LncRNA SNHG15 contributes to proliferation, invasion and autophagy in osteosarcoma cells by sponging miR-141
© The Author(s). 2017
Received: 19 December 2016
Accepted: 12 July 2017
Published: 18 July 2017
LncRNA small nucleolar RNA host gene 15 (SNHG15) was reported to play an oncogenic role in tumors. However, the role of SNHG15 and its molecular mechanism in osteosarcoma (OS) cells are largely unknown.
qRT-PCR was performed to evaluate the expression levels of SNHG15 and miR-141 in OS tissues and cells. Cell transfection with different siRNAs, miRNAs or pcDNAs into U2OS and MG63 cells were carried out by Lipofectamine 2000. The effects of SNHG15 and miR-141 on OS cell proliferation, invasion and the levels of autophagy-related proteins were analyzed by MTT assay, Transwell invasion/migration assay and western blot, respectively. Luciferase reporter assay was used to confirm whether SNHG15 could directly interact with miR-141.
We found that up-regulation of SNHG15 was inversely correlated with miR-141 expression in OS tissues. SNHG15 knockdown and miR-141 overexpression significantly suppressed cell proliferation, invasion, migration and autophagy while SNHG15 overexpression and miR-141 repression exhibited the opposite effects on OS cells. Besides, SNHG15 could directly interact with miR-141 and regulate its expression. Furthermore, miR-141 suppressing significantly overturned the inhibition on proliferation, invasion, migration and autophagy mediated by SNHG15 knockdown while miR-141 overexpression remarkably attenuated SNHG15 overexpression-induced proliferation, invasion, migration and autophagy in OS cells.
Our data showed that SNHG15 contributes to proliferation, invasion, migration and autophagy in OS by negatively regulating miR-141, providing a new potential target and prognostic biomarker for the treatment of OS.
Osteosarcoma (OS) is the most frequent primary malignant tumor of the skeleton worldwide that predominately affects the rapid growth of bones in children and adolescents, comprising approximately 15% of all bone malignancies . The 5-year survival rate of patients diagnosed with OS has significantly increased to approximately 60–70% due to the advent of adjuvant and neoadjevant chemotherapy . However, numerous OS patients are still insensitive to chemotherapy and the overall survival rate of OS patients has reached a plateau due to local relapse or distant metastasis even after curative excision of the primary tumor and intensive chemotherapy [3, 4]. Thus, it is urgently needed to develop novel molecular therapeutic targets for OS and better understand the underlying molecular mechanism of OS pathogenesis and progression.
Recently, advances in human genome sequencing have shown that more than 90% of the human genome is extensively transcribed but only approximately 2% of it serves as protein-coding genes . The majority of the remaining transcripts are non-coding RNAs (ncRNAs) with no protein-coding capacity, including small ncRNAs, especially microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) based on their transcript size. miRNAs have been demonstrated to be involved in regulating various pathological processes, such as cellular proliferation, differentiation, migration, as well as apoptosis . It is well documented that miRNAs function as either oncogenes or tumor suppressors via specifically regulating protein-coding genes . In recent years, mounting evidences have indicated that multiple miRNAs have been identified as key epigenetic regulators in development and progression of tumors, including OS . miR-141, belonging to miR-200 family, was found to be down-regulated in OS and contributed to OS tumorigenesis [9, 10]. Overexpression of miR-141 not only inhibited osteosarcoma cell proliferation but also induced cell apoptosis . Mei et al. reported that activating the miR-200 family might have an anti-osteosarcoma effect and miR-200 family might be the targets for osteosarcoma treatment . He et al. reported that miR-141 as a tumor-suppressor and miR-141 overexpression could suppress proliferation but induced apoptosis through down-regulating H19 or miR-675 in osteosarcoma . Besides, miR-141 also served as a tumor suppressor in nasopharyngeal carcinoma , hepatocellular carcinoma , and gastric cancer .
LncRNAs are evolutionarily conserved ncRNAs with more than 200 nucleotides in length . In recent years, many reports have shown that lncRNAs play an oncogenic or tumor suppressive role in various cancers, such as breast, gastric, colorectal cancers and hepatocellular carcinoma [17–19]. Emerging evidences have demonstrated that dysregulation of lncRNAs has been implicated in multiple cellular biological processes, such as cell proliferation, apoptosis, cancer metastasis, as well as tumorigenesis [20, 21]. Furthermore, several lncRNAs were reported to be involved in OS progression and tumorigenesis . For example, Zheng et al. found that lncRNA HOTAIR, one of the well-studied lncRNAs, functioned as a carcinogenic lncRNA, promoting proliferation and inhibiting apoptosis of OS MG-63 cells in vitro . Zhao et al. reported that lncRNA HNF1A-antisense 1 (HNF1A-AS1) was markedly up-regulated and promoted OS progression by regulating the activity of the Wnt/β-catenin pathway . Li et al. showed that lncRNA urothelial carcinoma associated 1 (UCA1) was overexpressed in OS tissues and cells and contributed to OS initiation and progression . Small nucleolar RNA host gene 15 (SNHG15) is located on chromosome 7p13 but does not encode protein. Previous studies reported that overexpression of SNHG15 sensitized human cells to death in response to various environmental stresses, such as cisplatin, cycloheximide, and mercury (II) oxide and has been identified as surrogate indicators for stress response [26, 27]. Up to now, no study reported the clinical role of SNHG15 and its biological functions in OS.
Autophagy is a process in which subcellular membranes undergo dynamic morphological changes that lead to the degradation of cellular proteins and cytoplasmic organelles . Autophagy is an important cellular response to stress or starvation. Many studies have showed that autophagy played a significant role in human cancers including osteosarcoma [29, 30]. Recently, several studies revealed that miRNAs could regulate autophagy in various cancer types. For example, miR-22 could inhibit autophagy in osteosarcoma cells during chemotherapy . miR-200 family microRNAs could regulate autophagy in vitro and in vivo in osteosarcoma . Moreover, miR-141 was reported to regulate autophagy in many human diseases [32, 33]. In our study, we investigated the expressions of SNHG15 and miR-141 and their biological functions in OS, then performed bioinformatics-based target prediction analysis and found that SNHG15 contains one conserved target site of miR-141. Based on these findings, we hypothesize that SNHG15 might regulate proliferation, invasion and autophagy in osteosarcoma cells by sponging miR-141.
Patients and tumor samples
Thirty-five paired cancer tissues and the adjacent normal tissues were collected from primary OS patients underwent resection during initial surgery at the Henan Provincial People’s Hospital between 2014 and 2015. Surgically resected tissues samples were immediately frozen in liquid nitrogen and subsequently stored at −80 °C until use. The resected nodules of OS were histopathologically confirmed. The present study was performed with written informed consent from all participants for the use of their tissues.
Cell lines and cell culture
The human OS cell lines (143B, U2OS, HOS, MG63, and SaOS2) and osteoblastic cell line HFOB1.19 were all purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). These OS cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), as well as 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified chamber with 5% CO2 at 37 °C. HFOB1.19 cells were maintained in DMEM/Ham’s F-12 (1:1) medium supplemented with 10% FBS and 0.3 mg/mL G418 (Gibco) under the same conditions.
The siRNAs specifically targeting SNHG15 (si-SNHG15–1, si-SNHG15–2 and si-SNHG15–3), scrambled negative control (si-control), pcDNA-SNHG15, empty pcDNA vector (vector), miR-141 mimics (miR-141), mimics negative control (miR-control), anti-miR-141 and anti-miR-control were commercially synthesized by Genepharma (Shanghai, China). For transient transfection, U2OS and MG63 cells were plated into six-well plates (2 × 105/well) and routinely maintained for 24 h at 37 °C. Then the cells were transfected with siRNAs, pcDNA-SNHG15, vector, miR-141, miR-control, anti-miR-141, anti-miR-control, si-SNHG15 + anti-miR-141, si-SNHG15 + anti-miR-control, pcDNA-SNHG15 + miR-141, or pcDNA-SNHG15 + miR-control by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Subsequent experiments were performed at 48 h post transfection. The sequences of si-RNAs, anti-miR-141 are as follows: si-SNHG15–1 (Sense: 5′-CAG GTA GAC CGT GCA CGT AA-3′, Anti-sence:3′-CCT TGA TGC GTT GCC AGC AGA-5′), si-SNHG15–2 (Sense: 5′-CCG TGC GTA AAC GTT TGC CA-3′, Anti-sence: 3′-TGG CGG TAA CGT AAA TGC G-5′), si-SNHG15–3 (Sense: 5′-ACG GTG GCA ACG TGC GTG GCC A-3′, Anti-sence: 3′-GCC TGC AAC GGT GCA AAT GCG-5′), anti-miR-141 (CCA UCU UUA CCA GAC AGU GU UA).
Quantitative real-time PCR
Total RNA was extracted from tissues specimens and cultured cells using Trizol reagent (Invitrogen). Total RNA concentration and purity were detected by the ratio of absorbance at 260/280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (2 μg) was reversely transcribed into cDNA using the Reverse Transcription System Kit (Takara; Dalian, China). The expression levels of SNHG15 and miR-141 were determined by RT-PCR using SYBR Premix ExTaq II kit (Takara) and mirVanaTM qRT-PCR miRNA Detection Kit (Ambion, Austin, TX, USA) on an ABI 7500 PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA), respectively. The relative expression levels of SNHG15 and miR-141 were calculated by the 2-ΔΔCt method and normalized to GAPDH expression and the relative expression levels of miR-141 were normalized to U6. PCR conditions for SNHG15 were as follows: 2 min at 50 °C, 10 min at 95 °C, 45 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 45 s; PCR conditions for miR-141 was 10 min at 95 °C, 45 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 12 s. Each experiment was performed at least three times. The primer sequences used are as follows: SNHG15 (Sense: 5′-CAA CCA TAG CGG TGC AAC TGT GC-3′, Anti-sence: 3′-GGC TGA ACC AAG TTG CAA GTC ATG-5′); miR-141 (Sense: 5′-GGG CAT CTT CCA GTA CAG T-3′, Anti-sence: 3′-CAG TGC GTG TCG TGG AGT-5′); GAPDH (Sense: 5′-CAG TGC CAG CCT CGT CTA T-3′, Anti-sence: 3′-AGG GGC CAT CCA CAG TCT TC-5′); U6 (Sense: 5′-CTCGCTTCGGCAGCACATATACT-3′, Anti-sence: 3′-ACG CTT CAC GAA TTT GCG TGT C-5′).
Cell proliferation ability was evaluated by MTT assay. Briefly, transfected U2OS and MG63 cells were seeded into 96-well plates at a density of 5 × 103/well and routinely cultured for 24 h, 48 h, 72 h and 96 h, respectively. At the indicated time, 20 μL MTT regents (5 mg/mL; Sigma-Aldrich, Irvine, Ayrshire, UK) was supplemented into each well at a final MTT concentration of 0.45 mg⁄mL and incubated for another 4 h at 37 °C in a humidified chamber. Subsequently, the medium was removed and 150 μL of dimethylsulfoxide (DMSO; Sigma-Aldrich) was added to each well to dissolve the blue formazan crystals for 30 min. The absorbance value at 490 nm was recorded on the Model 680 microplate reader (Bio-Rad, Hercules, CA, USA). The measurements for each sample were performed in triplicate.
Transwell invasion and migration assays
For the invasion and migration assays, 48 h after transfection, approximately 2 × 105 U2OS or MG63 cells in 200 μL serum-free media were transferred to the upper chamber (8.0 μm; Costar, Corning, NY, USA) with a porous membrane pre-coated with (invasion) or without (migration) Matrigel solution (BD, Franklin Lakes, NJ, USA). Meanwhile, DMEM Medium (500 μL) containing 10% fetal bovine serum was added to the lower chamber as a chemoattractant. After 24 h of incubation at 37 °C, the non-invading or non-migration cells on the upper membrane were removed mechanically. Cells invading or migrating to the lower surface of the chamber were fixed in methanol, stained with 4 g/L crystal violet for 2 h. The numbers of invasive or migration cells in each well were counted from five random fields using a microscope (Olympus Corp., Tokyo, Japan). Experiments were independently performed at least three times.
Luciferase reporter assay
Online softwares including Diana Tools (http://diana.imis.athena-innovation.gr/), Starbase (http://starbase.sysu.edu.cn/) and TargetScan (http://www.targetscan.org) showed that sequences of SNHG15 and miR-141 have binding domain as showed in Fig. 4a. The mRNA sequence of SNHG15 containing the putative binding sites of miR-141 was separately inserted into the KpnI and BglII sites of pGL3-Basic (Promega, Madison, WI, USA) to form the luciferase reporter vector pGL3-SNHG15-wild-type (pGL3-SNHG15-WT, the whole SNHG15 cDNA fragment). To mutate the putative binding site of miR-141 in SNHG15 gene, the sequence of putative binding site was replaced as indicated to construct pGL3-SNHG15-mutant (pGL3-SNHG15-MUT). U2OS and MG63 cells (3 × 104) were plated in 24-well plates and cultured for overnight at 37 °C. Subsequently, cells were cotransfected with pGL3-SNHG15-WT or pGL3-SNHG15-MUT (100 ng) and miR-141 or miR-control (50 nM) by Lipofectamine 2000 (Invitrogen). Each sample was cotransfected with the pGL3-TK plasmid (Promega) containing Renilla luciferase gene to monitor transfection efficiency. After 24 h of cotransfection, the cells were collected for analyzing the luciferase activities by a Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activity of each sample was normalized to Renilla luciferase activity. The assays were independently performed three times.
Western blot analysis
Transfected U2OS and MG63 cells were harvested and lysed using a lysis buffer (Beyotime, Shanghai, China) with protease inhibitor (Sigma-Aldrich). Following incubation on ice for 30 min, cell lysates were centrifugated at 12,000 rpm for 5 min. Total protein concentrations in different samples were measured by a BCA protein assay kit (Beyotime). Equal amounts of protein (20 μg) were denatured after heating in boiling water for 10 min and separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then proteins were transferred onto polyvinylidene fluoride (PVDF) (Millipore, Billerica, MA, USA) at 100 V for 2 h at 4 °C and subsequently blocked in 5% non-fat milk for 1 h at room temperature. Membranes were immunoblotted with the indicated primary antibodies: anti-Atg5, anti-LC3-I, anti-LC3-II, anti-p62, and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washed three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody anti-IgG for 1 h at room temperature. Finally, the proteins were visualized using an enhanced chemiluminescence detection system (Millipore) and quantified by densitometry using Quantity One software. β-actin was used for the internal control.
All data were presented as the mean ± standard error (S.E.M.) from at least 3 independent experiments. Statistical significances were assessed by two-tailed Student’s t-test or one-way multivariate analysis of variance (ANOVA) in SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA). P value less than 0.05 was considered statistically significant.
SNHG15 was negatively correlated with miR-141 expression in OS tissues
SNHG15 promoted OS cell proliferation, invasion, migration and autophagy
miR-141 inhibited OS cell proliferation, invasion and autophagy
SNHG15 could directly interact with miR-141 and regulate its expression
SNHG15 promoted OS cell proliferation, invasion and autophagy in by sponging miR-141
Recently, increasing evidences have demonstrated that lncRNAs play considerable functional roles in a wide variety of physiological and pathological processes involved in tumorigenesis, invasion and metastasis in human malignant cancers . Changes in the primary structure, secondary structure and expression levels of lncRNAs are closely related to cancer formation and progression . As a newly found lncRNA, SNHG15 was reported to be up-regulated in gastric cancer cells in comparison with normal gastric tissues and positively correlated with invasion depth, advanced tumor node metastasis (TNM) stage, lymph node metastasis and poor overall survival . Besides, knockdown of SNHG15 significantly inhibited cell proliferation and invasion and promoted apoptosis, whereas forced expression of SNHG15 exhibited the opposite effects on gastric cancer cells via regulating MMP2 and MMP9 protein expression . Furthermore, SNHG15 may serve as an efficient prognostic biomarker for hepatocellular carcinoma . In our study, we found that SNHG15 was dramatically highly expressed in OS tissues and cells, suggesting that SNHG15 played a pivotal role in OS progression. The biological functions of SNHG15 in OS cells were further investigated by conducting loss or gain of function of SNHG15 assays on OS cellular processes including cell proliferation, invasion, migration and autophagy. The results showed that SNHG15 knockdown markedly suppressed proliferation, invasion, migration and autophagy, while elevated SNHG15 expression contributed to proliferation, invasion, migration and autophagy, indicating that SNHG15 played an oncogenic role in OS cells.
A growing number of studies have demonstrated that abnormal expression of miR-141 played tumor suppressive roles in many cellular processes during tumor occurrence and progression [40, 41]. For example, Peng et al. reported that miR-141 expressed lower in glioma cells and tissues and functioned as tumor suppressor by targeting TGF-β2 . Yao et al. found that miR-141 expression was significantly lower in hepatocellular carcinoma (HCC) and elevated expression of miR-141 inhibited proliferation, invasion and migration of HCC cells . In addition, it was reported that miR-141 was down-regulated in OS and played an OS-suppressing role . Consistent with previous studies, our studies implied that miR-141 was dramatically down-regulated in OS tissues and cells. Besides, we proved that miR-141 overexpression resulted in a significant inhibition on OS cell proliferation, invasion and autophagy, while miR-141 suppressing showed the opposite effects on OS cells, confirming the tumor suppressive roles in OS cells.
Besides, emerging evidences have proved that lncRNAs can sponge miRNA to completely inhibit miRNA function in OS . For example, lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was highly expressed in OS tissues and cells and promoted OS cell growth and tumor progression by inhibiting miR-376a . Besides, tumor-suppressor miR-141 overexpression suppressed osteoblastic cell proliferation by down-regulating lncRNA H19 in OS . In our study, we first disclosed that SNHG15 was inversely correlated with miR-141 expression. Notably, bioinformatics analysis and luciferase reporter system revealed that miR-141 could directly bind to SNHG15. Our study further revealed that SNHG15 negatively regulated miR-141 expression. Meanwhile, rescue experiments showed that repression of miR-141 completely reversed the inhibition of OS cell proliferation, invasion, migration and autophagy by SNHG15 knockdown. In contrast, miR-141 overexpression significantly alleviated SNHG15 overexpression-induced OS cell proliferation, invasion, migration and autophagy. Taken together, these findings suggested that SNHG15 directly interacted with miR-141 and exerted its oncogenic roles by negatively regulating miR-141.
In conclusion, our study first found that SNHG15 was up-regulated in OS tissues and cells and functioned as an oncogene in regulating OS malignancy by directing interacting with miR-141 and negatively regulating its expression. These results showed that SNHG15 may be a new potential target and prognostic biomarker for the treatment of OS.
We are immensely grateful to all the members in our lab for their discussions on this project.
There are no funding sources.
Availability of data and materials
All data and materials are available.
KL and JZ participated project design, data analysis and manuscript. YH and YL performed major experiments. All authors read and approved the final manuscript.
Ke Liu, the first author. Jia Zheng, the corresponding author.
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