Knockdown of circ-ABCB10 promotes sensitivity of lung cancer cells to cisplatin via miR-556-3p/AK4 axis
Due to the acquired drug resistance, the potency of cisplatin-based chemotherapy is limited in lung cancer, which is a big obstacle in clinical treatment of lung cancer. Abundant evidence has revealed that circular RNAs (circRNAs) exerted facilitating or suppressive function on the tumorigenesis of multiple cancers. The oncogenic role of circ-ABCB10 in breast cancer and clear cell renal cell carcinoma has been validated in recent researches. However, the regulatory mechanism of circ-ABCB10 and its relation to cellular sensitivity to cisplatin in lung cancer is poorly understood.
The expression and characteristic of circ-ABCB10 were analyzed by RT-qPCR and nucleic acid electrophoresis. CCK-8, colony formation, TUNEL and transwell assays were applied to probe the role of FOXD3-AS1 in lung cancer. The interactions of miR-556-3p with circ-ABCB10 and AK4 were testified by luciferase reporter and RIP assays.
Circ-ABCB10 was markedly upregulated and featured with loop structure in lung cancer. Circ-ABCB10 depletion suppresses lung cancer progression and sensitizes lung cancer cells to cisplatin. Molecular mechanism assays manifested that circ-ABCB10 bound with miR-556-3p and negatively modulated miR-556-3p expression. Additionally, AK4 was testified to be the downstream target of miR-556-3p. More importantly, rescue assays clarified that upregulation of AK4 could reverse the cisplatin-sensitizing and tumor-suppressing effect of circ-ABCB10 knockdown on lung cancer cells.
Circ-ABCB10 knockdown enhances sensitivity of lung cancer cells to cisplatin by targeting miR-556-3p/AK4 axis.
KeywordsCirc-ABCB10 Cisplatin miR-556-3p AK4 Lung cancer
Adenylate kinase 4
Competing endogenous RNA
Long noncoding RNAs
RNA-induced silencing complex
Lung cancer is diagnosed as the most prevalent malignancy globally, with high incidence and death rate. For lung cancer, nearly 1.8 million new cases are diagnosis and 1.6 million cases died each year, and the deaths triggered by lung cancer take up 19% of all cancer-associated death cases . Known as a dominating cause of cancer-related death, lung cancer with a steady rise in occurrence rate has become a big obstacle for human health . High rates of recurrence and metastasis have been referred to as the major factors contributing to poor prognosis of patients with lung cancer . In spite of the improvement of diagnostic and therapeutic methods, the 5-year overall survival rate of patients suffering from lung cancer is less than 20% . More importantly, resistance to lung cancer treatment is closely related to abnormal expression of oncogenic or anti-tumor genes, including changes in the biological features of malignancies, cell proliferation, metastasis, apoptosis, etc. . Though cisplatin has already come into use as a kind of anti-cancer chemotherapy agent, multiple cancers (lung cancer included), may develop the acquired resistance to cisplatin, which is a stumbling block on the way to improving the efficacy of chemotherapy . In addition, cisplatin cytotoxicity remains a prevalent side-effect of cisplatin . Therefore, studying the mechanism underlying the cellular sensitivity to cisplatin in lung cancer is of extreme importance to enhance the efficacy of chemotherapy for lung cancer based on combined agents with specific molecular mechanisms.
Circular RNAs (circRNAs), microRNAs (miRNAs) as well as long noncoding RNAs (lncRNAs) belong to noncoding RNA (ncRNAs), among which circRNAs are featured with loop structure. Much attention has been paid to the function of circRNAs recently. A number of studies have verified that circRNAs are implicated in multiple human malignancies, including lung cancer [8, 9, 10]. Furthermore, recent researches have also revealed the critical effect of circRNAs exert on cellular sensitivity to cisplatin in different cancer types, such as osteosarcoma, gastric cancer and bladder cancer [11, 12, 13]. Therefore, identification of the circRNAs involved in regulating the sensitivity of lung cancer cells to cisplatin are of great value. Recently, existing literatures have uncovered the cancer-promoting role of circ-ABCB10 in clear cell renal cell carcinoma and breast cancer [14, 15]. However, the critical role of it in lung cancer and its association with cellular sensitivity to cisplatin in lung cancer are unclear, which therefore are worthy of exploring.
This study mainly focused on probing the regulatory mechanism of circ-ABCB10 and its influence on cell sensitivity to cisplatin in lung cancer. The results of this study elucidate that knockdown of circ-ABCB10 sensitized lung cancer cells to cisplatin via miR-556-3p/AK4 axis, which indicated that targeting circ-ABCB10 might be a new thought to improving the efficacy of cisplatin in lung cancer.
Materials and methods
Cell culture and treatment
Human bronchial epithelial cell (HBE) and human NSCLC cells (H-1299, H-125, NCI-H292, A549) were purchased from Chinese Academy of Sciences (Beijing, China). The RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin (Sigma-Aldrich, Milan, Italy) was applied for culturing cells, and cells were cultured in an incubator with 5% CO2 at 37 °C. To study cellular sensitivity to drugs, 2 μg/ml of 5-fluorouracil (5-Fu), 1 μM of cisplatin, 10 μM of Sorafenib and 1 μM of Sunitinib were all utilized for treating A549 or NCI-H292 cells, all from Sigma-Aldrich. 0.1% DMSO (Sigma-Aldrich) was added to culture medium as a solvent-only negative control group.
A549 and NCI-H292 cells were transfected with specific shRNAs against circ-ABCB10 (sh-circ-ABCB10#1#2), AK4 (sh-AK4#1#2), negative control (sh-NC), pcDNA3.1/AK4 or the empty pcDNA3.1 vector (GenePharma, Shanghai, China), separately. The miR-556-3p mimics and NC mimics were gained from GenePharma. Each plasmid was transfected into cells by Lipofectamine 2000 (Invitrogen).
Quantitative real time polymerase chain reaction (RT-qPCR)
TRIzol reagent (Takara, Otsu, Japan) was utilized for extracting total RNA from cultured A549 or NCI-H292 cells, and RNA concentration was measured with bicinchoninic acid (BCA) Kit (Invitrogen). Total RNA was reverse-transcribed into cDNA utilizing Reverse Transcription Kit (Applied Biosystems, Foster City, USA) for RT-qPCR. SYBR Green real-time PCR Kit (Takara, Tokyo, Japan) was then used on Bio-Rad CFX96 (Bio-Rad, Hercules, CA). Fold changes of gene expression were calculated by 2−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/U6 as endogenous control.
Actinomycin D (actD) and Rnase R treatment assays
2 mg/ml of actinomycin D (Sigma-Aldrich) was cultivated with A549 or NCI-H292 cells for 0, 4, 8, 12 h to block mRNA transcription, with DMSO as control. For treating cells with Rnase R, 2 μg of total RNA was cultivated for 30 min with or without 3 U/μg of Rnase R (Epicentre Technologies, Madison, WI, USA) at 37 °C, while cells without Rnase R treatment were seen as control group. RT-qPCR was employed for determining the expression levels of circABCB10 and ABCB10 mRNA.
Nucleic acid gel electrophoresis assay
After amplifying ABCB10 mRNA and circ-ABCB10, the cDNA and genomic DNA (gDNA) were obtained as templates, their PCR products were assessed by the agarose gels with TE buffer (Thermo Scientific, Waltham, MA, USA). GAPDH was used as a negative control.
The A549 and NCI-H292 cells (1 × 104) were inoculated into 96-well plates, and then incubated with 10 mg/ml of CCK-8 (Sigma-Aldrich) for another 4 h. The absorbance at 450 nm was measured with a plate reader (Bio-Tek Instruments, Hopkinton, MA, USA).
Colony formation assay
1 × 103 cells were cultured in 6-well plates and the culture medium was changed every 3 days. After 2 weeks, cells were fixed in ethanol (Sigma-Aldrich), and were then dyed using crystal violet (Sigma-Aldrich). Finally, the visible colonies containing ≥50 cells were then counted manually under a microscope (Olympus, Tokyo, Japan).
Using TUNEL Apoptosis Kit (Invitrogen), the apoptosis of A549 and NCI-H292 cells were determined. The above cells were stained by utilizing DAPI (Koritai Biotechnology, Beijing, China). Then, the TUNEL positive cells were captured under a fluorescence microscopy (Olympus).
The migration abilities of 1 × 103 A549 or NCI-H292 cells were examined using transwell chambers (Millipore, Billerica, MA, USA). Serum-free medium containing transfected cells was added to top compartment, while medium with 10% FBS was plated into bottom chamber. 48 h later, methanol (Sigma-Aldrich) and crystal violet (Solarbio, Beijing, China) were applied for fixing and dying cells, severally. 5 chosen fields at random under a microscope were used for counting migratory cells.
Total protein from cells were extracted by RIPA lysis buffer with protease inhibitors (Beyotime, Shanghai, China) and quantified through BCA Kit (Invitrogen). Then, total protein was isolated by SDS-PAGE and was moved to PVDF membranes (Millipore). After being blocked with skimmed milk, membranes were cultivated overnight with specific primary antibodies for AK4 (ab232888, Cambridge, USA) and GAPDH (ab8245). Later, secondary antibodies were added for cultivating for 1 h. The amount of protein was detected via chemiluminescence detection system.
The fractions of cytoplasmic and nuclear were isolated via NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Invitrogen) and gathered by RNeasy Midi Kit (Qiagen, Hilden, Germany) to determine the cellular localization of circ-ABCB10. The RT-qPCR was used for exploring the expression levels of circ-ABCB10, U6 (nuclear control) and GAPDH (cytoplasmic control).
Fluorescence-conjugated circ-ABCB10 probes were produced by Bersinbio Company (Guangzhou, China). NSCLC cells were immobilized in 4% paraformaldehyde (Sigma-Aldrich). After being cleaned using PBS and dehydrated in ethanol, the air-dried cells were denatured for further cultivation with FISH probes by hybridization reaction buffer. Later, cells were washed with × 2 saline-sodium citrate (SSC). Finally, DAPI was used for dying cells and results were visualized through a confocal laser microscopy (Olympus).
RIP assay was completed using an Imprint RNA immunoprecipitation kit (Sigma-Aldrich). A549 and NCI-H292 cells were reaped and lysed in RIP lysis buffer. Cell lysate was then incubated with magnetic beads which were conjugated with anti-Ago2 antibody or with a negative control normal anti-IgG antibody. The relative enrichment of circ-ABCB10, miR-556-3p and AK4 were determined by RT-qPCR analysis.
Luciferase reporter assay
The wild-type (WT) reporter vector psiCHECK-circ-ABCB10-WT (Promega, Madison, WI, USA) were constructed based on the bioinformatics-predicted binding sites of miR-556-3p, while binding sequences were mutated to form psiCHECK-circ-ABCB10-Mut vector. The two vectors were co-transfected with miR-556-3p mimics or NC mimics into A549 and NCI-H292 cells. The wild-type and mutant interacting sequences of miR-556-3p in AK4 3′-UTR fragments were obtained to generate psiCHECK-AK4-WT/Mut vectors, then co-transfected into cells with indicated transfection plasmids. The luciferase activity was examined at 48 h post-transfection via Dual-Luciferase Reporter Assay System (Promega, MA, USA).
Statistical analyses were carried out by GraphPad Prism 7.0 software (Graph Pad Software, La Jolla, CA, USA). Data from more than two independent experiments, were showed as mean ± SD. Student’s t-test or one-way ANOVA was employed for the assessing group difference. P < 0.05 had statistical significance in requirements.
Circular RNA circ-ABCB10 is highly expressed in NSCLC and circ-ABCB10 knockdown represses lung cancer progression and improves the sensitivity of lung cancer cells to cisplatin
Circ-ABCB10 sponges miR-556-3p in lung cancer
Circ-ABCB10 regulates AK4 expression by sponging miR-556-3p in lung cancer
Circ-ABCB10 contributes to cisplatin resistance of lung cancer by targeting miR-556-3p/AK4 axis
Due to the highest incidence and death rate globally, lung cancer is diagnosed as the most prevalent malignancy . Although cisplatin has been recently used as a kind of anti-cancer chemotherapy agent, multiple cancers, including lung cancer, may develop the acquired resistance to cisplatin . Therefore, a further understanding of the mechanism behind the cellular sensitivity of lung cancer cells might help identify potential target to enhance cisplatin efficacy in lung cancer.
A growing number of researches have revealed the critical function of circRNAs on cisplatin resistance of diverse cancers [11, 12, 13]. Therefore, identification and exploration of the circRNAs associated with cisplatin efficacy are of great value. Previous findings showed that circ-ABCB10 was upregulated and served as an oncogene in several cancers such as clear cell renal cell carcinoma and breast cancer [14, 15]. Also, a study by Tian X. et al. validated that circ-ABCB10 promoted the proliferation and migration of non-small cell lung cancer (NSCLC) . In consistence, our data also validated that circ-ABCB10 was upregulated in non-small cell lung cancer cell lines, and that knockdown of circ-ABCB10 decreased proliferation, increased apoptosis, and hindered migration in NSCLC cells. Notably, our data firstly showed that cisplatin treatment led to a decrease of circ-ABCB10 in lung cancer cells, indicating the involvement of circ-ABCB10 in the cellular response to cisplatin in lung cancer. This was the first time for circ-ABCB10 to be related to drug sensitivity in cancer cells. Further, we demonstrated that knockdown of circ-ABCB10 can further enhance the anti-proliferation and pro-apoptosis effect of cisplatin on lung cancer cells, suggesting that targeting circ-ABCB10 sensitized lung cancer cells to cisplatin in vitro.
Mechanistically, increasing evidence has indicated that circRNAs sponge specific miRNAs to regulate tumor initiation and progression [17, 18]. Several studies also demonstrated that circ-ABCB10 can sponge miRNAs to regulate certain oncogenes in cancers including NSCLC [15, 16]. Concordantly, we showed that circ-ABCB10 was mainly distributed in cytoplasm of lung cancer cells, verifying that circ-ABCB10 played vital roles in lung cancer by post-transcriptionally regulating certain miRNA. We firstly predicted through bioinformatics tool and validated that miR-556-3p interacted with circ-ABCB10. Formerly, miR-556-3p has been reported to be lowly expressed in endometrial tissues . However, the association of miR-556-3p with circ-ABCB10 was first confirmed by our data. Also, we validated via rescue assays that miR-556-3p participated in the regulation of circ-ABCB10 on lung cancer progression and sensitivity to cisplatin resistance of lung cancer cells, which also firstly linked miR-556-3p to lung cancer and drug sensitivity of lung cancer cells.
Adenylate kinase 4 (AK4), has been validated to exert oncogenic function on lung cancer cell growth and metastasis . Besides, AK4 has been uncovered to be involved in the radio-resistance of esophageal cancer cells . More importantly, previous investigations have confirmed the involvement of AK4 in chemoresistance of cancers [22, 23]. Formerly, a study suggested the correlation of high AK4 level with poor survival in lung cancer , but the detailed role of AK4 in lung cancer progression and cell response to cisplatin was first revealed in our study. Herein, we validated that AK4 was highly expressed in lung cancer cells. We also confirmed that AK4 was negatively regulated by miR-556-3p but positively regulated by circ-ABCB10. Besides, we validated that circ-ABCB10 competed with AK4 to bind to miR-556-3p, which suggested that circ-ABCB10 sponged miR-556-3p to upregulate AK4 by serving as a ceRNA. Moreover, downregulation of AK4 restrained lung cancer progression whereas sensitizes lung cancer cells to cisplatin. Finally, rescue assays manifested that upregulating AK4 expression could offset the suppressive effect of circ-ABCB10 knockdown on the progression of lung cancer cells without cisplatin, and that AK4 overexpression counteracted the sensitizing effect of circ-ABCB10 knockdown on cells to cisplatin treatment, indicating that AK4 was a target for circ-ABCB10 to regulate lung cancer progression and cell sensitivity to cisplatin.
In summary, all these data from this study indicate that targeting circ-ABCB10/miR-556-3p/AK4 pathway enhance sensitivity of lung cancer cells to cisplatin. These findings suggested that targeting circ-ABCB10 might be a potential new thought to improve the sensitivity to cisplatin in lung cancer. However, studies based on clinical samples and in vivo models are required to validate the clinical significance of circ-ABCB10, which we will carry out in the future.
We appreciate all people involved in this study.
This research project was designed mainly by ZW. QG and YY performed the functional assays in our study, whereas ZW and JZ cooperated to complete the mechanistic experiments. Besides, ZW accomplished the writing of Result and Discussion sections of this manuscript, whereas WL was responsible for statistical analyses of all data and the reset part of this manuscript. All authors read and approved the final manuscript.
The study was supported by the Natural Science Foundation of Hunan Province (2017JJ4072), and this body took full responsibility for the design of the study and collection, analysis, and interpretation of data as well as the writing of this manuscript.
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The authors declare that they have no competing interests.
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