- Primary Research
- Open Access
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.
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This was supported by the National Research Foundation of Korea (NRF) grant funded by Basic Research Program (2015R1C1A1A02036842).
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