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FBXO22 axis
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis Xun-Kai Hou a, Jun-Sheng Mao b, * a b
Department of Orthopedic Surgery, Shandong Provincial Third Hospital, Jinan, 250031, Shandong, China Department of Orthopedic Surgery, Taishan Hospital of Shandong Province, Tai’an, 271000, Shandong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2019 Accepted 3 January 2020 Available online xxx
Long noncoding RNA small nucleolus RNA host gene 14 (SNHG14) has been shown to exert oncogenic functions in seceral cancers, but its role in osteosarcoma still unclear. In this present study, we found that SNHG14 was significantly upregulated in osteosarcoma tissues and cell lines. Knockdown SNHG14 expression significantly inhibited osteosarcoma cell proliferation through inducing apoptosis. Further functional assays revealed that SNHG14 knockdown dramatically suppressed cell migration and invasion. Bioinformatics analysis and luciferase assays identified that microRNA-433e3p (miR-433e3p) was a direct target of SNHG14, and directly targeted F-box only protein 22 (FBXO22). Mechanistic analysis demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR-433e3p to upregulate FBXO22 expression. We also observed that knockdown of FBXO22 and SNHG14 and overexpression of miR-433e3p both dramatically suppressed osteosarcoma cell proliferation, migration and invasion, but induced cell apoptosis. Moreover, the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR433e3p inhibitor. Our findings demonstrated that SNHG14 promoted osteosarcoma progression by acting as a ceRNA for miR-433e3p to regulate FBXO22 expression, suggesting that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients. © 2020 Published by Elsevier Inc.
Keywords: Osteosarcoma SNHG14 miR-433e3p/FBXO22 Proliferation Migration Invasion
1. Introduction Osteosarcoma originates from osteoid bone tissues and is prone to metastasis, which is a highly malignant bone cancer with a high mortality rate [1,2]. At present, surgical resection, and postoperative chemo-therapy are the primary therapeutic modalities for osteosarcoma patients, however, the rates of death and metastasis still maintains a high despite the effectiveness of the above approaches [3]. There have been no marked improvements in mortality of osteosarcoma patients with evidence of metastatic disease. The 5-year survival rate after recurrence and metastasis is less than 30% [4]. Therefore, it is vital to identify the underlying molecular mechanisms of osteosarcoma progression. Long noncoding RNA (lncRNA) is a type of noncoding RNA with a minimum length of 200 nucleotides [5]. Emerging evidence has observed the important regulatory effects of lncRNAs on various
* Corresponding author. Department of Orthopedic Surgery, Taishan Hospital of Shandong Province, No.3 Tianwaicun Street, Tai’an, 271000, Shandong, China. E-mail address: [email protected] (J.-S. Mao).
parameters of physiological and patho-logical processes, especially cancers [6]. Abnormal lncRNA expression can function as oncogenes or tumor suppressors in many types of cancers including osteosarcoma. For example, ITGB2-AS1 exerted oncogenic roles in osteosarcoma cells, promoted cell proliferation and metastasis by activating Wnt/b-catenin signalling [7]; NBAT1 functions as a tumor suppressor and negatively modulates growth and metastasis of osteosarcoma cells through suppression of miR-21 [8]. LncRNA small nucleolus RNA host gene 14 (SNHG14) exerts oncogenic functions in seceral cancers, such as breast cancer, non-small cell lung cancer, pancreatic cancer and colorectal cancer [9e12]. Nevertheless, its function and molecular mechanism in osteosarcoma progresssion remain unclear. Accumulating evidences have shown that certain specific lncRNAs may function as competitive endogenous RNAs (ceRNA) in tumorigenesis and development [13]. It has been demonstrated that SNHG14 plays the same role in gastric cancer and clear cell renal cell carcinoma. For instance, SNHG14 contributes to gastric cancer development through targeting miR-145/SOX9 axis [14]; SNHG14 is a critical lncRNA that promotes clear cell renal cell
https://doi.org/10.1016/j.bbrc.2020.01.016 0006-291X/© 2020 Published by Elsevier Inc.
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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carcinoma migration and invasion via sponging miR-203 and elevating N-WASP [15]. In our study, we found that SNHG14 was significantly upregulated in osteosarcoma tissues and cell lines. We demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR433e3p to upregulate FBXO22 expression. Moreover, the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR-433e3p inhibitor. Our findings suggested that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients. 2. Materials and methods 2.1. Patients and tissue samples A total of 31 pairs of osteosarcoma tissues and the adjacent noncancerous tissues were collected from Shandong Provincial Third Hospital. Ethical approval was granted from the Ethical Review Committees of Shandong Provincial Third Hospital, and written informed consent was obtained from all the patients. Matched osteosarcoma samples and adjacent non-tumor tissues were obtained during the operation and immediately stored at-80 C until use. 2.2. Cell culture Human osteosarcoma cell lines (143B, MG-63, U2OS, HOS and Saos2) and immortalized hFOB (hFOB1.19) osteoblast cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), and maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37 C with 5% CO2. 2.3. Oligonucleotides and transfection SNHG14 short hairpin RNA (shSNHG14) and FBXO22 shRNA (shFBXO22) were chemically synthesized by GenePharma (Shanghai, China). miR-433e3p mimic, miR-433e3p inhibitor and related negative controls were purchased from GenePharma (Shanghai, China). The oligonucleotides were transfected into osteosarcoma cells using Lipofectamine 3000 (Invitrogen, USA). 2.4. RNA extraction and qRT-PCR assays Total RNA was extracted in both tissues and cells using TRIZOL reagent (Invitrogen), and then was reverse transcribed into cDNA using a Reverse Transcription Kit (Takara, Dalian, China). SYBR Premix Ex Taq (Takara, Dalian, China) was used for qRT-PCR analysis, which were conducted on an Applied Biosystems 7500 RealTime PCR System. Results were normalized to GAPDH expression. For analysis the miR-433e3p expression, U6 was used for normalization. For analysis the SNHG14 and FBXO22 expression, GAPDH was used for normalization.
cell culture plate. After cultured for two weeks, the colonies on the plates were fixed in 4% paraformaldehyde, stained with 1% crystal violet, and the numbers of colonies were then counted to evaluate cell proliferation. The experiments were conducted in triplicate. 2.7. Cell apoptosis Flow cytometric methods were performed to analyze cell apoptosis in accordance with the Annexin V-FITC/PI apoptosis detection kit. Cultured cells were harvested, washed, re-suspended and then incubated with Annexin V-FITC and propidium iodide. Flow cytometry was used to detect cell apoptosis rate on a FACSAria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). 2.8. Cell invasion and migration assays The transfected 143B and Saos2 cells were harvested, and a cell suspension was prepared at 106 cells/ml, respectively. Then, the migration and invasion of these cells were measured by the QCM Laminin Migration Assay and the Cell Invasion Assay kit, respectively. Finally, the absorbance was measured at 560 nm for migration and invasion assays in accordance with the manufacturer’s protocols. 2.9. Western blotting The extracted proteins in the cell lysates were separated by electrophoresis in an SDS-polyacrylamide gel and electrophoretically transferred to PVDF membrane. After blocked with 5% blocking buffer, the membranes was incubated with primary and secondary antibodies. The protein bands were visualized by ECL Kit and imaged on a Tanon-5200 Chemiluminescent Imaging System. The antibodies against FBXO22 and b-actin were purchased from Abcam (Cambridge, MA, USA). 2.10. Luciferase reporter assay We constructed the plasmid carrying a wild-type binding site for miR-433e3p named pmirGLO-SNHG14 wild-type (WT), and the plasmid carrying mutant binding site for miR-433e3p named pmirGLO-SNHG14 mutant (MT). The Saos-2 and 143B cells were cotransfected with pmirGLO-SNHG14-WT (or pmirGLO-SNHG14MT) and either the miR-433e3p mimic or miR-NC mimic using Lipofectamine 2000. The FBXO22 30 UTR-WT and -MT were constructed and transfected into cells along miR-433e3p mimic or miR-NC mimic. Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) 48 h later, the firefly luciferase activity was measured and normalized by Renilla luciferase activity.
2.5. Cell proliferation 2.11. Statistical analysis The 143B and Saos2 cells were plated into a 96-well plate and cell proliferation was detected using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) in accordance with the manufacturer’s protocol. At 1, 2, 3 and 4 days, 10 ml CCK-8 solution was added into each well. The absorbance at 450 nm was measured using a microplate reader. 2.6. Colony formation assay Cells (5 102 cells/well) were plated in each well of a six-well
The data are expressed as the mean ± SD, and all analyses were performed using GraphPad Prism, version 5.0 (GraphPad Prism Software, GraphPad, San Diego, CA). The statistical significance of the data was evaluated by Student’s t-test or one-way ANOVA. The relationship between SNHG14 and miR-433e3p or FBXO22 expression, and between miR-433e3p and FBXO22 expression was assessed by Spearman Pearson correlation analysis. P value < 0.05 was considered as statistically significant.
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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3. Results 3.1. Knockdown of SNHG14 suppresses osteosarcoma cell proliferation, migration and invasion To investigate the role of SNHG14 in osteosarcoma, we first determined its expression level in 31 pairs of osteosarcoma tissues and the adjacent non-cancerous tissues. The highly upregulated expression of SNHG14 in osteosarcoma tissues against adjacent normal tissues was shown in Fig. 1A. Furthermore, the qRT-PCR analysis was carried out to further confirm SNHG14 expression in osteosarcoma cell lines. As expected, SNHG14 was highly expressed in 143B, MG-63, Saos-2, HOS and U2OS cell lines compared to the normal human osteoblastic cell line (HFOB1.19) (Fig. 1B). These results suggested that SNHG14 was upregulated in osteosarcoma. The Saos-2 and 143B cells were transfected with shSNHG14 and scrambled negative control (shNC) to explore the functions of SNHG14 in osteosarcoma tumorigenesis. The results showed that SNHG14 expression was significantly reduced in Saos-2 and 143B cells transfected with shSNHG14 compared with shNC (Fig. 1C). Subsequently, we carried out the CCK-8 assay to detect cell proliferation and its result observed that knockdown of SNHG14 obviously suppressed cell proliferation compared with scrambled
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negative control group (Fig. 1D). SNHG14 knockdown also markedly decreased the anchor-independent growth ability of osteosarcoma cells as seen in the colony forming assay (Fig. 1E). In addition, the flow cytometric analysis was performed to determine whether the effect of SNHG14 on osteosarcoma cell proliferation is regulated by altering cell apoptosis. The Saos-2 and 143B cells were stained with Annexin V-FITC and PI following transfection of cells with shSNHG14 and shNC. As shown in Fig. 1F and G, the apoptosis rates of Saos-2 and 143B cells were increased in shSNHG14 group compared with shNC group. Moreover, the results of transwell assays suggested that knockdown SNHG14 expression inhibited cell migration and invasion (Fig. 1H and I). These results suggested that SNHG14 knockdown inhibited osteosarcoma cell proliferation, migration and invasion.
3.2. SNHG14 functioned as a sponge for miR-433e3p in osteosarcoma cells Increasing studies indicated lncRNA could function as a sponge for miRNAs and regulate its expression and activity. Herein, the candidate miRNAs that could be inactivated by SNHG14 were predicted via starBase 2.0. The results showed that SNHG14 contains one conserved binding site for miR-433e3p (Fig. 2A). Luciferase
Fig. 1. SNHG14 knockdown inhibits osteosarcoma cell proliferation, migration and invasion. (A) Relative expression of SNHG14 in human osteosarcoma tissues and the adjacent normal tissues. (B) Relative expression of SNHG14 in human osteosarcoma cell lines (143B, MG-63, HOS, U2OS and Saos-2) and the normal human osteoblastic cell line (HFOB1.19). (C) Relative expression of SNHG14 in 143B and Saos-2 cells transfected with shSNHG14 and shNC. (D) Cell viability, (E) colony formation ability, (F, G) Cell apoptosis rate, (H) cell migration ability and (I) cell invasion ability of 143B and Saos-2 cells transfected with shSNHG14 and shNC. *P < 0.05.
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Fig. 2. SNHG14 acted as a sponge for miR-433-3p in osteosarcoma cells. (A) The WT- or MT- SNHG14 target sequences of miR-433e3p. (B) Relative luciferase activity of 143B and Saos-2 cells after cotransfection with the WT- or MT- SNHG14 reporter genes along with miR-433e3p mimic or control. (C) Relative expression of miR-433e3p in osteosarcoma tissues and the adjacent normal tissues. (D) An negative correlation between SNHG14 and miR-433e3p expression levels in osteosarcoma tissues was analyzed by Spearman’s correlation analysis. (E) Expression of miR-433e3p were measured in 143B and Saos-2 cells transfected with shSNHG14 or shSNHG14 and miR-433e3p inhibitor. (F) Cell proliferation, (G) colony formation ability, (H) Cell apoptosis rate, (I) cell migration ability and cell invasion ability of 143B and Saos-2 cells were detected in these groups. *P < 0.05 vs. miR-NC group or NC group; &P < 0.05 vs. shSNHG14 group.
reporter assays were performed to further determine the relation between SNHG14 and miR-433e3p in 143B and Saos-2 cells. miR433e3p mimic transfection-mediated upregulation of miR-433e3p obviously reduced the luciferase activity of the SNHG14-WT plasmid, and the luciferase activity of SNHG14-MT had no significant change (Fig. 2B). Additionaly, we observed a lower expression of miR-433e3p in osteosarcoma tissues and was negatively related to SNHG14 expression in the osteosarcoma tissues (Fig. 2C and D). We next detected the expression of miR-433e3p in osteosarcoma cells transfected with shSNHG14 or cotransfected with shSNHG14 and miR-433e3p inhibitor, and explored cell activities in these different groups. As shown in Fig. 2EeI, decreased SNHG14 expression dramatically suppressed osteosarcoma cell proliferation, migration and invasion, and promoted cell apoptosis, and cotransfection of miR-433e3p inhibitor abolished the shSNHG14 mediated effects on osteosarcoma cells. 3.3. SNHG14 promotes FBXO22 expression through sponging miR433-3p Nextly, we predicted that FBXO22 was a target of miR-433e3p using Targetscan 7.0 (Fig. 3A). We cotransfected the reporter plasmid containing the FBXO22 30 UTR-WT or eMT and miR-
433e3p mimic or miR-NC into143B and Saos-2 cells to measure the luciferase activity. The results showed that miR-433e3p mimic reduced the luciferase activity of the FBXO22 30 UTR-WT plasmid, and the luciferase activity of FBXO22 30 UTR-MT had no significant change (Fig. 3B). Overexpression of miR-433e3p significantly suppressed FBXO22 expression at mRNA and protein level in 143B and Saos-2 cells (Fig. 3C and D). We next determined FBXO22 expression was obviously upregulated in osteosarcoma tissues, and was negatively related to miR-433e3p expression and positively related to SNHG14 expression in osteosarcoma tissues (Fig. 3EeG). Knockdown of SNHG14 also reduced FBXO22 mRNA and protein expression in osteosarcoma cells, and this inhibition was reversed by cotransfection with miR-433e3p inhibitor (Fig. 3H and I). These results revealed that SNHG14 promotes FBXO22 expression by competitive binding SNHG14 in osteosarcoma. 3.4. SNHG14 promotes the osteosarcoma cell invasion and migration through miR-433-3p/FBXO22 axis To investigate whether the oncogenic effects of SNHG14 on the malignancy of osteosarcoma cells were mediated by its influence on the miR-433-3p-FBXO22 axis, rescue experiments were carried out. The shSNHG14, shFBXO22, miR-433e3p mimic, shSNHG14
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Fig. 3. SNHG14 promotes FBXO22 expression through sponging miR-433-3p. (A) The miR-433e3p target sequences of FBXO22 30 UTR-WT or eMT. (B) Relative luciferase activity of 143B and Saos-2 cells after cotransfection with the FBXO22 30 UTR-WT or eMT reporter genes along with miR-433e3p or miR-NC. (C, D) The mRNA and protein expression of FBXO22 were detected in osteosarcoma cells transfected with miR-433e3p or miR-NC. (E) Relative expression of miR-433e3p or miR-NC in osteosarcoma tissues and the adjacent normal tissues. (F) An negative correlation between miR-433e3p and FBXO22 expression levels in osteosarcoma tissues was analyzed by Spearman’s correlation analysis. (G) A positive correlation between SNHG14 and FBXO22 expression levels in osteosarcoma tissues was analyzed by Spearman’s correlation analysis. (H, I) The mRNA and protein expression of FBXO22 were detected in osteosarcoma cells transfected with shSNHG14 or both shSNHG14 and miR-433e3p inhibitor. *P < 0.05 vs. miR-NC group or control group; & P < 0.05 vs. shSNHG14 group.
together with miR-433e3p inhibitor were transfected into 143B and Saos-2 cells, and FBXO22 expression were detected by western blotting (Fig. 4A). We found that knockdown of FBXO22 and SNHG14 and overexpression of miR-433e3p both dramatically suppressed osteosarcoma cell proliferation, migration and invasion, but induced cell apoptosis (Fig. 4BeF). Moreover, the suppresive effect of SNHG14 knockdown on the cell proliferation, invasion and migration of osteosarcoma cells was attenuated by miR-433e3p inhibitor (Fig. 4BeF). All data demonstrated that SNHG14 modulates osteosarcoma cell proliferaion, migration and invasion by sponging miR-433e3p to promote FBXO22 expression. 4. Discussion A few lncRNAs involved in the malignant progression of osteosarcoma have been identified. A identified lncRNA SNHG14 has been shown to be upregulated in some malignant tumors. SNHG14 facilitates hepatocellular carcinoma progression through regulating miR-4673/SOCS1 [16]; SNHG14 was upregulated in diffuse large B cell lymphoma and its depletion retarded proliferation, migration and epithelial-to-mesenchymal transition [17]; SNHG14 facilitates
colorectal cancer metastasis through targeting EZH2-regulated EPHA7 [12]. Although some studies have reported that SNHG14 is upregulated in several cancers, but the function and mechanism of SNHG14 in osteosarcoma remain unknown. In our study, we found that SNHG14 was upregulated in osteosarcoma tissues and cell lines. Knockdown SNHG14 expression suppressed osteosarcoma cell proliferation, migration and invasion, and induced cell apoptosis. A number of studies have shown that lncRNAs may function as ceRNAs in carcinogenesis. ceRNAs can sponge miRNAs and reduce the binding of miRNAs to their target genes, thereby regulating gene expression [18]. SNHG14 has been identified as miRNA ceRNA in some tumors [16,19]. Our study, bioinformatics analysis observed that SNHG14, miR-433e3p and FBXO22 have underlying correlation in osteosarcoma. The results of luciferase reporter assay demonstrated that SNHG14 directly binds to miR-433e3p. Emerging evidence has shown that miR-433e3p is downregulated and plays a role of tumor suppressor in many tumors. For example, miR-433 inhibits cervical cancer progression by directly targeting metadherin to regulate the AKT and b-catenin signalling pathways [20]; miR-433 represses proliferation and invasion of colon cancer
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Fig. 4. SNHG14 promotes the osteosarcoma cell invasion and migration through miR-433-3p/FBXO22 axis. (A) The shSNHG14, shFBXO22, miR-433e3p mimic, shSNHG14 together with miR-433e3p inhibitor were transfected into 143B and Saos-2 cells, and FBXO22 expression were detected by western blotting. (BeF) Cell proliferation, colony formation ability, cell apoptosis rate, cell migration ability and cell invasion ability of 143B and Saos-2 cells were detected in these groups. *P < 0.05 vs. control group; &P < 0.05 vs. shSNHG14 group.
cells by targeting Homeobox A1 [21]; miR-433 inhibits breast cancer cell growth via the MAPK signaling pathway by targeting Rap1a [22]. In this study, we determined miR-433e3p expression was downregulated in osteosarcoma tissues, and was negatively related to SNHG14 expression. We also demonstrated that the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR433e3p inhibitor. We also found that FBXO22 is a direct target of miR-433e3p in osteosarcoma. FBXO22, an F-box E3 ligase, is a member of the F-box protein family, which has been reportedly to be upregulated in some malignant tumors [23]. For example, Zhao et al. observed that FBXO22 mediates polyubiquitination and inactivation of LKB1 to promote lung cancer cell growth [24]; Zheng et al. reported that knockdown of FBXO22 inhibits melanoma cell migration, invasion and angiogenesis via the HIF-1a/VEGF pathway [25]; Zhang et al. showed that FBXO22 promotes the development of hepatocellular carcinoma by regulating the ubiquitination and degradation of p21 [26]; Sun et al. reported that FBXO22 was upregulated in primary breast tumors and promoted cell proliferation and colony formation and xenograft tumorigenicity [27]. In our study, We determined FBXO22 expression was upregulated in osteosarcoma tissues, and was negatively related to miR-433e3p expression and positively related to SNHG14 expression in osteosarcoma tissues. Knockdown SNHG14 decreased the FBXO22 mRNA and protein levels, and this inhibitory effect was reversed by miR-433e3p inhibitor. These results revealed that SNHG14 promotes FBXO22 expression by competitive binding miR-433e3p in osteosarcoma. In conclusion, we found that SNHG14 was upregulated in osteosarcoma tissues and cell lines. We demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR-433e3p to upregulate FBXO22 expression. These results suggested that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients.
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Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis Xun-Kai Hou a, Jun-Sheng Mao b, * a b
Department of Orthopedic Surgery, Shandong Provincial Third Hospital, Jinan, 250031, Shandong, China Department of Orthopedic Surgery, Taishan Hospital of Shandong Province, Tai’an, 271000, Shandong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2019 Accepted 3 January 2020 Available online xxx
Long noncoding RNA small nucleolus RNA host gene 14 (SNHG14) has been shown to exert oncogenic functions in seceral cancers, but its role in osteosarcoma still unclear. In this present study, we found that SNHG14 was significantly upregulated in osteosarcoma tissues and cell lines. Knockdown SNHG14 expression significantly inhibited osteosarcoma cell proliferation through inducing apoptosis. Further functional assays revealed that SNHG14 knockdown dramatically suppressed cell migration and invasion. Bioinformatics analysis and luciferase assays identified that microRNA-433e3p (miR-433e3p) was a direct target of SNHG14, and directly targeted F-box only protein 22 (FBXO22). Mechanistic analysis demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR-433e3p to upregulate FBXO22 expression. We also observed that knockdown of FBXO22 and SNHG14 and overexpression of miR-433e3p both dramatically suppressed osteosarcoma cell proliferation, migration and invasion, but induced cell apoptosis. Moreover, the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR433e3p inhibitor. Our findings demonstrated that SNHG14 promoted osteosarcoma progression by acting as a ceRNA for miR-433e3p to regulate FBXO22 expression, suggesting that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients. © 2020 Published by Elsevier Inc.
Keywords: Osteosarcoma SNHG14 miR-433e3p/FBXO22 Proliferation Migration Invasion
1. Introduction Osteosarcoma originates from osteoid bone tissues and is prone to metastasis, which is a highly malignant bone cancer with a high mortality rate [1,2]. At present, surgical resection, and postoperative chemo-therapy are the primary therapeutic modalities for osteosarcoma patients, however, the rates of death and metastasis still maintains a high despite the effectiveness of the above approaches [3]. There have been no marked improvements in mortality of osteosarcoma patients with evidence of metastatic disease. The 5-year survival rate after recurrence and metastasis is less than 30% [4]. Therefore, it is vital to identify the underlying molecular mechanisms of osteosarcoma progression. Long noncoding RNA (lncRNA) is a type of noncoding RNA with a minimum length of 200 nucleotides [5]. Emerging evidence has observed the important regulatory effects of lncRNAs on various
* Corresponding author. Department of Orthopedic Surgery, Taishan Hospital of Shandong Province, No.3 Tianwaicun Street, Tai’an, 271000, Shandong, China. E-mail address: [email protected] (J.-S. Mao).
parameters of physiological and patho-logical processes, especially cancers [6]. Abnormal lncRNA expression can function as oncogenes or tumor suppressors in many types of cancers including osteosarcoma. For example, ITGB2-AS1 exerted oncogenic roles in osteosarcoma cells, promoted cell proliferation and metastasis by activating Wnt/b-catenin signalling [7]; NBAT1 functions as a tumor suppressor and negatively modulates growth and metastasis of osteosarcoma cells through suppression of miR-21 [8]. LncRNA small nucleolus RNA host gene 14 (SNHG14) exerts oncogenic functions in seceral cancers, such as breast cancer, non-small cell lung cancer, pancreatic cancer and colorectal cancer [9e12]. Nevertheless, its function and molecular mechanism in osteosarcoma progresssion remain unclear. Accumulating evidences have shown that certain specific lncRNAs may function as competitive endogenous RNAs (ceRNA) in tumorigenesis and development [13]. It has been demonstrated that SNHG14 plays the same role in gastric cancer and clear cell renal cell carcinoma. For instance, SNHG14 contributes to gastric cancer development through targeting miR-145/SOX9 axis [14]; SNHG14 is a critical lncRNA that promotes clear cell renal cell
https://doi.org/10.1016/j.bbrc.2020.01.016 0006-291X/© 2020 Published by Elsevier Inc.
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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carcinoma migration and invasion via sponging miR-203 and elevating N-WASP [15]. In our study, we found that SNHG14 was significantly upregulated in osteosarcoma tissues and cell lines. We demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR433e3p to upregulate FBXO22 expression. Moreover, the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR-433e3p inhibitor. Our findings suggested that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients. 2. Materials and methods 2.1. Patients and tissue samples A total of 31 pairs of osteosarcoma tissues and the adjacent noncancerous tissues were collected from Shandong Provincial Third Hospital. Ethical approval was granted from the Ethical Review Committees of Shandong Provincial Third Hospital, and written informed consent was obtained from all the patients. Matched osteosarcoma samples and adjacent non-tumor tissues were obtained during the operation and immediately stored at-80 C until use. 2.2. Cell culture Human osteosarcoma cell lines (143B, MG-63, U2OS, HOS and Saos2) and immortalized hFOB (hFOB1.19) osteoblast cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), and maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37 C with 5% CO2. 2.3. Oligonucleotides and transfection SNHG14 short hairpin RNA (shSNHG14) and FBXO22 shRNA (shFBXO22) were chemically synthesized by GenePharma (Shanghai, China). miR-433e3p mimic, miR-433e3p inhibitor and related negative controls were purchased from GenePharma (Shanghai, China). The oligonucleotides were transfected into osteosarcoma cells using Lipofectamine 3000 (Invitrogen, USA). 2.4. RNA extraction and qRT-PCR assays Total RNA was extracted in both tissues and cells using TRIZOL reagent (Invitrogen), and then was reverse transcribed into cDNA using a Reverse Transcription Kit (Takara, Dalian, China). SYBR Premix Ex Taq (Takara, Dalian, China) was used for qRT-PCR analysis, which were conducted on an Applied Biosystems 7500 RealTime PCR System. Results were normalized to GAPDH expression. For analysis the miR-433e3p expression, U6 was used for normalization. For analysis the SNHG14 and FBXO22 expression, GAPDH was used for normalization.
cell culture plate. After cultured for two weeks, the colonies on the plates were fixed in 4% paraformaldehyde, stained with 1% crystal violet, and the numbers of colonies were then counted to evaluate cell proliferation. The experiments were conducted in triplicate. 2.7. Cell apoptosis Flow cytometric methods were performed to analyze cell apoptosis in accordance with the Annexin V-FITC/PI apoptosis detection kit. Cultured cells were harvested, washed, re-suspended and then incubated with Annexin V-FITC and propidium iodide. Flow cytometry was used to detect cell apoptosis rate on a FACSAria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). 2.8. Cell invasion and migration assays The transfected 143B and Saos2 cells were harvested, and a cell suspension was prepared at 106 cells/ml, respectively. Then, the migration and invasion of these cells were measured by the QCM Laminin Migration Assay and the Cell Invasion Assay kit, respectively. Finally, the absorbance was measured at 560 nm for migration and invasion assays in accordance with the manufacturer’s protocols. 2.9. Western blotting The extracted proteins in the cell lysates were separated by electrophoresis in an SDS-polyacrylamide gel and electrophoretically transferred to PVDF membrane. After blocked with 5% blocking buffer, the membranes was incubated with primary and secondary antibodies. The protein bands were visualized by ECL Kit and imaged on a Tanon-5200 Chemiluminescent Imaging System. The antibodies against FBXO22 and b-actin were purchased from Abcam (Cambridge, MA, USA). 2.10. Luciferase reporter assay We constructed the plasmid carrying a wild-type binding site for miR-433e3p named pmirGLO-SNHG14 wild-type (WT), and the plasmid carrying mutant binding site for miR-433e3p named pmirGLO-SNHG14 mutant (MT). The Saos-2 and 143B cells were cotransfected with pmirGLO-SNHG14-WT (or pmirGLO-SNHG14MT) and either the miR-433e3p mimic or miR-NC mimic using Lipofectamine 2000. The FBXO22 30 UTR-WT and -MT were constructed and transfected into cells along miR-433e3p mimic or miR-NC mimic. Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) 48 h later, the firefly luciferase activity was measured and normalized by Renilla luciferase activity.
2.5. Cell proliferation 2.11. Statistical analysis The 143B and Saos2 cells were plated into a 96-well plate and cell proliferation was detected using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) in accordance with the manufacturer’s protocol. At 1, 2, 3 and 4 days, 10 ml CCK-8 solution was added into each well. The absorbance at 450 nm was measured using a microplate reader. 2.6. Colony formation assay Cells (5 102 cells/well) were plated in each well of a six-well
The data are expressed as the mean ± SD, and all analyses were performed using GraphPad Prism, version 5.0 (GraphPad Prism Software, GraphPad, San Diego, CA). The statistical significance of the data was evaluated by Student’s t-test or one-way ANOVA. The relationship between SNHG14 and miR-433e3p or FBXO22 expression, and between miR-433e3p and FBXO22 expression was assessed by Spearman Pearson correlation analysis. P value < 0.05 was considered as statistically significant.
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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3. Results 3.1. Knockdown of SNHG14 suppresses osteosarcoma cell proliferation, migration and invasion To investigate the role of SNHG14 in osteosarcoma, we first determined its expression level in 31 pairs of osteosarcoma tissues and the adjacent non-cancerous tissues. The highly upregulated expression of SNHG14 in osteosarcoma tissues against adjacent normal tissues was shown in Fig. 1A. Furthermore, the qRT-PCR analysis was carried out to further confirm SNHG14 expression in osteosarcoma cell lines. As expected, SNHG14 was highly expressed in 143B, MG-63, Saos-2, HOS and U2OS cell lines compared to the normal human osteoblastic cell line (HFOB1.19) (Fig. 1B). These results suggested that SNHG14 was upregulated in osteosarcoma. The Saos-2 and 143B cells were transfected with shSNHG14 and scrambled negative control (shNC) to explore the functions of SNHG14 in osteosarcoma tumorigenesis. The results showed that SNHG14 expression was significantly reduced in Saos-2 and 143B cells transfected with shSNHG14 compared with shNC (Fig. 1C). Subsequently, we carried out the CCK-8 assay to detect cell proliferation and its result observed that knockdown of SNHG14 obviously suppressed cell proliferation compared with scrambled
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negative control group (Fig. 1D). SNHG14 knockdown also markedly decreased the anchor-independent growth ability of osteosarcoma cells as seen in the colony forming assay (Fig. 1E). In addition, the flow cytometric analysis was performed to determine whether the effect of SNHG14 on osteosarcoma cell proliferation is regulated by altering cell apoptosis. The Saos-2 and 143B cells were stained with Annexin V-FITC and PI following transfection of cells with shSNHG14 and shNC. As shown in Fig. 1F and G, the apoptosis rates of Saos-2 and 143B cells were increased in shSNHG14 group compared with shNC group. Moreover, the results of transwell assays suggested that knockdown SNHG14 expression inhibited cell migration and invasion (Fig. 1H and I). These results suggested that SNHG14 knockdown inhibited osteosarcoma cell proliferation, migration and invasion.
3.2. SNHG14 functioned as a sponge for miR-433e3p in osteosarcoma cells Increasing studies indicated lncRNA could function as a sponge for miRNAs and regulate its expression and activity. Herein, the candidate miRNAs that could be inactivated by SNHG14 were predicted via starBase 2.0. The results showed that SNHG14 contains one conserved binding site for miR-433e3p (Fig. 2A). Luciferase
Fig. 1. SNHG14 knockdown inhibits osteosarcoma cell proliferation, migration and invasion. (A) Relative expression of SNHG14 in human osteosarcoma tissues and the adjacent normal tissues. (B) Relative expression of SNHG14 in human osteosarcoma cell lines (143B, MG-63, HOS, U2OS and Saos-2) and the normal human osteoblastic cell line (HFOB1.19). (C) Relative expression of SNHG14 in 143B and Saos-2 cells transfected with shSNHG14 and shNC. (D) Cell viability, (E) colony formation ability, (F, G) Cell apoptosis rate, (H) cell migration ability and (I) cell invasion ability of 143B and Saos-2 cells transfected with shSNHG14 and shNC. *P < 0.05.
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Fig. 2. SNHG14 acted as a sponge for miR-433-3p in osteosarcoma cells. (A) The WT- or MT- SNHG14 target sequences of miR-433e3p. (B) Relative luciferase activity of 143B and Saos-2 cells after cotransfection with the WT- or MT- SNHG14 reporter genes along with miR-433e3p mimic or control. (C) Relative expression of miR-433e3p in osteosarcoma tissues and the adjacent normal tissues. (D) An negative correlation between SNHG14 and miR-433e3p expression levels in osteosarcoma tissues was analyzed by Spearman’s correlation analysis. (E) Expression of miR-433e3p were measured in 143B and Saos-2 cells transfected with shSNHG14 or shSNHG14 and miR-433e3p inhibitor. (F) Cell proliferation, (G) colony formation ability, (H) Cell apoptosis rate, (I) cell migration ability and cell invasion ability of 143B and Saos-2 cells were detected in these groups. *P < 0.05 vs. miR-NC group or NC group; &P < 0.05 vs. shSNHG14 group.
reporter assays were performed to further determine the relation between SNHG14 and miR-433e3p in 143B and Saos-2 cells. miR433e3p mimic transfection-mediated upregulation of miR-433e3p obviously reduced the luciferase activity of the SNHG14-WT plasmid, and the luciferase activity of SNHG14-MT had no significant change (Fig. 2B). Additionaly, we observed a lower expression of miR-433e3p in osteosarcoma tissues and was negatively related to SNHG14 expression in the osteosarcoma tissues (Fig. 2C and D). We next detected the expression of miR-433e3p in osteosarcoma cells transfected with shSNHG14 or cotransfected with shSNHG14 and miR-433e3p inhibitor, and explored cell activities in these different groups. As shown in Fig. 2EeI, decreased SNHG14 expression dramatically suppressed osteosarcoma cell proliferation, migration and invasion, and promoted cell apoptosis, and cotransfection of miR-433e3p inhibitor abolished the shSNHG14 mediated effects on osteosarcoma cells. 3.3. SNHG14 promotes FBXO22 expression through sponging miR433-3p Nextly, we predicted that FBXO22 was a target of miR-433e3p using Targetscan 7.0 (Fig. 3A). We cotransfected the reporter plasmid containing the FBXO22 30 UTR-WT or eMT and miR-
433e3p mimic or miR-NC into143B and Saos-2 cells to measure the luciferase activity. The results showed that miR-433e3p mimic reduced the luciferase activity of the FBXO22 30 UTR-WT plasmid, and the luciferase activity of FBXO22 30 UTR-MT had no significant change (Fig. 3B). Overexpression of miR-433e3p significantly suppressed FBXO22 expression at mRNA and protein level in 143B and Saos-2 cells (Fig. 3C and D). We next determined FBXO22 expression was obviously upregulated in osteosarcoma tissues, and was negatively related to miR-433e3p expression and positively related to SNHG14 expression in osteosarcoma tissues (Fig. 3EeG). Knockdown of SNHG14 also reduced FBXO22 mRNA and protein expression in osteosarcoma cells, and this inhibition was reversed by cotransfection with miR-433e3p inhibitor (Fig. 3H and I). These results revealed that SNHG14 promotes FBXO22 expression by competitive binding SNHG14 in osteosarcoma. 3.4. SNHG14 promotes the osteosarcoma cell invasion and migration through miR-433-3p/FBXO22 axis To investigate whether the oncogenic effects of SNHG14 on the malignancy of osteosarcoma cells were mediated by its influence on the miR-433-3p-FBXO22 axis, rescue experiments were carried out. The shSNHG14, shFBXO22, miR-433e3p mimic, shSNHG14
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Fig. 3. SNHG14 promotes FBXO22 expression through sponging miR-433-3p. (A) The miR-433e3p target sequences of FBXO22 30 UTR-WT or eMT. (B) Relative luciferase activity of 143B and Saos-2 cells after cotransfection with the FBXO22 30 UTR-WT or eMT reporter genes along with miR-433e3p or miR-NC. (C, D) The mRNA and protein expression of FBXO22 were detected in osteosarcoma cells transfected with miR-433e3p or miR-NC. (E) Relative expression of miR-433e3p or miR-NC in osteosarcoma tissues and the adjacent normal tissues. (F) An negative correlation between miR-433e3p and FBXO22 expression levels in osteosarcoma tissues was analyzed by Spearman’s correlation analysis. (G) A positive correlation between SNHG14 and FBXO22 expression levels in osteosarcoma tissues was analyzed by Spearman’s correlation analysis. (H, I) The mRNA and protein expression of FBXO22 were detected in osteosarcoma cells transfected with shSNHG14 or both shSNHG14 and miR-433e3p inhibitor. *P < 0.05 vs. miR-NC group or control group; & P < 0.05 vs. shSNHG14 group.
together with miR-433e3p inhibitor were transfected into 143B and Saos-2 cells, and FBXO22 expression were detected by western blotting (Fig. 4A). We found that knockdown of FBXO22 and SNHG14 and overexpression of miR-433e3p both dramatically suppressed osteosarcoma cell proliferation, migration and invasion, but induced cell apoptosis (Fig. 4BeF). Moreover, the suppresive effect of SNHG14 knockdown on the cell proliferation, invasion and migration of osteosarcoma cells was attenuated by miR-433e3p inhibitor (Fig. 4BeF). All data demonstrated that SNHG14 modulates osteosarcoma cell proliferaion, migration and invasion by sponging miR-433e3p to promote FBXO22 expression. 4. Discussion A few lncRNAs involved in the malignant progression of osteosarcoma have been identified. A identified lncRNA SNHG14 has been shown to be upregulated in some malignant tumors. SNHG14 facilitates hepatocellular carcinoma progression through regulating miR-4673/SOCS1 [16]; SNHG14 was upregulated in diffuse large B cell lymphoma and its depletion retarded proliferation, migration and epithelial-to-mesenchymal transition [17]; SNHG14 facilitates
colorectal cancer metastasis through targeting EZH2-regulated EPHA7 [12]. Although some studies have reported that SNHG14 is upregulated in several cancers, but the function and mechanism of SNHG14 in osteosarcoma remain unknown. In our study, we found that SNHG14 was upregulated in osteosarcoma tissues and cell lines. Knockdown SNHG14 expression suppressed osteosarcoma cell proliferation, migration and invasion, and induced cell apoptosis. A number of studies have shown that lncRNAs may function as ceRNAs in carcinogenesis. ceRNAs can sponge miRNAs and reduce the binding of miRNAs to their target genes, thereby regulating gene expression [18]. SNHG14 has been identified as miRNA ceRNA in some tumors [16,19]. Our study, bioinformatics analysis observed that SNHG14, miR-433e3p and FBXO22 have underlying correlation in osteosarcoma. The results of luciferase reporter assay demonstrated that SNHG14 directly binds to miR-433e3p. Emerging evidence has shown that miR-433e3p is downregulated and plays a role of tumor suppressor in many tumors. For example, miR-433 inhibits cervical cancer progression by directly targeting metadherin to regulate the AKT and b-catenin signalling pathways [20]; miR-433 represses proliferation and invasion of colon cancer
Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Fig. 4. SNHG14 promotes the osteosarcoma cell invasion and migration through miR-433-3p/FBXO22 axis. (A) The shSNHG14, shFBXO22, miR-433e3p mimic, shSNHG14 together with miR-433e3p inhibitor were transfected into 143B and Saos-2 cells, and FBXO22 expression were detected by western blotting. (BeF) Cell proliferation, colony formation ability, cell apoptosis rate, cell migration ability and cell invasion ability of 143B and Saos-2 cells were detected in these groups. *P < 0.05 vs. control group; &P < 0.05 vs. shSNHG14 group.
cells by targeting Homeobox A1 [21]; miR-433 inhibits breast cancer cell growth via the MAPK signaling pathway by targeting Rap1a [22]. In this study, we determined miR-433e3p expression was downregulated in osteosarcoma tissues, and was negatively related to SNHG14 expression. We also demonstrated that the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR433e3p inhibitor. We also found that FBXO22 is a direct target of miR-433e3p in osteosarcoma. FBXO22, an F-box E3 ligase, is a member of the F-box protein family, which has been reportedly to be upregulated in some malignant tumors [23]. For example, Zhao et al. observed that FBXO22 mediates polyubiquitination and inactivation of LKB1 to promote lung cancer cell growth [24]; Zheng et al. reported that knockdown of FBXO22 inhibits melanoma cell migration, invasion and angiogenesis via the HIF-1a/VEGF pathway [25]; Zhang et al. showed that FBXO22 promotes the development of hepatocellular carcinoma by regulating the ubiquitination and degradation of p21 [26]; Sun et al. reported that FBXO22 was upregulated in primary breast tumors and promoted cell proliferation and colony formation and xenograft tumorigenicity [27]. In our study, We determined FBXO22 expression was upregulated in osteosarcoma tissues, and was negatively related to miR-433e3p expression and positively related to SNHG14 expression in osteosarcoma tissues. Knockdown SNHG14 decreased the FBXO22 mRNA and protein levels, and this inhibitory effect was reversed by miR-433e3p inhibitor. These results revealed that SNHG14 promotes FBXO22 expression by competitive binding miR-433e3p in osteosarcoma. In conclusion, we found that SNHG14 was upregulated in osteosarcoma tissues and cell lines. We demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR-433e3p to upregulate FBXO22 expression. These results suggested that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients.
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Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016
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Please cite this article as: X.-K. Hou, J.-S. Mao, Long noncoding RNA SNHG14 promotes osteosarcoma progression via miR-433-3p/FBXO22 axis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.016