Background
According to global cancer statistics, about 782,500 new liver cancer cases occurred worldwide in 2012, together with about 745,500 deaths [
1]. Seventy to 90 % of primary liver cancers are hepatocellular carcinoma (HCC). Since patients are asymptomatic at the early stages of the disease, HCC is mostly diagnosed at an advanced stage [
2]. Moreover, the effectiveness of anti-cancer treatment varies considerably among different HCC patients. Therefore, it is necessary to identify targets for enhancing the sensitivity to chemotherapeutic management of HCC.
In recent years, a growing number of studies have shown that protein phosphatase 2A (PP2A) is an important tumor suppressor. As a crucial member of the serine/threonine protein phosphatase family widely expressed in eukaryotic cells, PP2A is involved in the regulation of the signal transduction, cell cycle, proliferation, differentiation, apoptosis, and other processes [
3]. PP2A-B55δ subunit, encoded by the
PPP2R2D gene, is one of four isoforms (α, β, γ, and δ) of the PP2A B55 regulatory subunit family [
4]. The interaction between B55δ and cyclin-dependent kinase 1 (CDK1) is reported to play a critical role in cell cycle progression [
5]. However, it is still unclear whether B55δ enhances chemotherapy sensitivity of HCC cells by regulating the cell cycle.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression either through mRNA degradation or translational repression [
6]. The interaction with the 3’-untranslated region (3’UTR) of the targeted mRNAs via base pairing is thought to be the main mechanism of miRNA function [
7]. As nodes of signaling networks, miRNAs play a role in the regulation of metabolic homeostasis and cancer development [
8‐
10]. Recent studies suggest a number of clinically significant miRNAs that may target PP2A [
11]. In view of the lack of conclusive information on the miRNA regulation of
PPP2R2D, this study aimed to characterize the functional role of miRNA targeting of
PPP2R2D in the chemotherapy of HCC
.
As a key drug in the treatment of advanced HCC [
12], cisplatin (cDDP) was selected as a representative chemotherapeutic drug of HCC in this study. We explored the role of PP2A-B55δ both in regulating the cell cycle, migration, colony formation, apoptosis, and proliferation of human hepatoblastoma HepG2 cells and in tumor growth in xenograft mice in the presence of cDDP, and we investigated the details of the microRNA-133b (miR-133b)/
PPP2R2D signaling pathway. We concluded that PP2A-B55δ, under the regulation of miR-133b, could serve as a promising target for increasing chemotherapy sensitivity of HCC.
Methods
Cell culture and reagents
The human hepatic cell line L02, HCC cell lines HepG2, MHCC97H, MHCC97L, Hep3B, Huh7 and human embryonic kidney 293T cells (HEK-293T) were obtained from the Cancer Center of Sun Yat-sen University (Guangzhou, China). The human hepatic cell line QSG7701 and HCC cell line QGY7703 were obtained from the College of Chemistry and Chemical Engineering, Xiamen University (Xiamen, China). L02, QSG7701, HepG2, Hep3B, and QGY7703 were cultured in RPMI-1640 medium (Gibco, NY, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco) and 1 % penicillin-streptomycin (P&S, Gibco). MHCC97H, MHCC97L, and Huh7 were cultured in DMEM (Gibco) with 10 % FBS and 1 % P&S. All cell lines were maintained at 37 °C in a 5 % CO2 humidified incubator (Thermo, CO, USA). cDDP was obtained from Hansoh Pharmaceutical Co., Ltd (Jiangsu, China).
Antibodies
The primary antibodies for PP2A-B55δ were purchased from Abcam (MA, USA) and Santa Cruz (CA, USA). PP2A-Aα subunit antibody was from Covance (NJ, USA). PP2A-C subunit, phosphorylated CDK1 (p-CDK1 Tyr15), CDK1, and cleaved Caspase-3 antibodies, and the secondary antibodies anti-rabbit IgG and anti-mouse IgG were from Cell Signaling Technology (MA, USA). PP2A-B55α antibody was from Merck Millipore (MA, USA). PP2A-B56γ antibody was obtained from Thermo. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and B-cell lymphoma 2-related protein (Bcl-2) antibodies were from Beyotime (Shanghai, China). Antibodies for Cyclin B1, Cyclin E1, Bcl-2-associated X protein (Bax), and proliferating cell nuclear antigen (PCNA) were from Ruiying Bioalogical (Jiangsu, China). The secondary antibodies anti-rat IgG and anti-goat IgG were from Proteintech (IL, USA). The fluorescein isothiocyanate-conjugated (FITC) rabbit anti-goat IgG was from Liankebio (Zhejiang, China).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIzol® Reagent (Ambion, TX, USA) and reverse-transcribed into cDNA using PrimeScript™ RT reagent kit (TaKaRa, Otsu, Japan). qRT-PCR was performed with SYBR®
Premix Ex Taq™ II kit (TaKaRa) in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, CA, USA) using the primers (Sangon Biotech, Shanghai, China) shown in Additional file
1: Table S1. Cycling conditions were 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. The mRNA levels of genes were evaluated using the 2
-△△Ct relative quantification method.
ACTB (encoding β-actin) was used as a reference control. Quantitative analysis of miRNA expression was performed with the Bulge-Loop™ hsa-miR-133b qRT-PCR primer set (Ribobio, Guangzhou, China). U6 snRNA was used as a reference control.
Western blotting (WB) analysis
Cells were lysed in whole-cell lysate buffer. For phosphorylated protein, 1 % phosphatase inhibitor cocktail was added to the whole-cell lysate buffer. Protein lysates were resolved by 10 % or 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to poly vinylidene fluoride (PVDF) membranes (Pall, NY, USA). After blocking with 5 % nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C, and then incubated with the corresponding secondary antibodies at room temperature for 1 h. Protein bands were visualized with an enhanced chemiluminescence kit (Pierce, IL, USA). The blot intensities of each band were analyzed by ImageJ software (NIH, MD, USA). GAPDH was used as a loading control.
PP2A activity assay
A Serine/Threonine Phosphatase Assay System (Promega, WI, USA) was used for measuring PP2A activities. Following the instruction manual, collected cell lysates were centrifuged at 1 × 105
g for 1 h at 4 °C in phosphatase storage buffer. Sephadex® G-25 spin columns were used to remove endogenous phosphate. The treated lysates were added to a mixture containing PP2A reaction buffer and phosphopeptide, and then incubated for 1 h at 37 °C. The reaction was stopped with molybdate dye/additive mixture. The optical density (OD) of the samples was read using a Multiskan™ FC microplate photometer (Thermo) at 600 nm. PP2A activity was measured in three parallel experiments.
Immunofluorescence assay
The cells, seeded on coverslips in 12-well plates, were fixed with freshly-prepared 4 % paraformaldehyde and permeabilized with 0.5 % Triton X-100. After blocking with phosphate-buffered saline (PBS) containing 1 % BSA, the cells were treated with primary antibody overnight and incubated with fluorescent secondary antibody for 1 h in the dark. After extensive washing, cell nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) for 1 min. The cells were photographed using a confocal microscope (Olympus, Tokyo, Japan). Three independent assays were conducted; representative images are shown.
Establishment of stable PPP2R2D-knockdown cell lines
The short hairpin RNA (shRNA) targeting the
PPP2R2D mRNA sequence (GenBank Accession No. NM_018461.4) was designed using the Genetic Perturbation Platform (
http://www.broadinstitute.org/rnai/public/). The sense and antisense oligonucleotides of sh
2R2D (Additional file
1: Table S1) were annealed and then ligated into lentiviral vector pLKO.1-puro (a kind gift from Dr. Wen Chen, Sun Yat-sen University, China) to construct the pLKO.1-sh
2R2D recombinant plasmid. The corresponding control plasmid was pLKO.1 bearing shRNA targeting green fluorescent protein (pLKO.1-sh
GFP) [
13]. The lentiviral plasmid (pLKO.1-sh
GFP or pLKO.1-sh
2R2D), packaging plasmid (pCMV-delta 8.9), and envelope plasmid (VSVG) were co-transfected into HEK-293T cells using X-tremeGENE HP DNA transfection reagent (Roche, IN, USA). Lentiviruses packaged in HEK-293T cells were purified and then introduced into HepG2 cells using polybrene (Sigma, MO, USA). The established cell lines (HepG2-sh
GFP and HepG2-sh
2R2D) were screened with 0.6 μg/ml puromycin.
Establishment of stable PPP2R2D-overexpression cell lines
PPP2R2D full-length coding sequence (
2R2Dc) was obtained using the primers shown in Additional file
1: Table S1 and incorporated into retroviral vector pBabe-puro (a kind gift from Dr. Wen Chen) to construct the pBabe-
2R2Dc recombinant plasmid. HEK-293T cells were co-transfected with retroviral plasmid (pBabe or pBabe-
2R2Dc) and pCL-Ampho vector in a ratio of 1:1. HepG2 cells were then infected with retroviruses produced by HEK-293T cells. The established cell lines (HepG2-pBabe and HepG2-
2R2Dc) were screened with 0.6 μg/ml puromycin.
Cell cycle analysis
Cells were collected, fixed in 70 % ice-cold ethanol, incubated with RNase, and dyed with propidium iodide (PI). Flow cytometry (FCM, Beckman, CA, USA) was used to analyze DNA content. The data were analyzed using ModFit LT 3.1 (Verity Software House, ME, USA) and cell cycle distribution was calculated. Each experiment was repeated at least three times.
Cell migration assay
The migratory ability of cells was evaluated using 6.5 mm Transwell chambers with 8.0 μm pore polycarbonate membrane insert (Corning, NY, USA). Cells prepared in FBS-free medium were seeded onto the upper chambers, and medium with 10 % FBS was added to the bottom chambers as a chemoattractant. cDDP was given to the cells 30 min later. After 12 h, migrated cells located on the lower surface of chambers were fixed and stained with crystal violet. The cells were photographed using an inverted microscope and counted in 10 randomly selected fields at a 200× magnification using Image Pro-Plus software 6.0 (IPP 6.0, Media Cybernetics, MD, USA). The assay was carried out three times.
To evaluate the self-renewal capacity of different cell types, 800 cells were seeded in 6-well plates. After 24 h, cells were treated with cDDP for 12 h and maintained in culture for another 6 days. On day 7, the cells were fixed, stained, and drained for taking photographs. The colony formation areas were calculated by ImageJ. The assay was carried out in triplicate.
Apoptosis assay
The Annexin V‑FITC Apoptosis Detection kit (Beyotime) was used for apoptosis assays. Briefly, cells were harvested, resuspended in Annexin V-FITC binding buffer, and stained with Annexin V-FITC and PI in the dark. Cells were then analyzed by FCM. FlowJo v.7.6.5 (FlowJo, OR, USA) was used to analyze the data. The experiment was repeated three times separately.
Cell proliferation assay
Cells were plated at a density of 8 × 103 cells/well in 96-well plates and exposed to cDDP for 24 h. Then, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well. The OD was measured spectrophotometrically at 570 nm using a Multiskan™ FC microplate photometer 4 h later. The cell growth inhibition (GI) rate was calculated as the following formula: GI (%) = [1 − (OD570Sample − OD570Blank)/(OD570Ctrl − OD570Blank)] × 100 %. This assay was repeated at least three times.
In vivo xenograft studies
BALB/c nude mice (5–7 weeks old) were obtained from the Xiamen University Laboratory Animal Center (Xiamen, China). All experimental procedures were approved by the Experimental Animal Ethics Committee of Xiamen University. The mice were randomly allocated to four groups: pBabe-Ctrl group, 2R2Dc-Ctrl group, pBabe-cDDP group, and 2R2Dc-cDDP group (n = 6 in each group). A total of 5 × 106 cells (HepG2-pBabe or HepG2-2R2Dc) were suspended in PBS with matrigel (BD Biosciences, CA, USA) in a 1:1 ratio. These cell suspensions were subcutaneously inoculated into the right flank of each mouse, and the tumors were allowed to grow for a week. Tumor width (w) and length (l) and body weights were measured every other day. Tumor volume (V) was calculated using the formula: V = (w2 × l)/2. A week later, the mice in the 2R2Dc-cDDP group or the pBabe-cDDP group were injected with 1 mg/kg body weight cDDP via tail vein every other day for three times, respectively. All mice were sacrificed and tumors were excised on day 14. Tumor weight was recorded, and tumor tissues were subjected to immunohistochemistry (IHC), WB, and qRT-PCR assays.
Immunohistochemistry (IHC)
The dissected tumor tissues were fixed, embedded in paraffin, and serially sectioned at 4 μm thickness. Sections were dewaxed, rehydrated, and antigen retrieved. UltraSensitive™ SP (Mouse/Rabbit) IHC Kit (Maixin Biotech, Fuzhou, China) was used according to the manufacturer’s instructions. Sections were incubated with primary antibodies overnight followed by a secondary antibody. Next, sections were stained with 3, 3’-diaminobenzidine, counterstained with hematoxylin, and dehydrated through graded ethanol solutions. Finally, sections were photographed using a microscope and analyzed using IPP 6.0.
Transient transfection and luciferase reporter assay
The
PPP2R2D 3’UTR fragment was amplified using the primers shown in Additional file
1: Table S1, ligated into the pGL3-control luciferase reporter vector (pGL3c, a kind gift from Dr. Shimei Zhuang, Sun Yat-sen University, China), and confirmed by sequencing. Transfection was performed in 96-well plates using Lipofectamine® 2000 transfection reagent (Invitrogen, CA, USA) according to the standard protocol. Briefly, cells were co-transfected with 50 ng pRL-TK vector and 200 ng of either pGL3c or pGL3c-
2R2D-3’UTR for 4 h. After treatment with cDDP for 6 h, cells were analyzed using Dual Luciferase® reporter assay system (Promega). The luciferase activities were normalized against the activity of pGL3c.
miRNA mimic or inhibitor transfection
Cells were transfected with miRNA mimic or inhibitor (Ribobio) according to the manufacturer’s instructions. Briefly, cells were plated in 6-well plates and grown to 70 % confluence. For each well, 50 nM miR-133b mimic or 100 nM inhibitor in riboFECT™ CP Buffer (Ribobio) was mixed gently with riboFECT™ CP Reagent and added to the cells for 12 h.
Statistics
Statistical analyses were carried out using the Statistical Package for Social Sciences (SPSS) version 16.0 (SPSS, IL, USA). All data were expressed as mean ± standard deviation (SD). The statistical significance was determined using two-tailed unpaired Student’s t-test for the comparison of two groups and using one-way analysis of variance (ANOVA) for the comparison of multiple groups, following which the statistical significance was determined by Dunnett’s t-test. Correlation between variables was evaluated with Pearson’s correlation for normal distribution or Spearman’s correlation for non-normal distribution. A P < 0.05 was considered statistically significant.
Discussion
HCC is one of the most lethal forms of cancer in the world [
18]. In recent years, molecule-targeted drugs, such as Sorafenib and Brivanib, have been developed for the treatment of liver cancer [
19]. However, there are few drugs designed for HCC and most targeted therapeutic drugs are less effective [
20]. cDDP is an old drug that is commonly used as a chemotherapeutic agent against advanced HCC [
12]. However, the sensitivity to cDDP varies among HCC patients. Thus, the identification of effective targets for enhancing chemotherapeutic sensitivity is urgently required.
As a key tumor suppressor, PP2A has emerged as a novel target in alternative therapeutic strategies in many cancers [
21‐
23]. Chien et al. reported that PP2A activation is crucial for deceleration of pancreatic tumorigenesis [
24]. Furthermore, PP2A activation shows a promising anti-tumor effect in breast cancer [
25]. Our previous studies have shown that the genetic variant -241 (-/G) (rs11453459) in the 5’-flanking region of
PPP2R1A contributes to the decreased risk of HCC in a southern Chinese Han population [
26]. Moreover, other studies have demonstrated that the regulation of regulatory B subunits of PP2A (PP2A-Bs), such as B55β and B56γ, play important roles in cancer development or treatment [
27]. In the current study,
PPP2R2D expression was found to be down-regulated in both HCC tumors and HCC cell lines. The difference in
PPP2R2D expression between normal and tumor cells suggested it as a potential biomarker for HCC. Following treatment with cDDP, B55δ was increased. We hypothesize that increased B55δ enhances the sensitivity of HCC to cDDP chemotherapy. The current study conducted in stable
PPP2R2D-knockdown and -overexpression cell lines validated this conclusion. The results showed that knockdown of B55δ markedly decreased the effect of cDDP, while overexpression of B55δ promoted the chemosensitivity of HepG2 cells and that of HCC xenograft tumors
. Thus, our study has shown for the first time that PP2A holoenzyme containing the B55δ subunit is a potential molecular target which might enhance chemotherapy sensitivity of HCC. Further exploitation of B55δ might help patients overcome insensitivity of anti-cancer agents and advance the development of tailor-made treatments for HCC.
PP2A-B55δ is critical for the control of the entry into and exit from mitosis. On one hand, overexpression of B55δ blocks CDK1 activation and delays mitotic progression; on the other hand, depletion of B55δ accelerates entry into mitosis [
28]. As an essential phosphatase, PP2A appears to act as a central modulator of phosphorylation to generate interphase and mitosis [
29]. In our study, when B55δ was knocked down, CDK1 was partially increased, which reversed the G2/M reduction and G1 arrest induced by cDDP. In contrast, overexpression of B55δ held HepG2 cells in G1 phase by the phosphorylation of CDK1, increasing cDDP’s suppression of cell migratory ability, self-renewal capacity, and proliferation. The activation of PP2A plays a key initiating role in various pathways that lead to apoptosis in cancer cells [
30,
31]. We also observed that overexpression of B55δ in the presence of cDDP resulted in down-regulation of the anti-apoptotic molecule Bcl-2 and up-regulation of pro-apoptotic molecules Bax and cleaved Caspase-3. Moreover, it was noteworthy that B55δ inhibited tumor growth while reducing the expression of the proliferation-related protein PCNA. These observations shed light on the underlying molecular mechanism of B55δ in the regulation of the cell cycle, and confirm the pivotal role that B55δ plays in increasing the sensitivity to chemotherapy in HCC.
Our previous studies showed that the transcriptional activities of
PPP2R2D and
PPP2R1A were regulated by the polymorphism and methylation through genetic and epigenetic mechanisms [
26,
32]. To further investigate the miRNA-mediated epigenetic mechanisms of
PPP2R2D up-regulation induced by cDDP, various approaches, including
in silico analyses of putative mRNA/miRNA complexes, were applied. We confirmed for the first time that miR-133b regulates
PPP2R2D post-transcriptional expression and translation by binding to complementary sequences of the 3’UTR of
PPP2R2D mRNA. It has been postulated that miRNAs can function as either tumor suppressors or oncogenes by regulating protein-coding genes [
33,
34]. miR-133b has been revealed to be down-regulated in Parkinson disease, human non-small cell lung, bladder, gastric, and colorectal cancers [
35‐
39]. In this study, miR-133b was up-regulated in HCC cell lines, with a corresponding down-regulation of
PPP2R2D. Thus, we speculate miR-133b might act as an oncogenic miRNA (oncomiR) in HCC. Recently, miRNAs have been considered as good biomarkers for early diagnosis and therapy of HCC [
33]. Inhibition of oncomiRs has been introduced as a novel therapeutic strategy for cancer treatment [
40]. By mimicking or inhibiting miR-133b, our study elucidated the miR-133b/
PPP2R2D signaling pathway involved in cDDP chemotherapy. Taken together, these results suggest that miR-133b may serve as a gene-specific biomarker for estimating the prognosis of HCC. It would be of great interest to further investigate the functional characterization and regulatory mechanism of miR-133b and its derepression of PP2A-B55δ synthesis and function in the chemotherapy of HCC in both animal models and clinical samples.
Acknowledgements
We thank Drs. William Melchior and Lei Guo for English editing. This work was supported by grants from the National Natural Science Foundation of China (NSFC Nos. 81172705, 81573181, 81402648, 81472997), Key Program of NSFC (No. 81130052), Early-stage Project of the National Key Basic Research Program of China (No. 2014CB560710), the Natural Science Foundation of Fujian Province of China (Nos. 2014 J01372, 2014Y2004, 2015 J01344), the Education Scientific Research Project of Young Teachers in Fujian Province (No. JA14004), Project funded by the Science Foundation of Xiamen City (No. 3502Z20140045), Xiamen Municipal Bureau of Ocean and Fisheries (No. 14PYY051SF04), and the Shanhai Fund of Xiamen University (No. 2013SH007).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LZN, LYC, ZQY, and ZTJ conceived and designed the study. ZQY, ZTJ, and ZSL carried out experiments and analyzed the data. HCY, QY, and LB participated in statistical analyses and data interpretation. ZQY, ZTJ, HCY, ZR, LHC, LYC, and LZN drafted and revised the manuscript. All authors read and approved the final manuscript.