (Figure 4b), implying that a direct binding between miR-377-3p and SNHG4. In addition, qRT-PCR indicated that miR-377-3p expression in MG63 and HOS cells considerably increased when SNHG4 was downregulated ( Figure 4c). And osteosarcoma tissues exhibited significant downregulation of miR-377-3p (n = 24) (Figure 4d). Moreover, a negative correlation was detected between the SNHG4 expression and miR-377-3p expression in osteosarcoma tissues (r = −.4283) (Figure 4e), suggesting that miR-377-3p might be a tumor inhibitor in osteosarcoma.

| SNHG4 promotes the proliferation and migration of osteosarcoma cells by sponging miR-377-3p
SNHG4 knockdown and miR-377-3p inhibition were performed simultaneously in MG63 and HOS cells to further explore whether the cell proliferation and migration in osteosarcoma were enhanced by SNHG4 via negative regulation of miR-377-3p. CCK8, EdU, colony-formation assays, and Transwell migration assays showed that the cell proliferation and migration were remarkably suppressed by SNHG4 knockdown and reversed by miR-377-3p inhibition to a certain extent (Figure 5a-d).

| Upregulating and downregulating miR-337-3p could interfere with cell apoptosis rate and cell cycle in HOS cells
To explore whether upregulating and downregulating miR-337-3p would interfere with cell apoptosis rate and cell cycle, we performed flow cytometry. The results showed that upregulating miR-337-3p markedly promoted the apoptotic ratio in HOS cells, while downregulating miR-337-3p inhibited it F I G U R E 5 SNHG4 promotes osteosarcoma cell proliferation and migration as miR-377-3p sponge. (a-c) Inhibition effects of SNHG4 knockdown on HOS and MG63 cell proliferation and partial reversion by miR-377-3p inhibition from CCK8, Colony formation, and EdU assays.

| DISCUSSION
Osteosarcoma is a highly genetically unstable tumor with poor prognosis (Du, Yang, Yang, Tian, & Zhu, 2014). Nevertheless, we know little with regard to the mechanisms of the regulation on the development of osteosarcoma. New molecular markers are required for the diagnosis and prognosis of osteosarcoma. Here, we found significant upregulation of SNHG4 in osteosarcoma. Additionally, SNHG4 was crucial for the proliferation, migration, cycle, and apoptosis of osteosarcoma cells, suggesting that SNHG4 might be a novel marker for osteosarcoma. Previous evidence have shown that lncRNAs function as ceRNA to sponge miRNAs (Militello et al., 2017;Paraskevopoulou & Hatzigeorgiou, 2016). For further exploration of the molecular mechanism underlying the regulation of SNHG4 on osteosarcoma, a predication was made to identify miR-377-3p as a candidate. The functions of miR-377-3p have been proven in some cancer cells. For instance, El Baroudi, Machiels, and Schmitz (2017) have reported that miR-377-3p acts as a prognostic biomarker in mutated TP53 squamous cells of head and neck cancer. MicroRNA-377-3p suppresses growth and invasion as a JAG1 sponge in ovarian cancer (Tang, Yang, et al., 2019). Based on the above studies, miR-377-3p may be a tumor depressor. So far, the roles of miR-377-3p in osteosarcoma are still elusive.
In the present study, miR-377-3p downregulation was observed in osteosarcoma tissues. Luciferase reporter assays verified that SNHG4 directly bond to miR-377-3p. CCK8, EdU, colony-formation assays, and Transwell migration assays were conducted for further determination whether the cell proliferation and migration were enhanced by SNHG4 through inhibiting miR-377-3p, and indicated the reverse effects of miR-377-3p inhibition on the effects on SNHG4 knockdown-induced cell proliferation and migration. This research has several deficiencies. First, larger sample size is required to further explore the clinical value of SNHG4 in osteosarcoma. Second, the downstream target of miR-337-3p in osteosarcoma should be further investigated.
Collectively, our results indicated that the oncogene SNHG4 enhanced the osteosarcoma progression via negative regulation of miR-377-3p. The SNHG4/miR-377-3p may function as a new target for osteosarcoma.