MicroRNA 200c-3p regulates autophagy via upregulation of endoplasmic reticulum stress in PC-3 cells
© The Author(s) 2018
Received: 20 September 2017
Accepted: 25 December 2017
Published: 3 January 2018
Autophagy is a response to cellular and environmental conditions and facilitates cell survival. Here, we investigated the role of ectopic expression of microRNA (miRNA) 200c-3p in autophagy.
miRNA mimics were used to overexpress miRNAs. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed to analyze miRNA expression. RT-qPCR and western blotting were performed to determine the expression levels of inositol requiring protein-1 (IRE1α), activating transcription factor-6 (ATF6), C/EBP homologous protein (CHOP), and light chain-3 (LC3).
Western blotting and RT-qPCR analysis revealed that ectopic expression of miR-200c-3p increased the expression of IRE1α, ATF6, and CHOP in PC-3 prostate cancer cells. Furthermore, the level of miR-200c-3p was enhanced by treatment with the endoplasmic reticulum (ER) stress inducer thapsigargin. In addition, ectopic expression of miR-200c-3p led to an increase in LC3-II expression, and formed puncta of green fluorescent protein-fused LC3-II in PC-3 cells. Interestingly, starvation stress induced by Hank’s balanced salt solution buffer increased the level of miR-200c-3p and conversely miR-200c-3p inhibitor blocked the increased expression of LC3-II induced by starvation in PC-3 cells. In addition, silencing of IRE1α by transfection of short interfering RNA attenuated the expression of LC3-II induced by upregulation of miR-200c-3p in PC-3 cells.
Overall, our findings suggest that miR-200c-3p regulates autophagy via upregulation of ER stress signaling.
Autophagy is characterized by the degradation of cellular components in lysosomes . The initial step of autophagy involves the formation of dysfunctional organelles, misfolded/aggregated proteins, or autophagosomes [2, 3]. In the late stage, autophagosomes fuse with lysosomes to generate autolysosomes and substrates are degraded by lysosomal hydrolases. This programmed cell destruction plays a role in cancer, cell death, survival, and adaptive responses [4–7].
Endoplasmic reticulum (ER) stress, which functions in protein folding, is considered an inducer of autophagy . Intracellular and extracellular stimuli can substantially affect ER functions, leading to the accumulation of unfolded or misfolded proteins in the ER lumen . To avoid cell damage, accumulation of unfolded or misfolded proteins activates the unfolded protein response (UPR), which involves the three major transducers of ER stress, activating transcription factor-6 (ATF6), inositol requiring protein-1 (IRE1α), and protein kinase RNA-like ER kinase (PERK). ER stress can induce apoptosis by activating the UPR in cancer, and may be a target for anticancer treatment .
MicroRNAs (miRNAs) are small non-coding RNAs that serve as negative regulators of gene expression involved in cell growth, cancer, apoptosis, and aging [11, 12]. The role of miRNAs as novel regulators of autophagy and ER stress has been well documented [13, 14]. Thus, in this study, we investigated the role of miR-200c-3p in autophagy. Here, we demonstrate that overexpression of miR-200c-3p promotes ER stress signaling to induce autophagy via light chain-3 (LC3)-II activation and autophagosome formation in PC-3 prostate cancer cells.
Materials and methods
PC-3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Welgene, Daegu, Korea), 2 μM l-glutamine, and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) in a 5% CO2 atmosphere at 37 °C.
To assess cytotoxicity, PC-3 cells were transfected with miR-200c-3p mimics (Genolution, Korea). Two days after transfection, the cells were treated with or without 0.5 mM thapsigargin (TG) (Sigma, St. Louis, MO, USA) for 24 h and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma) was performed according to the manufacturer’s instructions.
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis
Total RNA from PC-3 cells transfected with control, miR-200c-3p mimic, or miR-200c-3p inhibitor was isolated using QIAzol (Invitrogen). One microgram of total RNA was used to generate complementary DNA (cDNA) by superscript reverse transcriptase (Invitrogen). RT-qPCR was performed with the LightCycler instrument (Roche Applied Sciences, Indianapolis, IN, USA) with the following primers: LC3-II-forward, 5′-GCC TTC TTC CTG CTG GTG AAC-3′ and reverse, 5′-AGC CGT CCT CGT CTT TCT CC-3′; Beclin 1-forward, 5′-GGA TGG ATG TGG AGA AAG GCA AG-3′ and reverse, 5′-TGA GGA CAC CCA AGC AAG ACC-3′; ATF6-forward, 5′-AACAAGACCACAAGACCAA-3′ and reverse, 5′-AGGAGGAACTGACGAACT-3′; C/EBP homologous protein (CHOP)-forward, 5′-CTCCTTCGGGACACTGTCCA-3′ and reverse, 5′-CTTTCTCCTTCATGCGCTGC-3′; PERK-forward, 5′-CGATGAGACAGAGTTGCGAC-3′ and reverse, 5′-TGCTTTCACGGTCTTGGTC-3′; eukaryotic initiation factor (eIF) 2α-forward, 5′-CTCTTGACAGTCCGAGGATC-3′ and reverse, 5′-GTATCCCAGCTGTGCCATCT-3′; and glyceraldehyde 3-phosphate dehydrogenase (GADPH)-forward, 5′-AGGGCTGCTTTTAACTCTGGT-3′; and reverse, 5′-CCCCACTTGATTTTGGAGGGA-3′.
PC-3 cells were transfected with miR-135a, miR-1290, miR-200c-3p, miR-374b, miR-3195, and control mimic plasmids (Genolution). Two days after transfection, the cells were lysed in radioimmunoprecipitation buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid, 1 M EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail). Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred onto a Hybond enhanced chemiluminescence (ECL) transfer membrane (Amersham Pharmacia, Piscataway, NJ, USA). After blocking, the membrane was incubated with the following primary antibodies: LC3-II (1:1000; #2775, Cell Signaling Technology, Danvers, MA, USA), PERK (1:1000; #5683, Cell Signaling Technology), IRE1α (1:1000; #3294, Cell Signaling Technology), 78 kDa glucose-regulated protein (GRP78) (1:500; #sc-376768, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ATF6 (1:1000; #65880, Cell Signaling Technology), Beclin (1:1000, #3738, Cell Signaling Technology), and β-actin (1:5000; #4970, Cell Signaling Technology). After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (1:5000; AbD Serotec, Kidlington, UK). An ECL system (Amersham Pharmacia) was used to visualize the protein bands.
Short interfering RNA transfection assay
PC-3 cells were transiently transfected with control, IRE1α, or PERK short interfering RNA (siRNA; Bioneer, Korea) using interferin transfection reagent (Polyplus-transfection Inc., New York, NY, USA). Briefly, the mixture of siRNA (40 nM) and interferin transfection reagent were incubated for 10 min, and added to PC-3 cells.
miRNA transfection assay
miR-200c-3p and control mimic plasmids (200 nM; Genolution) were transfected into PC-3 cells using interferin transfection reagent (Polyplus-transfection Inc.) according to the manufacturer’s instructions. To evaluate the expression of miR-200c-3p, total RNA from PC-3 cells was isolated with QIAzol following treatment with TG (Invitrogen). To construct miRNA cDNA, a GenoExplorer miRNA cDNA kit (GenoSensor Corporation, Tempe, AZ, USA) was used according to the manufacturer’s instructions. To measure miRNA levels, RT-qPCR analysis was performed using the LightCycler instrument (Roche Applied Sciences). miRNA primers of miR-200c-3p were purchased from GenoExplorer (GenoSensor Corporation), and the U6 small nuclear ribonucleoprotein (snRNA) primer was used to normalize miRNA levels. Sequences of the U6 snRNA primers were as follows: forward, 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-TTCACGAATTTGCGTGTCAT-3′.
PC-3 cells grown on LAB-TEK II chamber slides (Nalge Nunc International, Rochester, NY, USA) following transfection of miR-200c-3p mimic (100 nM) were washed and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Fixed cells were washed with PBS and permeabilized with 1% Triton-X 100 in PBS for 5 min. The primary antibody, anti-LC3-II (1:1000; Cell Signaling Technology), diluted in 1% bovine serum albumin in PBS, was added and incubated overnight at 4 °C. Fixed cells were washed and stained with the corresponding Alexa Fluor fluorescent antibody (1:2000; ab150077, Abcam, Cambridge, UK) for 30 min at room temperature. After washing, cell nuclei were counterstained with 1 μg/mL 4′,6-diamidino-2-phenylindole and mounted. Images were obtained using a Delta Vision imaging system (Applied Precision, Issaquah, WA, USA).
Statistical analyses of the data were conducted using Prism software (La Jolla, CA, USA). All data were expressed as the mean ± standard error of the mean. Statistically significant differences between the control and treatments were determined by Student’s t test.
Ectopic expression of miR-200c-3p induced ER stress markers
miR-200c-3p was regulated by ER stress
Ectopic expression of miR-200c-3p induced autophagy in PC-3 cells
Effect of starvation on the levels of miR-200c-3p
miR-200c-3p induced autophagy in an IRE1α-dependent manner
The role of the miR-200 family in cancer has been extensively investigated. miR-200 family members are frequently downregulated in metastases, with low expression of miR-200 inducing aggressive, invasive, or chemoresistant phenotypes in several cancer types, including non-small-cell lung cancer and female reproductive cancers [18, 19]. miR-200 family members also regulate the epithelial-mesenchymal transition (EMT), which is important in the metastatic progression of many types of cancer. miR-200 family members have been shown to regulate the EMT via the transcriptional repressors ZEB1 and SIP1/ZEB2 [20–22]. Although the role of the miR-200c family in the EMT has been investigated extensively, its role in autophagy has not been studied. Here, we report a potential role for miR-200c-3p in autophagy-mediated ER stress in prostate cancer cells.
Evidence suggests that miRNA expression can regulate or be regulated by the UPR of the ER. In addition, ER stress induces miR-30c-2* , and reduces the expression of the miR-199a/214 cluster in human hepatocellular cancer . Chitnis et al. reported that miR-211 was enhanced in mammary carcinoma in a PERK-dependent manner while increased expression of miR-211 attenuated the accumulation of CHOP . A recent study reported that miR-200c exerted an anticancer effect via ER stress in H460 cells . Similarly, in this study, we demonstrated that ectopic expression of miR-200c-3p resulted in increased expression of IRE1α, ATF6, and CHOP, and ER stress regulated the expression of miR-200c-3p. This suggests that miR-200-3p regulates or is regulated by ER stress.
Several studies have revealed that miRNAs are linked to autophagy, including evidence that miR-23b can sensitize pancreatic cancer cells to radiation treatment by blocking radiation-induced autophagy , and miR-155 and miR-31 inhibit interferon γ-induced autophagy . Inhibition of miR-142-3p, miR-376A, and miR-376B induced autophagy-mediated starvation . It is also known that miR-101 and miR-30a are potent inhibitors of autophagy [30, 31]. miRNAs are thought to be negative regulators of autophagy, with the exception of miRNA-155, which enhanced autophagy to eliminate intracellular mycobacteria  and miR-18a, which enhanced autophagy in colon cancer cells . In this study, we demonstrated that overexpression of miR-200c-3p with a mimic increased the expression of LC3-II and the accumulation of LC3 puncta in PC-3 cells. In addition, the miR-200c-3p inhibitor blocked starvation-induced LC3-II expression. Thus, our data suggest that miR-200c-3p enhances autophagy in PC-3 cells.
Overall, our data demonstrated that miR-200c-3p increased the expression of ER stress genes as well as the expression of LC3-II. Furthermore, the level of miR-200c-3p was increased in starvation-induced autophagy. Thus, our data suggest that miR-200c-3p-mediated autophagy, which is induced via ER stress, might be a potential target for the treatment of prostate cancer cells.
The authors declare that they have no competing interests.
Availability of data and materials
All data is included in the manuscript.
Consent for publication
The authors consent for publication.
Ethics approval and consent to participate
This article does not contain any studies with human participants and animals performed by any of the authors.
This was supported by the National Research Foundation of Korea (NRF) grant funded by Basic Research Program (2015R1C1A1A02036842).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6(4):463–77.View ArticlePubMedGoogle Scholar
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19(21):5720–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 2004;36(12):2503–18.View ArticlePubMedGoogle Scholar
- Cuervo AM. Autophagy: in sickness and in health. Trends Cell Biol. 2004;14(2):70–7.View ArticlePubMedGoogle Scholar
- Levine B. Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell. 2005;120(2):159–62.PubMedGoogle Scholar
- Hönscheid P, Datta K, Muders MH. Autophagy: detection, regulation and its role in cancer and therapy response. Int J Radiat Biol. 2014;90(8):628–35.View ArticlePubMedGoogle Scholar
- Levine B. Cell biology: autophagy and cancer. Nature. 2007;446(7137):745–7.View ArticlePubMedGoogle Scholar
- Yorimitsu T, Nair U, Yang Z, Klionsky DJ. Endoplasmic reticulum stress triggers autophagy. J Biol Chem. 2006;281(40):30299–304.View ArticlePubMedPubMed CentralGoogle Scholar
- Kadowaki H, Nishitoh H, Ichijo H. Survival and apoptosis signals in ER stress: the role of protein kinases. J Chem Neuroanat. 2004;28(1–2):93–100.View ArticlePubMedGoogle Scholar
- Wang WA, Groenendyk J, Michalak M. Endoplasmic reticulum stress associated responses in cancer. Biochim Biophys Acta. 2014;1843(10):2143–9.View ArticlePubMedGoogle Scholar
- Zagryazhskaya A, Zhivotovsky B. miRNAs in lung cancer: a link to aging. Ageing Res Rev. 2014;17:54–67.View ArticlePubMedGoogle Scholar
- Lynam-Lennon N, Maher SG, Reynolds JV. The roles of microRNA in cancer and apoptosis. Biol Rev Camb Philos Soc. 2009;84(1):55–71.View ArticlePubMedGoogle Scholar
- Frankel LB, Lund AH. MicroRNA regulation of autophagy. Carcinogenesis. 2012;33(11):2018–25.View ArticlePubMedGoogle Scholar
- Maurel M, Chevet E. Endoplasmic reticulum stress signaling: the microRNA connection. Am J Physiol Cell Physiol. 2013;304(12):C1117–26.View ArticlePubMedGoogle Scholar
- Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem. 1995;270(5):2320–6.View ArticlePubMedGoogle Scholar
- Kouroku Y, Fujita E, Tanida I, Ueno T, Isoai A, Kumagai H, Ogawa S, Kaufman RJ, Kominami E, Momoi T. ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell Death Differ. 2007;14(2):230–9.View ArticlePubMedGoogle Scholar
- Talloczy Z, Jiang W, Virgin HWT, Leib DA, Scheuner D, Kaufman RJ, Eskelinen EL, Levine B. Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc Natl Acad Sci USA. 2002;99(1):190–5.View ArticlePubMedGoogle Scholar
- Ceppi P, Mudduluru G, Kumarswamy R, Rapa I, Scagliotti GV, Papotti M, Allgayer H. Loss of miR-200c expression induces an aggressive, invasive, and chemoresistant phenotype in non-small cell lung cancer. Mol Cancer Res. 2010;8(9):1207–16.View ArticlePubMedGoogle Scholar
- Cochrane DR, Howe EN, Spoelstra NS, Richer JK. Loss of miR-200c: a Marker of Aggressiveness and Chemoresistance in Female Reproductive Cancers. J Oncol. 2010;2010:821717.View ArticlePubMedGoogle Scholar
- Wang X, Chen X, Wang R, Xiao P, Xu Z, Chen L, Hang W, Ruan A, Yang H, Zhang X. microRNA-200c modulates the epithelial-to-mesenchymal transition in human renal cell carcinoma metastasis. Oncol Rep. 2013;30(2):643–50.View ArticlePubMedGoogle Scholar
- Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10(5):593–601.View ArticlePubMedGoogle Scholar
- Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 2008;22(7):894–907.View ArticlePubMedPubMed CentralGoogle Scholar
- Byrd AE, Aragon IV, Brewer JW. MicroRNA-30c-2* limits expression of proadaptive factor XBP1 in the unfolded protein response. J Cell Biol. 2012;196(6):689–98.View ArticlePubMedPubMed CentralGoogle Scholar
- Duan Q, Wang X, Gong W, Ni L, Chen C, He X, Chen F, Yang L, Wang P, Wang DW. ER stress negatively modulates the expression of the miR-199a/214 cluster to regulates tumor survival and progression in human hepatocellular cancer. PLoS ONE. 2012;7(2):e31518.View ArticlePubMedPubMed CentralGoogle Scholar
- Chitnis NS, Pytel D, Bobrovnikova-Marjon E, Pant D, Zheng H, Maas NL, Frederick B, Kushner JA, Chodosh LA, Koumenis C, Fuchs SY, Diehl JA. miR-211 is a prosurvival microRNA that regulates chop expression in a PERK-dependent manner. Mol Cell. 2012;48(3):353–64.View ArticlePubMedPubMed CentralGoogle Scholar
- Bai T, Dong DS, Pei L. Synergistic antitumor activity of resveratrol and miR-200c in human lung cancer. Oncol Rep. 2014;31(5):2293–7.View ArticlePubMedGoogle Scholar
- Wang P, Zhang J, Zhang L, Zhu Z, Fan J, Chen L, Zhuang L, Luo J, Chen H, Liu L, Chen Z, Meng Z. MicroRNA 23b regulates autophagy associated with radioresistance of pancreatic cancer cells. Gastroenterology. 2013;145(5):1133–1143e1112.View ArticlePubMedGoogle Scholar
- Holla S, Kurowska-Stolarska M, Bayry J, Balaji KN. Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling. Autophagy. 2014;10(2):311–30.View ArticlePubMedGoogle Scholar
- Korkmaz G, Tekirdag KA, Ozturk DG, Kosar A, Sezerman OU, Gozuacik D. MIR376A Is a Regulator of Starvation-Induced Autophagy. PLoS ONE. 2013;8(12):e82556.View ArticlePubMedPubMed CentralGoogle Scholar
- Frankel LB, Wen J, Lees M, Hoyer-Hansen M, Farkas T, Krogh A, Jaattela M, Lund AH. microRNA-101 is a potent inhibitor of autophagy. EMBO J. 2011;30(22):4628–41.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu H, Wu H, Liu X, Li B, Chen Y, Ren X, Liu CG, Yang JM. Regulation of autophagy by a beclin 1-targeted microRNA, miR-30a, in cancer cells. Autophagy. 2009;5(6):816–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang J, Yang K, Zhou L, Wu Y, Zhu M, Lai X, Chen T, Feng L, Li M, Huang C, Zhong Q Q, Huang X. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. PLoS Pathog. 2013;9(10):e1003697.View ArticlePubMedPubMed CentralGoogle Scholar
- Qased AB, Yi H, Liang N, Ma S, Qiao S, Liu X. MicroRNA-18a upregulates autophagy and ataxia telangiectasia mutated gene expression in HCT116 colon cancer cells. Mol Med Rep. 2013;7(2):559–64.View ArticlePubMedGoogle Scholar