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Abstract

We aimed to study the anti-tumor effects of triptolide on osteosarcoma and the related molecular mechanisms. The cell viability, apoptosis portion, tumor size, tumor weight, and invasion of osteosarcoma cells were determined. The relative level of microRNA-181 in osteosarcoma tissues and the adjacent tissues was determined by quantitative real-time reverse transcription polymerase chain reaction. The target gene of microRNA-181a was determined and verified by luciferase report assay. At last, osteosarcoma cells were treated with triptolide and triptolide + microRNA-181a mimics to verify the relationship between triptolide and microRNA-181a. Triptolide inhibited the cell viability, promoted the apoptosis, decreased the tumor size and weight, and reduced the invasion of osteosarcoma cells. The level of microRNA-181a in osteosarcoma cells decreased significantly after treating with triptolide, and the relative level of microRNA-181a in osteosarcoma tissues was markedly higher than that in the adjacent tissues. PTEN was reported and verified the direct target gene of microRNA-181a. The overexpression of microRNA-181a decreased the inhibition of triptolide on osteosarcoma proliferation and promotion on osteosarcoma apoptosis. In conclusion, triptolide inhibited cell growth and invasion of osteosarcoma by regulating microRNA-181a via targeting PTEN gene in vivo and vitro.

Keywords

Triptolide osteosarcoma miR-181a PTEN

Introduction

Osteosarcoma (OS) is a locally aggressive malignant tumor of mesenchymal origin, and patients with OS are susceptible to early systemic metastasis.¹ It arises primarily in adolescents and is a leading cause of cancer death in adolescents; it also has a second peak of incidence in those aged over 50 years.² The current treatment of OS is still based on surgery and chemotherapy.³ In recent years, although a lot of research in the mechanism of OS development and metastasis had been done, the molecular mechanisms are still elusive, especially in certification and functional research of molecules, which are specifically expressed in OS.⁴ Therefore, it is urgent to elucidate the potential mechanism that mediates the initiation and progression of OS and develop better prognosis, new therapeutic targets, and approaches for OS treatment.

MicroRNAs (miRs) are small RNA molecules, modulating the messenger RNA (mRNA) translation and degradation. miRs could bind to complementary sequences in the 3′-untranslated regions (UTRs) of their target mRNAs, promoting mRNA degradation or translational repression.⁵ miR has been associated with mounting biological process, including apoptosis, invasion, and proliferation, which directly targets tumor suppressor genes or oncogenes, regulating tumorigenesis.⁶ Triptolide, a purified diterpenoid, is the major active component of the traditional Chinese medicine (TCM) herb Tripterygium wilfordii Hook.f. (TWHF) which belongs to the Celastraceae family. Triptolide is purported to have anti-tumor activity property.⁷ Currently, triptolide has been reported to inhibit proliferation and induce apoptosis against a variety of tumors in vitro and in vivo, including cholangiocarcinoma, breast cancer, melanoma, and gastric cancer.⁸ So far, little is known about the effect of triptolide on herpesvirus-associated malignancies. In this study, we aimed to explore the effect of triptolide on OS and the related molecular mechanism.

Material and methods

Cell culture

Primary OS tissues and the adjacent non-tumor tissues were obtained from 20 patients treated at Affiliated Hospital of Weifang Medical University. All patients provided informed consent for the use of their biological samples. This study was approved by the Ethics Committees of our institutes. Those patients who received chemo- or radiotherapy were excluded from this study. All tissues were snap-frozen in liquid nitrogen for further analysis.

OS cell lines (SAOS2 and U2OS) and normal cell lines were obtained from Prof. K. Lan (Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China). Cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA). All cells were cultured at 37°C with 5% CO₂ and 100% humidity. Triptolide (purity 98%) was purchased from Xi’an Kai Lai Biological Engineering (Xi’an, China) and dissolved in dimethyl sulfoxide (DMSO).

Cell viability assay

The cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Differentiated SAOS2 and U2OS cells were cultured in 96-well plates for 24 h with the purpose of stabilization. Then cells were incubated with 0, 1.5, 5, 15, 50, and 150 µmol/L triptolide for 24, 48, and 72 h. Each group of cells was cultured at 37°C. After incubation, MTT was added to each culture well at a final concentration of 2 mg/mL, and the cells were incubated for 4 h at 37°C. Then 100-µL DMSO was added to dissolve the formazan crystals and the amount of the formazan was measured by determining the absorbance at 570 nm.

Flow cytometry assays

Flow cytometry assay was used to clarify cells apoptosis. Cells were collected with trypsinisation before and after treating with triptolide and then washed twice with phosphate-buffered saline (PBS), fixed in cold 80% ethanol, and finally stored at 4°C overnight. The cells were washed with PBS and RNase A was administrated. Propidium iodide was added to tubes and then incubated for 20 min at 4°C in the dark. Fluorescein isothiocyanate (FITC)-labeled Annexin V/PI staining was applied. A total of 1 × 10⁶ cells in each well were suspended with buffer containing FITC-conjugated Annexin V/PI. Samples were then analyzed via flow cytometry.

Invasion assays

Cell invasion assay was performed using 24-well transwell chambers containing 8-mm-pore-diameter polycarbonate membrane (Corning, New York, NY, USA). A total of 200-mL cell suspension containing 4 × 10⁴ cells was added into the upper chamber, and 500-mL culture medium containing 20% (v/v) FBS was added to the lower chamber. After incubation at 37°C under 5% (v/v) CO₂ for 24 h, the non-filtered cells were gently removed with a cotton swab, and the migrated cells were fixed with 100% methanol, stained with 0.5% crystal violet, and washed with PBS (Gibco). The invaded cell number was counted under the microscope.

Transfection

Cells were seeded in six-well plates and cultured for overnight. miR-181a mimics, corresponding negative control (mimic NC), the siRNAs targeting ROCK1 (miR-181a inhibitor), and corresponding negative control (inhibitor-NC) were synthesized and purified by GenePharma (Shanghai, China). miR-181a overexpressed plasmid (pCDNA3.1-miR-181a) and blank vector pCDNA3.1 were purchased from Chinese Academy of Sciences (Changchun, China). Cells were transfected with these oligonucleotides using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction.

RNA isolation and quantitative real-time reverse transcription polymerase chain reaction

TRIzol reagent (Invitrogen) was used to isolate total RNA from tissues or cells. Total RNA was used to synthesize complementary DNA (cDNA) with PrimeScript reverse transcription-PCR kit (TaKaRa, Shiga, Japan) and subjected to quantitative real-time polymerase chain reaction (PCR). The relative expression levels of miR-181a were measured using a SYBR PrimeScript miRNA quantitative real-time polymerase chain reaction Kit (TaKaRa) following the manufacturer’s instructions, with U6 as an internal control. miR-181a mRNA expression levels were quantified using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. The reaction was performed on ABI 7500 Sequence Detection System (ABI, Vernon, CA, USA).

Western blot

Total proteins from the cells were extracted by ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1 mM protease inhibitor PMSF (phenylmethylsulfonyl fluoride; Sigma, St. Louis, MO, USA). The protein concentration was quantified with a bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF; Millipore, Bedford, MA, USA) membrane, and then blocked with 5% non-fat milk in Tris-buffered saline. The membranes were incubated with primary antibodies, mouse anti-human monoclonal ZEB1, E-cadherin, Vimentin and N-cadherin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse anti-human monoclonal β-acitin antibody (Santa Cruz Biotechnology), at 4°C overnight. The membranes were washed and subsequently probed with secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) at a 1/4000 dilution for 1 h at room temperature. Proteins were visualized with chemiluminescent detection system (ECL; Beyotime). β-acitin was used as internal control.

Luciferase report assay

The pmiR-181a-3′UTR wild-type (Wt) and pmiR-181a-3′UTR mutant-type (Mut) reporter vectors were synthesized and purified by GenePharma. Cells were seeded in 24-well plates at a density of 1 × 10⁶ cells/well. After incubation overnight, luciferase reporter vectors, and miR-181a mimics, miR-181a inhibitor, or NC were transfected into cells using Lipofectamine 2000. Firefly and Renilla luciferase activities were detected using Dual-Luciferase Reporter Assay System (Promega, Madison, WI, Germany) following the manufacturer’s instructions, 48 h after transfection. All transfection assays were carried out in triplicate.

Statistical analysis

The results were expressed as mean ± standard deviation (SD). All data was analyzed using SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA). Differences were considered significant at p value <0.05.

Results

Triptolide inhibits cell growth of OS in vitro and in vivo

To investigate the effect of triptolide in OS cell proliferation and apoptosis, SAOS2 and U2OS cells were treated with 0, 1.5, 5, 15, 50, and 150 µmol/L triptolide for 24, 48, and 72 h. Cell proliferation assays revealed that SAOS2 and U2OS cell viability decreased with the increase in triptolide concentration and treating time (Figure 1(a)). In apoptosis assay, we found that the apoptosis portion of SAOS2 and U2OS cells was much higher than that in the control. The apoptosis portion of SAOS2 cells in triptolide group was seven times that in the control and the apoptosis portion of U2OS cells in triptolide group was nine times that in the control (Figure 1(b)). To further verify the inhibition of triptolide on OS proliferation, we calculated the tumor size and weight after treating with triptolide for 5 weeks. Results showed that tumor size increased with increase in treating time from 1 to 5 weeks at control group. The use of triptolide reduced the increase in tumor size (Figure 1(c)). The results of tumor weight were almost the same with tumor size. The tumor weight increased with the increase in time from 1 to 5 weeks in control. After using triptolide, the degree of growth decreased (Figure 1(d)).

Figure 1.

Inhibition of proliferation by triptolide. (a) Control: SAOS2 and U2OS cells cultured in RPMI-1640 containing 10% FBS; others incubated with indicated concentrations of triptolide for 24, 48, or 72 h. Survival probability was determined by MTT assay. (b) SAOS2 and U2OS cells proliferation after treatment with 0 or 150 nM triptolide, by flow cytometry. (c) Tumor size of SAOS2 and U2OS cells after treatment with 150 nM triptolide for 1, 2, 3, 4, and 5 weeks. (d) Tumor weight of SAOS2 and U2OS cells after treatment with 150 nM triptolide for 1, 2, 3, 4, and 5 weeks.

Triptolide inhibits invasion of OS

To study the effect of inhibition of triptolide on the invasion of OS, transwell assay was performed to determine the invasion ability of SAOS2 and U2OS cells after treating with triptolide. Results showed the invade ability of SAOS2 and U2OS cells became weaker and invaded cell number reduced significantly after treating with triptolide (Figure 2(a)). To explore the molecular mechanism, the levels of epithelial-to-mesenchymal transition (EMT)-related proteins were determined by western blot after treating with triptolide. Results showed the protein expression levels of ZEB1 and E-cadherin decreased after treating with triptolide compared with the control, and the levels of Vimentin and N-cadherin increased after treating with triptolide (Figure 2(b)).

Figure 2.

Inhibition of invasion by triptolide. (a) SAOS2 and U2OS cells invasion after treatment with 0 or 150 nM triptolide, by transwell assay and (b) the protein expression values of EMT-related proteins (ZEB1, E-cadherin, Vimentin and N-cadherin) by western blot.

Triptolide downregulates miR-181a

To research the molecular mechanism about the effect of triptolide on OS, the relative levels of 5 miRs (miR-199a, miR-34a, miR-133a, miR-181a, and miR-128) which were related with glioma growth before and after treating with triptolide were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results showed that the levels of miR-199a, miR-34a, miR-133a, and miR-128 almost had no change after treating with triptolide compared with the control, while the level of miR-181a in SAOS2 and U2OS cells decreased significantly after treating with triptolide (Figure 3(a)–(e)). Then, relative expression of miR-181a in OS tissues and the adjacent OS tissues was determined by qRT-PCR. Results showed the relative level of miR-181a in cancer tissues was significantly higher than that in the adjacent tissues (Figure 3(f)).

Figure 3.

Triptolide-regulated microRNA-181a. (a–e) Relative levels of five miRs (miR-199a, miR-34a, miR-133a, miR-181a, and miR-128) which were related with glioma growth by qRT-PCR and (f) relative expression of miR-181a in OS tissues and the adjacent tissues.

PTEN was a direct target of miR-181a

To explore the potential target of miR-181a, miRanda (http://www.microrna.org) and TargetScan 7.0 (http://www.targetscan.org/) were used to predicate miR-181a target genes. Among these candidate targets, PTEN attracted our attention (Figure 4(a)). To verify the transfection effect, the expression level of miR-181a after transfection with miR-181a mimics or NC, miR-181a inhibitor or NC was determined. Results showed that miR-181a mimics markedly raised the level of miR-181a after transfection with miR-181a mimics (Figure 4(b)), while the level of miR-181a was reduced significantly by miR-181a inhibitor (Figure 4(c)).

Figure 4.

PTEN was a direct target of miR-181a. (a) Sequence of PTEN-3′UTR carrying wild-type and mutant miR-181a binding site. (b and c) The relative expression of miR-181a in SAOS2 cells after transfection with miR-181a mimics or NC and miR-181a inhibitor or NC. (d) Relative luciferase activities of the pmiR-PTEN-3′UTR Wt and pmiR-PTEN-3′UTR mt after transfection with miR-181a mimics or NC and miR-181a inhibitor or NC. (e–g) The protein expression levels of PTEN after transfection with miR-181a mimics or NC and miR-181a inhibitor or NC.

To validate whether miR-181a could directly target 3′UTR of PTEN, luciferase reporter assays were performed. After cotransfection with the luciferase report vectors and miR-181a mimics or NC, overexpression of miR-181a by miR-181a mimics resulted in a significant downregulation in the luciferase activities of the pmiR-PTEN-3′UTR Wt. However, knockdown of miR-181a by miR-181a inhibitor resulted in a significant upregulation in the luciferase activities of the pmiR-PTEN-3′UTR Wt (Figure 4(d)). Western blot assay was also used to determine the protein expression levels of PTEN in SAOS2 and U2OS cells after transfection with miR-181a mimics or NC and miR-181a inhibitor or NC. Results showed that the protein expression of PTEN decreased significantly after transfection with miR-181a mimics and increased markedly after transfection with miR-181a inhibitor (Figure 4(e)–(g)).

Triptolide treating and overexpression of miR-181a inhibited the promotion of triptolide on OS cell apoptosis

From the above results, we speculated triptolide could promote the apoptosis of OS by reducing the level of miR-181a. Western blot was used to determine the protein level of PTEN in SAOS2 and U2OS cells after treating with triptolide and miR-181a mimics, respectively. Results showed triptolide significantly increased the level of PTEN, while miR-181a mimics decreased the value (Figure 5(a) and (b)). Then, to further verify this conclusion, we treated OS cells with triptolide and triptolide + miR-181a mimics and studied the changes of OS cell proliferation ability. Results showed triptolide significantly decreased the proliferation of SAOS2 and U2OS cells at 24, 48, and 72 h. However, the proliferation ability of SAOS2 and U2OS cells was markedly higher after treating with triptolide + miR-181a mimics compared with triptolide group, and the proliferation ability was almost the same with the control (Figure 5(c)). The apoptosis assay showed triptolide increased the apoptosis cell portion in SAOS2 and U2OS cells. However, the apoptosis portion decreased after miR-181a mimics was transfected (Figure 5(d)). Moreover, invasion assay showed that the treating on OS cells with triptolide + miR-181a mimics significantly increased the invaded cell number compared with those treating with only triptolide (Figure 5(e)).

Figure 5.

(a and b) The protein expression levels of PTEN in SAOS2 cells after treating with triptolide or triptolide+miR-181a mimics. (c) The proliferation of SAOS2 and U2OS cells after treating with triptolide or triptolide+miR-181a mimics at different time. (d) The apoptosis of SAOS2 and U2OS cells after treating with triptolide or triptolide+miR-181a mimics. (e) The invaded cell number of AOS2 and U2OS cells after treating with triptolide or triptolide+miR-181a mimics.

Discussion

OS is the most common malignant bone tumor in children and adolescents.⁹ It derives from primitive bone-forming mesenchymal cells. The incidence rates and 95% confidence intervals of OS for all races and both sexes are 4.0 (3.5–4.6) for the range 0–14 years.¹⁰ The complex molecular mechanisms underlying OS tumorigenesis and progression remain largely unclear.¹¹ Therefore, searching for a molecular therapeutic target for preventing OS cells from migration becomes one of the important methods to break this “bottle neck” of the treatment of OS.

Triptolide is an extract of the herb T. wilfordii, which has been used to treat autoimmune disorders in TCM.¹² It has anti-inflammatory, antiproliferative, and pro-apoptotic properties.¹³ Triptolide is known to suppress the proliferation of a number of pancreatic cancer cell lines in vitro.¹⁴ It can sensitize lung cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inhibition of nuclear factor-κB (NF-κB) activation.¹⁵ Moreover, triptolide had anti-tumor effect on growth inhibition and could promote the apoptosis of OS cells.¹⁶ Zhao et al.¹⁷ reported triptolide could reduce the viability of OS cells by reducing mitogen-activated protein kinase phosphatase-1 (MKP-1) and heat shock protein 70 (Hsp70) expression. In this study, our results showed triptolide inhibited the proliferation and promoted the apoptosis of OS cells. OS tumor size and weight decreased after treating with triptolide compared with the control. It was accordant with the results reported before that triptolide had anti-tumor effect on OS cells. The invasion assay showed the invaded cell number decreased significantly after treating with triptolide. Moreover, the protein expression values of ZEB1 and E-cadherin decreased, and the values of EMT-related protein Vimentin and N-cadherin increased after treating with triptolide. EMT is implicated in the progression of primary tumors toward metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development.¹⁸ ZEB1 is a crucial inducer of EMT in various human tumours.¹⁹ It promotes tumorigenicity by repressing stemness-inhibiting microRNAs.²⁰ Overexpression of ZEB1 in cancer leads to EMT and increased metastasis.²¹ E-cadherin plays a central part in the process of epithelial morphogenesis.²² Transcriptional downregulation of E-cadherin appears to be an important event in the progression of various epithelial tumors.²³ Vimentin is a major constituent of the intermediate filament family of proteins. It shows dynamically altered expression patterns during different developmental stages and high sequence homology throughout all vertebrates.²⁴ Therefore, we speculated triptolide could inhibit the invasion of OS cells by regulating EMT.

miRs are a small (about 22 nucleotides), highly conserved noncoding class of regulatory RNA molecules expressed in a tissue- and development-specific manner 23.²⁵ Huang et al.²⁶ reported triptolide could inhibit the proliferation of multiple myeloma (MM) cells via microRNAs. We speculated triptolide may inhibit the proliferation of OS cells via some miRs. Therefore, the relative levels of five miRs (miR-199a, miR-34a, miR-133a, miR-181a, and miR-128) which were related to glioma growth in OS cells before and after treating with triptolide were determined. Results showed the level of miR-181a decreased after treating with triptolide. Moreover, the relative level of miR-181a in OS tissues was significantly higher than that in the control. Therefore, we speculated triptolide may inhibit the proliferation of OS cells via miR-181a. However, the molecular mechanism about how triptolide inhibits the proliferation of OS cells via miR-181a is unclear. So, we further explore the molecular mechanism about the effect of miR-181a on OS.

miR-181a is an intrinsic modulator of T-cell sensitivity and selection.²⁷ Ke et al.²⁸ reported miR-181a confers resistance of cervical cancer to radiation therapy through targeting the pro-apoptotic PRKCD gene. miR-181a mediates metabolic shift in colon cancer cells via the PTEN/AKT pathway.²⁹ In this study, we found that miR-181a directly targeted 3′UTR of PTEN and luciferase report assay verified that PTEN is a target gene of miR-181a. The tumor suppressor PTEN is a phosphatase with sequence similarity to the cytoskeletal protein tensin.³⁰ It is located at chromosome 10q23, is mutated in a variety of sporadic cancers and in two autosomal dominant hamartoma syndromes.³¹ Therefore, we speculated triptolide may influence the proliferation of OS via miR-181a by targeting PTEN gene. To verify the relationship between triptolide and miR-181a, we treated OS cells with triptolide and triptolide + miR-181a mimics and then determined the proliferation and apoptosis of OS cells. Results showed the proliferation of OS cells was inhibited and apoptosis was promoted by triptolide, while the transfection of miR-181a mimics decreased the inhibition and the promotion.

In conclusion, we speculated from the above results that triptolide inhibits the growth of OS in vivo and vitro by regulating miR-181a via targeting PTEN gene.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research was supported by the National Natural Science Foundation of China (Grants No.: 81472511), Zhejiang Provincial Natural Science Foundation of China (Grant No.: LY16H160044), and the Medicine and Health Foundation of Zhejiang Province (Grant No.: 2015KYB285).

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Triptolide inhibits the growth of osteosarcoma by regulating microRNA-181a via targeting PTEN gene in vivo and vitro

Abstract

Keywords

Introduction

Material and methods

Cell culture

Cell viability assay

Flow cytometry assays

Invasion assays

Transfection

RNA isolation and quantitative real-time reverse transcription polymerase chain reaction

Western blot

Luciferase report assay

Statistical analysis

Results

Triptolide inhibits cell growth of OS in vitro and in vivo

Triptolide inhibits invasion of OS

Triptolide downregulates miR-181a

PTEN was a direct target of miR-181a

Triptolide treating and overexpression of miR-181a inhibited the promotion of triptolide on OS cell apoptosis

Discussion

Footnotes

Declaration of conflicting interests

Funding

References