Background
Prostate cancer (PC) is one of the most common malignancies in male, and its incidence is increasing every year in the world. Organ-confined PC can be effectively treated through radical prostatectomy or radiation therapies [
1]. However, for advanced prostate carcinoma, androgen deprivation therapy (ADT) is the first line of therapeutic intervention [
2,
3]. Once hormone resistance develops, advanced PC is typically fatal within approximately 1 year [
4]. Currently, docetaxel (Doc)-based chemotherapy is considered to be therapeutically efficacious for metastatic castration-resistant PC [
5]. Unfortunately, many patients often encounter several undesirable side effects [
6], and drug resistance often leads to treatment failure [
7]. Thus, there is an urgent need to identify factors that influence efficacy of docetaxel therapy. Although recent studies suggest that some microRNAs (miRNAs), such as miR-375 [
8], miR-200c and miR-205 [
6], might be involved in docetaxel resistance of PC, the molecular mechanisms of the acquired docetaxel resistance are largely unknown.
miRNAs play a critical role in tumor progress by regulating gene expression at the posttranscriptional level. Several miRNAs, including miR-34a [
9], miR-375 [
8], miR-124 [
10], miR-205 [
11] and miR-21 [
12] have been implicated in tumor drug resistance. Recent studies showed that miR-193a-5p but only suppresses tumor growth, but also promotes tumor progression through regulating cell proliferation [
13,
14] and apoptosis, as well as through inducing drug resistance [
15,
16]. A previous study reported that miR-193a-3p, another mature miRNA of miR-193a precursor family, regulates the multi-drug resistance of bladder cancer by targeting the LOXL4 gene [
17]. However, it remains unclear whether miR-193a-5p is involved in the resistance of PC cells to docetaxel-induced apoptosis.
Heme oxygenase-1 (HO-1), a cytoprotective enzyme, exerts antioxidant, anti-inflammatory, and anti-apoptotic effect [
18]. HO-1 overexpression is known to be associated with PC progression and poor clinical outcomes [
19] . Under oxidative stress conditions caused by chemotherapeutic agents, cancer cells upregulate antioxidant factors, such as HO-1, and enhance their anti-apoptotic capacity to protect against oxidative injury induced by anticancer agents [
20]. However, the precise mechanism underlying anticancer agent-induced HO-1 upregulation remains largely unclear.
Previous studies have demonstrated that HO-1 gene transcription is highly inducible, and its expression is regulated by the different transcription factors, such as Nrf2 [
21], Bach1 [
22], activator protein-1(AP-1) [
23] and PPARα [
24]. Furthermore, Bach2 has been shown to transcriptionally repress HO-1 expression in chronic myeloid leukemia (CML) cells, which induces apoptosis in response to oxidative stress [
25]. Although low Bach2 expression was reported to be associated with high leukemic cell proliferation, unfavorable clinical features, and poor clinical outcome in acute lymphoblastic leukemia (ALL) [
25,
26], there are only a few reports about the role of Bach2 in solid tumors. Moreover, the specific contribution of Bach2 to the resistance of PC cells to docetaxel-induced apoptosis has not been investigated.
In the present study, we detected the apoptosis-associated gene (Bcl-2, Bax and cleaved caspase-3) expression in human PC tissues and PC cell line in the context of docetaxel treatment. Our findings provide the evidence that regulatory crosstalk between miR-193a-5p, Bach2 and HO-1 is responsible for the resistance of PC cells to docetaxel-induced apoptosis. Furthermore, our results have linked miR-193a-5p to the regulation of Bach2 and HO-1 expression in human PC.
Methods
Patients
Patients (median age 65 years, range 52 to 79) underwent radical prostatectomy for localized PC (
n = 62) and benign prostatic hyperplasia (n = 62) underwent underwent transurethral resection of the prostate (TURP) at the department of urology, the second hospital of Hebei medical university, China from July 2014 to October 2017. No treatment was administered prior to surgery. All the tissue specimens were confirmed by two experienced pathologists. Pathological grading was judged by Gleason points-scoring system. The patient characteristics are summarized in Additional file
1: Table S1. The study protocol was approved by the Ethics Committee of Second Hospital of Hebei Medical University and Verbal consent was obtained from each patient.
Cell culture and transfection
PCa cell lines (LNCap, PC3 and DU145), bladder cancer cell lines (T24, UM-UC-3) and the human normal prostate epithelial cell line (RWPE-1) were originally obtained from the American Type Culture Collection (ATCC, Manassas, USA). The LNCap, PC3, DU145 and UM-UC-3 cells were cultured in RPMI 1640 medium (Gibco Life Technologies, Rockville, MD) containing 10% fetal bovine serum (FBS) (Foundation, Gemini, CR/US), and RWPE-1 cells were grown in K-SFM supplemented with 10% FBS; T24 cells were grown in McCoy’s 5A (Modified) Medium (Thermo Fisher, 16,600,082). All kinds of cells were incubated at 37 °C in a humidified incubator with 5% CO2. According to the manufacturer’s protocol, the transfection of all cells was carried out using Lipofectamine 2000 (Invitrogen). The miR-193a-5p mimics, mimic NC, miR-193a-5p inhibitors, inhibitor NC and Bach2 siRNA were purchased from GenePharma Co., Ltd. (Shanghai, China). After 24~48 h of transfection, the cells were harvested and lysed for Western blotting, and the total RNA was extracted for qRT-PCR.
Xenograft animal model
All animal studies were approved by the Institutional Animal Care and Use Committee of Hebei Medical University (approval ID: HebMU 20,080,026), and all efforts were made to minimize suffering. Xenograft model was performed as described previously [
27]. In brief, male BALB/c nude mice at 4–6 weeks of age (18–22 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, china). 5 × 10
6 LV-Ctl- or LV-miR-193a-5p-infected PC3 cells were harvested by trypsinization and resuspended in 0.2 mL PBS mixed with 50% Matrigel (Collaborative Research Inc., Bedford, MA, USA); this suspension was injected subcutaneously into the right dorsal flanks. When the average volume of the tumors reached 180 mm
3, mice were randomly divided into PBS control group or 10 mg/kg Doc group (Cayman Chemicals, Ann Arbor, MI). Mice were given intraperitoneal injection once per week for four weeks. The length and width of mouse tumor were measured twice a week with calipers. Then the following formula was used to calculate tumor volume (volume = [(length × width
2)/2]). At the end of this experiment, the mice were euthanized by Carbon dioxide asphyxiation. At last, the tumor tissues were fixed in 4% formalin solution or flash frozen in liquid nitrogen immediately, and stored at −80 °C until further use.
RNA extraction and quantitative real-time PCR
Clinical and xenograft tissues were homogenized with a gentle MACSTM Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), and cultured cells were lysed using QIAzol Lysis Reagent (79306). The concentration and purity of the RNA were determined by using NanoDrop 2000 (Thomer Fisher). For microRNA, the miScripIIRT kit (QIAGEN GmbH, D-40724 Hilden, GERMANY) was used for reverse transcription, and the miScript SYBR
® Green PCR kit was used for qRT-PCR with specific primers for miR-193a-5p, and the RNU6b (U6) was used as internal control. For large mRNA analysis, reverse transcription of RNA was performed by using the M-MLV First Strand Kit (Life Technologies). The Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen) was used for the qRT-PCR of mRNAs. The real-time PCR experiments were carried on a CFX96™ Real-Time System (Bio-Rad). All data were normalized with GAPDH and analyzed by adopting 2
-ΔΔCt method as described previously [
8].
Western blot analysis
Western blotting was performed as described previously [
28]. In brief, frozen tissue samples were homogenized in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate and 0.1% SDS), and cultured cells were lysed with lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM Na
3VO
4, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% NP-40). Equal amounts of protein were run on 10% SDS-PAGE, and electro-transferred to a polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked with 5% milk in TTBS at room temperature for 2 h and then incubated with primary antibodies overnight at 4 °C. The antibodies that were used were as follows: anti-HO-1 (1:500, ab13248), anti-Bach2 (1:500, ab83364), anti-caspase 3 (1:1000, ab13847), anti-Nrf2 (1:1000, ab31163), anti-Bcl-2 (1:1000, 12,789–1-AP), anti-Maf (1:500, 55,013–1-AP), anti-Bax (1:1000, 50,599–2-Ig) or anti-β-actin (1:1000, sc-47,778). Membranes were then incubated with the HRP-conjugated secondary antibody (1:5000, Rockland) for 1 h at room temperature. The blots were treated with the Immobilo™ Western (Millipore), and detected by ECL (enhanced chemiluminescence) Fuazon Fx (Vilber Lourmat). Images were captured and processed by FusionCapt Advance Fx5 software (Vilber Lourmat). All experiments were replicated three times.
In situ hybridization
In situ hybridization was performed as described previously [
28]. In brief, according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon), paraffin cross-sections (5-μm thick) from clinical PC tissues were deparaffinized and rehydrated for fluorescence in situ hybridization. Hybridization was performed using fluorescence-labeled miR-193a-5p probes with hybridization buffer (Exiqon) by incubation at 56 °C for 1 h in a thermo-block (Labnet, USA). After stringent washing with SSC buffer, nonspecific binding sites were blocked with 10% normal goat serum (710,027, KPL, USA). According to need, the sections were then incubated for 1 h at 37 °C with anti-HO-1 primary antibody (ab13248, Abcam) or anti-Bach2 (ab83364, Abcam) diluted 1:50 in PBS or incubated with secondary antibody directly. After washing with PBS, the sections were incubated with a rhodamine-labeled secondary antibody (031506, KPL, USA) at 37 °C for 30 min. Images were acquired by using a Leica microscope (Leica DM6000B, Switzerland) and digitized with a software of LAS V.4.4 (Leica).
Vector construction and luciferase reporter assay
All plasmids were constructed using restriction-enzyme digestion and one-step cloning (ClonExpress II One Step Cloning Kit, C112–02; Vazyme Biotech Co., Ltd., Nanjing, PR China) or recombinant methods. The 3′ untranslated region (UTR) sequences of Bach2 containing wild-type or mutant forms of the miR-193a-5p target site were inserted into the
Xho1 and
Sal1 digested-pmir-GLO Dual-Luciferase miRNA Target Expression Vector (Promega Corp., Madison, WI, USA). 4.9 kb HO-1 promoter sequence was obtained by PCR with primer (Additional file
2: Table S3) and inserted into the
Mlu1 and
Xho1 digested-pGL3-basic vector (Promega Corp., Madison, WI, USA). Luciferase assay was performed as described previously [
29]. In brief, PC3 cells were seeded into a 24-well plate, Bach2 reporter construct (wild-type or mutant) or the empty reporter vector was co-transfected with miR-193a-5p mimic and pRL-TK, or co-transfected with mimic ctl and pRL-TK, or PC3 cells were co-transfected with pGL3-HO-1-luc vector and si-Bach2. After 24 h of transfection, luciferase activity was measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI) with a Flash and Glow (LB955, Berthold Technologies) reader. The specific target activity was expressed as the relative activity ratio of firefly luciferase to Renilla luciferase.
Immunofluorescence staining
Cells were fixed with 4% formaldehyde and pre-incubated with 10% normal goat serum (710,027, KPL, USA), and then incubated with primary antibodies anti-Bach2 (ab83364, Abcam) and anti-HO-1 (ab13248, Abcam). Secondary antibodies were fluoresce-labeled antibody to rabbit IgG (021516, KPL, USA) and rhodamine-labeled antibody to mouse IgG (031806, KPL, USA). DAPI (157,574, MB biomedical) was used for nuclear counter staining. Images were captured by confocal microscopy (DM6000 CFS, Leica) and processed by LAS AF software.
Immunohistochemistry (IHC) analysis
Five-micrometer paraffin cross-sections of the tissues were deparaffinized in xylene solution and rehydrated by using gradient ethanol concentrations. Sections were subjected to antigen retrieval with citrate buffer. After hydrogen peroxide and protein blocking, the sections was incubated with HO-1 primary antibody at 4 °C overnight, and then was incubated in streptavidin (HRP)-biotin labeled secondary antibody. 3, 3′-diaminobenzidine was used to detect the peroxidase. Images were acquired using a Leica microscope (Leica DM6000B, Switzerland) and digitized with LAS V.4.4 (Leica). Positively stained cells were counted in at least five fields from each area with 400 × magnification.
Chromatin immunoprecipitation (ChIP) assay
The chromatin immunoprecipitation (ChIP) assay was performed as described previously [
29]. Briefly, PC3 cells were treated with docetaxel after transfected with anti-miR-ctl or anti-miR-193a-5p for 24 h. According to the manufacturer’s protocol of EZ-CHIP™ Chromatin Immunoprecipitation Kit (Millipore, #17–371), cells were crosslinked with 1% formaldehyde and sonicated to an average size of 400–600 bp. Bach2 antibody (ab83364, Abcam) and normal mouse IgG control were used for ChIP, respectively. The precipitated DNA was purified and analyzed by qRT-PCR amplification using primers specific for the HO-1 promoter.
Cell apoptosis
TUNEL staining was performed to evaluate cell apoptosis as previously described [
28]. In brief, PC3 cells were treated with 10 nM docetaxel combined with 20 μM Hemin or Znpp for 24 h and fixed by using 4% formaldehyde. Paraffin cross-sections (5-μm thick) of xenograft tissues were deparaffinized and rehydrated for TUNEL staining according to the manufacturer’s instructions (Vazyme, TUNEL Bright-Red Apoptosis Detection Kit, A113). TUNEL-positive cells were counted under fluorescence microscopy (DMI4000B, Leica).
Statistical analysis
All of the data were represented as the means ± S.E.M. Independent Student’s
t-test was used for comparisons of differences between two groups. The correlation between miR-193a-5p and Bach2 mRNA expression was evaluated using Spearman’s correlation analysis. Results were considered statistically significant at
p < 0.05. Observer variation in immunohistochemical staining was analyzed by interclass correlation coefficient (ICCC) and κ statistics (κ) [
32].
Discussion
In this study, we found that 1) miR-193a-5p is upregulated in PC tissues and cell lines, 2) miR-193a-5p promotes HO-1 expression through downregulating Bach2 level, 3) HO-1 upregulation leads to resistance of PC3 cells to docetaxel-induced apoptosis, 4) miR-193a-5p, Bach2 and HO-1 constitute a regulatory axis and coordinate docetaxel-induced apoptosis in PC3 cells, and 5) Silencing of miR-193a-5p enhances sensitization of PC3 cells to docetaxel-induced apoptosis and reduces PC xenograft growth in vivo.
Previous studies have demonstrated that STAT1 [
37], PIM-1 [
38], and β3-tubulin [
39] might be implicated in docetaxel resistance of PC. Mechanistically, STAT1 mediates the resistance of DU145 cells to docetaxel through inducing the expression of clusterin that is involved in cell survival in the presence of docetaxel, blockage of STAT1 expression by siRNA decreases clusterin expression and inhibits PC cell proliferation by re-sensitizing drug-resistant tumor cells to docetaxel [
38]. Serine/threonine kinase PIM-1 protects PC cells from apoptosis induced by docetaxel through phosphorylating transmembrane drug efflux pump BCRP/ABCG2 [
39]. Functional overexpression or knockdown of β3-tubulin modulates PC cell line sensitivity to docetaxel presumably through altering cell morphology and the rate of cell proliferation [
40].
In the recent decades, several microRNAs have been identified to be involved in tumor development and progression through acting either as tumor suppressors or oncogenes [
40]. For example, a previous study reported that the expression of miR-200 family members miR-200c and miR-205, which function as key regulators of EMT, was significantly reduced in docetaxel-resistant cells [
6]. Transfection of miR-200c and miR-205 restored E-cadherin expression level, accompanied by increased apoptosis, in docetaxel-resistant cells, suggesting that reduced miR-200c and miR-205 levels during chemotherapy are responsible for cancer cell survival and drug resistance [
6]. Our previous study showed that miR-146a functioned as a tumor suppressor in PC cells, and increased miR-146a expression in both LNCaP and PC3 cells by 5-Aza-2′-deoxycytidine correlated with delayed progression of castration-resistant PC [
27]. In some studies, miR-193a-5p was downregulated in several types of cancers, therefore, miR-193a-5p is believed to be an important tumor inhibitor. However, miR-193a-5p was also reported to be upregulated in certain cancer types including PC [
33,
41]. Thus, miR-193a-5p could play a dual role in tumor development and progression, depending on the type of cancers or anticancer drugs used in cancer therapy. In this study, we found that miR-193a-5p was upregulated in PC tissues and PC cell lines, and the upregulation of miR-193a-5p was closely associated with PC development. We further confirmed that miR-193a-5p expression was significantly decreased in H
2O
2-treated PC3 cells, and miR-193a-5p mimic reduced, whereas its antagomir increased PC3 cell apoptosis induced by oxidative stress. Importantly, miR-193a-5p upregulation also attenuated PC3 cell apoptosis induced by docetaxel, while depletion of miR-193a-5p enhanced sensitization of PC cells to docetaxel-induced apoptosis.
Apoptosis is a physiological process that eliminates abnormal or nonfunctional cells and is critical for maintenance of tissue homeostasis, and failure of apoptosis results in accumulation of abnormal cells, potentially leading to tumor development [
42]. Cell apoptosis is regulated at multiple levels and involves anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax. Various chemotherapeutic drugs have been shown to induce apoptosis in both in vitro and in vivo studies, suggesting that apoptosis plays a crucial role in cancer treatment [
43]. It is well known that the ability of docetaxel to kill tumor cells depends partly on its ability to induce apoptosis in tumor cells [
7]. HO-1 has an anti-apoptotic effect on certain cancer cells by regulating cellular homeostasis and promoting cell survival [
44], these effects would be relevant to resistance to chemotherapy [
45]. Indeed, HO-1 upregulation was observed in different human cancers [
46], and HO-1 expression level was closely related to the disease severity of cancers. Sacca et al. revealed that the degree of HO-1 expression in the nuclei of PC cells was positively correlated with Gleason score, the higher the Gleason score, the more the number of nuclear HO-1-positive staining [
47]. These results clearly suggest that HO-1 upregulation facilitates the progression of cancers. Consistent with these reports, we found that expression level of HO-1 was not only correlated with Gleason grades, but also with aggressive pathologic features, such as tumor stage and PSA level. Importantly, we showed that miR-193a-5p overexpression further increased HO-1 expression level induced by docetaxel and attenuated PC3 cell apoptosis. Reversely, depletion of miR-193a-5p markedly reduced docetaxel-induced HO-1 expression and promoted PC3 cell apoptosis. These results suggest that miR-193a-5p mediates docetaxel regulation of HO-1 expression. We further used HO-1 inhibitor or HO-1 inducer to treat PC3 cells and determined the effects of HO-1 on docetaxel-induced PC3 cell apoptosis. Experimental results revealed that HO-1 upregulation increased the resistance of PC3 cells to docetaxel-induced apoptosis.
Previous studies have suggested that the anti-apoptotic genes Bcl-2 and Bcl-xL were markedly upregulated in paclitaxel-resistant hepatoma cell line, and Bcl-xL expression was enhanced by paclitaxel treatment [
48]. In addition, Bcl-2 upregulation was observed in 30–60% of PC, as well as in nearly 100% of hormone-refractory PC [
49]. Bax is a member of the Bcl-2 family and counteracted the anti-apoptotic roles of Bcl-2 [
50]. Reagan-Shaw et al. reported that vitamin E and selenium induced apoptosis of the LNCaP, DU145 and PC3 cell lines through upregulating Bax, Bak and Bid, as well as through downregulating Bcl-2 [
51]. These observations suggest that the anti-tumor effects of chemotherapeutic drugs occur through their regulation of the Bcl-2 signaling pathway. But there are only few evidences to show the relationship between HO-1, Bcl-2 and Bax in PC. In this study, we demonstrated for the first time that there is a positive correlation between Bcl-2 and HO-1 mRNAs but a negative correlation between Bax and HO-1 mRNAs in PC3 cells. Based on the fact that docetaxel combined with HO-1 inhibitor repressed Bcl-2 expression and enhanced Bax expression, whereas docetaxel combined with HO-1 inducer increased Bcl-2 and reduced Bax expression, it can be concluded that HO-1 upregulation leads to resistance of PC3 cells to docetaxel-induced apoptosis by increasing Bcl-2 and decreasing Bax expression.
There is no binding site of miR-193a-5p in the 3′-UTR of HO-1 gene. Thus, we thought that HO-1 may not be a direct target of miR-193a-5p. Because the HO-1 promoter has multiple copies of the antioxidant-response element (ARE) [
35], and these elements can bind with the transcription factor Nrf2 [
21] and the transcriptional repressor Bach1 [
22], we determined whether miR-193a-5p suppresses the expression of transcription factors which regulate HO-1 expression. As expected,transfection of PC3 cells with miR-193a-5p mimic significantly decreased the expression of Bach2, but not Nrf2, Bach1 or Hif1α. Transfection of miR-193a-5p mimic combined with docetaxel treatment further attenuated Bach2 expression. Further, Bach2 3′-UTR-luciferase reporter assay revealed that miR-193a-5p downregulated Bach2 expression by directly targeting the miR-193a-5p-binding site in the Bach2 3′-UTR. Moreover, we found that Bach2 expression was significantly lower in PC than in BPH tissues, and expression level of miR-193a-5p closely correlated with Bach2 level in human PC tissues. A recent study revealed that Bach2 functioned in a variety of cellular lineages that can either promote or suppress immune responses against tumors [
52]. Consistent with this, we showed that Bach2 induced PC cell apoptosis through repressing HO-1 expression. Notably, we found that a modest increase of HO-1 induced by docetaxel facilitated docetaxel-induced cell apoptosis. However, docetaxel-induced miR-193a-5p upregulation, which in turn inhibits Bach2 expression and thus enhances HO-1 expression, partly counteracts docetaxel-induced apoptosis. Therefore, silencing of miR-193a-5p can increase the sensitization of PC cells to docetaxel-induced apoptosis.