Background
Breast cancer (BC) is the most common cause of cancer mortality among women, accounting for 16% of cancer deaths in adult women [
1]. Despite improvements in the prognosis of BC seen in recent decades, additional therapeutic advances are needed, particularly for patients with metastatic/advanced disease [
2]. Furthermore, the drug resistance in the course of chemotherapy has brought great threat to breast cancer patients [
3], especially as chemoresistance limits the effectiveness of chemotherapeutic agents to a large extent. Therefore, it is imperative to clarify the underlying molecular mechanism during BC progression.
Long non-coding RNAs (LncRNAs) are defined as a class of non-protein coding transcripts over 200 nucleotides, and are now emerging as crucial regulators of cellular processes and diseases, and their aberrant transcription can lead to altered expression of target genes involved in cancer pathways and functions [
4]. By binding to the associated gene of cancer, lncRNAs function as oncogenes or tumor suppressors in human cancer. For example, lncRNA PTENP1, the pseudogene of PTEN tumor suppressor, contains a highly homologous region upstream of the 3′UTR of PTEN, can regulate PTEN expression, thus exerts an effect on the process of carcinogenesis [
5]. PTENP1 also participates in repressing cell proliferation, inhibiting migration, and promoting apoptosis [
6,
7]. Recent studies have shown that lncRNAs, as competing endogenous RNAs (ceRNA), play important roles in modulating miRNA function through binding sites [
8]. Enhanced PTENP1 could inhibit BC cell growth, metastasis and tumourigenicity by inhibiting miR-19b and facilitating PTEN in BC [
6]. Consequently, miRNAs further affect the downstream protein-coding genes by binding to its 3′-UTR. Wei et al. showed that miR-130a as an oncogenic miRNA that targets PTEN to drive malignant cell survival and tumor growth [
9]. MiR-20a acts as a negative regulator of PTEN, and mediates the proliferation, migration and apoptosis of multiple myeloma [
10]. Similarly, we have found that PTENP1 influences the biological function of miR-20a in BC progression.
The PI3K/Akt pathway is activated subsequent to RTK activation. Hyperactivation of PI3K/Akt signaling has been reported in many types of human cancers, thus targeting the regulators in this pathway has attractive therapeutic potential [
11]. Ectopic expression of PTENP1 resultes in the upregulation of PTEN, accompanies by the blockage of PI3K/Akt pathway and growth inhibition in prostate and renal cancer cells [
12,
13]. MiR-106b and miR-93 regulate BC cell migration, invasion and proliferation by suppression of PTEN via PI3K/Akt pathway, which could be blocked by upregulation of PTEN [
14]. MiR-130b targets PTEN to reduce drug resistance, proliferation and apoptosis of BC cells via the PI3K/Akt pathway [
15]. However, PTENP1 and miR-20a affect PTEN, an important process in BC progression, is not clear.
In the present study, the association of downregulated PTENP1 and PTEN and BC progression was examined. LncRNA PTENP1 was evaluated as a molecular sponge for miR-20a, and these ncRNAs further regulated PTEN. Relative function mechanism assays revealed that PTENP1/miR-20a/PTEN axis exerted its function in BC partly through PI3K/Akt signaling pathway.
Materials and methods
Samples from BC patients
A total of 52 previously diagnostic BC patients who received surgical operation at the First Affiliated Hospital of Dalian Medical University from April 2014 to January 2018 were enrolled in this study. The study and its informed consent have been examined and certified by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University (YJ-KY-FB-2017-32). In accordance with the International Union against Cancer (UICC), the samples were identified BC tissues and their adjacent noncancerous tissues. The samples were maintained in liquid nitrogen for later experiments.
Cell culture
The human BC cell lines MDA-MB-231, T-47D and MCF-7 were obtained from KeygenBiotech Co. Ltd. (Nanjing, China). The mammary epithelium MCF-10A was purchased from ATCC cell banking. The BC cells were cultured in DMEM, supplied with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37 °C in a humidified and 5% CO2 incubator. MCF-10A cells were maintained in DMEM-F12 media supplemented with hydrocortisone (0.5 μg/ml), insulin (10 μg/ml), hEGF (20 ng/ml) and 10% (v/v) FBS. Adriamycin (Sigma, St Louis, MO, USA) was added to parental cell cultures in stepwise increasing concentrations from 1 mg/l to 5 mg/l for 2 months to develop an adriamycin-resistant (ADR) subline, named MCF-7/ADR and T47D/ADR, correspondingly. To maintain the resistant phenotype, MCF-7/ADR and T47D/ADR cells were kept in the medium containing 1 mg/l adriamycin (ADR) and were cultured in drug-free medium for 48 h before the experiments.
Real-time PCR analysis
Total RNA was isolated from tissues and BC cell lines by RNeasy Mini Kit (Qiagen, Valencia, CA). RNA was reverse transcribed to cDNA using Reverse Transcription Kit (Invitrogen, CA, USA). The qRT-PCR was performed under an ABI Prism7500 fast real-time PCR system (Applied Biosystems, Foster City, CA) with mixing a QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA). Relative RNA expression was calculated by ΔΔCt method with normalization to U6 small nuclear RNA.
Western blot analysis
Total cell lysates were prepared with RIPA lysis buffer (KeyGEN, Nanjing, China). The proteins were separated by 10% SDS-PAGE gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). TBST with 5% skim milk powder was used for blocking the PVDF membranes. Then, the blots were incubated with different primary antibodies at 4 °C overnight. After incubating with secondary antibodies, the bands were detected by an ECL Western blot kit (Thermo Fisher Scientific, USA) and analyzed by LabWorks (TM ver4.6, UVP, BioImaging Systems, NY, USA). GAPDH was used as control.
Cell transfection and RNA interference
PCR production of PTEN ampliation was cloned into pmirGLO vector (Promega). MiR-20a mimic, inhibitor and miR-NC were synthesized by GenePharma Co.Ltd. (Suzhou, China). LncRNA PTENP1 pcDNA3.1 vector (PTENP1, Invitrogen, CA, USA), LV-NC, LV-PTENP1, siPTENP1, shPTENP1, siAkt, siSCR and shSCR were obtained from GenePharma Co.Ltd. (Suzhou, China). The transfection assay was conducted with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for incubation. The transfected efficiency was measured by qRT-PCR.
Dual luciferase reporter gene assay
A pmirGLO Dual-Luciferase miRNA Target Expression Vector was purchased from GenePharma Co.Ltd. (Suzhou, China). Firefly luciferase functioned as primary reporter to regulate mRNA expression, and renilla luciferase was used as a normalized control. Co-transfection was conducted with lipofectamine 2000 for 48 h, the dual luciferase reporter assay system (Promega) was utilized according to the manufacturer’s instruction. The relative luciferase activity was calculated as the ratio of frefly luciferase activity versus renilla luciferase activity. Data were shown as the mean value ± SD and each experiment was performed thrice.
CCK8 assay
Cell proliferative ability was investigated by using cell counting kit-8 (CCK-8; Dojindo, Japan). Cells (1 × 103 per well) were plated into 96-well plate with the corresponding medium, and cultured in a humidified incubator at 37 °C. 11 μLCCK8 were added into the plate for 4 h. The spectrometric absorbance was measured by microplate reader (Model 680; Bio-199 Rad, Hercules, CA, USA) at 490 nm.
The drug resistance to ADR was detected by CCK-8. Different concentration of ADR was added into 96-well plate after seeding the cells. Similarly, the absorbance was then measured to evaluate the drug resistance to ADR of BC cells. Each experiment was performed thrice.
Single-cell suspension was obtained and then seeded in 6-well plate with 1 × 103 per well. The medium was changed every 4 days. 12 days later, the foci were formed obviously. The colonies were fixed by 4% paraformaldehyde for 20 min, and then stained with 0.2% crystal violet. The colonies were photographed and counted.
Cell invasion assay
Cell invasive ability was measured using ECMatrix gel (Chemicon)-coated transwell inserts, respectively (Trevigen, City of Gaithersburg, Maryland, USA). 5 × 104 cells were harvested in serum-free DMEM and added to the upper chamber. Medium containing 10% FCS was added to the bottom chamber, and cells were allowed to invade for 24 h at 37 °C. The upper side cells were removed by a cotton swab. The invading cells were counted to estimate the invasive capacity. Five random fields were analyzed for each chamber.
Flow cytometry analysis
Cells were incubated with different concentration of ADR for 48 h. Annexin-V-FITC apoptosis detection kit (BD, Franklin Lakes, NJ, USA) was used to measure cell apoptosis. 2 × 103 cells were harvested and adjusted in 100 μL binding buffer. Annexin V and propidium iodide were used to stain for 10 min avoiding lights, and 400 μL binding buffer was added into the cell suspension. The apoptosis cells were detected by FACS Calibur (Becton-Dickinson, CA, USA).
Immunofluorescence staining
BC cells were added in culture dishes and fixed with 4% paraformaldehyde for 20 min. BC cells were treated with 0.2% Triton X-100 for 3 min, and incubated with 5% BSA for 1 h. The primary antibody was added into the dish overnight at 4 °C. Slides were then washed three times with PBS and incubated 1 h with secondary antibody. The cells were stained by 4, 6-diamino-2-phenylindole (DAPI, Sigma-Aldrich, St Louis, MO, USA) in PBS for nuclear staining. Images were taken in a Carl Zeiss fluorescent microscope (Carl Zeiss Microscopy).
TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick end labelling)
TUNEL assay was carried out to measure the fragmented DNA of apoptotic cells. Apoptotic cells were induced by ADR, and fixed by 4% formaldehyde for 25 min at 4 °C. The cells were permeabilized by 0.2% TritonX-100 for 5 min. Then the cells were equilibrated with 100 μL Equilibration buffer for 10 min at room temperature. Cellswere labeled with 50 μL TdT reaction mix at 37 °C for 1 h. SSC buffer was used to stop the reaction and stained nuclei with DAPI. The images were obtained by fluorescence microscopy.
RNA immunoprecipitation (RIP) assay
The Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (Millipore, USA) was used to conduct RNA immunoprecipitation (RIP) assay. The endogenous miR-20a which combined with PTENP1 was pulled down. The cell lysis were collected and incubated in RIP buffer containing magnetic bead conjugated to either a human anti-Ago2 antibody (Millipore) or negative control IgG and was used to precipitate the cell extracts. The expression levels of PTENP1 and miR-20a in the precipitates were analyzed by qPCR.
In vivo antitumor activity
4-week-old female athymic nude mice were purchased from the Model Animal Research Institute of Nanjing University. Approximately, 1 × 107 cells were injected subcutaneously into the right flank of each nude mouse, respectively. In addition, to evaluate the chemosensitivity effect of PTENP1, the treatment groups received 7 mg/kg ADR i.p. three times a week for 3 weeks. The mice were humanely killed and their tumors were photographed. The tumor volume was calculated. These experiments were approved by the Committee on the Ethics of Animal Experiments of the Dalian Medical University, China.
Immunohistochemistry (IHC) staining
Human BC samples and xenograft tumors were collected and performed on paraffin-embedded sections. 4 μm-thick sections were deparaffinized, rehydrated and then immersed with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase and labeled with antibodies at 4 °C overnight. The slides were stained with the secondary streptavidin-horseradish peroxidase-conjugated antibody (Santa Cruz Biotech, Santa Cruz, CA) for 1 h. The slides were then counterstained with hematoxylin for 30s and cover slipped.
Statistical analysis
Data were expressed as means ± standard deviation (SD). SPSS 17.0 software was used to analyze the experimental data. Student’s t-test was performed to compare two different groups. The one-way analysis of variance (ANOVA) was used to determine the significant difference of multiple groups. The survival curves were calculated by Kaplan-Meier method, and the difference was assessed by a log-rank test. Spearman’s correlation analysis was used to identify the association between miRNAs and mRNA expression. P < 0.05 was considered statistically signifcant.
Discussion
Metastasis and chemoresistance lead to the treatment failure of BC patients. Interestingly, lncRNAs are reported to be critical regulators involved in tumour-related progression. Thus, we investigated the ceRNA-dependent role of the lncRNA PTENP1 in the development of BC. This study provided us the first clarification into the potential mechanism that PTENP1-miR-20a-PTEN network modulated the BC progression via PI3K/AKT pathway.
Recently, lncRNA PTENP1 and PTEN expression were found to be decreased in some cancer types, including hepatocellular carcinoma (HCC) [
16], gastric cancer [
7] and head and neck squamous cell carcinoma (HNSCC) [
17]. We assessed PTENP1 and PTEN expression in BC tissues and cell lines, and the results showed down-regulation in BC tissues compared with adjacent normal tissues. In line with the results of our study, Li et al. reported decreased PTENP1 expression in BC cells, indicating such a decrease in expression may be important in oncogenesis [
6]. Furthermore, our study also found low expression of PTENP1 and PTEN to be closely related to advanced TNM stage and overall survival in BC. Low levels of PTENP1 have been correlated with worse overall survival and disease-free survival rates of HNSCC patients [
17], consistent with our results. Dysregulation of lncRNAs often led to the tumorigenesis and the malignant progression. PTENP1 over-expression resulted in the growth inhibition of cancer cells both in vitro and in vivo, suggesting that PTENP1 played a tumor suppressive role in HCC [
16]. In esophageal squamous cell carcinoma (ESCC), overexpression of PTENP1 resulted in inhibited proliferation [
18]. In the present study, we found that ectopic expression of PTENP1 led to inhibition of the tumor growth, colony formation, invasion and xenograft tumor growth of BC. Our results indicated that PTENP1 might function as potential therapy target of BC.
Recent evidence has suggested that competitive endogenous RNAs (ceRNAs) are important regulatory molecules in cancer and their dysregulation may contribute to cancer pathogenesis. For example, enhanced PTENP1 could inhibit BC cell growth, metastasis and tumourigenicity by inhibiting miR-19b and facilitating PTEN in BC [
6]. MiR-19b and miR-20a were members of crucial oncogene miR-17-92 clusters. Although similar oncogenetic effects of miR-20a and 19b were existed on tumor procession, the overall BC cell malignancy should be better verified. In gastric cancer, PTENP1 was confirmed by binding miR-106b/ miR-93, in a ceRNA modulation manner, further affected PTEN level [
7]. PTENP1 acted as a competing endogenous RNA to protect PTEN transcripts from being inhibited by miR-21, and consequently inhibited proliferation and colony formation in oral squamous cell carcinoma (OSCC) [
19]. MiRNAs also play an important role in the ceRNA network through combining with target mRNA, inhibiting the action of mRNA expression [
20]. MiR-20a has been shown to be aberrantly expressed in BC and regulated cancer aggressiveness by target genes. MiR-20a-5p was highly expressed in both triple-negative breast cancer (TNBC) tissues and cell lines, and promoted the growth of TNBC cells through targeting Runt-related transcription factor 3 (RUNX3) [
21]. High mobility group AT-hook 2 (HMGA2) was a target of miR-20a-5p, which significantly induced carcinogenesis of BC [
22]. Thus, the molecular mechanism was further clarified that PTENP1 acted as a ceRNA of miR-20a in BC progression. In the ceRNA network constructed in this paper, we found that PTENP1 expression was inversely correlated to miR-20a level in BC cell lines and patients. MiR-20a bound to PTENP1 in a sequence-specific manner and regulated PTENP1 expression. On the other hand, miR-20a was negatively correlated with PTEN level. Moreover, PTEN was a direct target of miR-20a and could be regulated by either miR-20a overexpression or inhibition. Additionally, altered level of PTENP1 and miR-20a was significantly associated with PTEN expression, and impacted BC malignancy. These results provide additional evidence to the reciprocal repression loop of PTENP1/miR-20a/PTEN in a functional aspect of BC development.
PI3K is responsible for coordinating a diverse range of cellular functions, including proliferation, cell survival, degradation and cell migration [
23]. As a key oncogentic signaling pathway, PI3K/Akt pathway plays a pivotal role in the development of many cancers, including BC [
24]. Additionally, NF-κB showed critical modulation on tumor cells malignancy and progression by regulating numerous genes transcriptionally. Although the involvement of PI3K/Akt pathway in BC has been declared, PTENP1-miR-20a-PTEN network mediated the signaling cascade has not been fully explained so far. In this present study, we found that PTEN level was regulated by PTENP1 and miR-20a, whereas PTEN participated in suppressing the proliferation of cancer cells via negatively regulating the PI3K/Akt pathway [
25,
26], which suggested that PTEN was important in malignant transformation of cancer cells. In agreement with these observations, this study showed that altered PTENP1 and miR-20a could effectively influence the expression of PI3K/Akt pathway molecules via PTEN levels. On the other hand, treatment with LY294002 or siAkt signifcantly inhibited the phosphorylation of PI3K/Akt. As expected, the cell colony formation and invasion were attenuated in BC cells treated with LY294002 or siAkt. It was reasonable to conclude that the regulatory effects of PTENP1/miR-20a/PTEN crosstalk on cell aggressiveness could at least be partially mediated via PI3K/Akt pathway.
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