Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway.

Hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer. Hypoxia induces upregulation of a specific set of microRNAs (miRNAs) in a variety of cell types. Here, we describe hypoxia-induced MIR155 as a potent inducer of autophagy. Enforced expression of MIR155 increases autophagic activity in human nasopharyngeal cancer and cervical cancer cells. Knocking down endogenous MIR155 inhibits hypoxia-induced autophagy. We demonstrated that MIR155 targets multiple players in MTOR signaling, including RHEB, RICTOR, and RPS6KB2. MIR155 suppresses target-gene expression by directly interacting with their 3' untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness. Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G 1/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.

Introduction

Oxygen deprivation or hypoxia is a common feature in various physiological or pathophysiological states, including exercise, development, or cancer. Hypoxia has emerged as a pivotal factor in solid tumors due to overwhelming growth, inadequate blood supply, tumor progression, and resistance to therapy. Responses to hypoxia are orchestrated in part through activation of HIF (hypoxia-inducible factor). By controlling the transcription of hundreds of target genes, HIF regulates many cellular processes, such as angiogenesis, metabolism, cell migration, and tumor invasion. Recent studies suggest that hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process by which cells capture intracellular proteins and organelles and eliminate them through delivery to lysosomes. Autophagy is vital in a range of physiological and pathological situations, and dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer, neurodegenerative diseases, and infection. -

The mechanistic target of rapamycin (MTOR), integrates and transmits signals from a diverse array of signaling pathways to regulate cell growth, metabolism, and ribosomal biogenesis, as well as autophagy. MTOR is part of 2 different protein complexes: rapamycin-sensitive MTORC1 and rapamycin-insensitive MTORC2, along with the regulatory-associated proteins RPTOR (regulatory-associated protein of MTOR, complex 1) and RICTOR (RPTOR independent companion of MTOR, complex 2). , Under nutrient-rich conditions, activation of MTORC1 triggers a cascade of anabolic processes for cell growth and proliferation, mainly mediated by RPS6KB2 (ribosomal protein S6 kinase, 70 kDa, polypeptide 2/p70 S6 kinase) and EIF4EBP1 (eukaryotic translation initiation factor 4E binding protein 1). Concomitantly, activation of MTORC1 also inhibits autophagosome formation through phosphorylating and inhibiting the ULK1-ATG13-RB1CC1-C12orf44/ATG101 complex. - By contrast, MTORC2 is less well understood. It has been reported that MTORC2 activates AKT via phosphorylation and enhances the expression of HIF1A. ,

MicroRNAs (miRNAs) are the most widely studied class of noncoding RNAs that regulate gene expression by blocking mRNA translation and/or mediating mRNA degradation. , miRNAs control diverse fundamental biological and pathological processes, including cell proliferation, apoptosis, differentiation, and metabolism, and are also associated with cancer. , - miRNA expression is often deregulated in cancer and miRNAs can act as oncogenes or tumor suppressors.

Recently, miRNAs have been well characterized in the modulation of different autophagic stages. Autophagy induction is initiated by the ULK1 complex. MIR20A and MIR106B impede autophagy induction by leucine deprivation in C2C12 cells via directly targeting Ulk1. Vesicle nucleation mainly involves the BECN1 (Beclin 1, autophagy related) complex. BECN1 itself is targeted by MIR30A, MIR376B, and MIR519A. - Additional regulators of this step are MIR101, MIR630, and MIR374A, which target RAB5A and UVRAG, respectively. , Vesicle elongation is controlled by two ubiquitin-like conjugation systems. Several miRNAs, including MIR30A, MIR181A, MIR374A, MIR630, MIR376B, MIR204, MIR375, MIR519A, MIR885–3P, and MIR-101, have been identified to regulate this process by targeting the expression of some ATG (autophagy-related) genes. , To date, most of the miRNAs primarily inhibit the autophagic process and function as negative regulators. MIR7 is the only example of an miRNA that may induce autophagy. MIR7 has been characterized as a tumor suppressive miRNA through negative regulation of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR pathway in hepatocellular carcinoma, which may account for the role in autophagy induction.

In the present study, we report that hypoxia-induced MIR155 is a potent inducer of autophagy. Overexpression of MIR155 increases autophagic activity, while knocking down endogenous MIR155 alleviates hypoxia-induced autophagy. Importantly, we demonstrated that 3 members of the MTOR pathway, RHEB, RICTOR and RPS6KB2, are direct targets of MIR155. By suppressing the activation of MTOR-AKT pathway, MIR155 induces autophagy, decelerates cell proliferation and G1/S cell cycle progression.

Results

Hypoxia induces MIR155 upregulation

To investigate the function of MIR155 in hypoxia-induced autophagy, first we examined the expression of MIR155 in CNE and HeLa cells under hypoxic stress. As shown in Figure 1A, the expression level of MIR155 was low in normal culture conditions (21% oxygen). Hypoxia (1% oxygen) treatment induced a sustained upregulation of MIR155 in a time-dependent manner in both cell types. At 48 h after hypoxia treatment, more than a 12-fold increase of the MIR155 expression level was detected.

Figure 1. Hypoxia-induced MIR155 promotes autophagosome accumulation. (A) Hypoxia induces MIR155 expression. CNE or HeLa cells were exposed to 1% oxygen for 24, 36 and 48 h. Cells were collected for qRT-PCR to quantify the expression of MIR155. (mean ± s.d. of independent experiments, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, Student 2-tailed t test). (B) MIR155 promotes GFP-LC3 translocation. CNE or HeLa cells stably expressing GFP-LC3 were transfected with negative control (NC), MIR155 or MIR203. Cells were fixed 48 h post transfection. Representative images are shown. Scale bar: 10 μm. (C) Quantitative analysis of GFP-LC3 puncta in (B). At least 200 cells were examined in each experimental group. Data shown are means ± s.d. of four independent experiments. **P < 0.01, Wilcoxon rank sum test.

Overexpression of MIR155 induces autophagy

To explore the role of MIR155 in autophagy, we performed a GFP-LC3 puncta-formation assay and an LC3 conversion assay. MIR155 was transfected into CNE or HeLa cells that stably expressing GFP-LC3 fusion protein, the localization of GFP-LC3 was examined by confocal microscopy. GFP-LC3 puncta appear in the cytoplasm reflects the recruitment of LC3 proteins to autophagosomes. As shown in Figure 1B, there was a significant increase of GFP-LC3 puncta in MIR155 transfected cells. MIR203, a control miRNA that has no predicted targets in the autophagy pathway, behaved similarly to the negative control (NC) miRNA. The quantification of GFP-LC3 dots per cell confirmed that overexpression of MIR155 induced autophagosome accumulation in both CNE and HeLa cells (Fig. 1C). Next, we detected the conversion of LC3-I [cleaved, cytosolic form of MAP1LC3 (LC3)] to LC3-II (subsequently lipidated and membrane-bound form) by western blot. Consistent with the GFP-LC3 puncta formation assay, MIR155 overexpression led to a significant upregulation of lipidated LC3-II (Fig. 2A and B). Thus, both assays suggest that overexpression of MIR155 induces autophagosome accumulation.

Figure 2. Overexpression of MIR155 induces autophagic activity. (A) Overexpression of MIR155 induces LC3 conversion and SQSTM1 degradation. Western blots of control (NC) and MIR155-transfected cells. (B) ImageJ densitometric analysis of the LC3-II/GAPDH or (C) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, ***P < 0.001, Student 2-tailed t test. (D) Overexpression of MIR155 increases autophagic flux. CNE or HeLa cells were transfected with MIR155 or NC, BAF was applied to the medium for 1 h at 47 h post transfection, cells were harvested for western blot. (E) ImageJ densitometric analysis of the LC3-II/GAPDH or (F) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, **P < 0.01, Student 2-tailed t test.

To distinguish whether autophagosome accumulation is due to autophagy induction or rather a block in downstream steps, we performed autophagic flux assays. SQSTM1/p62, a polyubiquitin binding protein is selectively incorporated into autophagosomes through direct binding to LC3 and efficiently degraded during autophagy, thus the total cellular levels of SQSTM1 reflects the autophagic activity. We examined the intracellular amount of SQSTM1 by western blot, overexpression of MIR155 resulted in 20% to 40% reduction of SQSTM1 proteins in CNE and HeLa cells, suggesting that MIR155 promotes autophagic degradation (Fig. 2A and C). Finally, we performed an LC3 turnover assay. Cells were treated with the lysosomotropic reagent bafilomycin A1 (BAF) to block autophagic degradation. BAF treatment caused significant increase of LC3-II in both NC and MIR155 transfected cells (Fig. 2D and E). In addition, the protein levels of SQSTM1 in MIR155 transfected cells were also upregulated by BAF (Fig. 2F). Therefore, these data demonstrate that overexpression of MIR155 increases autophagic activity.

Inhibition of endogenous MIR155 represses hypoxia-induced autophagy

To document the physiological relevance of MIR155 on autophagy, we inhibited the expression of endogenous MIR155 and repeated the above validation assays in both CNE and HeLa cells. LNA-derived MIR155 inhibitor was used to inhibit the high level of endogenous MIR155 during hypoxia treatment. Hypoxia-induced GFP-LC3 puncta accumulation was dramatically suppressed by LNA-155 in both HeLa and CNE cells (Fig. 3A and B). Compared with LNA-NC control, SQSTM1 degradation during hypoxia treatment was also reduced upon LNA-155 transfection, reflecting a decrease of autophagic activity (Fig. 3C). Hence, these results demonstrate the physiological relevance of endogenous MIR155 on regulating autophagy process during hypoxia treatment.

Figure 3. Knockdown of endogenous MIR155 inhibits hypoxia-induced autophagy. (A) Inhibition of MIR155 in hypoxia suppresses GFP-LC3 translocation. HeLa or CNE cells stably expressing GFP-LC3 were transfected with LNA-NC or LNA-155 and exposed to 1% oxygen. The number of GFP-LC3 puncta was quantified. (mean ± s.d. of independent experiments, n = 4, *P < 0.05,**P < 0.01, Wilcoxon rank sum test). (B) Representative images from the quantification are shown. Scale bar: 10 μm. (C) Knockdown of endogenous MIR155 inhibits autophagy. HeLa or CNE cells transfected with LNA-NC or LNA-155 were cultured in 1% oxygen for 36 h, cells were harvested for western blot. Protein ratio of SQSTM1/GAPDH was calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).

Experimental identification of MIR155 targets

Having established the role of MIR155 in autophagy, we next wanted to identify the direct targets of MIR155. We used FindTar, a prediction algorithm designed by our laboratory, to clarify the potential targets of MIR155 in regulating autophagy. FindTar predicted that several upstream or downstream regulators of the MTOR signaling pathway, including RHEB, RPTOR, RICTOR, RPS6KB2, and MTOR are potential MIR155 targets. Besides, BCL2 and ATG3 were also putative MIR155 targets. We performed qRT-PCR to examine the mRNA levels of MIR155 putative targets. MIR155 but not control miRNA, led to a significant attenuation of the mRNA levels of RHEB. Other putative MIR155 target genes, however, did not show significant changes in both HeLa and CNE cells (Fig. 4A and B). Immunoblots with specific antibodies showed that the cellular levels of RHEB, RICTOR, RPS6KB2, and ATG3 proteins were decreased in MIR155-overexpressing cells (Fig. 4C). RHEB and RICTOR are reported to be positive regulators of MTORC1 and AKT; thus, we used phosphorylation site-specific antibodies to directly measure the activation of MTOR and AKT, including a phospho-Ser2448 antibody for MTOR and a phospho-Ser473 antibody for AKT. MIR155 did not significantly change the total protein levels of MTOR and AKT; however, the phosphorylation status of these 2 proteins was significantly reduced (Fig. 4C). To validate, we also checked the mRNA and protein expression levels of MIR155 targets RHEB, RICTOR, RPS6KB2, and ATG3 in CNE and HeLa cells that stably expressing MIR155 by lentiviral infection. Similar to transient expression, stable transfection of MIR155 significantly suppressed RHEB mRNA levels in both CNE and HeLa cells. The mRNA expression of RPS6KB2 was also downregulated by MIR155 in HeLa cells (Fig. S1A). Stable transfection of MIR155 suppressed the protein levels of RHEB, RPS6KB2, and RICTOR, as well as the phosphorylation of MTOR and AKT (Fig. S1B). Therefore, immunoblots from both transient and stable expression of MIR155 confirmed that MIR155 suppresses the expression of RHEB, RICTOR, RPS6KB2 and also downregulates MTOR-AKT signaling. Stable expression of MIR155 did not change the protein levels of ATG3, thus subsequent studies will concentrate on RHEB, RICTOR, and RPS6KB2.

Figure 4. MIR155 downregulates multiple players of MTOR signaling. (A) qRT-PCR analysis of mRNA expression levels of predicted targets of MIR155. CNE or (B) HeLa cells were transfected with MIR155 or NC, cells were harvested at 48 h for qRT-PCR. (mean ± s.d. of independent experiments, n = 4, **P < 0.01, ***P < 0.001, Student 2-tailed t test). (C) Western blot analysis of RHEB, RICTOR, RPS6KB2, ATG3, MTOR, phospho-MTOR (Ser2448), RPTOR, BCL2, AKT, phospho-AKT (Ser473), and GAPDH proteins in CNE or HeLa cells transfected with MIR155, MIR203, or NC. Protein ratios were calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).

MIR155 is a hypoxia-inducible miRNA, thus we predicted that the protein levels of MIR155 targets will be downregulated by hypoxia. As shown in Figure S2, hypoxia treatment dramatically attenuated the protein expression levels of RHEB, RICTOR and RPS6KB2 in both CNE and HeLa cells. The total protein levels of MTOR also decreased 23% and 54% in HeLa and CNE cells under hypoxic stress. Similarly, the phosphorylation of MTOR was reduced by hypoxia treatment (Fig. S2).

We reasoned whether knocking down functionally important target of MIR155 could phenocopy the effect of MIR155 on autophagy. Small GTP-binding protein RHEB is indispensable for MTOR activity by direct binding and activating MTOR kinase. , Thus, we tested the effect of RHEB siRNA on autophagosome accumulation and LC3 conversion. Similar to MIR155 overexpression, depletion of endogenous RHEB induced a significant increase of GFP-LC3 puncta and LC3 conversion (Fig. S3).

MIR155 directly interacts with the 3′ UTRs of RHEB, RICTOR, and RPS6KB2

To clarify if MIR155 regulates the expression of target genes via direct interaction with their 3′ UTRs, we used 2 bioinformatics softwares TargetScan and FindTar to predict the potential binding sequences in the 3′ UTRs of target genes (Fig. 5A). Then we designed pRL-TK-luciferase reporter constructs containing either the wild-type (WT) or mutated (MUT) MIR155 binding sequences in the 3′ UTRs of RHEB, RICTOR and RPS6KB2. The pRL-TK-luciferase reporter constructs were cotransfected with either MIR155 or NC control. Luciferase activity was determined at 24 h after transfection. MIR155 significantly reduced luciferase activity when cells transfected with wild-type 3′ UTRs of RHEB, RICTOR and RPS6KB2. By contrast, the inhibitory effect of MIR155 on luciferase activity was completely restored by the mutants (Fig. 5B). Moreover, the luciferase activity was also susceptible to knockdown of endogenous MIR155. Inhibition of MIR155 could specifically relieve the repression of all 3 reporters (Fig. S4).

Figure 5. MIR155 directly targets RHEB, RICTOR and RPS6KB2. (A) Predicted binding sequences between MIR155 and seed matches in RHEB, RICTOR, and RPS6KB2 3′ UTRs. (B) Luciferase reporter vectors were generated by inserting the wild-type or mutated 3′UTR fragments of RHEB, RICTOR, and RPS6KB2 into pRL-TK plasmid. Luciferase reporter assays at 24 h after transfection with wild-type (WT) or mutated (MUT) plasmids, cotransfected with NC or MIR155. Data shown are means ± s.d. of independent experiments, n = 4, **P < 0.01, ***P < 0.001, Student 2-tailed t test.

Inhibition of MIR155 leads to an increase of target-gene expression

To test whether silencing endogenous MIR155 would have an impact on target protein expression, we transfected cells with antagomir-155 (LNA-155) or nonspecific control antagomir (LNA-NC). Then, we checked the protein expression levels of RHEB, RICTOR, and RPS6KB2 by western blot. We showed that hypoxia induced downregulation of RHEB, RICTOR, and RPS6KB2 proteins. The amounts of all 3 proteins were increased when hypoxia-induced upregulation of MIR155 was suppressed by LNA-155. In addition, there was a clear increase of in the phosphorylation of MTOR (Ser2448) (Fig. 6; Fig. S5). In the light of these results, we demonstrate that endogenous MIR155 targets the expression of MTOR signaling, and hence the autophagic activity.

Figure 6. Blockage of endogenous MIR155 led to an increase in RHEB, RICTOR, and RPS6KB2 protein levels. Western blot analysis of RHEB, RICTOR, RPS6KB2, MTOR, phospho-MTOR (Ser2448), and GAPDH in CNE or HeLa cells transfected with LNA-NC or LNA-155. Protein ratios were calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).

MIR155 attenuates cell proliferation and cell cycle progression

Numerous studies demonstrate that MTOR signaling is essential for cell growth and proliferation. In this study, we discovered that overexpression of MIR155 could suppress the activation of MTORC1 and AKT. Thus, it is interesting to check whether MIR155 regulate cell proliferation and cell cycle progression. We observed that overexpression of MIR155 attenuated cell proliferation in both CNE and HeLa cells (Fig. 7A). Next, we examined the effect of MIR155 on cell cycle progression. HeLa cells transfected with NC or MIR155 were synchronized at G1/S boundary by treatment with hydroxyurea. At 4 h after release from G1 block, more than 70% of the control cells finished G1 and entered S phase. Similarly, MIR885-5P, a control miRNA had no effect on G1/S cell cycle progression. By contrast, both MIR155 and RHEB siRNA induced significant G1 phase arrest (Fig. 7B). Then we tested whether the effect of MIR155 on autophagy contributes to G1 phase cell cycle arrest. Cells were cotransfected with MIR155 and ATG5 siRNA. Interestingly, depletion of ATG5 relieved the inhibitory effect of MIR155 on G1/S cell cycle progression (Fig. 7B; Fig. S6). These data indicate a possible link between autophagy and cell cycle progression.

Figure 7. MIR155 decelerates cell proliferation and cell cycle progression. (A) Overexpression of MIR155 inhibits cell proliferation. CNE or HeLa cells transfected with MIR155 or NC were cultured for 24, 48, and 72 h to determine cell proliferation rates. Data shown are means ± s.d. of independent experiments, n = 4, **P < 0.01, Student 2-tailed t test. (B) MIR155 induces G1/S cell cycle arrest. HeLa cells transfected with indicated miRNAs or siRNAs were synchronized at G1/S boundary by treatment with hydroxyurea. Cells were released from hydroxyurea block for 4 h, fixed and stained with propidium iodide (PI) for flow cytometry. Percentage of G1, S, and G2/M phase cells was quantified from flow cytometry analysis. Data shown are means ± s.d. from 3 independent experiments. (C) Proposed model of hypoxia-induced MIR155 in regulating autophagy, cell proliferation, and cell cycle progression. MIR155 is a hypoxia-inducible miRNA. MIR155 targets the expression of RHEB, RICTOR, and RPS6KB2, which in turns suppresses the activation of AKT and MTORC1. By deregulating the MTOR-AKT pathway, MIR155 induces autophagy and decelerates cell proliferation and cell cycle progression.

Discussion

The tumor microenvironment plays a critical role in tumor cell proliferation and progression. As the tumor grows, it rapidly outgrows its blood supply, leaving tumor cells been deprived of oxygen. HIF is a key transcription factor that allows rapid adaptation to hypoxic stress. Hypoxia regulates the expression of a group of miRNAs in a cell type- and tissue-specific manner. - In the present study, we identified hypoxia-induced MIR155 as a potent inducer of autophagy. We discovered that multiple players of MTOR signaling are novel MIR155 targets, including RHEB, RICTOR, and RPS6KB2. By suppressing the activation of MTOR-AKT pathway, MIR155 induces autophagy, and attenuates cell proliferation and cell cycle progression.

MTOR functions as a central sensor of cellular energy that responses to many oncogenic and metabolic events as well as the control of autophagy. Extensive studies indicate that regulation of MTOR pathway by miRNAs plays a major role in cancer progression. miRNAs either directly interact with MTOR itself or target key players within the pathway, such as IGF1R (insulin-like growth factor 1 receptor), PI3K, AKT, and PTEN. For instance, MIR99A, MIR100, MIR101, and MIR199-3P inhibit tumor cell migration and cell growth, induce G1 phase cell cycle arrest by directly binding to MTOR 3′ UTR and suppress MTOR expression. - By contrast, some miRNAs, including MIR21, MIR221, MIR222, MIR26, and MIR19 can function as oncomiR by targeting the expression of PTEN and promotes cancer cell growth, proliferation, and invasion. - MIR155 targets, RHEB and RICTOR, are essential upstream regulators of MTORC1. The activity of MTORC1 is controlled through RHEB, a GTP-binding protein, which stimulates MTORC1 when it is in the GTP-bound state. , RICTOR is scaffold protein that regulating the assembly and substrate binding of MTORC2. It has been reported that MTORC2 activates AKT via phosphorylation and enhances the expression of HIF1A. , In addition, AKT activates RHEB by negative phosphorylation of TSC1/TSC2 complex. A recent study by Wang et al. also reports that AKT-mediated BECN1 phosphorylation functions in autophagy inhibition. Thus, MIR155 is a potent autophagy inducer by suppressing RICTOR- and RHEB-mediated MTORC1-AKT activity (Fig. 7C).

Deregulation of MIR155 has been reported in a wide range of malignancies, including various forms of lymphoma and carcinomas of breast, lung, pancreas, head and neck, nasopharyngeal, and kidney. - Several lines of evidence demonstrate that MIR155 functions as an “OncomiR” and plays a role in tumor development. In human breast cancer cells, MIR155 induces cell proliferation and chemosensitivity by targeting the SOCS1 (suppressor of cytokine signaling 1) and FOXO3 (forkhead box O3) genes. , In human lung cancer cells, MIR155 targets the expression of APAF1 (apoptotic peptidase activating factor 1) and FOXO3, and downregulation of MIR155 enhances the sensitivity of lung cancer cells to cisplatin treatment or radiotherapy. However, contradictory results exist concerning the oncogenic feature of MIR155. In some circumstances, MIR155 could exert a tumor suppression function. For example, MIR155 is downregulated in the majority of melanoma cell lines, overexpression of MIR155 significantly inhibits proliferation and induces apoptosis. MIR155 inhibits the growth of lung cancer 95D cells through the induction of G0/G1 cell cycle arrest. MIR155 also inhibits proliferation and migration in human extravillous trophoblast derived HTR-8/SVneo cells via negative regulation of the CCND1-CDKN1B pathway. Here we demonstrated that overexpression of MIR155 inhibits cell proliferation and G1/S cell cycle progression. This tumor-suppressive effect could be due to MIR155-mediated downregulation of RPS6KB2. Therefore, the function of MIR155 in physiological processes such as tumor development and tumor growth might be determined by its wide variety of targets in a cell type and tissue-specific manner.

There is growing evidence for the role of autophagy in the control of cell proliferation, however, less is known about the relationship between autophagy and the cell cycle. It has been demonstrated autophagy regulatory protein ATG7 could bind to the tumor suppressor TP53 to regulate the transcription of cell cycle inhibitor CDKN1A [cyclin-dependent kinase inhibitor 1A (p21, Cip1)]. Starved MEFs lacking the Atg7 gene have impaired TP53-mediated cell cycle arrest. In U87 glioma cells, pharmacological inhibition of resveratrol-induced autophagy abrogates S-G2/M cell cycle arrest and significantly reduces the levels of CCNA, CCNE, CCNB, and phosphorylation of RB1 (Ser807/811) and CDC2 (Y15). Here, we show that knockdown of the essential autophagy regulator ATG5 could reverse MIR155-induced G1 phase arrest, indicating that MIR155-induced delay of cell cycle progression might be related to autophagy. More work is required to define the mechanisms of autophagy in the control of cell cycle progression.

Materials and Methods

Cell culture and hypoxia treatment

CNE (human nasopharyngeal cancer) or HeLa (human cervical cancer) cells were grown in Dulbecco’s modified Eagle’s medium (Gibco/Invitrogen Ltd, 12800-017) containing 10% fetal bovine serum (PAA, A15-101), 10 U/ml penicillin-streptomycin (Gibco/Invitrogen Ltd, 15140-122) in a 5% CO2-humidified incubator at 37 °C. Hypoxic conditions were induced by incubating cells in a modular incubator chamber flushed with a gas mixture containing 1% O2, 5% CO2, and 94% N2 at 37 °C. For experiments involving hypoxia, cells were maintained in culture medium supplemented with 25 mM HEPES buffer.

Target prediction

The conventional online programs, including Targetscan (http://www.targetscan.org) and Findtar (http://bio.sz.tsinghua.edu.cn) were used to predict the targets of MIR155. The targets predicted by these 2 programs were further analyzed and demonstrated by the following biological experiments.

MiRNAs, LNAs, siRNAs, and transfections

All synthetic miRNAs (MIR155, MIR203, MIR885-5P), siRNAs and the negative control (NC) were purchased from Shanghai GenePharma Co. Ltd. LNA-NC and LNA-155 were purchased from Exiqon. For transfection, HeLa or CNE cells were transiently transfected with lipofectamine™ 2000 (Invitrogen, 11668-019) according to the manufacturer’s protocol. The sequences of siRNAs are as follows: ATG5: 5′-GCAACUCUGG AUGGGAUUGTT-3′, 5′-CAAUCCCAUC CAGAGUUGCTT-3′, RHEB: 5′-GAAAGGGUGA UCAGUUAUGTT-3′, 5′-CAUAACUGAU CACCCUUUC TT-3′.

Plasmid construction and lentivirus production

A fragment of pre-MIR155 encompassing the stem-loop was amplified from CNE genomic DNA and cloned into lentiviral vector pBabi-globin-EGFP, a plasmid developed by our laboratory. MicroRNA fragment was inserted into the globin intron flanking the promoter and EGFP. A lentiviral vector expressing a scrambled RNA was used as control. To produce viruses, the pre-MIR expression plasmid and the backbone plasmids were cotransfected into 293LTV cells using the calcium phosphate method. Supernatant containing the viruses were harvested at 48 h. Cells were infected with the viruses along with 8 ug/ml polybrene. The transfection efficiency was determined by monitoring EGFP expression using a fluorescence microscope (Leica DMI 6000B).

GFP-LC3 stable cell lines and quantitative GFP-LC3 analyses

HeLa GFP-LC3 stable cell line was established by transient transfection of pEGFP-LC3 plasmid following screening with G418 (Shanghai Sangon Biotech Co. Ltd, GDJ958). To obtain CNE GFP-LC3 stable cell line, we cloned CMV-GFP-LC3 to the pMSCV-neor and generated lentiviruses, CNE cells were infected with the viruses and selected stable clones with G418. CNE GFP-LC3 or HeLa GFP-LC3 stable cell lines were transfected with individual miRNAs or LNAs and fixed in dry methanol. GFP-LC3 puncta formation in normoxia or hypoxia conditions was determined by capturing images using Olympus FV1000 confocal microscope (Olympus). The number of GFP-LC3 dots per cell was counted from at least 200 cells.

qRT-PCR for mRNA and miRNA quantification

Total RNA was isolated using RNA iso Plus (Takara, D9108B). Real-time qRT-PCR was performed using reverse transcription kit (Takara, D6130) and SYBR Green PCR Master Mix (Toyobo, QPK-201). mRNA level were normalized to GAPDH. miRNA qPCR were performed according to manufacturer’s instructions using the Hairpin-it™ miRNA qPCR kit (Shanghai GenePharma Co. Ltd, QPM-091 for U6 snoRNA, QPM-040 for MIR155). Briefly, 4 µg total RNAs were reverse transcribed using mRNA specific RT primer, miRNA qPCR were performed using miRNA specific primer set to detect expression of mature MIR155. miRNA expression levels were normalized by Livak’s method and U6 snoRNA was used as endogenous control. Primers used in the present study were listed in Table S1.

Construction of luciferase plasmids and reporter assay

The 3′ UTRs of RHEB, RICTOR, and RPS6KB2 were amplified by PCR from CNE genomic DNA and cloned into pRL-TK reporter vector (Promega, E2241). The predicted target site was mutated by site-directed mutagenesis. For luciferase reporter assays, the WT or MUT luciferase plasmids and miRNAs were cotransfected into HeLa cells and each experiment was repeated in triplicate. Transfected cells were lysed 30 h after transfection and luciferase activities were assayed by a Dual-Luciferase Reporter System (Promega, E1960). Total protein concentration of cell lysate was determined using Bradford assay (Bio-Rad, 500-0006) at 595 nm on a spectrophotometer (Thermo Scientific). The luciferase activity was normalized by total protein content.

Cell synchronization and flow cytometry

For synchronization, cells were treated with 2 mM hydroxyurea (Sigma, H8627) for 16 h. The synchronous cells were washed 3 times with PBS to release from G1 block. At 4 h after release, cells were fixed in 70% ethanol in PBS and counterstained with 10 µg/ml propidium iodide (Sigma, P4170) and analyzed for DNA content by use of a BD Influx™ flow cytometer (BD Biosciences).

Western blotting

Cells were lysed in ice-cold whole cell extract buffer (50 mM TRIS-HCl, pH 8.0, 4 M urea and 1% Triton X-100), supplemented with complete protease inhibitor mixture (Roche Diagnostics, 04693132001). Cell extracts were resolved by SDS-PAGE, and analyzed by western blotting. Antibodies used for western blotting were as follows: MAP1LC3B/LC3B (Sigma, L7543), SQSTM1/p62 (BD Bioscience, 610832), HIF1A (BD Bioscience, 610958), GAPDH (Proteintech, 10494-1-AP), RHEB (Cell Signaling, 4935), RPS6KB2/p70S6K (Cell Signaling, 2708), RICTOR (Cell Signaling, 2114), phospho-MTOR (Ser2448) (Cell Signaling, 5536), MTOR (Cell Signaling, 2972), ATG3 (Cell Signaling, 3415), ATG5 (Cell Signaling, 8540), phospho-AKT (Ser473) (Cell Signaling, 9271), AKT (Cell Signaling, 4691), BCL2 (Cell Signaling, 2870), RPTOR (Cell Signaling, 2280). Following incubation with horseradish peroxidase-coupled secondary anti-mouse (KPL, 074-1806) or anti-rabbit antibodies (KPL, 474-1506), protein bands were visualized using ECL Blotting Detection Reagents (KPL, 54-61-00).

Statistical analyses

Statistical significance of differences observed in samples was determined using the Wilcoxon rank sum test or the Student 2-tailed t test. Data were shown as means ± s.d. of at least 3 independent experiments. Values of P < 0.05 were considered significant.

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