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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 5  |  Page : 68-71

Inhibitory roles of miR-320 in osteosarcoma via regulating E2F1


Department of Orthopedics, Henan University Huaihe Hospital, Kaifeng 475000, Henan, P.R. China

Date of Web Publication7-Oct-2016

Correspondence Address:
Xiao Wang
Department of Orthopedics, Henan University Huaihe Hospital, Kaifeng 475000, Henan
P.R. China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.191635

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 > Abstract 


Objectives: Osteosarcoma (OsC) is the most common primary bone malignant tumor with lower incidence and high degree of malignancy, but the exact mechanism remains unknown. More evidence demonstrated microRNAs (miRNAs) could contribute to tumor progression. In this study, we investigated the expression and functions of miR-320 in OsC cells.
Materials and Methods: miR-320 expression levels in several human OsC cell lines and human normal osteoblastic cell line were tested by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). U2OS cells were transfected with miR-320 mimics or negative control oligos. MTT assay and cell flow cytometry assay by PI staining were performed to access the cell growth rate. Bioinformatic prediction and luciferase assays were used to identify the predicted target E2F1. qRT-PCR and Western blot were performed to access the molecular alteration of E2F1.
Results: miR-320 was decreased in human OsC cell lines. Heterogeneous expression of miR-320 inhibited cell proliferation and induced cell cycle arrest. Besides, we proved that miR-320 could directly regulate the expression of E2F1 in U2OS cells.
Conclusion: These data suggested that miR-320 regulates the proliferation and cell cycle by targeting E2F1 in human OsC progression.

Keywords: Cell cycle, E2F1, miR-320, osteosarcoma, proliferation


How to cite this article:
Wu H, Li W, Zhang M, Zhu S, Zhang D, Wang X. Inhibitory roles of miR-320 in osteosarcoma via regulating E2F1. J Can Res Ther 2016;12:68-71

How to cite this URL:
Wu H, Li W, Zhang M, Zhu S, Zhang D, Wang X. Inhibitory roles of miR-320 in osteosarcoma via regulating E2F1. J Can Res Ther [serial online] 2016 [cited 2018 May 22];12:68-71. Available from: http://www.cancerjournal.net/text.asp?2016/12/5/68/191635




 > Introduction Top


Osteosarcoma (OsC) is the most frequent type of primary bone tumor in children and adolescents, accounts for approximately 20% of all primary bone cancers.[1],[2] Currently, the incidence of OsC is around 0.2–3/100,000 per year, which is even higher in the age group of 15–19 years (0.8–11/100,000 per year); however, there is a second incidence peak among individuals aged >60 years.[3] Despite several improvements had been made in the curative protocols, OsC remains as a devastating disease with poor early diagnosis and low long-term survival rate. Approximately 35% of patients will die within 5 years.[4],[5] Hence, future comprehensive studies on abnormal gene regulation networks are urgent and essential for deeply elucidating the mechanisms governing the development and progression of OsC, as well as for designing novel treatment methods of this disease in a long run.

MicroRNAs (miRNAs) are a subtype of small noncoding RNA molecules with 19–25 nucleotides in length that are abundant in eukaryotic organisms and function in a transcriptional/posttranscriptional manner to regulate target gene expression.[6],[7] miRNAs are demonstrated to be widely involved in the physiological and pathological process, including cell proliferation, apoptosis, transformation, and metastasis of cancer cells.[8] miRNAs have been intensively studied in recent years, including in OsC research, in which several studies have demonstrated the involvement of miRNAs in the pathogenesis of OsC with the potential for development in disease diagnostics and therapeutics.[9],[10]

miR-320 is well known as a tumor-suppressive miRNA, and previous studies have shown that miR-320 is significantly decreased in multiple types of cancers, including breast cancer, colon cancer, and acute myelogenous leukemia.[11],[12],[13] Specifically, miR-320 was shown to target transferrin receptor 1 (CD71) and inhibit cell proliferation in human leukemia cells.[11] Therefore, miR-320 might have the potential to be developed as an anticancer drug, which requires more evidence and clears underlined mechanisms being characterized in cancer research field.

In the present study, we analyzed the expression pattern and biological functions of miR-320 in OsC. We found that in line with the previous study,[14] miR-320 was truly decreased in OsC cell lines. Furthermore, we showed that ectopic expression of miR-320 inhibited cell proliferation and induced cell cycle arrest of U2OS cells in vitro. We finally suggested that miR-320 may act as a tumor suppressor for OsC via negatively regulating E2F1, a cell cycle regulator.


 > Materials and Methods Top


Cell culture

Human normal osteoblastic cell line hFOB1.19 and other OsC cell lines HOS, MG63, SaoS2, and U2OS were purchased from the American Type Culture Collection. All these cell lines were cultured in 1640 or Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in a humidified 5% CO2.

Quantitative real-time polymerase chain reaction

Total RNAs were extracted from TRIzol (Life Technologies) according to the manufacturer's instruction and eluted by 50 µl diethylpyrocarbonate-treated water. Complementary DNAs (cDNAs) were synthesized using 0.5 µg total RNAs and PrimeScript RT Reagent Kit (Takara). Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed to quantify the relative expression of miR-320. U6B was used as normalization control. All real-time amplifications were measured thrice and performed with the ABI7900 system. The fold change of miR-320 was calculated using the 2ΔΔCT method.

Western blotting

The protein lysate was sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Protein expression was analyzed by Western blotting (WB) using primary antibodies against E2F1 or GAPDH (Santa Cruz), followed by incubation with secondary antibodies. Specific proteins were detected by ECL Plus WB Detection Reagents (GE Healthcare Biosciences).

Luciferase assays

E2F1 3'-UTR was amplified by PCR from human cDNA. The DNA segments produced from these amplifications were inserted into the pmirGLO vector (Promega). U2OS cells were co-transfected with pmirGLO vectors containing either E2F1 3'-UTR or the control vector with miRNA mimics. Renilla luciferase activity was used as internal controls. Transfections were performed by Lipofectamine 2000. Moreover, miR-320 mimics (miR-320) and negative control (miR-C) were purchased by GenePharma (Shanghai, China).

MTT assay

Cell growth ability was determined by the MTT assay. Briefly, 2 × 103 U2OS cells/well were seeded into 96-well plates. At the indicated time point, 5 µl MTT solution (5 mg/ml) was added into each well and incubated 2 h at room temperature. The reaction was terminated by 100 µl DMSO and the absorbance at 490 nm was measured by a microplate reader.

Cell cycle analysis

U2OS Cells were 70% ethanol fixed and stained for 30 min with propidium iodide and immediately analyzed by cell flow cytometry. Histograms represent the percentage of cells in each phase of the cell cycle (G0/G1 phase, S phase, and G2/M phase).

Statistic analysis

Data are presented as the mean ± standard deviation of at least triplicate experiments. Statistical analyses were performed using SAS version 9.2 (http://www.sas.com/en_us/home.html) and GraphPad Prism Software (http://www.graphpad.com/scientific-software/prism/). For all statistical analyses, P < 0.05 was considered statistically significant.


 > Results Top


The expression of miR-320 in osteosarcoma cells

Although it was already shown that miR-320 was downregulated in human OsC tissues compared with pair-matched adjacent noncancerous tissues,[14] its expression pattern in OsC cell lines remained unclear. We first applied qRT-PCR to test miR-320 expression levels in several human OsC cell lines and human normal osteoblastic cell line. As shown in [Figure 1], the expression of miR-320 was significantly decreased in a panel of OsC cells (HOS, MG63, SaoS2, and U2OS) compared to the normal hFOB1.19 cell line. This observation was quite consistent with the findings of Cheng's report that indicated a putative suppressing role of miR-320 in OsC cell.
Figure 1: The expression of hsa-miR-320 in osteosarcoma cell lines. Quantitative reverse transcription-polymerase chain reaction analysis was performed to detect expression of miR-320 expression in human normal osteoblastic cell line hFOB1.19 and other human osteosarcoma cell lines. miR-320 expression levels were normalized to U6B (*P < 0.05)

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miR-320 induced cell cycle arrest and inhibited cell proliferation

To further reveal the functions of miR-320 in OsC cells, we transfected mimics of miR-320 into U2OS cells in vitro, with the negative control oligos as the internal control group. Overexpression effect was analyzed by qRT-PCR as described above [Figure 2]a. 24 h after transfection, cell cycle attribution was also determined by flow cytometry followed by PI staining. We found that miR-320 could also induce G1 phase arrest in U2OS cells [Figure 2]b. In addition, cells were seeded into 96-well plates and MTT assay was performed to analyze the effect of miR-320 on proliferation. As shown in [Figure 2]c, we found that miR-320 overexpression could significantly inhibit cell proliferation. Taken together, our functional assays demonstrated that miR-320 played as a tumor suppressing miRNA by limiting cell proliferation and cell cycle progression in OsC cells.
Figure 2: miR-320 induced cell cycle arrest and inhibited cell proliferation in human osteosarcoma. (a) Quantitative reverse transcription polymerase chain reaction analysis of miR-320 expression in U2OS cells transfected with miR-320 mimics (miR-320) or negative control (miR-C). (b) The cell cycle phase of U2OS cells transfected with miR-320 mimics (miR-320) or negative control (miR-C) was analyzed by cell flow cytometry. (c) Cell proliferation was measured in the control or miR-320-transfected U2OS cells using MTT assay (**P < 0.01, ***P < 0.001)

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miR-320 directly targeted E2F1 by base pairing to its 3'-UTR

Although Cheng et al. had shown that miR-320 could directly fatty acid synthase (FASN), thus to impact on malignant behaviors of OsC cells,[14] there are still other explanations or mechanisms to interpret the growth-inhibiting activity of miR-320 in OsC cells. We sought to identify its regulated targets through bioinformatics software (TargetScan) and found that gene encoding E2F1, a cell cycle regulator, harbored a potential miR-320 binding site among its 3'-UTR [Figure 3]a. Afterward, luciferase reporter assay was performed to confirm this interaction and the results showed that E2F1 was another direct target of miR-320 in U2OS cells [Figure 3]b. In addition, transfection of miR-320 mimics in U2OS cells resulted in a reduced E2F1 mRNA and protein expression, respectively, as shown in [Figure 3]c and [Figure 3]d.
Figure 3: E2F1 might be a potential target of hsa-miR-320 in human osteosarcoma. (a) Putative binding sites for hsa-miR-320 are predicted in the 3'-UTR of E2F1 mRNA. (b) Relative luciferase expression levels obtained from pmirGLO vectors containing E2F1 3'-UTRs following co-transfection with precursors of miR-C or miR-320. It was calculated as the ratio of firefly/Renilla luciferase activities in the cells and normalized to those of the control. (c and d) Overexpression of miR-320 inhibits E2F1 mRNA and protein expression (*P < 0.05)

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 > Discussion Top


As the most common primary bone malignant tumor in children and young adults, OsC becomes a second leading cause of cancer-related death in this age group.[15] Since reliable diagnostic biomarkers and effective targeted therapeutic agents are still lacking, the 5-year survival rate and prognosis of OsC patients remain poor. Therefore, keeping on investigating the molecular mechanisms of OsC development and progression is urgently required. Currently, miRNAs are intensively studied and emerge as a new frontier of translational research from molecular biology to clinical applications.

miR-320 has been uncovered as a tumor suppressing miRNA in various cancers, including OsC. Cheng et al. showed that miR-320 was decreased in clinical OsC tissues, and it could inhibit cell proliferation both in vitro and in vivo via directly targeting FASN.[14] However, a single miRNA might harbor multiple different mRNA targets. The targeting of E2F1 by miR-320 was also proved by other groups: (a) miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development;[16] (b) miR-320 inhibited cell proliferation in glioma by targeting E2F1.[17]

Here in our study, we similarly showed that its downregulation in selected OsC cell lines, compared to the normal control cells. We also found that after miR-320 was forced expressed in U2OS cells, the proliferation rate and cell cycle progression were both inhibited. Moreover, we found that E2F1 was another direct target of miR-320 by interaction with its 3'-UTR. Although short of more evidence, our findings still provide another possibility to characterize the tumor suppressing role of miR-320 in OsC cells, via negatively regulating E2F1. Further understanding the inhibitory roles of miR-320 in OsC tumorigenesis will provide novel potential therapeutic agents for malignant tumor.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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  [Figure 1], [Figure 2], [Figure 3]



 

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