MiR–20a-5p promotes radio-resistance by targeting Rab27B in nasopharyngeal cancer cells
© The Author(s) 2017
Received: 5 September 2016
Accepted: 28 January 2017
Published: 1 March 2017
MicroRNAs (miRNAs) was reported to be involved in cancer radio-resistance, which remains a major obstacle for effective cancer therapy.
The differently expressed miRNAs were detected by RNA-seq experiment in nasopharyngeal cancer (NPC) cells. MiR-20a-5p was selected as our target, which was subject to finding its target gene Rab27B via bioinformatics analysis. The qRT-PCR, western blot and the luciferase reporter assays were performed to confirm Rab27B as the target of miR-20a-5p. In addition, the roles of miR-20a-5p in NPC radio-resistance were detected by transfection of either miR-20a-5p-mimic or miR-20a-5p-antagomiR. The involvement of Rab27B with NPC radio-resistance was also detected by the experiments with siRNA-mediated repression of Rab27B or over-expression of GFP-Rab27B. Wound healing and invasion assays were performed to detect the roles of both miR-20a-5p and Rab27B.
MiR-20a-5p promotes NPC radio-resistance. We identified that its target gene Rab27B negatively correlates with miR-20a-5p-mediated NPC radio-resistance by systematic studies of a radio-sensitive (CNE-2) and resistant (CNE-1) NPC cell lines. Repression of Rab27B by siRNA suppresses cell apoptosis and passivates CNE-2 cells, whereas over-expression of Rab27B triggered cell apoptosis and sensitizes CNE-1 cells.
MiR-20a-5p and its target gene Rab27B might be involved in the NPC radio-resistance. Thus the key players and regulators involved in this pathway might be the potential targets for developing effective therapeutic strategies against NPC.
KeywordsRab27B miR-20a-5p Radio-resistance Nasopharyngeal cancer
The malignant tumor nasopharyngeal carcinoma (NPC) occurs in the lining of nasopharynx with a multifactorial etiology . Beyond the chemotherapy , radiation therapy is the other major methods against cancer due to its excellent local control and increased overall survival rates [3–5]. However, owing to the high sensitivity, radiation therapy often fails in various cancers, such as NPC. The main reason is that radiation treatment can intrinsically induce radio-resistant tumor cells, which show enhanced DNA repair ability . To overcome the problem of radio-resistance, it is urgently needed to elucidate the mechanisms of radio-resistance and develop new radiosensitizers.
MicroRNAs (miRNAs) are non–coding regulatory RNAs, post-transcriptionally regulate gene expression through targeting to a panel of target genes. As the critical roles reported , their dysregulation is associated with human diseases, including cancer biology [8, 9]. Notably, the emerging studies have shown that miRNAs are associated with the development of radio-resistance in different type of cancers [10, 11], such as prostate cancer , esophageal cancer . As one of the well-studied miRNAs, miR-20a has been shown to function as an oncomiR in many cancers, including lung cancer , hepatocellular carcinoma , and gastric cancer . Notably, miR-20a was also found to be involved in cancer irradiation treatment . For example, miR-20a was shown to induce cell radio-resistance by activating the PTEN/PI3 K/Akt signaling pathway in hepatocellular carcinoma .
In the present study, we performed an RNA-seq assay to detect differentially expressed genes in radio-sensitive (CNE-2) versus radio-resistant (CNE-1) NPC cell lines. We showed that miR-20a-5p promoted NPC radio-resistance via repression of Rab27B, a newly identified target of miR-20a-5p. We further performed a systematic analysis of Rab27B and miR-20a-5p for their roles in the NPC radio-resistance. The regulatory effect of miR-20a-5p on NPC cell survival and apoptosis was also detected upon irradiation.
Human nasopharyngeal cancer cell lines, CNE-1 and CNE-2 were supplied by the department of radiation oncology of Sun Yat-sen University, Guangzhou, China . Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) in a humid atmosphere containing 5% CO2 at 37 °C.
RNA-seq analysis was performed by BGI-Tech (Shenzhen, China). RNA was purified and fragmented to construct the RNA-seq library for sequencing. The sense and anti-sense cDNA molecules were synthesized. After agarose gel electrophoresis, suitable fragments were used as templates for PCR amplification. Real-Time PCR System was used in quantification and qualification of the sample library. Finally, the library was subjected to sequencing using Illumina HiSeq 2000 (Illumina, USA). The single-end library was prepared following the protocol of the IlluminaTruSeq RNA Sample Preparation Kit (Illumina) .
The Homo sapien miR–20a-5p mimics, miR–20a-5p antagomiRs and miR–20a-5p scrambled nega-tive control (NC) were obtained from Guangzhou Ribobio, China. All the transfection experiments were performed using the Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies), which was described previously . Western blot and qRT–PCR assays were performed to confirm the effect of Rab27Bon the expression of miR–20a-5p. The sequences used in this study are as follows:
5′- CAGUAGGAAUAGACUUUCG dTdT-3′
Irradiation and clonogenic assay
Cells treated with miRNAs were seeded on 6-well plates in triplicate and exposed to radiation at the doses indicated using a 6-MV x-ray generated by a linear accelerator (Varian trilogy at a dose rate of 200 cGy/min). After incubation at 37 °C for 14 days, cells were fixed in 100% methanol and stained with 0.1% crystal violet. Colonies containing >50 cells were counted under a light microscope. The surviving fraction was calculated as described previously [13, 18]. At least three independent experiments were performed to calculate the means and standard deviations.
Total RNA was extracted using Trizol (Vazyme). For the mRNA analysis, the cDNA primed by oligo-dT was made with RT reagent kit (Tiangen, China), and the mRNA level of Rab27B was quantified by a duplex-qRT-PCR analysis where the TaqMan probes with a different fluorescence for β-actin (Shing Gene, China) were used in the FTC-3000P PCR instrument (Funglyn, Canada). The miRNA expression level was normalized using U6 small nuclear RNA (HmiRQP9001) as an internal control, as previ-ously described . Using the 2−ΔΔCt method, the β-actin level was normalized before comparing the relative level of the target genes. The sequences of primers and probes used for the qRT-PCR analysis are as follows:
hRab27B probe, 5′-ROX-CGGTTCCGGAGTCTCACCACTGC-3′;
hACTB F: 5′-GCCCATCTACGAGGGGTATG-3′
hACTB R: 5′-GAGGTAGTCAGTCAGGTCCCG-3′
hACTB probe: 5′-CY5-CCCCCATGCCATCCTGCGTC-3′
Western blotting assays
Total proteins were extracted from cultured cells with cell lysis buffer (60 mM Tris–HCl, pH 6.8, 2% SDS, 20% glycerol, 0.25% bromophenol blue, and 1.25% 2-mercaptoethanol) and heated at 95 °C for 10 min. The heated proteins were separated by 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk in TBST for 2 h, the membranes were incubated overnight at 4 °C with diluted Anti-Rab27B primary antibody (13412-1-AP; SanYing, China). Followed by washing with TBST buffer three times, the membranes were incubated with secondary antibody (SA00001-2; SanYing, China) at 37 °C while shaking on a rotary for 2 h. The relative density (level) of proteins over the GAPDH (10494-1-AP; SanYing, China) band was quantified with the Gel-Pro Analyzer (Media Cybernetics).
Cell apoptosis analysis
Apoptosis was analyzed using Annexin V/PI double staining. After transfection for 48 h, the cells in the logarithmic growth phase were harvested and rinsed twice with ice-bathed PBS, then FITC-labeled enhanced annexin V (3 μl) and propidium iodide (3 μl) were added to the cell suspension at the final volume of 150 μl. After incubation for 30 min, flow cytometry was performed on a FACS Calibur instrument. The number of apoptotic and necrotic cells were calculated by flow cytometry (Becton–Dickinson, USA) and analyzed by Flowjo Software. The ratio of early apoptosis was used for the test results. The experiments were performed three times independently, and a representative is shown.
Luciferase reporter assay
Cells were seeded in 24-well plate at a concentration of 2 × 105 cells/per well and co-transfected 24 h later with pGL3-luc-Rab27B UTR WTand miR-20a-5pmimic/antagomir or NC. After transfection for 48 h, cells were collected, and the relative luciferase activity was performed using Dula-Luciferase Reporter Assay Kit (Promega). The relative firefly luciferase activities of the UTR construct was analyzed as previously reported .
For cell motility assays, cells stably expressing mimics, antagomiRs or NC were seeded in 24-well plates and cultured to near confluence. After culture for 6 h in DMEM without FBS, a linear wound was carefully made using a sterile 10 µl pipette tip across the confluent cell monolayer, and the cell debris was removed by washing with phosphate-buffered saline. The cells were incubated in DMEM plus 10% FBS, and the wounded monolayers were then photographed at 0, 8, 24 and 48 h after wounding.
According to the manufacture’s description, cell invasion assays were performed in a 24-well Transwell Chambers with 8 mm pore size chamber inserts (Corning, USA). In the assay, 1 × 104 cells were seeded into the upper chamber with 200 µl of DMEM without FBS. In the lower chamber, 600 µl of DMEM supplemented with 10% FBS was added. After incubation for 40 h at 37 °C and 5% CO2, the non-invading cells were removed from the plate with cotton stick.The cells that moved to the bottom surface were stained with 0.1% crystal violet for 30 min at 37 °C. The cells were then imaged and counted in at least 5 random fields using a CKX41 inverted microscope (Olympus, Tokyo, Japan). The assays were conducted three independent times.
Immunofluorescent staining for γ-H2AX
Twenty-four hours following transfection with miR-20a-5p mimic or miRNA mimic negative control, 1 × 105 cells were seeded in chamber slides and incubated overnight. The cells were subsequently exposed to 4 Gy irradiation (IR). Twenty-four hours following IR, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 (Sigma), blocked in 2% bovine serum albumin and incubated with a primary antibody against γ-H2AX (SanYing, China) overnight at 4 °C. The primary antibody was subsequently washed off, and a secondary antibody conjugated to fluorescein isothiocyanate was applied to the slides. Cells were washed with phosphate-buffered saline and counterstained with DAPI. The γ-H2AX foci were observed under a fluorescence microscope (Olympus). For each group, the γ-H2AX foci were counted in ≥50 cells.
The data are presented as the mean, and the error bars indicate the SD. All statistical analyses were performed with Excel (Microsoft, Redmond, WA, USA). Two-tailed Student’s t test, a one-way analysis of variance or Mann–Whitney U test was used to calculate statistical significance. A P-value of <0.05 was considered significant.
Rab27B negatively regulates the NPC radio-resistance
The Rab27B gene is a target of miR-20a-5p in NPC cells
Sequence analysis revealed that 3′-UTR region of Rab27B contains one potential binding motif (from 627 to 649 bp) for miR-20a-5p (Fig. 2d). To further conclude whether Rab27B is a direct target of miR-20a-5p, we put the wild-type Rab27B gene at the downstream of the Renilla luciferase gene of pGL3-control vector (Promega) to create pGL3-Rab27B UTR WT (Fig. 2d). These constructs were transfected into CNE-2 and CNE-1 cells, respectively, to compare the luciferase activity. The pGL3-Rab27B-UTR WT gave the relative luciferase activity of 0.78 (Fig. 2e). The transfection of miR-20a-5p-mimic into CNE-2 cells significantly brought down the luciferase activity of pGL3-Rab27B-UTR WT construct, whereas the control cells showed almost the same activity upon the transfection of miR-20a-5p-mimic (Fig. 2e). Meanwhile, the transfection of miR-20a-5p-antagomiR into CNE-1 cells drastically raised the luciferase activity of pGL3-Rab27B-UTR WT construct (Fig. 2e). Getting together, our results strongly indicate that Rab27B is indeed a target of miR-20a-5p.
The Rab27B expression negatively correlates with the miR-20a-5p’s promoting effect on the NPC radio-resistance
MiR-20a-5p promotes cell migration and invasion of NPC cells
Radiation therapy is one of the major treatments against tumors as it has the advantages of being non-invasive and well tolerated. However, radiotherapy resistance is also a common occurrence that blocks the effective therapy . The emerging studies have focused on the resistance mechanisms and the involved biological factors [28, 29]. Among the factors involved, miRNAs are reported to be closely associated with tumor radiosensitivity [30–32]. The miR-20a-5p studied here is dysregulated in many human cancers [33–35], and the high level of miR-20a-5p was considered as an indicator of advanced stage, poor prognosis and chemo-therapy resistance. Of note, miR-20a was also found to induce cell radio-resistance by activating the PTEN/PI3 K/Akt signaling pathway in hepatocellular carcinoma . However the knowledge of miR-20a-5p on cancer radio-resistance is still limited, especially in NPC. In this study, we found that miR-20a-5p was involved in NPC radio-resistance, probably by targetingRab27B 3′-UTR. Furthermore we demonstrated that miR-20a-5p-mediated Rab27B repression promoted the invasion and metastasis of NPC cells. Both role and mechanisms of miR-20a-5p and Rab27B in NPC radio-resistance were systematically investigated in cultured cells. Furthermore, the influence of miR-20a-5p and Rab27B on the growth of tumor xenografts was also addressed in nude mice.
Rab27B is a member of Ras-like small GTPases that modulate endocytosis and exocytosis vesicle-trafficking control [36–39]. Rab27B is normally expressed in a large number of secretory cells to regulate secretory pathways . In addition, it is reported that aberrant expression of Rab27B is associated with several types of cancers. For examples, the increased Rab27B expression correlates with lymph node metastasis and is a marker for breast cancer progression [41, 42]. Rab27B can also be recognized as a valuable prognostic indicator for hepatocellular carcinoma patients. In addition, Rab27B regulates invasive tumor growth of colorectal cancer , hepatocellular carcinoma  and breast cancer [42, 45, 46]. Based on the above studies, Rab27B demonstrates oncogenic function and plays important roles in cancer development. However, the expression of Rab27B, as well as its role in NPC, has barely been investigated. In this study, our data suggest that Rab27B might facilitate the invasive/metastatic phenotypes of NPC, and thus might be treated as a novel marker for clinical diagnosis. We showed that the expression of Rab27B is associated with the radio-resistance of NPC cell lines, which is mediated by miR-20a-5p. Despite that the expression level of γ-H2AX is correlated with the cell apoptosis of NPC cells, other pathways may be activated upon the exposure to the radiation, such as JNK signal pathway . The detailed mechanism for the miR-20a-5p-mediated Rab27B repression of NPC radio-resistance remains to be elucidated.
member RAS oncogene family
Conception and design: DBH and SH. Acquisition of data (provided animals, provided facilities, etc.): DBH, GB, YYP, XHH, YBS, YW analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): GB, GDS, MC, XF. Writing, review, and/or revision of the manuscript: DBH, GB and SH. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
Consent for publication
The authors consent for publication.
Animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal research was approved by the biomedical ethics committee of Anhui Medical University, when we applying for the National Natural Science Foundation of China (No. 81471552). The animal study proposal was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Science and Technology of China. All of the mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.
This work was supported by the National Natural Science Foundation of China (No. 81471552 granted to Min Chen), Anhui Provincial Natural Science Foundation (Nos. 1408085MH156 and 1408085MH167 granted to Min Chen, Guodong Shen, respectively), and Anhui Provincial science and technology key projects (Nos. 1303063017, 1301042094 granted to Shilian Hu, Guodong Shen, respectively). The funders had no role in the study design, data collection and analysis, decision to publish, or the preparation of the manuscript.
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