MicroRNA 200c-3p regulates autophagy via upregulation of endoplasmic reticulum stress in PC-3 cells

Background 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. Methods 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). Results 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. Conclusions Overall, our findings suggest that miR-200c-3p regulates autophagy via upregulation of ER stress signaling.


Background
Autophagy is characterized by the degradation of cellular components in lysosomes [1]. 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][5][6][7].
Endoplasmic reticulum (ER) stress, which functions in protein folding, is considered an inducer of autophagy [8]. Intracellular and extracellular stimuli can substantially affect ER functions, leading to the accumulation of unfolded or misfolded proteins in the ER lumen [9]. 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 [10].
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
Cell culture 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% CO 2 atmosphere at 37 °C.

Statistical analyses
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
To determine whether miRNAs were involved in ER stress, we randomly selected several miRNAs, including miR-135a, miR-1290, miR-200c-3p, miR-374b, and miR-3195. After transfection with miRNA mimics, we determined the expression levels of the ER stress markers IRE1α, PERK, CHOP, ATF6, and GRP78. As shown in Fig. 1a, western blotting revealed that the levels of CHOP, ATF6, and IRE1α, but not GRP78 and PERK, were increased following transfection with miR-1290, miR-200c-3p, and miR-374b mimics. Consistent with this, RT-qPCR analysis showed that ectopic expression of the miR-200c-3p mimic in PC-3 cells increased the mRNA level of ATF6, elF2α, and CHOP, but not PERK (Fig. 1b), suggesting that miR-200c-3p positively regulates ER stress. Therefore, we focused on miR-200c-3p in this study.

miR-200c-3p was regulated by ER stress
To determine whether miR-200c-3p was regulated by ER stress, we treated PC-3 cells with the ER stress inducer TG. As shown in Fig. 2a, miR-200c-3p expression was determined by RT-qPCR in TG-treated PC-3 cells. After 6-h treatment with TG (0.5 mM), the level of miR-200c-3p was increased for 24 h (Fig. 2a). We also determined the expression of eIF2α and ATF6 mRNA as a positive control of ER stress (Fig. 2b). To determine the role of miR-200c-3p in cytotoxicity induced by ER stress, miR-200c-3p was transfected into cells, followed by treatment with TG for 48 h. As shown in Fig. 2c, the miR-200c-3p mimic enhanced the TG-induced cytotoxicity in PC-3 cells.

Ectopic expression of miR-200c-3p induced autophagy in PC-3 cells
ER stress induces autophagy [8]. Therefore, we predicted that autophagy might be induced by ectopic expression of miR-200c-3p in PC-3 cells. RT-qPCR was performed following transfection of miR-200c-3p mimic or inhibitor in PC-3 cells to determine the effects on LC3-II and Beclin mRNA levels, which are markers of autophagy. As shown in Fig. 3, ectopic expression of miR-200c-3p increased the mRNA levels of LC3-II and Beclin, while the miR-200c-3p inhibitor attenuated their expression (Fig. 3a, b). Next, western blotting was performed to evaluate the conversion of LC3-I to LC3-II following overexpression of miR-200c-3p. Similar to the RT-qPCR results, western blotting revealed that ectopic expression of the miR-200c-3p mimic increased LC3-II expression, while the miR-200c-3p inhibitor slightly attenuated LC3-II expression (Fig. 3c). In addition, we treated miR-200c-3p mimic-or inhibitor-transfected PC-3 cells with or without NH 4 Cl, an inhibitor of acidification that blocks fusion of the lysosome. This treatment had no effect on LC3-II expression compared with NH 4 Cl alone, suggesting that miR-200c-3p might block autophagic flux (Fig. 3c). Furthermore, we observed that the miR-200c-3p mimic increased protein levels of Beclin while the miR-200c-3p inhibitor attenuated the level of Beclin (Fig. 3d). Next, we evaluated autophagic activity by fluorescence microscopy and found that overexpression of the miR-200c-3p mimic resulted in the formation of puncta of endogenous LC3-II outside the autophagosomes (Fig. 4a). Additionally, following transfection with a green fluorescent protein (GFP)-fused LC3-II plasmid, the formation of GFP-fused LC3-II puncta was increased in miR-200c-3p mimictransfected PC-3 cells (Fig. 4b).

Effect of starvation on the levels of miR-200c-3p
To further investigate the role of miR-200c-3p in autophagy, nutrient starvation was induced in PC-3 cells for 2, 4, or 6 h using Hank's balanced salt solution (HBSS) and the level of miR-200c-3p was examined by RT-qPCR, which showed that starvation led to an increase in the level of miR-200c-3p (Fig. 6a). To determine whether miR-200c-3p was responsive to autophagy induced by starvation, PC-3 cells were transfected with a control, miR-200c-3p mimic, or miR-200c-3p inhibitor, and 2 days after transfection, starvation by HBSS was induced for 3 or 6 h. After starvation for 3 h, the miR-200c-3p inhibitor blocked the increase in LC3-II compared with the control inhibitor, while the miR-200c-3p mimic increased LC3-II slightly (Fig. 6b).

miR-200c-3p induced autophagy in an IRE1α-dependent manner
Several lines of evidence suggest that ER stress causes autophagy. Specifically, IRE1α and PERK are important mediators of ER stress-induced autophagy [16,17]. To determine whether miR-200c-3p induced autophagy via ER stress, we transfected control or miR-200c-3p mimic in IRE1α-or PERK-silenced PC-3 cells with siRNA. As shown in Fig. 7, transfection of the miR-200c-3p mimic in control siRNA-treated cells enhanced the level of LC3-II, while LC3-II expression was attenuated in IRE1αsilenced PC-3 cells. However, there was no attenuation of LC3-II in PERK siRNA-treated PC-3 cells. Our results indicate that miR-200c-3p induces autophagy in an IRE1α-dependent manner.

Discussion
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][21][22]. Although the role of the Fig. 2 miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p < 0.001, **p < 0.01, and *p < 0.05 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 autophagymediated 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* [23], and reduces the expression of the miR-199a/214 cluster in human hepatocellular  [25]. A recent study reported that miR-200c exerted an anticancer effect via ER stress in H460 cells [26]. 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 [27], and miR-155 and miR-31 inhibit interferon γ-induced autophagy [28]. Inhibition of miR-142-3p, miR-376A, and miR-376B induced autophagy-mediated starvation [29]. 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  [32] and miR-18a, which enhanced autophagy in colon cancer cells [33]. 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 starvationinduced LC3-II expression. Thus, our data suggest that miR-200c-3p enhances autophagy in PC-3 cells.

Conclusion
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.  Western blotting was performed to determine the levels of LC3-II and β-actin. Data are presented as the mean ± SEM of triplicate samples. ***p < 0.001, **p < 0.01, and *p < 0.05

Fig. 7
Silencing of IRE1α blocked the conversion of LC3-I to LC3-II in miR-200c-3p-transfected PC-3 cells. PC-3 cells were transfected with control, IRE1α, or PERK short interfering RNAs (siRNAs). The following day, control or miR-200c-3p mimics were transfected into siRNAtreated cells. One day after the second transfection, western blotting was performed to determine the levels of IRE1α, PERK, LC3-II, and β-actin