Long noncoding RNA GAS5 modulates α-Solanine-induced radiosensitivity by negatively regulating miR-18a in human prostate cancer cells

Biomedicine & Pharmacotherapy 112 (2019) 108656

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Long noncoding RNA GAS5 modulates α-Solanine-induced radiosensitivity by negatively regulating miR-18a in human prostate cancer cells

T

⁎

Jinhui Yang, Tongtong Hao, Jiantao Sun , Pengtao Wei, Han Zhang Department of Urology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, Henan, China

A R T I C LE I N FO

A B S T R A C T

Keywords: α-Solanine Radiosensitivity GAS5 miR-18a Prostate cancer

Radiotherapy is an adjuvant treatment of surgery in prostate cancer, while radioresistance has been the challenge of treatment. It has been reported that α-Solanine exhibits anti-cancer activity and enhances the chemoand radio-sensitivity in several human cancers, whereas the role of α-Solanine on radiosensitivity to PCa remains to be uncovered yet. We found α-Solanine decreased cell viability in human PCa cells rather than normal prostate epithelial cells in vitro. Functional experiments showed that cell viability and colonies formation were declined & apoptosis rate and DNA double strand breaks (DSBs) marker γ-H2AX expressions were elevated by αSolanine in PCa cells treated with X-ray irradiation, compared with X-ray irradiation treatment only. GAS5 was down-regulated & miR-18a was up-regulated in PCa cells, which was reversed in the presence of α-Solanine. Effects of ectopic GAS5 on inhibiting cell viability and survival & promoting apoptosis and DNA damage were reversed by miR-18a overexpression in PCa cells. Moreover, GAS5 regulated miR-18a expression by target binding during α-Solanine treatment. Collectively, α-Solanine suppresses cell proliferation and promotes radiosensitivity through up-regulating GAS5/miR-18a pathway in PCa. Our results provide a novel mechanism of α-Solanine treatment in human prostate cancer and help to develop a new approach to sensitizing radioresistant prostate cancer cells by targeting GAS5/miR-18a.

1. Introduction Prostate cancer (PCa), a relatively common malignant cancer, is the second leading cause of cancer related deaths among male patients [1,2]. Radiotherapy is an adjuvant treatment of surgery and is also a therapeutic choice for the patients with regionally unresectable advanced PCa [3]. In that regard, tumor radiosensitivity underlies the successful therapeutic effect, which are challenged by the side-effect and the emergence of resistance, unfortunately. Therefore, it becomes important to enhance tumor radiosensitivity and to develop novel alternative therapeutic strategy of PCa. In recent years, increasing interests have been devoted into exploring the use of small-molecule anticancer compounds for prevention or treatment of tumors. α-Solanine, molecular formula C45H73NO15, is mainly found in the potato tuber and the nightshade plant [4]. Like other alkaloid steroid [5], α-Solanine has been reported to be an important bioactive component of steroidal glycoalkaloids, and exhibits anti-metastasis activity in different human cancers [6–8], chemoprotective and chemotherapeutic effects in mice breast cancer [9]. The study showed that radiosensitivity of esophageal cancer cells were

enhanced by α-Solanine [10]. Therefore, α-Solanine may possess a potential effect on radiotherapy in PCa, and the associated molecular mechanisms remain to be further elucidated. α-Solanine takes part in the regulation of miRNA-138 (tumor suppressor) in cancers, including esophageal cancer [8,11] and lung adenocarcinoma [12]. miR-21 (tumor promoter) has also been involved in α-Solanine-induced biological functions [7]. However, this is the only available evidence that gives us a clue of the relationship between miRNAs and α-Solanine-induced tumor suppressive effects at posttranscriptional level. Furthermore, previous studies announced that miR-18a enhanced the radiosensitivity of cervical cancer [13] and nonsmall cell lung cancer [14]. Considering the abnormal expression of miR-18a in PCa [15–17], we aimed to reveal that whether miR-18a participates in α-Solanine-induced biological effects. Long noncoding RNAs (LncRNAs) refer to a group of RNAs with length more than 200 nucleotides and have limited protein-coding potential. Accumulating evidences indicate LncRNAs play a widespread role in regulating biological processes, such as cell differentiation, proliferation, apoptosis, and migration [18]. Growth arrest-specific 5 (GAS5) gene, first isolated in 1988, is a recently identified tumor

⁎ Corresponding author at: Department of Urology, Luoyang Central Hospital Affiliated to Zhengzhou University, Unit 20, Building 6, No. 288, Middle Zhongzhou Road, Xigong District, 471000, Luoyang, Henan, China. E-mail address: [email protected] (J. Sun).

https://doi.org/10.1016/j.biopha.2019.108656 Received 24 November 2018; Received in revised form 23 January 2019; Accepted 1 February 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Biomedicine & Pharmacotherapy 112 (2019) 108656

J. Yang, et al.

measured at 450 nm. All operations were performed in triplicate.

suppressor involved in several cancers [19], like breast, prostate, lung, and colorectal cancer. Existing studies mirror that GAS5 inhibits cell proliferation and promotes apoptosis of multiple cell types [20,21]. Moreover, GAS5 has been announced to confer to chemosensitivity such as cisplatin [22,23], tamoxifen [24] and Adriamycin [25] in various cancers. Although α-Solanine has been shown anti-carcinogenic potential against various cancer cell lines, the underlying mechanism involved in its inhibition on tumor growth and radiosensitivity remain to be further elucidated. This work figured out the role of α-Solanine in PCa cells under X-ray irradiation insults in vitro. Functional experiments declared that GAS5 participated in α-Solanine action in PCa cells by sponging miR-18a.

2.5. Colony formation assay

2. Materials and methods

Clonogenic survival is the ability of cells to maintain clonogenic capacity to form colonies. Treated cells with X-ray irradiation were immediately collected, followed by transplanting of 200 cells onto 10 cm dishes (Corning). The colony formation was visible and occurred after incubation of 12 d. Then, the colonies formed were fixed with ethanol and stained with crystal violet. Colonies with over 50 cells were scored as surviving colonies. The blank group was treated with PBS and X-ray irradiation insult or not, and the control group was treated with 1‰ DMSO with X-ray irradiation insult or not. The survival fractions = the number of colonies/ (number of inoculated cells × plating efficiency), and the radiation survival curve was drawn.

2.1. Cells and cell culture

2.6. Flow cytometry assay

Human normal prostate epithelial cell line RWPE-1 and PCa cell lines DU145 and PC-3 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% FBS (Gibco) and 1% penicillin/streptomycin at 37 °C in 5% CO2. α-Solanine and its solvent, dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich (St. Louis, MO, USA). α-Solanine at concentration of 1 mM was stored at −20 °C after dissolution. The medium was replaced with medium containing 4, 8, 12 and 16 μM of α-Solanine when cells achieve 90% cell confluence. And each cell culture was refreshed every 2 d. The blank group was treated with PBS, and the control group was treated with 0 μM of α-Solanine containing 1‰ DMSO.

Cells with different processing methods were analyzed by Annexin V-FITC/PI kit (Beyotime, Shanghai, China) on flow cytometry. Apoptotic cells were labelled complying with the protocol. Fluorescence was analyzed on cytoFLEX LX flow cytometer (BeckmanCounter Electronics, Jiangsu, China) using CytExpert software. The blank group was treated with PBS and 4 Gy of X-ray irradiation insult or not, and the control group was treated with 1‰ DMSO with X-ray irradiation insult or not. Apoptosis rate was calculated in the formula: apoptosis rate (%) = apoptotic cells (Annexin V+/ PI-, Annexin V+/ PI+) / total cells ×100%. 2.7. Quantitative real time PCR (qPCR) Total RNA from cultured cells was isolated using TRIzol reagent (Thermo, Waltham, MA, USA) following the protocol. The first strand of cDNAs was synthesized depending on total RNA using Reverse transcription kit (Abcam, Cambrige, UK) and the amplification of cDNA was performed by SYBR Premix Ex Taq Master Mix (Invitrogen). qPCR was conducted on Applied biosystem 7500 real-time PCR system (Thermo), and the expressions of GAS5 and miR-18a were calculated according to the comparative threshold cycle value (2−ΔΔCt) method, compared with U6 small nuclear RNA(U6, for miRNA). PCR primers as follows: GAS: 5′cttctgggctcaagtgatcct-3′ (sense) and 5′-ttgtgccatgagactccatcag-3′ (antisense); miR-18a: 5′-acgtaaggtgcatctagtgcagata-3′ (sense) and 5′-gtgcagggtccgaggt-3′ (antisense); U6: 5′-ctcgcttcggcagcaca-3′ (sense) and 5′-aacgcttcacgaatttgcgt-3′ (antisense); All operations were carried out at least 3 times.

2.2. Irradiation treatment Cells were plated into six-well plates (Corning, NY, USA) and cultured for 24 h to settle down. Cells were then exposed to different doses of X-ray irradiation (0, 2, 4, 6, and 8 Gy) from a linear accelerator (RadSource, Suwanee, GA, USA) with a 6-MV photon beam at a dose rate of 3.2 Gy/min. 2.3. Cell transfection For ectopic expression, GAS5 was amplified and cloned into the multiple cloning site (MCS) of the pcDNA3.1 vector (Invitrogen, Shanghai, China). Small interfering RNA (siRNA) against GAS5 (siGAS5), miR-18a/NC mimic were purchased from Ribobio Co. (Guangzhou, China). Cell transfection with oligonucleotides or plasmids into PC-3 and DU145 cells was performed by Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Cells were subsequently cultured for further study. Cells were seeded into 6-well plates (Corning) and grown to 80% cell confluence on the day of transfection, and harvested for further experiments after transfection for 48 h.

2.8. Western blotting Treated cells were extracted with total protein with RIPA lysis buffer (Beyotime) to measure the protein expressions. Western blotting was performed according to standard procedures, and β-actin on the same membrane was used as a loading control. The primary antibodies were from Cell Signaling Technology (CST; Danvers, Massachusetts, USA) and as follows: γ-H2AX (CST, #9718, 1:2000); H2AX (CST, #7631, 1:2000); Anti-β-actin antibody (#9484, 1:5000) was purchased from Abcam (Cambridge, UK). The proteins were visualized using ECL procedure.

2.4. CCK-8 assay Cells in logarithmic phase growth were seeded into 96-well plate (Corning) at a density of 1 × 104 cells/well for 24 h. The cells were treated with 4, 8, 12 and 16 μM of α-Solanine for 24 h, and exposed to 2, 4, 6, and 8 Gy of X-ray irradiation. The blank group was treated with PBS and X-ray irradiation insult or not, and the control group was treated with 1‰ DMSO with X-ray irradiation insult or not. The viability of cells was determined according to Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) manufactures. Cells were cultured with 10% CCK-8 for another 3 h, and the optical density was

2.9. Luciferase reporter assay Plasmids of pGL3-GAS5 WT or pGL3-GAS5 MUT was co-transfected with Renilla luciferase reporter plasmid (pTK-Green Renilla Luc, invitrogen) and miR-18a/NC mimic into 293FT cells. All transfection procedures were performed by Lipofectamine 2000 (Invitrogen). After 30 h-transfection, the luciferase activity were measured using dual2

Biomedicine & Pharmacotherapy 112 (2019) 108656

J. Yang, et al.

and D, 4–8 Gy of X-ray radiation cause obviously decreased survival fraction in PC-3 and DU145 cells, which subsequently was aggravated by α-Solanine treatment. Besides, 2 Gy of X-ray radiation failed to impair survival fraction. These outcomes suggested a promotion role of αSolanine on radiosensitivity to human PCa.

luciferase reporter system (Promega, Madison, WI, USA) according to the protocol. The ratio of Firefly to Renilla luciferase activity was used as the relative luciferase activity and was normalized to control (miRNC). All operations were repeated 3 times. 2.10. RNA immunoprecipitation (RIP) and RNA pulldown assay

3.3. α-Solanine promoted radiation-induced cell apoptosis and DNA damage in PCa cells

The RNA immunoprecipitation (RIP) and RNA pulldown assay were performed with PC-3 cell extract. For RIP, miR-18a/NC mimic was transfected into PC-3 cells, and Magna RIP™ RNA-binding protein immunoprecipitation kit (MilliporeSigma, Billerica, MA, USA) was chose to detect expression of GAS5 from the samples bound to the Ago2 antibody or IgG; for RNA pulldown, GAS5-WT/MUT was overexpressed in PC-3 cells, and GAS5 levels were examined using qPCR in samples pulled down by biotin-labelled miR-18a or NC. All operation obeyed the standard instructions.

Continued, the physiological mechanism of α-Solanine promoting radiosensitivity was explored in PCa cells. PC-3 and DU145 cells were treated with α-Solanine and X-ray irradiation, followed by measurement of cell apoptosis and DNA damage. As shown in Fig. 3A and B, apoptosis rate did not altered by α-Solanine without X-ray irradiation insult; whereas, α-Solanine dramatically elevated apoptosis rate when exposed to 4 Gy of radiation. Similar results were drawn on the expression of DNA double strand breaks (DSBs) biomarker. γ-H2AX levels were abundantly raised with treatment of α-Solanine on condition of 4 Gy of radiation. Of note, 8 μM of α-Slanine caused higher levels of apoptosis rate and γ-H2AX expression than 4 μM of α-Solanine in PC-3 and DU145 cells (Fig. 3A–D). These data provided a preliminary interpretation of how α-Solanine promoted radiosensitivity in PCa cells.

2.11. Statistical analyses Data given were the means ± SD. Statistical significance was determined by two-tailed Student’s t test. The correlation between the expression of miR-18a and GAS5 was examined by Spearman rank analysis using SPSS 16.0 software (Chicago, IL, USA). P < 0.05 was considered as significant difference.

3.4. Expressions of GAS5 in PCa cells GAS5 has been reported as a key indicator for the diagnosis and prognosis prediction of PCa [27,28]. However, the expression level of GAS5 was controversial in PCa [29,30]. In this study, first of all, expressions of GAS5 in PCa cells were uncovered. We observed lower expression of GAS5 in PC-3 and DU145 cells compared with control RWPE-1 cells (Fig. 4A). Whereas, GAS5 upregulation was stimulated by α-Solanine both in PC-3 and DU145 cells (Fig. 4B and C). 4 μM of αSolanine induced expression of GAS5 up to over 1.8 fold; what’s more, 8 μM of α-Solanine led to more than 3.2 fold. These results indicated the potential role of GAS5 and α-Solanine-mediated biological effects in PCa.

3. Results 3.1. α-Solanine suppressed cell proliferation in PCa cells It has been well known that α-Solanine modulates tumor growth and invasion [26]. The role of α-Solanine in PCa was uncovered in this study. We measured the cell proliferation by cell viability assay (CCK-8) in PC-3 and DU145 cells (Fig. 1A and B). 4–12 μM of α-Solanine didn’t make a difference on cell viability according to OD450 values both in PC-3 and DU145 cells, however 16 μM of α-Solanine declined the cell viability to less than 50%. Simultaneously, 4–16 μM of α-Solanine treatment failed to affect OD450 values in RWPE-1 cells (Fig. 1C). All data were compared with 0 μM of α-Solanine (1‰ DMSO) group. These results showedα-Solanine suppressed cell proliferation in PCa cells rather than normal prostate epithelial cells.

3.5. GAS5 regulated miR-18a expression by sponging Enhancement of miR-18a on radiosensitivity has been reported in several cancers [13,14,31,32], we wondered whether exist a relationship between GAS5 and miR-18a. Potential target sites (red) was predicted on Starbase software (Fig. 5A). The site of GCACCTTA on GAS5 3′ UTR was mutated into AGCTCCGT, and full length of GAS5 3′ UTR wide type (GAS5-WT) and mutant (GAS5-MUT) was cloned into pGL3 vector. Luciferase reporter assay (Fig. 5B) showed relative declined luciferase activity after co-transfected with GAS5-WT and miR-18a mimic; no significant difference of luciferase activity occurred in co-transfection of GAS5-MUT and miR-18a mimic. The relative luciferase activity was compared to miR-NC groups. Besides, RIP and RNA pull-down assay

3.2. α-Solanine contributed to radiosensitivity in PCa cells The effects of α-Solanine in PCa cells were explored. PC-3 and DU145 cells were exposed to 2–8 Gy of X-ray irradiation to induce cell injury, and 4–8 μM of α-Solanine was added to investigate its role on radiosensitivity. Cell viability and colony formation were measured. We explained that 2–8 Gy of X-ray irradiation caused decreased OD450 values, and cell viability was even lower in the presence of α-Solanine, compared with X-ray irradiation only (Fig. 2A–B). As shown in Fig. 2C

Fig. 1. α-Solanine suppressed cell proliferation in PCa cells. Cell viability was performed using CCK-8 assay. (A, B) Cell viability was measured on OD450 values in PCa cell lines, PC-3 and DU145 cells. (C) Cell viability was measured on OD450 values in normal prostate epithelial cell line RWPE-1. *P < 0.05 compared with 0 μM of α-Solanine. 3

Biomedicine & Pharmacotherapy 112 (2019) 108656

J. Yang, et al.

Fig. 2. α-Solanine contributed to radiosensitivity in PCa cells. PC-3 and DU145 cells were treated with α-Solanine and X-ray irradiation. (A, B) Cell viability was measured by CCK-8. (C, D) Cell survival was detected using colony formation assay. *P < 0.05 compared with the empty group (PBS treated cells).

cut down miR-18a levels to 0.4-fold. In consideration of α-Solanine upregulating GAS5 in PC-3 and DU145 cells, the high expression of GAS5 was even promoted by ectopic expression of GAS5 and was decreased by GAS5 silencing (Fig. 7D and E). Level of miR-18a was decreased by α-Solanine, and was even lower to about 0.2-fold in presence of ectopic GAS5; moreover, GAS5 silencing reversed miR-18a level near to 0.8fold by α-Solanine (Fig. 7F and G).

were performed (Fig. 5C and D). Dramatically high expression of GAS5 was obtained from Ago2 after ectopic expression of biotin labelled miR18a in PC-3 cells. PC-3 cells were aberrantly expressed GAS5 (Fig. 5E) to explore the regulatory effect on its downstream target miR-18a. The results exhibited that miR-18a was down-regulated (0.2-fold) by GAS5 overexpression, and up-regulated (4.0-fold) when GAS5 knockdown (Fig. 5F). These information illustrated GAS5 regulated miR-18a expression by directly binding, indicating GAS5 probably displayed effects through miR-18a in PCa cells.

4. Discussion

3.6. Effects of ectopic expression of GAS5 were inversed by miR-18a mimic in PCa cells

In recent years, the incidence of prostate cancer was significantly increased, and researches on new treatments and new drugs for prostate cancer have been of important significance. This work supported the antitumor efficacy of α-Solanine on inhibition of cell proliferation and promotion of radiosensitivity in PCa cells. Furthermore, we uncovered that GAS5 was induced by α-Solanine, and high expression of GAS5 mediated the α-Solanine effect by down-regulating miR-18a, suggesting GAS5/miR-18a could serve as state-of-the-art candidates for molecular diagnosis and treatment for human prostate cancer. α-Solanine serves as tumor suppressor in cancers through several molecular mechanism. α-Solanine, the most important and active component of Solanum nigrum, was found to have anti-cancer activity on multiple cancer cells [33]. More interestingly, S. nigrum and its components exerted inhibitory effects on different pathways including PI3K/AKT, JAK-STAT, VEGF/VEGFR, and matrix metalloproteinases in different cancers [33]. Wu J et al. [8,11] believed that α-Solanine modulated the radiation- and chemo-sensitivity of esophageal cancer (EC) cells by inducing miR-138, during which α-Solanine could downregulate Survivin expression level by up-regulating miR-138 expression in EC cells. That is miR-138/Survivin pathway was the underlying molecular mechanism of α-Solanine on enhancing chemosensitivity and radiosensitivity in EC cells. In this study, we concluded that GAS5/miR18a axis contributed to PCa cell radiosensitivity by promoting X-ray irradiation-induced apoptosis and DNA damage. However, the effect of

GAS5 biological effects was investigated on cell proliferation, apoptosis and radiation sensitivity in PC-3 and DU145 cells, relatively. As shown, ectopic GAS5 caused GAS5 up regulation more than 9-fold (Fig. 6A). Cell viability was declined with ectopic expression of GAS5, which subsequently was rescued by miR-18a mimic (Fig. 6B); apoptosis rate was greatly induced by GAS5 over 22%; however it was decreased to about 13% in the presence of ectopic expression of both GAS5 and miR-18a (Fig. 6C). Ectopic GAS5 deteriorated the survival fraction induced by X-ray irradiation, and miR-18a mimic greatly reversed it (Fig. 6D). Consistent with those results, miR-18a mimic abolished ectopic GAS5-induced γ-H2AX up regulation (Fig. 6E). All these data demonstrated that the effects of ectopic GAS5 on inhibiting cell viability and survival, facilitating apoptosis and DNA damage were inversed by miR-18a mimic in PCa cells. 3.7. GAS5 mediated α-Solanine-induced down regulation of miR-18a in PCa cells Above all, miR-18a was up-regulated in PC-3 and DU145 cells (Fig. 7A). As shown in Fig. 7B and C, expression of miR-18a could be significantly down-regulated with α-Solanine, and 8 μM of α-Solanine 4

Biomedicine & Pharmacotherapy 112 (2019) 108656

J. Yang, et al.

Fig. 3. α-Solanine promoted X-ray irradiation-induced apoptosis and DNA damage in PCa cells. PC-3 and DU145 cells treated with α-Solanine and X-ray irradiation. (A, B) Cell apoptosis was examined with flow cytometry. (C, D) Expressions of γ-H2AX were monitored by western blotting. *P < 0.05 compared with the empty group (PBS treated cells).

α-Solanine/GAS5/miR-18a in PCa and associated signaling pathway remains to be further elucidated. GAS5 is firstly reported to serve a protective effect of radiosensitivity in human prostate cancer in the present study. It is increasingly acknowledged that LncRNAs play essential regulatory roles in fundamental biological processes. Dysregulation of LncRNAs may consequently contribute to major human diseases, including cancer

[34,35]. Of particular interest in this regard is that GAS5 LncRNA was down-regulated in multiple cancers [36–38]. Besides, expression levels of GAS5 were related to both clinico-pathological characteristics and patient prognosis. Whereas, GAS5 was found to be high expression in mesothelioma patient tissues [39]. Research reported that GAS5 knockdown reduced the chemosensitivity of none-small cell lung cancer (NSCLC) cell to cisplatin through miR-21/PTEN axis [22] and

Fig. 4. Expressions of GAS5 in PCa cells. (A) Lower expression of GAS5 in PC-3 and DU145 cells was observed using qPCR. *P < 0.05 compared with control cells (RWPE-1). (B, C) Higher expression of GAS5 in PC-3 and DU145 cells in the presence ofα-Solanine. Both 4 μM and 8 μM of α-Solanine induced the up regulation of GAS5. *P < 0.05 compared with 0 μM of α-Solanine. 5

Biomedicine & Pharmacotherapy 112 (2019) 108656

J. Yang, et al.

Fig. 5. GAS5 regulated miR-18a expression by sponging. (A) Potential target sites (red) was predicted on Starbase software. (B) Luciferase reporter assay was conducted to identify the binding sites. Relative luciferase activity was significantly reduced in cells transfected with GAS 3′ UTR wild type (GAS5-WT) and miR-18a mimic (miR-18a). (C, D) RIP and RNA pull-down assay were performed. Dramatically high expression of GAS5 was obtained from Ago2 and biotin labelled miR-18a (bio-miR-18a, miR-18a) in PC-3 cells. (E, F) Levels of GAS5 and miR-18a during ectopic expression of GAS5 (GAS5) and GAS5 silencing (si-GAS5) in PC-3 cells. *P < 0.05 compared with miR-NC, pcDNA or si-NC. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

biomarker. Functional studies, including our study, have shown that inhibition of proliferation and promotion of apoptosis, together with these molecular mechanisms, are likely to form the basis of tumor suppressor action of GAS5. GAS5 was aberrantly regulated with lower expression pattern among the overwhelming majority of human cancers, which conferred poor prognosis and patient survival [44,45]. At the same time, advances have been made in our understanding of the molecular mechanisms of GAS5 action in recent years, including riborepression of certain steroid hormone receptors, sequestration of miRNAs, and direct interaction with CDK6, YBX1 or other proteins [45]. When it comes to target genes, expressions of miR-18a [46], miR21 [23] and miR-103 [43] were modulated by GAS5 in PCa cells. In

autophagy pathway [40], and conferred tamoxifen resistance by miR222 in breast cancer [24]. Moreover, GAS5 regulated cisplatin resistance via sponging miR-21 in cervical cancer [23] and inhibited tumorigenesis and enhanced radiosensitivity by suppressing miR-135b expression in NSCLC [41]. In PCa, reciprocal regulation of GAS5 levels were recorded to exert similar outcome with mTOR inhibitor [42]. Information of GAS5 on inhibiting PCa cell proliferation and progression has been announced and miR-103, a target gene of GAS5, took part in AKT/mTOR signaling pathway [43]. However, this work provides the first-hand data about the radiosensitivity-enhancing effect of GAS5 in PCa cells. GAS5 promises to be a novel therapy target, as well as prognostic

Fig. 6. Effects of ectopic expression of GAS5 were inversed by miR-18a mimic in PCa cells. GAS5 effects were studied in PC-3 and DU145 cells. Transfected cells were divided into four groups: pcDNA, pcDNA-GAS5 (GAS5), GAS5+miR-NC mimic (miR-NC) and GAS5+miR-18a mimic (miR-18a). (A) GAS5 levels were examined by qPCR. (B) Cell viability was measured with CCK-8 assay. (C) Cell apoptosis was measured on flow cytometry. (C) Cell survival was detected using colony formation assay. (D) DNA damage was examined using western blotting. *P < 0.05 compared with pcDNA group or GAS5+miR-NC group. 6

Biomedicine & Pharmacotherapy 112 (2019) 108656

J. Yang, et al.

Fig. 7. GAS5 mediated α-Solanine-induced down regulation of miR-18a in PCa cells. Expressions of GAS5 and miR-18a were studied utilizing qPCR in PC-3 and DU145 cells. (A) High expression levels of miR-18a compared with that in RWPE-1 cells. (B, C) Reduced expressions of miR-18a in cells incubated with α-Solanine. (D, E) The expressions of GAS5 were measured when treated with α-Solanine and either ectopic expression of GAS5 or silencing of GAS5. (F, G) The expressions of miR-18a were measured when treated with α-Solanine and either ectopic expression of GAS5 or silencing of GAS5. *P < 0.05.

underlying through more experiments in the near future. In conclusion, our results lay the foundation of opening-up a new and promising field in the use of GAS5/miR-18a and the molecular diagnosis and treatment for human prostate cancer, especially radiation-resistant prostate cancer. In this study, we mainly discussed the molecular mechanism of the tumor suppressive effect of α-Solanine on promoting human prostate cancer cell radiosensitivity. And, GAS5 enhances α-Solanine-induced radiosensitivity by negatively regulating miR-18a in human prostate cancer cells, indicating a potential therapeutic strategy (GAS5 overexpression and/or miR-18a knockdown) for treatment of human prostate cancer.

addition, GAS5 sponged miR-135b [37] in NSCLC cells, and miR-21 among ovarian [47], renal [48], lung [22], and hepatocellular cancer cells [49]. Indeed, it is now widely acknowledged that LncRNAs are likely to be of crucial importance in the pathogenesis of cancer. Therefore, increased understanding of GAS5 may lead to novel and better approaches for the diagnosis and treatment of PCa. Here, it is the first proof up to date that α-Solanine up-regulates GAS5 expression, and the modulation of GAS5 is one of the mechanism of α-Solanine action. What’s more, accumulation of GAS5 contributes to human prostate cancer cell radiosensitivity by inhibiting cell survival and facilitating apoptosis and DNA damage. Not only that, the pathway of GAS5 functions has been identified to sponge miR-18a through directly binding in human prostate cells. What is noteworthy is that miR18a was down-regulated by α-Solanine and/or GAS5 and overexpression of miR-18a reversed GAS5-induced radiosensitivity in PCa cells. These data suggested miR-18a knockdown contributed to PCa radiosensitivity, which is just the opposite of that in other cancers [13,14,31,32]. It is imperative to dig out this difference and mechanism

Funding None.

7

Biomedicine & Pharmacotherapy 112 (2019) 108656

J. Yang, et al.

Availability of data and materials

[20] Y. Zhang, X. Su, Z. Kong, F. Fu, P. Zhang, D. Wang, H. Wu, X. Wan, Y. Li, An androgen reduced transcript of LncRNA GAS5 promoted prostate cancer proliferation, PLoS One 12 (8) (2017) e0182305. [21] M. Mourtada-Maarabouni, M.R. Pickard, V.L. Hedge, F. Farzaneh, G.T. Williams, GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer, Oncogene 28 (2) (2009) 195–208. [22] L. Cao, J. Chen, B. Ou, C. Liu, Y. Zou, Q. Chen, GAS5 knockdown reduces the chemo-sensitivity of non-small cell lung cancer (NSCLC) cell to cisplatin (DDP) through regulating miR-21/PTEN axis, Biomed. Pharmacother. 93 (2017) 570–579. [23] Q. Wen, Y. Liu, H. Lyu, X. Xu, Q. Wu, N. Liu, Q. Yin, J. Li, X. Sheng, Long noncoding RNA GAS5, which acts as a tumor suppressor via microRNA 21, regulates cisplatin resistance expression in cervical Cancer, Int. J. Gynecol. Cancer 27 (6) (2017) 1096–1108. [24] J. Gu, Y. Wang, X. Wang, D. Zhou, C. Shao, M. Zhou, Z. He, Downregulation of lncRNA GAS5 confers tamoxifen resistance by activating miR-222 in breast cancer, Cancer Lett. 434 (2018) 1–10. [25] N. Zhang, A.Y. Wang, X.K. Wang, X.M. Sun, H.Z. Xue, GAS5 is downregulated in gastric cancer cells by promoter hypermethylation and regulates adriamycin sensitivity, Eur. Rev. Med. Pharmacol. Sci. 20 (15) (2016) 3199–3205. [26] L. Wang, Q.Q. Sun, S.J. Zhang, Y.W. Du, Y.Y. Wang, W.Q. Zang, X.N. Chen, G.Q. Zhao, Inhibitory effect of alpha-solanine on esophageal carcinoma in vitro, Exp. Ther. Med. 12 (3) (2016) 1525–1530. [27] L.Z. Zhu, Q. Zhu, H.H. Wen, X. Huang, G. Zheng, Mutations in gas5 affect the transformation from benign prostate proliferation to aggressive prostate cancer by affecting the transcription efficiency of gas5, J. Cell. Physiol. (2018). [28] C.H. Ma, X.F. Shi, Q.Q. Zhu, Q. Li, Y.F. Liu, Y.W. Yao, Y. Song, The growth arrestspecific transcript 5 (gas5): A pivotal tumor suppressor long noncoding rna in human cancers, Tumour Biol. 37 (2) (2016) 1437–1444. [29] Y.Y. Zhang, X.Y. Su, Z. Kong, F.Q. Fu, P. Zhang, D. Wang, H. Wu, X.C. Wan, Y. Li, An androgen reduced transcript of lncrna gas5 promoted prostate cancer proliferation, PLoS One 12 (8) (2017) e0182305. [30] G. Luo, D. Liu, C. Huang, M. Wang, X.Y. Xiao, F.Q. Zeng, L. Wang, G.S. Jiang, Lncrna gas5 inhibits cellular proliferation by targeting p27(kip1), Mol. Cancer Res. 15 (7) (2017) 789–799. [31] L. Song, C. Lin, Z. Wu, H. Gong, Y. Zeng, J. Wu, M. Li, J. Li, miR-18a impairs DNA damage response through downregulation of ataxia telangiectasia mutated (ATM) kinase, PLoS One 6 (9) (2011) e25454. [32] X. Chen, L. Wu, D. Li, Y. Xu, L. Zhang, K. Niu, R. Kong, J. Gu, Z. Xu, Z. Chen, J. Sun, Radiosensitizing effects of miR-18a-5p on lung cancer stem-like cells via downregulating both ATM and HIF-1alpha, Cancer Med. 7 (8) (2018) 3834–3847. [33] G. Butt, M.A. Romero, F. Tahir, A.A. Farooqi, Emerging themes of regulation of oncogenic proteins by Solanum nigrum and its bioactive molecules in different cancers, J. Cell. Biochem. 119 (12) (2018) 9640–9644. [34] J.R. Evans, F.Y. Feng, A.M. Chinnaiyan, The bright side of dark matter: lncRNAs in cancer, J. Clin. Invest. 126 (8) (2016) 2775–2782. [35] Y. Mitobe, K.I. Takayama, K. Horie-Inoue, S. Inoue, Prostate cancer-associated lncRNAs, Cancer Lett. 418 (2018) 159–166. [36] M.R. Pickard, G.T. Williams, Regulation of apoptosis by long non-coding RNA GAS5 in breast cancer cells: implications for chemotherapy, Breast Cancer Res. Treat. 145 (2) (2014) 359–370. [37] X. Shi, M. Sun, H. Liu, Y. Yao, R. Kong, F. Chen, Y. Song, A critical role for the long non-coding RNA GAS5 in proliferation and apoptosis in non-small-cell lung cancer, Mol. Carcinog. 54 (Suppl. 1) (2015) E1–E12. [38] L. Liu, T. Meng, X.H. Yang, P. Sayim, C. Lei, B. Jin, L. Ge, H.J. Wang, Prognostic and predictive value of long non-coding RNA GAS5 and mircoRNA-221 in colorectal cancer and their effects on colorectal cancer cell proliferation, migration and invasion, Cancer Biomark. 22 (2) (2018) 283–299. [39] A. Renganathan, J. Kresoja-Rakic, N. Echeverry, G. Ziltener, B. Vrugt, I. Opitz, R.A. Stahel, E. Felley-Bosco, GAS5 long non-coding RNA in malignant pleural mesothelioma, Mol. Cancer 13 (2014) 119. [40] N. Zhang, G.Q. Yang, X.M. Shao, L. Wei, GAS5 modulated autophagy is a mechanism modulating cisplatin sensitivity in NSCLC cells, Eur. Rev. Med. Pharmacol. Sci. 20 (11) (2016) 2271–2277. [41] Y. Xue, T. Ni, Y. Jiang, Y. Li, Long noncoding RNA GAS5 inhibits tumorigenesis and enhances radiosensitivity by suppressing miR-135b expression in non-small cell lung cancer, Oncol. Res. 25 (8) (2017) 1305–1316. [42] K. Yacqub-Usman, M.R. Pickard, G.T. Williams, Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells, Prostate 75 (7) (2015) 693–705. [43] D. Xue, C. Zhou, H. Lu, R. Xu, X. Xu, X. He, LncRNA GAS5 inhibits proliferation and progression of prostate cancer by targeting miR-103 through AKT/mTOR signaling pathway, Tumour Biol. (2016) [Epub ahead of print]. [44] H.P. Qiao, W.S. Gao, J.X. Huo, Z.S. Yang, Long non-coding RNA GAS5 functions as a tumor suppressor in renal cell carcinoma, Asian Pac. J. Cancer Prev. 14 (2) (2013) 1077–1082. [45] M.R. Pickard, G.T. Williams, Molecular and cellular mechanisms of action of tumour suppressor GAS5 LncRNA, Genes (Basel) 6 (3) (2015) 484–499. [46] Q. Liu, W. Yu, S. Zhu, K. Cheng, H. Xu, Y. Lv, X. Long, L. Ma, J. Huang, S. Sun, K. Wang, Long noncoding RNA GAS5 regulates the proliferation, migration, and invasion of glioma cells by negatively regulating miR-18a-5p, J. Cell. Physiol. 234 (1) (2018) 757–768. [47] N. Ma, S. Li, Q. Zhang, H. Wang, H. Qin, S. Wang, Long non-coding RNA GAS5 inhibits ovarian cancer cell proliferation via the control of microRNA-21 and SPRY2 expression, Exp. Ther. Med. 16 (1) (2018) 73–82. [48] L. Liu, X. Pang, W. Shang, H. Xie, Y. Feng, G. Feng, Long non-coding RNA GAS5 sensitizes renal cell carcinoma to sorafenib via miR-21/SOX5 pathway, Cell Cycle (2018) [Epub ahead of print]. [49] L. Hu, H. Ye, G. Huang, F. Luo, Y. Liu, Y. Liu, X. Yang, J. Shen, Q. Liu, J. Zhang, Long noncoding RNA GAS5 suppresses the migration and invasion of hepatocellular carcinoma cells via miR-21, Tumour Biol. 37 (2) (2016) 2691–2702.

All data generated or analyzed during this study are included in this article. Consent for publication Not applicable. Competing interests The authors declare that there are no financial competing interests. Acknowledgements Not applicable. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.108656. References [1] C. Chen, K. Wang, Q. Wang, X. Wang, LncRNA HULC mediates radioresistance via autophagy in prostate cancer cells, Braz. J. Med. Biol. Res. 51 (6) (2018) e7080. [2] M.L. Lindenberg, B. Turkbey, E. Mena, P.L. Choyke, Imaging locally advanced, recurrent, and metastatic prostate Cancer: a review, JAMA Oncol. 3 (10) (2017) 1415–1422. [3] S. Evans-Axelsson, O.V. Timmermand, A. Bjartell, S.E. Strand, J. Elgqvist, Radioimmunotherapy for prostate cancer–current status and future possibilities, Semin. Nucl. Med. 46 (2) (2016) 165–179. [4] M. Friedman, Chemistry and anticarcinogenic mechanisms of glycoalkaloids produced by eggplants, potatoes, and tomatoes, J. Agric. Food Chem. 63 (13) (2015) 3323–3337. [5] X.Y. Gu, X.F. Shen, L. Wang, Z.W. Wu, F. Li, B. Chen, G.L. Zhang, M.K. Wang, Bioactive steroidal alkaloids from the fruits of Solanum nigrum, Phytochemistry 147 (2018) 125–131. [6] M.K. Lu, Y.W. Shih, T.T. Chang Chien, L.H. Fang, H.C. Huang, P.S. Chen, alphaSolanine inhibits human melanoma cell migration and invasion by reducing matrix metalloproteinase-2/9 activities, Biol. Pharm. Bull. 33 (10) (2010) 1685–1691. [7] K.H. Shen, A.C. Liao, J.H. Hung, W.J. Lee, K.C. Hu, P.T. Lin, R.F. Liao, P.S. Chen, alpha-Solanine inhibits invasion of human prostate cancer cell by suppressing epithelial-mesenchymal transition and MMPs expression, Molecules 19 (8) (2014) 11896–11914. [8] Y. Wang, J. Wu, W. Guo, Q. Sun, X. Chen, W. Zang, Z. Dong, G. Zhao, alphaSolanine modulates the radiosensitivity of esophageal cancer cells by inducing MicroRNA 138 expression, Cell. Physiol. Biochem. 39 (3) (2016) 996–1010. [9] M. Mohsenikia, A.M. Alizadeh, S. Khodayari, H. Khodayari, S.A. Kouhpayeh, A. Karimi, M. Zamani, S. Azizian, M.A. Mohagheghi, The protective and therapeutic effects of alpha-solanine on mice breast cancer, Eur. J. Pharmacol. 718 (1-3) (2013) 1–9. [10] W.F. Zhong, S.P. Liu, B. Pan, Z.F. Tang, J.G. Zhong, F.J. Zhou, [Solanine inhibits prostate cancer Du145 xenograft growth in nude mice by inducing cell cycle arrest in G1/S phase], Nan Fang Yi Ke Da Xue Xue Bao 36 (5) (2016) 665–670. [11] J. Wu, L. Wang, X. Du, Q. Sun, Y. Wang, M. Li, W. Zang, K. Liu, G. Zhao, alphasolanine enhances the chemosensitivity of esophageal cancer cells by inducing microRNA138 expression, Oncol. Rep. 39 (3) (2018) 1163–1172. [12] F. Zhang, R. Yang, G. Zhang, R. Cheng, Y. Bai, H. Zhao, X. Lu, H. Li, S. Chen, J. Li, S. Wu, P. Li, X. Chen, Q. Sun, G. Zhao, Anticancer function of alpha-solanine in lung adenocarcinoma cells by inducing microRNA-138 expression, Tumour Biol. 37 (5) (2016) 6437–6446. [13] S. Liu, X. Pan, Q. Yang, L. Wen, Y. Jiang, Y. Zhao, G. Li, MicroRNA-18a enhances the radiosensitivity of cervical cancer cells by promoting radiation-induced apoptosis, Oncol. Rep. 33 (6) (2015) 2853–2862. [14] Z. Shen, X. Wu, Z. Wang, B. Li, X. Zhu, Effect of miR-18a overexpression on the radiosensitivity of non-small cell lung cancer, Int. J. Clin. Exp. Pathol. 8 (1) (2015) 643–648. [15] E. Shajari, H. Mollasalehi, Ribonucleic-acid-biomarker candidates for early-phase group detection of common cancers, Genomics (2018) [Epub ahead of print]. [16] C.J. Song, H. Chen, L.Z. Chen, G.M. Ru, J.J. Guo, Q.N. Ding, The potential of microRNAs as human prostate cancer biomarkers: a meta-analysis of related studies, J. Cell. Biochem. 119 (3) (2018) 2763–2786. [17] T.I. Hsu, C.H. Hsu, K.H. Lee, J.T. Lin, C.S. Chen, K.C. Chang, C.Y. Su, M. Hsiao, P.J. Lu, MicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo, Oncogenesis 3 (2014) e99. [18] C. Ma, X. Shi, Q. Zhu, Q. Li, Y. Liu, Y. Yao, Y. Song, The growth arrest-specific transcript 5 (GAS5): a pivotal tumor suppressor long noncoding RNA in human cancers, Tumour Biol. 37 (2) (2016) 1437–1444. [19] X. Yu, Z. Li, Long non-coding RNA growth arrest-specific transcript 5 in tumor biology, Oncol. Lett. 10 (4) (2015) 1953–1958.

8