Long Noncoding RNA LINC01133 Functions as an miR-422a Sponge to Aggravate the Tumorigenesis of Human Osteosarcoma.

Long noncoding RNAs (lncRNAs) have been verified to participate in various types of malignant tumors, including osteosarcoma (OS), which is the most common primary bone tumor with outstanding morbidity. Although an increasing number of lncRNAs have been reported to mediate the occurrence of OS, the potential mechanisms are still unclear. This study intends to uncover the mechanism by which lncRNA LINC01133 functions as an miRNA sponge to mediate OS tumorigenicity. In this study, we found that the expression level of LINC01133 was statistically upregulated in OS tumor tissue and cell lines compared to noncancerous tissues and a normal human osteoplastic cell line. LINC01133 silencing could also observably suppress the proliferation, migration, and invasion of OS cells (HOS and U2-OS). Bioinformatics analysis predicted that LINC01133 specifically targeted miR-422a, which was validated by dual-luciferase reporter assay. Furthermore, functional experiments revealed that miR-422a played a tumor-suppressive role in OS progression and could effectively reverse the function of LINC01133. In summary, our study discovered that lncRNA LINC01133 aggravates the proliferation, migration, and invasion of OS by sponging miR-422a, which provides a novel insight in the tumorigenesis of OS.

INTRODUCTION

Among the primary malignant bone tumors, osteosarcoma (OS) is the most common, and it mainly occurs in adolescence or childhood with an incidence of nearly 3 million. OS is characterized by highly aggressive and early systemic metastasis, including approximately 10%–25% metastasis to the lung1. There are abundant differences between localized disease compared to distant metastasis. Although diagnostic methods and therapeutic strategies are progressing, effective therapeutic approaches are still lacking, and the 5-year survival rate of OS patients is still unsatisfactory. Therefore, exploration of high diagnostic and therapeutic value methods is urgently needed2.

Long noncoding RNAs (lncRNAs), composed of more than 200 nucleotides without protein-coding capacity, have become the most attractive hotspots in multiple diseases, including atherosclerosis, glioblastoma, gastric carcinoma, and OS3,4. At present, studies have uncovered the fact that lncRNAs exert a biological function in multiple diseases, particularly tumors, through mediating oncogenic occurrence and tumor-suppressing pathways (e.g., MALAT1, H19, HOTAIR)5–7. Because they lack protein translation capacity, one of the major patterns of lncRNAs that regulate biological function is as a competing endogenous RNA (ceRNA) and sponge for microRNAs (miRNAs), regulating the expression of target mRNA8,9. Studies have validated that patients having squamous cell lung cancer with a higher expression level of LINC01133 show a shorter survival time, which indicates that LINC01133 promotes oncogenesis and could serve as a potential biomarker10.

miRNAs have been identified as pivotal regulators in the occurrence and progression of OS. The mediated fields contain almost all of the functions in the tumorigenesis of OS, including proliferation, migration, epithelial–mesenchymal transition, and angiogenesis11,12. miR-451 inhibited cell proliferation, migration, and angiogenesis and promoted apoptosis of human OS cells by inhibiting migration inhibitory factor expression13. miR-193a-3p and miR-193a-5p suppress OS metastasis through the downregulation of the Rab27B and SRR genes14. miR-422a overexpression suppresses OS cell proliferation and invasion and facilitates paclitaxel- and cisplatin-mediated apoptosis15. In the tumorigenesis of OS, miRNAs have been verified as playing a vital function in physiological processes, including cell proliferation, metastasis, and apoptosis.

For OS, many of the potential functions of lncRNAs need to be discovered, and more effective therapeutic methods need to be probed. The goal of this study was to verify the functions of lncRNA LINC01133 and the lncRNA/miRNA pathway in the metastasis of OS.

MATERIALS AND METHODS

Patient Samples and RNA Extraction

A total of 27 human primary OS samples and their corresponding matched adjacent noncancerous tissue samples were obtained from the surgical resection tissue of OS patients of the Zhejiang Provincial People’s Hospital. None of the patients received any chemotherapy or radiotherapy before the surgery. All the enrolled patients were notified of the research purpose and signed informed consent. The study obtained approval and supervision from the ethics committee of Zhejiang Provincial People’s Hospital.

All tissue samples were frozen in liquid nitrogen immediately and stored at −80°C until further use. Total RNA was extracted from samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified using a NanoDrop ND-1000, and RNA integrity was assessed using standard denaturing agarose gel electrophoresis.

Osteosarcoma Cell Lines and Culture

OS cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), including MG63, Saos-2, HOS, and U2-OS. The normal human osteoplastic cell line (NHOst) and HEK-293 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, P.R. China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% glutamine at 37°C in 5% CO2.

Cell Transfection

All the involved oligonucleotides were supplied and synthesized by GenePharma (Shanghai, P.R. China), including mimics, siRNAs, and scramble sequence negative control (NC). The sequences were as follows: si-LINC01133-1, 5′-GUGAAAUCAACAGACUCAAUU-3′; si-LINC01133-2, 5′-CCACCGCCAUGAACAGAGAUU-3′; si-LINC01133-3, 5′-UAACAGAAAGACCGCUAGGUU-3′; si-miR-422a, 5′-AGCGAGGGACGUUGCUGCAGUU-3′. Cell transfections were conducted using the Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.

Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR)

Total RNAs were isolated using the TRIzol reagent (Tiangen, Beijing, P.R. China). cDNA was first synthesized with specific stem-loop primers by the isolated RNA using a SYBR Green PCR Kit (TaKaRa, Dalian, P.R. China). The involved primers were as follows: LINC01133, 5′-CTAACTAATGGGGTCAAGAGAG-3′ (forward) and 5′-AAACAGCCACTCGTCGTACTAC-3′ (reverse); miR-422a, 5′-GCATACCGCTATGCCTAATGGTG-3′ (forward) and 5′-GTGCAGGAGGTTCCGGT-3′ (reverse); U6, 5′-CGCTAGCACATATCGGCTA-3′ (forward) and 5′-TTCTGCGACGAATTTGTCAT-3′ (reverse). Amplification conditions were performed according to the manufacturer’s instructions. The relative gene expression results were quantified by normalization to U6 and expressed as fold change compared with the internal standard using the 2−ΔΔCT method.

Luciferase Assays

For luciferase assay, in order to establish pGL3-luc-LINC01133, the full-length 3′-UTR of LINC01133 and containing miR-422a binding sites was cloned into the downstream of the firefly luciferase gene in pGL3 (Invitrogen). Cells were seeded into 96-well plates at a density of 1 × 104 cells/well and then transfected with luciferase vector. Cells were transiently cotransfected with luciferase reporter plasmids (Promega, Madison, WI, USA) with Lipofectamine 2000 according to the manufacturer’s instructions. After 24 h of transfection, luciferase activity was measured using the dual-luciferase reporter gene assay kit (Promega) according to the manufacturer’s instructions. The relative firefly luciferase activities were normalized with the Renilla luciferase activities as a control for transfection efficiency.

Cell Proliferation Assay

Measurement of cell proliferation was performed with the cell counting kit-8 (CCK-8; Beyotime, Jiangsu, P.R. China). The transfected cells were seeded into 96-well plates at a density of 2,000/well. CCK-8 (20 μl) was then added after 48 h and incubated at 37°C for 2 h, and the absorbance was assessed at 450 nm.

Migration and Invasion Assays

For the migration and invasion assays, 1.0 × 105 OS cells were seeded in FBS-free DMEM into the upper chamber of a 24-well Transwell (Corning, Corning, NY, USA) with an 8-μm pore size polycarbonate membrane (Corning) according to the manufacturer’s instructions. The upper chambers were precoated with Matrigel solution (BD Biosciences, San Jose, CA, USA), and the harvested cells were resuspended in serum-free medium. After 24 h of incubation, cells in the upper chamber were removed, and the membrane was fixed with methanol and glacial acetic acid and then stained with crystal violet. All experiments were performed at least three times.

Statistical Analysis

The data are presented as the means and standard deviation (SD) values. All statistical analyses were performed with Student’s t-test, and one-way ANOVA was used to evaluate the difference. A value of p < 0.05 was considered to indicate a statistically significant result.

RESULTS

Expression of LINC01133 in OS Patients and Cell Lines

At the preliminary stage of study, we assessed the expression levels of LINC01133 in 27 human primary OS samples and their corresponding matched adjacent noncancerous tissue samples, as well as OS cell lines, using qRT-PCR. LINC01133 expression levels were significantly increased in OS tissues compared to their corresponding matched adjacent noncancerous tissues (Fig. 1A). qRT-PCR showed that the expression of LINC01133 was significantly increased in HOS and U2-OS cell lines among all the OS cell lines (MG63, Saos-2, HOS, and U2-OS) (Fig. 1B). The expression of LINC01133 indicated that it was significantly increased in both OS tissue and cell lines. Furthermore, Kaplan–Meier analysis and log-rank test were performed to assess the prognosis of OS patients. Data indicated that patients with higher LINC01133 expression had a lower overall survival and a poorer prognosis than those with lower LINC01133 expression (Fig. 1C). Our data showed that LINC01133 was authentically overexpressed in OS tissue and cell lines and suggested a poor prognosis, which powerfully proved the oncogenic and diagnostic functions of LINC01133.

Figure 1

Overexpression of LINC01133 in osteosarcoma (OS) tissues and cell lines and its clinical prognosis. (A) Relative expression of LINC01133 examined by real-time quantitative polymerase chain reaction (qRT-PCR) in OS tissues and corresponding matched adjacent noncancerous tissues. (B) LINC01133 significantly overexpressed in OS cell lines (MG63, Saos-2, HOS, and U2-OS) compared to a normal human osteoplastic cell line (hFOB1.19). (C) Kaplan–Meier analysis and log-rank test were performed to assess the prognosis of OS patients. Data are presented as the mean ± standard deviation (SD). *p < 0.05, **p < 0.01 compared to hFOB1.19 or the noncancerous group.

LINC01133 Knockdown Suppressed Osteosarcoma Cell Proliferation, Migration, and Invasion

Our study uncovered that LINC01133 was upregulated in both OS tissues and cell lines, which implied a possible cancerogenic role in the physiological process. In subsequent assays, we knocked down the expression level of LINC01133 with a specific interference nucleotide sequence to measure the effect on tumorigenicity. We entrusted GenePharma to design and synthesize three types of oligonucleotides to induce LINC01133 silencing, and the interference efficiency is shown in Figure 2A. Cell viability, measured by the CCK-8 assay, showed that oligonucleotide interference sequence could observably suppress proliferation activity (Fig. 2B). Moreover, the colony formation assay showed similar suppression of oligonucleotides on the HOS and U2-OS cell lines (Fig. 2C). The above results indicated that, among the three types of interference sequences, the third sequence had a higher interference efficiency, and thus we chose it to knock down LINC01133 expression in subsequent experiments. Migration and invasion assays manifested that LINC01133 silencing markedly inhibited migration and invasion abilities (Fig. 2D and E). Overall, our results indicate that LINC01133 silencing suppresses the proliferation, migration, and invasion of OS cells, which implies a potential carcinogenic role.

Figure 2

Regulation of LINC01133 on the proliferation, migration, and invasion of OS cells. (A) LINC01133 silencing interfered with three types of oligonucleotides, and the interference efficiency is shown. (B) Cell viability was measured by the cell counting kit-8 (CCK-8) assay. (C) Colony formation assay observed after 48 h of transfection. (D) Migration ability of HOS and U2-OS cell lines detected by Transwell assay. (E) Invasion ability of HOS and U2-OS cell lines. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 compared to the si-NC group.

LINC01133 Functions as a miR-422a Sponge in OS

Our study validated that LINC01133 silencing suppressed the malignant progression of OS cells and exerted a carcinogenic role in the regulation process. Increasing evidence has illustrated that lncRNAs act as miRNA sponges to mediate the physiology; thus, we predicted potential conjugated miRNAs, a total of 19, using bioinformatics analysis (starBase, www.starbase.sysu.edu.cn). Interfered with si-LINC01133, the variant expression levels of possible miRNAs in the HOS cell line were detected by qRT-PCR (Fig. 3A). Among these miRNAs, we focused on the most significantly upregulated miRNA, miR-422a, as our research object to probe complementary bindings. The alignment of the complementary binding of miR-422a and 3′-UTR of LINC01133 was shown and verified by dual-luciferase reporter assay (Fig. 3B and C). The expression level of miR-422a was memorably decreased in the HOS and U2-OS cell lines (Fig. 3D). Furthermore, the expression level of miR-422a in the OS tissue was similarly decreased, which was negatively associated with the expression of LINC01133 assessed by Pearson’s correlation analysis (Fig. 3E and F). In this part, we screened the potential target miRNAs and fortunately validated that LINC01133 functions as an miR-422a sponge, which provided orientation to investigate the ceRNA pathway in OS.

Figure 3

LINC01133 functions as an miR-422a sponge in OS. (A) Expression levels of 21 predicted microRNAs (miRNAs) in LINC01133 silencing of the HOS cell line detected by qRT-PCR. (B) Putative complementary sites within miR-422a and 3′-UTR of LINC01133 predicted using starBase. (C) Dual-luciferase reporter assay verified the binding of miR-422a with LINC01133. (D) The expression level of miR-422a was confirmed in the HOS and U2-OS cell lines. (E) The expression level of miR-422a in OS tissues. (F) Pearson’s correlation analysis revealed the correlation between LINC01133 and miR-422a expression in OS tissues. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 compared to the control group.

LINC01133 Regulated OS Proliferation, Migration, and Invasion by Targeting miR-422a

Our study validated that LINC01133 functions as an miR-422a sponge, so we investigated its effect on proliferation, migration, and invasion through functional trials. In the HOS cell line, miR-422a mimics and inhibitors could effectively increase or decrease its expression levels. Moreover, LINC01133 silencing could upregulate miR-422a expression, whereas the miR-422a inhibitor could reverse the upregulation (Fig. 4A). Proliferation activity and colony formation assays jointly showed that miR-422a mimics and si-LINC01133 repressed the proliferation and colony formation activities and that the miR-422a inhibitor rescued the suppression (Fig. 4B and C). Transwell assays showed LINC01133 knockdown and miR-422a mimics inhibited the migration and invasion abilities of the OS cell line, while the miR-422a inhibitor reversed the inhibition (Fig. 4D–G). The above functional experiments revealed that LINC01133 silencing suppressed OS proliferation, migration, and invasion, which indicates that LINC01133 exerts a carcinogenic role by targeting miR-422a.

Figure 4

LINC01133 regulated OS proliferation, migration, and invasion by targeting miR-422a in the HOS cell line. (A) Expression of miR-422a was measured in multiple treated groups. (B) Cell proliferation viability of HOS cells was detected by the CCK-8 assay. (C) Cell colony formation assay. (D, E) Migration capacity was detected by the Transwell assays. (F, G) Invasion ability was detected by the Transwell assays. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 compared to the control group.

DISCUSSION

The lncRNA LINC01133 has been reported to be overexpressed in squamous cell lung cancer and to participate in oncogenesis, which could serve as a potential biomarker10. Our study first uncovered that LINC01133 is likewise upregulated in OS tissues and cell lines. Moreover, we performed a series of functional experiments and validated the oncogenic role of LINC01133 in OS tumorigenesis and progression by targeting miR-422a.

OS is one of the most aggressive malignant tumors and has a very low 5-year survival rate. In addition, local advancement or metastasis and recurrence often occur in OS patients, which increasingly draws the public’s attention16,17. The poor prognosis and therapeutic efficacy strongly suggest that the exploration of more effective diagnostic and therapeutic methods is needed. An increasing number of studies reveal that lncRNAs have a close correlation with OS tumorigenicity. For example, TUG1 is identified as an oncogenic lncRNA in OS and may act as a prognostic biomarker for tumors18. Downregulation of lncRNA MEG3 is related to poor overall survival, clinical stage, and distant metastasis in OS patients19. Moreover, lncRNA BCAR4 overexpression is markedly correlated with large tumor size, advanced Enneking stage, and lung metastasis, and BCAR4 knockdown suppresses OS tumorigenesis and lung metastasis in vivo20. Therefore, lncRNAs could act as promising biomarkers in the progression and prognosis of OS.

In our study, we found that lncRNA LINC01133 is overexpressed in OS tissues and cell lines. Kaplan–Meier analysis and log-rank test showed that patients with a higher LINC01133 expression level had a lower overall survival and poorer prognosis than those with lower LINC01133 expression. Loss-of-function experiments showed that LINC01133 silencing represses the proliferation, migration, and invasion of OS cells, which implies an oncogenic role for LINC01133 in OS tumorigenesis and progression. Therefore, our study uncovered the tumor promoting effect of LINC01133 in OS, which is in accordance with existing literature10,21. LINC01133 exerts an oncogenic role in multiple tumors, including squamous cell lung cancer10, non-small cell lung cancer21, and colorectal cancer22.

lncRNAs play a critical role in many biological processes by silencing miRNAs, which is a canonical ceRNA regulating pattern. Our study indicates that LINC01133 promotes the oncogenesis of OS cell lines by directly targeting miR-422a. Accumulating evidence indicates that ceRNA regulation widely exists in the tumor pathological mechanism and plays an important role in diverse cellular processes, including proliferation, differentiation, apoptosis, and invasion23. lncRNA PVT1 promotes OS development by acting as a sponge of miR-19524. TUG1 acts as a ceRNA by sponging miR-9-5p and decreases POU2F1 expression and facilitates the tumorigenesis of OS25.

In this study, we investigated the effects of lncRNA LINC01133 and miR-422a on the tumorigenesis of OS. LINC01133 aggravates the oncogenesis of OS cell lines by sponging miR-422a. Hence, the LINC01133/miR-422a regulation pathway plays a key role in pathophysiological processes, which provides a novel insight for OS.