Background
Osteosarcoma (OS) is the most common primary malignant bone tumor with the highest incidence in adolescents, often with early metastasis and lung metastases [
1,
2]. To date, OS patients regularly suffer from poor clinical prognosis [
3‐
5]. Therefore, it is significant to illustrate the molecular mechanism of OS to help provide new directions and methods for the treatment of OS patients.
About 70%–80% of the genome can be transcribed into RNAs in humans, but only 2%–3% of RNAs can be transcribed to encode proteins [
6]. LncRNA refers to RNA larger than 200 nucleotides in length that do not have the ability to code protein [
7,
8]. LncRNAs influence various biological processes, including chromatin organization, epigenetic regulation, gene transcription and translation, RNA turnover, and genome defense [
9,
10]. In the recent years, increasing studies have found that lncRNAs play an extremely important role in the occurrence and development of tumors [
11‐
13]. At present, a significant number of lncRNAs have been discovered to play an important role in OS. For instance, the upregulation of lncRNA MALAT1, TUG1, HULC, and SNHG12 promote the tumorigenesis of OS [
14‐
17]. Conversely, lncRNA loc285194, TUSC7, and HIF2PUT serve as tumor suppressor genes in OS [
18‐
20].
MicroRNAs (miRNAs) are small non-coding RNA molecules, generally 20–22 nucleotides long, that can mediate the inhibition of transcription and degradation of mRNA via their 3′-untranslated region (3′-UTR) [
21]. Several miRNAs have been reported as a suppressor gene or tumor-promoting gene that regulates the proliferation, migration, and invasion of tumors [
22,
23]. Existing research shows that the interaction between lncRNA and miRNA is critically important in the progression of cancer. LncRNAs have been also confirmed to regulate the progression of cancers by sponging miRNA [
24,
25]. Currently, lncRNA small nucleolar RNA host gene 5 (SNHG5) has been found to act as a tumor-promoting gene in bladder cancer and colorectal cancer [
26,
27] and a suppressor gene in gastric cancer [
28]. Moreover, SNHG5 can also modulate the progression of cancer by competitively binding to miRNA [
29]. These studies show that SNHG5 plays a significant role in a variety of different cancers [
26‐
28,
30‐
32]. However, the function of SNHG5 in osteosarcoma remains unclear, thus driving us to explore the role of SNHG5 in OS.
In the present study, our results demonstrate that the overexpression of SNHG5 can promote the migration, invasion, and proliferation of OS, whereas knockdown of SNHG5 reduces the migration, invasion, and proliferation of OS cells. Subsequently, our study found that the SNHG5/miR-212-3P/SGK3 axis is critical in the progression of OS.
Materials and methods
Cell culture
The human OS cell lines (143B, U2OS, U2R, MG63) were kindly provided by professor Kang [
33]. All of the OS cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco). 293T cells were cultured in DMEM medium supplemented with 10% FBS (BI). All cells grew in a 37 °C humidified incubator with 5% CO
2.
Cell transfection
The overexpression plasmid of SNHG5 (pcDNA-SNHG5) was constructed by inserting the full-length SNHG5 sequences into the pcDNA3.1 vector. The sequence of sh-SNHG5 was cloned into pLKO.1 vector and constructed a SNHG5 knockdown stable cell line for further experiments. Primers of sh-SNHG5 are listed in Table
1; SNHG5 siRNA, SGK3 siRNA, miRNA mimics and inhibitors were purchased from GenePharma Co. Ltd. (SuZhou, China). Lipofecmine 2000 and Lipofectamine
® RNAiMAX were used as the transfection reagents according to the manufacturer’s instructions.
Table 1
The primer sequences were used in this article
sh-SNHG5-1-F | 5′-CCGGGAGGCCAGATTGTCTTGGACTCGAGTCCA AGACAATCTGGCCTCTTTTTTGGTACC-3′ |
sh-SNHG5-1-R | 5′-AATTGGTACCAAAAAAGAGGCCAGATTGTCTTG GACTCGAGTCCAAGACAATCTGGCCTC-3′ |
sh-SNHG5-2-F | 5′-CCGGGCAACGATTTCTGGCTAGTCTCGAGACTAG CCAGAAATCGTTGCTTTTTTGGTACC-3′ |
sh-SNHG5-2-R | 5′-AATTGGTACCAAAAAAGCAACGATTTCTGGCTAG TCTCGAGACTAGCCAGAAATCGTTGC-3′ |
SNHG5-F | 5′-CGCTTGGTTAAAACCTGACACT-3′ |
SNHG5-R | 5′-CCAAGACAATCTGGCCTCTATC-3′ |
SGK3-F | 5′-CCAGGAGTGAGTCTTACAG-3′ |
SGK3-R | 5′-CCAGCCACATTAGGATTA-3′ |
β-Actin -f | 5′-GCCCTGGCACCCAGCACAAT-3′ |
β-Actin -R | 5′-GGAGGGGCCGGACTCGTCAT-3′ |
ZEB1-f | 5′-CAGGCAGATGAAGCAGGATG-3′ |
ZEB1-R | 5′-CAGCAGTGTCTTGTTGTTGTAG-3′ |
Twist-f | 5′-CCAGGTACATCGACTTCCTCTA-3′ |
Twist-R | 5′-CCATCCTCCAGACCGAGAA-3′ |
FLII-F | 5′-CCTCCTACAGCTAGCAGGTTATCAAC-3 |
FLII-R | 5′-GCATGTGCTGGATATATACCTGGCAG-3 |
BAD-F | 5′-ATGTTCCAGATCCCAGAGTTTG-3′ |
BAD-R | 5′-ATGATGGCTGCTGCTGGTT-3′ |
bcl-xl-F | 5′-GCATATCAGAGCTTTGAACAGG-3′ |
bcl-xl-R | 5′-GAAGGAGAAAAAGGCCACAATG-3′ |
bim-F | 5′-AAGGTAATCCTGAAGGCAATCA-3′ |
bim-R | 5′-CTCATAAAGATGAAAAGCGGGG-3′ |
AIP4-F | 5′-GCAGCAGTTTAACCAGAGATTC-3′ |
AIP4-R | 5′-GTGTGTTGTGGTTGACGAAATA-3′ |
GSK3-β-F | 5′-AGGAGAACCCAATGTTTCGTAT-3′ |
GSK3-β-R | 5′-ATCCCCTGGAAATATTGGTTGT-3′ |
Transwell assay
Transwell chambers were inserted in 24-well plates. Firstly, cells were washed with serum free media and treated with mitomycin c (10 µg/ml for 30 min). The upper chamber of each well was seeded with 1 × 105 cells with serum-free DMEM medium. DMEM containing 10% fetal bovine serum was added to the lower chamber. To assess cell invasion, 50 μl diluted Matrigel (BD Biosciences, Franklin Lakes, NJ) was added to the upper chamber of the transwell. The 143B, MG63, U2R and U2OS cells were allowed to migrate for 22 h and invade for 24 h. At the specified time, the cells that had migrated or invaded were fixed, stained, and counted.
MTT assay
MTT assay was used to detect the viabilities of OS cells. Cells (about 1 × 104 cells/well) were placed in 96-well plates and seeded for 24 h, 48 h, 72 h. Subsequently, 4 h before the specified time, MTT solution was added before adding DMSO to dissolve. Finally, cell viability was detected at a wavelength of 450 nm according to the manufacturer’s instructions. Each group was repeated three times to ensure accuracy of the results.
Cell spreading assay
Cells were centrifuged and resuspended, before allowing to spread on a matrigel-coated plate in a 37 °C humidified incubator with 5% CO2. After 1.5 h, cells that did not adhere were washed out with PBS, and the adherent cells were fixed, stained, and counted.
Flow cytometry
Cell apoptosis was determined using Annexin V-FITC/PI kit (Cat. no: KGA108, Keygen, China). After 48 h the transfection, OS cells were first washed with PBS and resuspended in 500 μl of 1 × binding buffer. Next, 5 μl of Annexin V-FITC and 5 μl of PI were added for 20 min in the dark at room temperature. Finally, flow cytometry (Becton–Dickinson, USA) was performed to detect the number of apoptotic cells according to the Manufacturer’s instructions.
After transfection of siSNHG5 or pcDNA-SNHG5 for 12 h, OS cells were seeded in 6-well plates at a density of 500 cells per well and maintained for 10 days. Cells were immobilized with paraformaldehyde for 20 min, stained with crystal violet for 30 min and washed with PBS for 3 times. The stained cell colonies were counted.
Wound healing assays
Wound-healing assays were performed to examine the migratory ability of cells. Firstly, cells were washed with serum free media and treated with mitomycin c (10 µg/ml for 30 min). Transfected cells were cultured in 12-well plates for 24 h. The cell monolayer was scratched using a 20-µl pipette tip, and the cells were cultured for an additional 24 h. The progression of migration was observed and photographed at 0, 12, and 24 h after wounding. The distance between the two edges of the scratch was measured and calculated.
Real-time PCR
Total RNA was extracted from OS cell lines with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, China) was used in order to obtain cDNA. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Select Master Mix for CFX (Invitrogen) and the CFX Connect Real-time PCR system (BioRad) at 95 °C for 15 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. The data were analyzed with the 2
−ΔΔCt method. All of primers are listed in Table
1.
Western blot analysis
Cells were homogenized in RIPA protein lysis buffer supplemented with protease inhibitors on ice for 30 min before centrifuging at 12,000 g for 20 min at 4 °C. The BCA protein assay kit (Pierce, Rockford, IL, USA) was used to measure protein concentration. The total protein extracts were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk for 2 h. Afterwards, the membranes were incubated overnight at 4 °C with the corresponding antibody. Subsequently, the membranes were washed with PBST three times and incubated with secondary antibodies (anti-Rabbit, anti-Mouse) for 2 h. After incubating the anti-Rabbit and anti-Mouse antibodies, the membranes were washed with PBST 3 times and then developed with ECL Western Blotting Substrate. β-actin was used as a control.
Luciferase assay
The SNHG5 3′-UTRs were constructed into pMIR-reporter plasmids. The SNHG5 and miR-212-3p mimics or NC mimics were co-transfected into 293T cells with Lipofectamine 2000 (Invitrogen), respectively. The Luciferase Reporter Assay System was used for to detecting the luciferase activity.
Statistical analysis
All of statistical analyses were executed using SPSS 16 software (SPSS, Inc, Chicago, IL, USA). The P-values were calculated using a one-way analysis of variance (ANOVA). A P-value of < 0.05 was considered to indicate a statistically significant result.
Discussion
OS is the most common malignant bone tumor in adolescents. Between 70 and 80% of patients are 10 to 25 years old, with an annual incidence of 1–3/100,000 [
46,
47]. Currently, the 5-year survival rate of OS patients is still in the range of 60% to 70% due to the unclear pathogenesis of OS [
48]. Thus, it is vital that the molecular mechanism of OS is evident to improve treatment.
Many previous studies have elaborated on the functions of lncRNA SNHG5 in detail. For example, SNHG5 promotes colorectal cancer cell survival by counteracting STAU1-mediated mRNA destabilization [
27]. SNHG5 is also associated with poor prognosis of bladder cancer, and promotes bladder cancer cell proliferation through targeting P27 [
26]. Long non-coding RNA SNHG5 also suppresses gastric cancer progression by trapping MTA2 in the cytosol [
28], in addition to being a new biomarker in malignant melanoma [
31]. These studies demonstrate the importance of SNHG5 in cancers. In our study, we found that overexpression of SNHG5 promoted OS cell growth. Yet, knockdown of SNHG5 inhibited OS cell proliferation and induced apoptosis through activating cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP. However, it has been demonstrated that mesenchymal stromal cells, from which osteosarcoma cells originate. Interestingly, Alessio et al. [
49] found cells are prone to senescence rather than apoptosis even after high exogenous stress. This is also worth further exploring. In addition, EMT plays a critical role in the migration and invasion of tumors, as it promotes tumor cell migration and invasion [
34,
35]. E-cadherin acts as an epithelial marker that is lowly expressed in tumors, whereas vimentin and β-catenin are interstitial markers highly expressed in tumors [
50]. As key proteins of EMT, upregulation of E-Cadherin and downregulation of vimentin and β-catenin are considered characteristic expressions of EMT. In the present study, western blotting revealed that the knockdown of SNHG5 increased the expression of E-cadherin and reduced the expression of Vimentin and β-catenin in OS cells. The overexpression of SNHG5 produced an opposite result.
In recent years, the competing endogenous RNA (ceRNA) hypothesis has been extensive promoted, and some studies have confirmed the interaction of lncRNA and miRNA in multiple cancers [
24,
25]. In previous studies, lncRNA SNHG5 was found to be able to interact with miRNA. For example, long non-coding RNA SNHG5 regulates gefitinib resistance in lung adenocarcinoma cells by targeting the miR-377/CASP1 axis [
32]. The lncRNA SNHG5/miR-32 axis regulates gastric cancer cell proliferation and migration by targeting KLF4 [
29], in addition to regulating imatinib resistance in chronic myeloid leukemia via MiR-205-5p [
30]. To examine whether existing miRNA can bind to lncRNA SNHG5 in OS cells, we used an online bioinformatics software, Starbase, to predict the sites of lncRNA SNHG5 and miRNA, in which miR-212-3p was chosen for further study. Interestingly, we found that researcher [
51] evaluated the expression of miR-212-3p in OS tissues by RT-qPCR with the result showing a substantial decrease of miR-212-3p expression in OS tissues compared to that in normal tissues, which was consistent with our results.
Subsequently, luciferase report assays and RT-PCR results showed that miR-212-3p can be combined with SNHG5 to reduce the activity of SNHG5. However, rescue assays revealed that miR-212-3p mimics suppress the growth and metastasis of pcDNA-SNHG5, and that its the restoration can be induced by co-transfecting the si-SNHG5-mixture with miR-212-3p inhibitors.
SGK3 has been found in others cancers as a carcinogenic gene [
42,
52‐
55]. A recent report revealed that SGK3 plays a vital role in glioblastoma as the target gene of miR-212-3p [
42]. To explore whether SGK3 could also promote the proliferation and metastasis of OS cells, we performed a series of assays to detect the function of SGK3. MTT assays showed that knockdown of SGK3 reduced the growth of OS cells. Transwell assays and western blotting indicated that knockdown of SGK3 suppressed the migration and invasion of OS cells via the EMT process.
Conclusions
Overall, our current study demonstrated SNHG5 is involved in the regulation of OS cell progression. What is more meaningful is that SNHG5 regulated the proliferation, migration, and invasion of OS cells via the miR-212-3p/SGK3 axis. Consequently, these conclusions showed that lncRNA SNHG5 plays as a critical role in OS tumorigenesis—indicating that SNHG5 may be a potential therapeutic target for osteosarcoma.
Authors’ contributions
CJ and RZ carry out most experiments of the study and drafted the manuscript. JS, FZ, KKC and XFT clone the plasmids and perform luciferase, and qRT-PCR experiments. JZ, XL and SL carried out the statistical analysis and organized the figures. ZZ and XBL designed the study and associated with all the steps of the study. All authors read and approved the final manuscript.