Triptolide inhibits benign prostatic epithelium viability and migration and induces apoptosis via upregulation of microRNA-218.

Benign prostatic hypertrophy (BPH) has become a troublesome disease for elder men. Triptolide (TPL) has been reported to be a potential anticancer agent. However, the potential effects of TPL on BPH have not been shown out. BPH-1 cells were treated with different concentrations of TPL and/or transfected with microRNA-218 (miR-218) inhibitor, pc-survivin, sh-survivin, or their corresponding controls (NC). Thereafter, cell viability was determined by CCK-8 assay. Cell migration was accessed by modified two-chamber migration assay. Cell apoptosis was checked by propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated Annexin V staining. In addition, messenger RNA (mRNA) and protein levels were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. BPH-1 cell viability and migration were significantly decreased, while cell apoptosis and expression of miR-218 were statistically enhanced by TPL ( P < 0.05 or P < 0.01). However, downregulation of miR-218 increased cell viability and migration, while decreased cell apoptosis compared with the negative control group ( P < 0.05 or P < 0.01). Furthermore, the expression of cell cycle-related proteins and cell apoptosis-related proteins were also led to the opposite results with NC. In addition, we found that miR-218 negatively regulated the expression of survivin ( P < 0.01) and suppression of survivin significantly enhanced cell apoptosis ( P < 0.01). Moreover, the results demonstrated that TPL could inactivate mammalian target of rapamycin (mTOR) pathway, while inhibition of miR-218 alleviated the effects. TPL inhibits viability and migration of BPH-1 cells and induces cell apoptosis and also inactivates mTOR signal pathway via upregulation of miR-218. This study provides evidence for the further studies representing triptolide as a potential agent in the treatment of human BPH.

Introduction

Benign prostatic hyperplasia (BPH), also known as prostatic hypertrophy (prostate enlargement), is a common benign disease among middle-aged and elderly men and can also influence quality of daily life and sleeping patterns.[1,2] There are various kinds of treatment strategies for the patients with BPH, such as surgical, prostatectomy, and laser.[3,4] Except these, nowadays some medicines exert notable effects on BPH, such as α-adrenergic receptor antagonists, inhibitors of the 5-α reductase enzyme, and various phytotherapies.[5] Even though most of these treatments are available and effective, many patients are tormented by these side effects or the surgery complications.[6] Therefore, new medicine or therapy is urgently needed for the treatment of BPH.

Triptolide (TPL), a diterpenoid triepoxide extracted from the traditional Chinese herb Tripterygium wilfordii, has been widely used in inflammatory and autoimmune diseases, such as myeloid leukemia,[7] colon carcinoma,[8] pancreatic cancer,[9] nephritis, and rheumatoid arthritis.[10] Due to the effects of TPL on multiple biological and pharmacological activities, such as antioxidant,[11] anti-tumor,[12] anti-proliferative,[13] immunosuppressive,[14] and anti-inflammatory properties,[10,15] TPL is currently under clinical trials.[16] Previous studies demonstrated that TPL effectively inhibited the development of BPH induced by testosterone in a rat model.[17] However, the effect of TPL on the treatment of BPH is still unclear.

Considerable research works have been devoted to determine the effect of TPL on diseases through regulation of microRNAs (miRNAs).[18,19] MiRNAs which refer to a class of small (~22 nucleotides) non-coding RNAs can regulate gene expression by directing their target mRNAs and exert various effects.[20] Previous studies have demonstrated that deregulation of miRNAs affected various activities, such as cell viability, migration, and apoptosis in many cancers.[21] Another interesting finding is that microRNA-218 (miR-218) downregulated expression in human malignancies and it has been treated as a suppressor of tumor metastasis and is correlated with clinical stage.[14] Therefore, we hypothesized that miR-218 might affect the response of BPH-1 cells to TPL.

In our study, we aimed to determine the effects of TPL on the prostatic epithelial BPH-1 cells through regulation of miR-218. The treatment with TPL resulted in regulation of cell growth in the human benign prostatic epithelium cell line tested, representing the first use of this approach on prostate cancer cell lines in vitro. This study provides support for the further studies representing triptolide as a potential therapeutic pharmaceutical agent in the treatment of human BPH.

Materials and methods

Cell culture and treatment

Cells of a BPH epithelial cell line (BPH-1) were provided by the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in BPH-1 culture medium consisting of RPMI 1640 medium supplemented with testosterone 20 ng/mL, transferrin 5 µg/mL, sodium selenite 5 ng/mL, insulin 5 µg/mL, 1% penicillin/streptomycin, and 20% fetal bovine serum (FBS; Life Science, Logan, UT, USA) and maintained at 37°C with air of 5% CO2. The above chemicals and TPL used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA). TPL was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and then was diluted in phosphate-buffered saline (PBS) solution into different concentrations in cell culture medium.

CCK-8 assay

Cell viability was measured by a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA). In brief, BPH-1 cells were seeded in 96-well plates with 5000 cells/well. After stimulation, 10-μL CCK-8 solution was added to each culture medium with different concentrations of TPL (0, 50, 100, 150, and 200 nM), and then results were, respectively, determined at different time points after treatment (24 and 48 h) at 37°C in humidified 95% air and 5% CO2. The absorbance was detested by Microplate Reader (Bio-Rad; Hercules, CA, USA) under the optical density (OD) at 450 nm.

Apoptosis assay

Propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated Annexin V staining purchased from Sigma-Aldrich were used for cell apoptosis analysis. BPH-1 cells were washed in PBS and then fixed in 70% ethanol (Sigma-Aldrich). After that, fixed cells were washed twice in PBS and stained in PI/FITC-Annexin V in the presence of 50 μg/mL RNase A (Sigma-Aldrich). After incubation for 1 h at room temperature in the dark, FACS (Beckman Coulter, Fullerton, CA, USA) was used for flow cytometry analysis according to the manufacturer’s instructions. The data were analyzed using FlowJo software.

Migration assay

Cell migration was determined using a modified two-chamber migration assay with a pore size of 8 μm (Bedford, MA, USA). Cells suspended in 200 μL of serum-free medium were seeded on the upper compartment of 24-well Transwell culture chamber, and 600 μL of complete medium was added to the lower compartment. After incubation at 37°C, cells were fixed with methanol. Non-traversed cells were carefully removed from the upper surface of the filter with a cotton swab. Traversed cells on the lower side of the filter were stained with crystal violet and counted.

MiRNA transfection

MiR-218 inhibitor and the negative control (NC) were synthesized by GenePharma Co. (Shanghai, China). Cell transfections were conducted using Lipofectamine 3000 reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s protocol. The primer sequence of miR-218 inhibitor is as follows: 5′-ACAUGGUUAGAUCAAGCACAA-3′. The sequence of the NC is 5′-CAGUACUUUUGUGUAGUACAA-3′.

Transfection and generation of stably transfected cell lines

Short-hairpin RNA-directed against human survivin and the full length of survivin were ligated into the pcDNA3.1 plasmid (GenePharma Co.) and were referred as sh-survivin and pc-survivin, respectively. The Lipofectamine 3000 reagent (Life Technologies Corporation) was used for the cell transfection according to the manufacturer’s instructions. The plasmid carrying a non-targeting sequence was used as NC of sh-survivin and pcDNA 3.1 was used as control of pc-survivin. The stably transfected cells were selected by the culture medium containing 0.5 mg/mL G418 (Sigma-Aldrich). After approximately 4 weeks, G418-resistant cell clones were established.

qRT-PCR

Total RNA was extracted from cells using Trizol reagent (Life Technologies Corporation) according to the manufacturer’s instructions. The TaqMan MicroRNA Reverse Transcription Kit and TaqMan Universal Master Mix II with the TaqMan MicroRNA Assay were used for measuring expression of miR-218, and U6 (Applied Biosystems, Foster City, CA, USA) was used as internal control.

Western blot

The protein used for western blot was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Guangzhou, China). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The western blot system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions. Primary antibodies included anti-p16 antibody (ab51243), anti-p21 antibody (ab109520), anti-Cyclin D1 antibody (ab134175), anti-Bcl-2 antibody (ab32124), anti-pro-caspase-3 antibody (ab32150), anti-cleaved-caspase-3-3 antibody (ab32042), anti-Bax antibody (ab182733), anti-survivin antibody (ab192675), anti-β-actin antibody (ab115777) from Abcam (Cambridge, UK); anti-totally 70 kDa ribosomal protein S6 kinase (t-p70S6K) antibody (2708), anti-phosphorylation of 70 kDa ribosomal protein S6 kinase (p-p70S6K) antibody (9234), anti-totally mammalian target of rapamycin (t-mTOR) antibody (2893), anti-phosphorylation of mTOR (p-mTOR) antibody (2976) from Cell Signaling Technology (Beverly, MA, USA). All these primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1000. Primary antibodies were incubated with the membrane at 4°C overnight, followed by wash and incubation with secondary antibody (goat anti-rabbit, IgG ab6721; Abcam) marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane carried blots and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS system, and then 200 μL Immobilon Western Chemiluminescent HRP Substrate (Millipore, Danvers, MA, USA) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad).

Statistical analysis

All data are shown as means ± standard deviation (SD). Statistical differences among at least three experiment groups were performed using Graphpad 6.0 statistical software (GraphPad Prism, San Diego, CA, USA). The P values were calculated using a one-way analysis of variance (ANOVA). If P < 0.05, it means statistically significant.

Results

TPL inhibits cell viability and induces cell apoptosis

In the first part, we tested the effects of TPL on the viability and apoptosis of BPH-1 cells. As shown in Figure 1(a), the results showed that, for time internal 24 h, cell viability was significantly decreased when TPL concentration was or above 100 nM, while for time internal 48 h, the changing concentration point was 50 nM. The protein expression of p21 and p16 was significantly upregulated and Cyclin D1 was statistically increased after treatment with TPL compared with the control group (Figure 1(b); P < 0.05). Similar results were obtained using western blot (Figure 1(c)). In addition, the results in Figure 1(d) show that TPL significantly enhanced the apoptosis of BPH-1 cells compared with the control group (P < 0.01). The expression of cleaved-caspase-3 and Bax were markedly overexpressed, while Bcl-2 showed downregulated expression after treatment with TPL compared with the control (Figure 1(e)). The results indicated that TPL significantly inhibited cell viability in a dose- and time-dependent manner and enhanced cell apoptosis of BPH-1 cells.

Figure 1.

Effects of triptolide (TPL) on benign prostatic hypertrophy line 1 (BPH-1) cell viability and apoptosis. (a) Cell viability was determined with TPL treatment at various concentrations (0, 50, 100, 150, and 200 nM) at 24 and 48 h by CCK-8 assay. Cell viability was significantly decreased with the increasing concentration of TPL. (b, c) Western blot was used to determine the expression level of p16, p21, and Cyclin D1. p16 and p21 were upregulated, while Cyclin D1 was downregulated under the TPL treatment compared with control. (d) Cell apoptosis was detected by flow cytometry analysis. TPL significantly induced the apoptosis of BPH-1. (e) Western blot was used for exploring apoptosis-related protein expression level. Cleaved-caspase-3 and Bax were overexpressed, while the Bcl-2 showed downregulated expression under the TPL treatment compared with control. Each point represented the mean ± standard deviation (SD) of triplicates. Each experiment was performed three times. *P < 0.05; **P < 0.01.

TPL inhibits migration of BPH-1

Ma et al.[9] found that TPL can suppress the migration of human pancreatic cancer cells. To test whether TPL can also affect the migration of BPH-1 cells, we performed the effects of TPL on BPH-1 cell migration. As shown in Figure 2, the results demonstrated that the migration of BPH-1 cells was significantly decreased after treatment with TPL compared with the control group (P < 0.05). These results indicated that TPL inhibited cell migration in BPH-1 cells.

Figure 2.

Effects of TPL on BPH-1 cell migration. TPL inhibited the migration of BPH-1 cells. Each point represented the mean ± SD of triplicates. Each experiment was performed three times. *P < 0.05; **P < 0.01.

TPL upregulates the expression of miR-218

Previous studies showed that TPL had effects on the lymphocytic leukemia cell lines through the regulation of miRNAs.[22] Among these identified miRNAs, miR-218 has been previously implicated as a tumor suppressor.[23] In order to investigate whether effects of TPL on BPH-1 cells were through the regulation of miR-218, we measured the expression of miR-218 in BPH-1 cells under different concentrations of TPL. The results in Figure 3 demonstrated that compared with control group, treatment with TPL 100 and 200 nM statistically increased the expression of miR-218 (both P < 0.05). However, no significant difference was found at the lower concentration (50 nM) between the TPL treatment group and the control group. It means that TPL upregulated the expression of miR-218 in a dose-dependent manner.

Figure 3.

Effects of TPL on microRNA-218 (miR-218) expression. miR-218 expression was observed upregulation with TPL treatment in various concentrations (0, 50, 100, and 200 nM) by qRT-PCR. Each point represented the mean ± SD of triplicates. Each experiment was performed three times. *P < 0.05; **P < 0.01.

TPL inhibits the growth of BPH-1 and induces cell apoptosis via upregulation of miR-218

Transfecting miR-218 inhibitor was used to test whether the effects of TPL on BPH-1 cells was through regulation of miR-218. The results in Figure 4(a) show that miR-218 inhibitor significantly downregulated the expression of miR-218 in BPH-1 cells compared with the NC group (P < 0.01), implying high transfection efficiency. The cell viability was significantly increased by treatment with TPL plus miR-218 inhibitor compared with treatment with TPL plus NC (Figure 4(b); P < 0.05). In addition, the protein expression of p16 and p21 was significantly decreased, while expression of Cyclin D1 was statistically increased after treatment with TPL plus miR-218 inhibitor compared with treatment with TPL plus NC (Figure 4(c) and (d); P < 0.05 or P < 0.01). BPH-1 cell apoptosis was significantly decreased after treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC (Figure 4(e); P < 0.01). The related protein cleaved-caspase-3 and Bax were obviously downregulated, while the protein Bcl-2 was markedly upregulated in the treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC (Figure 4(f)). BPH-1 cells migration after treatment with TPL plus miR-218 inhibitor was significantly increased compared with the group treatment with TPL plus NC (Figure 4(g); P < 0.05). Moreover, TPL significantly decreased the expression of survivin compared with control (P < 0.01), while miR-218 downregulation enhanced the expression of survivin compared with NC (P < 0.05; Figure 4(h)). These results revealed that TPL could inhibit cell viability and migration and induce cell apoptosis through upregulation of miR-218 in BPH-1 cells.

Figure 4.

Effects of TPL and miR-218 on cell viability, migration, and apoptosis in BPH-1 cells. (a) The expression of miR-218 was significantly decreased under transfection of miR-218 inhibitor compared with transfection of the negative control (NC) group. (b) Cell viability was increased by treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL and NC by CCK-8 assay. (c, d) Western blot was used to determine the expression level of p16, p21, and Cyclin D1. The expression of p21 and p16 were upregulated, while the expression of Cyclin D1 was downregulated under the treatment with TPL plus miR-218 inhibitor compared with the treatment with TPL plus NC. (e) Cell apoptosis was detected by flow cytometry analysis. Cell apoptosis was significantly decreased under the treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC. (f) Western blot was used for exploring apoptosis-associated proteins in BPH-1 cells. Western blot of pro-caspase-3, cleaved-caspase-3, Bcl-2, and Bax were tested to β-actin, the loading control. TPL and miR-218 inhibitor inhibited the expression of caspase-3 and Bax, while induced the expression of Bcl-2 compared with the group treatment with TPL plus NC. (g) Cell migration was accessed by modified two-chamber migration assay. Cell migration was significantly increased under the treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC. Each point represented the mean ± SD of triplicates. (h) Survivin expression was significantly decreased by TPL, while enhanced by the group with TPL and transfection with miR-218 inhibitor. Each experiment was performed three times. *P < 0.05; **P < 0.01.

MiR-218 negatively regulates expression of survivin and suppression of survivin enhances cell apoptosis

Several studies have identified that survivin is one of the most important miR-218 key targets[24] and it was downregulated by miR-218.[25] This information hints us that miR-218 might play important roles in expression of survivin. The results in Figure 5(a) and (b) show that downregulation of miR-218 could promote expression of survivin compared to the NC group (P < 0.05). Furthermore, we overexpressed or downregulated the expression of survivin. As expected, the expression of survivin was significantly increased by transfection with pc-survivin and was significantly decreased by transfection with sh-survivin (Figure 5(c) and (d); P < 0.01). The further study in Figure 5(e) demonstrated that sh-survivin significantly induced cell apoptosis and the similar results from western blot demonstrated that cleaved-caspase-3 and Bax were remarkably overexpressed, while Bcl-2 showed downregulated expression by downregulation of survivin (Figure 5(f)). It means that miR-218 negatively regulates expression of survivin and suppression of survivin enhanced cell apoptosis.

Figure 5.

Effect of survivin on BPH-1 cell apoptosis. (a) qRT-PCR was used to determine the expression level of survivin. The cell group transfected with miR-218 inhibitor increased the expression of survivin compared with group NC. (b) Western blot was used for exploring protein expression level. Western blot results presented that after transfected with miR-218 inhibitor, expression of survivin overexpressed compared with NC. (c, d) Western blot was used to determine the expression level of survivin. Results showed that pc-survivin enhanced the expression of survivin, while sh-survivin downregulated the expression of survivin. (e) Cell apoptosis was detected by flow cytometry analysis. pc-survivin has no significantly effect on the cell apoptosis, while sh-survivin induces cell apoptosis significantly. (f) Western blot results showed that sh-survivin can enhance the apoptosis-related protein expression of caspase-3 and Bax. Each point represented the mean ± SD of triplicates. Each experiment was performed three times. *P < 0.05; **P < 0.01.

TPL inactivates mTOR signal pathway through upregulation of miR-218

The results in Figure 6(a) demonstrated that TPL significantly decreased the phosphorylation of mTOR and p70S6K compared with the control group (both P < 0.05), while treatment with TPL plus miR-218 inhibitor increased the expression of phosphorylation of mTOR and p70S6K compared with the group treatment with TPL plus NC (both P < 0.01). In addition, downregulation of miR-218 activated mTOR signal pathway. The western blot results also presented that the expression of p-mTOR and p-p70S6K was markedly downregulated in the treatment with TPL compared with the control group, while p-mTOR and p-p70S6K both obviously showed upregulated expression by treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC (Figure 6(b)).

Figure 6.

Effects of TPL on mammalian target of rapamycin (mTOR) signal pathway through upregulated expression of miR-218. (a) The expression of phosphorylation of mTOR, phosphorylation of 70 kDa ribosomal protein S6 kinase (p70S6K) were downregulated in the treatment with TPL compared with control, while the expression of phosphorylation of mTOR and phosphorylation of p70S6K were significantly increased after treated with TPL plus miR-218 inhibitor while compared with the group treatment with TPL plus NC. (b) Western blot was used for exploring protein expression level. The western blot results showed that the phosphorylation of mTOR and phosphorylation of p70S6K were obviously overexpressed after treatment with TPL plus miR-218 inhibitor compared with the transfection with TPL plus NC. Each point represented the mean ± SD of triplicates. Each experiment was performed three times. *P < 0.05; **P < 0.01.

Discussion

In this study, we investigated whether TPL has potential effects on the treatment of BPH, as well as the underlying mechanisms. We found that TPL decreased cell viability and migration and increased cell apoptosis through upregulation of miR-218. These effects might be by inactivation of mTOR signal pathway.

BPH has become a common disease for the elder men. However, the pathology of BPH is not clearly elucidated and the associated clinical symptoms are much complicated.[4] BPH surgery can cause other side effects, such as bleeding.[3,26] Therefore, more effective medicine and therapies are needed for the treatment of BPH. In recent years, traditional medicine has become popular for treatment of diseases. TPL, which is purified from a Chinese herb Tripterygium wilfordii was reported to display antitumor effects.[13] An increasing number of evidence demonstrated that TPL could inhibit cancer cell migration, invasion, and metastasis, such as in leukemia,[27] in oral cancer,[28] and in colon cancer.[29] Similarly, our results also demonstrated that TPL can inhibit cell viability and migration in BPH-1 cells and induce cell apoptosis compared with control (Figures 1(a) and (d) and 2). In addition, cell viability was significantly decreased with increasing concentrations of TPL (Figure 1(a)). p21 and p16 and Cyclin D1 are important cell cycle regulators. p16 and p21 act as cell inhibitors[30] and Cyclin D1 plays as a positive regulator and leads to cell cycle progression.[31,32] Western blot results from Figure 1(b) and (c) demonstrated that the expression of p16 and p21 increased, while expression of Cyclin D1 decreased by treatment with TPL. Increasing expression of p16 and p21 and reducing expression of Cyclin D1 indicated that TPL inhibited the BPH-1 cell viability.

Based on what we have found in the experiment, we further explored the potential possible mechanisms. Previous studies revealed TPL affected on cell growth through regulation of miRNAs expression, such as miR-142-5p and miR-181a,[18] miR-21,[33] and miR-30.[34] Among all these identified miRNAs, miR-218 has been found to play an important role in the cell growth and can be treated as a novel potential biomarker for gastric cancer detection.[35] Moreover, miR-218 inhibited invasion and metastasis of gastric cancer[36] and inhibition of miR-218 can increase cell viability.[37] Therefore, we hypothesized that TPL might affect BPH-1 cell growth through regulation of miR-218. Further experiments were performed to verify this hypothesis. Results in our studies demonstrated that the expression of miR-218 was upregulated in treatment with TPL (Figure 3). In addition, after transfection with miR-218 inhibitor, we found that the cell viability and the migration were increased, while the cell apoptosis was decreased compared with the group of TPL plus NC (Figure 4(b) and (g)). Our results were also supported by the western blot that the expression of p16 and p21 were reduced, while the expression of Cyclin D1 was increased after treated with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC (Figure 4(c) and (d)). Similar results were also found by the results from Xia et al.[37] who revealed that glioma cell viability was increased after transfection with miR-218 inhibitor. Moreover, BPH-1 cell apoptosis was significantly decreased after treatment with TPL plus miR-218 inhibitor (Figure 4(e)). This was consistent with the previous studies that miR-218 overexpression was observed to suppress glioma cell apoptosis.[37] Caspase-3 and Bax execute the program of cell apoptosis through several signal pathways.[38] In our study, the expression of cleaved-caspase-3 and Bax were observed downregulation, while expression of Bcl-2 was increased by treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC (Figure 4(f)). Therefore, we found that downregulation of miR-218 increased viability and migration and inhibited cell apoptosis.

Survivin, which belongs to the inhibitor of apoptosis protein family, is observed in most of the human tumors but is rarely found in terminally differentiated normal cells.[39] Survivin was found to be regulated by the expression of miR-218 in tumor cell lines.[24,25] Therefore, we detected the expression of survivin in BPH-1 cells according to its important role in apoptosis and its close factor related to miR-218. Consistent with previous reports,[39,40] we found that in our study that expression of survivin was upregulated by miR-218 inhibitor (Figure 5(a) and (b)). Then, we investigated the roles of survivin in BPH-1 cells. After overexpression or suppression of survivin in BPH-1 cells, cell apoptosis was analyzed. Apoptosis of BPH-1 cells (Figure 5(e)) and also the related protein cleaved-caspase-3 and Bax was significantly overexpressed, while the Bcl-2 was downregulated by downregulation of survivin (Figure 5(f)). The results demonstrated that suppressing the effects of miR-218 on BPH-1 cell apoptosis might be through regulation of survivin. Therefore, TPL can upregulate the expression of miR-218 and decrease the expression of survivin and induce apoptosis in BPH-1 cells.

mTOR signal pathway is often activated in cancer due to genetic alterations of the genes implicated in this pathway[41] and had also shown to cooperate in prostate cancer progression.[42] p70S6K is a serine/threonine kinase regulated by mTOR pathway, which plays an important role in controlling of cell cycle, growth, and survival.[41] Results of western blot revealed that phosphorylation of mTOR and p70S6K was inhibited by TPL, while phosphorylation of mTOR and p70S6K were enhanced by the treatment with TPL plus miR-218 inhibitor compared with the group treatment with TPL plus NC (Figure 6(a) and (b)). It demonstrated that TPL inhibited phosphorylation of mTOR and p70S6K and then through this to inactivate the signal pathway of mTOR. The result provided a possible explanation about how TPL can regulate BPH-1 cell growth through upregulation of the expression of miR-218.

In conclusion, TPL could inhibit the BPH-1 cells viability and migration and induce apoptosis through upregulation of the expression of miR-218 and inactivate mTOR signal pathway. Our data provided new evidence for the mechanism of the effects of TPL on the treatment of BPH. However, further research should be performed to examine the safety and side effects of TPL for the treatment of BPH to support the therapeutic choice and the clinical judgment.