Introduction
Non-coding RNAs (ncRNAs) include long non-coding RNAs (lncRNAs, longer than 200 nucleotides) and small non-coding RNAs (sncRNAs, shorter than 200 nucleotides). MicroRNAs (miRNAs) are small ncRNAs consisting of 20–24 nucleotides that negatively regulate the stability and translational efficiency of target mRNAs through binding to the 3′ untranslated regions [
1]. Many of them have been demonstrated to play crucial roles in post-transcriptional gene regulation and cancer biology. Genes of miRNAs were assumed to be incapable of encoding proteins. The biogenesis of miRNAs involves the processing of larger primary miRNAs (pri-miRNAs) into shorter pre-miRNAs, and the maturation of pre-miRNA to produce active miRNAs [
2]. Recent studies have demonstrated that pri-miRNAs harbor short open reading frames that can encode regulatory peptides, termed miRNA-encoded peptides (miPEPs).
miPEPs that regulate growth and development were first identified in plants. For example, miPEP171b of
Medicago truncatula and miPEP165a of
Arabidopsis thaliana are two plant miPEPs that positively regulate the levels of their corresponding miRNAs [
3]. The overexpressed miPEPs or synthetic peptides specifically increase the accumulation of the corresponding miRNAs and enhance the inhibition of the miRNA-targeted genes involved in root development. Therefore, it is hypothesized that miPEPs specifically stimulate the transcription of their associated miRNA to induce more pronounced silencing of miRNA-targeted genes. However, it is unclear how miPEPs regulate the transcription machinery. The molecular basis of miPEP specificity and activity are unknown.
In animals, very few miPEPs have been identified till date. Two peptides encoded by lncRNAs were identified as important regulators of muscle physiology [
4,
5]. Myoregulin (MLN) is encoded by a skeletal muscle-specific lncRNA. It finely controls calcium uptake by interacting with sarco/endoplasmic reticulum calcium ATPase (SERCA) and plays a critical role in regulating skeletal muscle performance. Another peptide of 34 amino acids named dwarf open reading frame (DWORF) also interacts with SERCA and enhances cardiac muscle contractility. A ncRNA-encoded microprotein named Cancer-Associated Small Integral Membrane Open reading frame 1 (CASIMO1) was recently identified as a 10 kDa protein encoded by a long non-coding RNA (NR_029453). CASIMO1 interacts with squalene epoxidase to regulate lipid droplet clustering and the proliferation of breast cancer cells [
6].
miR-34a is a tumor suppressor that inhibits the expression of about 700 target genes [
7]. It plays an important role in suppressing tumorigenesis and is downregulated in human cancers [
8‐
10]. The expression of miR-34a is mainly induced by p53 [
11‐
13]. However, accumulating evidence indicates that the level of miR-34a can be regulated in a p53-independent manner. Due to its anti-cancer functions, miR-34a has become one of the first targets of miRNA therapy entering clinical trials [
14,
15].
In this study, we report for the first time the identification and functional characterization of a miPEP encoded by the primary transcript of miR-34a. We named it miPEP133 (pri-miR encoded peptide 133) because it consists of 133 amino acid residues. Since miPEP133 is highly expressed in the normal pharynx and significantly downregulated in nasopharyngeal carcinoma (NPC), we utilized the in vitro and in vivo models of NPC to study the role of miPEP133 in tumorigenesis. NPC is a rare type of head and neck cancer with 80,000 incident cases and 50,000 deaths annually [
16]. It is more prevalent in southern China, southeastern Asian countries, northern and northeastern Africa, Alaska, and western Canada [
17]. NPC is a leading cancer type in Malaysia [
18]. NPC is usually sensitive to radiotherapy. If diagnosed at early stage it is considered curable with the 5-year survival rate of about 80% [
19]. However, more than 30% of patients will relapse with local recurrence or distant metastases [
20]. When NPC progresses into advanced disease, the prognosis is very poor. The clinical management of late stage NPC is extremely challenging. Research on the tumor biology and etiology of NPC is very limited comparing to other more common cancer types. Our findings on the regulation and functions of miPEP133 will advance our knowledge on the roles of miPEPs in tumorigenesis and tumor progression.
Methods
Human tissue samples
The NPC study protocol was approved by the Human Research Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. The procedures are in accordance with the Helsinki Decaration of 1975. Written informed consent was obtained from all participants. Normal nasopharyngeal tissues adjacent to the operation areas were collected from 8 patients. Ovarian cancer sample collection was carried out with patient consent and approved by the Human Investigations Committee of Yale University School of Medicine. Tissue samples were immediately frozen in liquid nitrogen after resection and stored at − 80 °C until use. Clinical information regarding the tissue samples is in Additional file
1.
Cell lines and transfection
Human NPC cell lines HNE3, C666–1, CNE2, CNE1, 5-8F, TWO3, and normal nasopharyngeal cell line NP69 were gifts from Guangxi Medical University Nasopharyngeal Cancer Research Laboratory. SKOV3 and Hela cell lines were obtained from NCI Repository of Tumors and Tumor Cell Lines. The ovarian cancer patient-derived cell lines were provided by Dr. Gil Mor (Yale University). Fallopian tube epithelial cell lines were generated previously [
21] and provided by Dr. Ron Drapkin (University of Pennsylvania). Cells were cultured in DMEM containing 10% fetal bovine serum, 100 IU/ml penicillin and 100 mg/ml streptomycin in humidified 5% CO
2 incubator at 37 °C. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for transfection according to the manufacturer’s instruction. Lentivirus was produced as previously described to establish stable cell lines with miPEP133 overexpression [
22]. Puromycin (1 μg/ml, Invitrogen) was used to maintain selective pressure. Details about plasmids used in this study are listed in Additional file
2.
Antibody production and evaluation
The production of anti-miPEP133 antibody was conducted by GenScript (Piscataway, NJ, USA) as described in Additional file
2. The antibody was evaluated by SDS-PAGE and Coomassie Brilliant Blue staining. The specificity of the produced antibody against miPEP133 was validated by ELISA.
Western blot and co-immunoprecipitation (co-IP)
Details of western blot and co-IP and the information of antibodies are described in Additional file
2.
Quantitative PCR (QPCR)
Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed using Bulge-LoopTM microRNA specific RTprimers (RiboBio, Guangzhou, China) and M-MLV reverse transcriptase (Promega, Madison, WI, USA). Real-time QPCR was performed on a CFX96TouchTM system (Bio-Rad, Hercules, CA, USA) using SYBR SuperMix (Bio-Rad). Mir-X miRNA qRT-PCR TB Green Kit (Takara Bio, San Francisco, CA) was used to detect miR-34a. RNU6B (U6) or GAPDH were used as internal controls. Relative expression levels were calculated using the 2 − ΔΔCT method. Primers are listed in Additional file
2.
Mass spectrometry
The gel piece containing the 15 kDa protein band from HEK293 cell lysate was excised and digested into peptide fragments for mass spectrometry analysis. Mass spectrometry was performed as previously described [
23]. For the identification of miPEP133-binging proteins, the protein lysate of HEK293 cells expressing flag-labeled miPEP133 or control empty vector was incubated with anti-flag-tag antibody in the co-IP assay. Proteins in the co-IP products were identified by mass spectrometry analysis. The proteins that were identified in the IP product of cells transfected with empty vector were excluded from the list of protein hits as background signals (Additional file
4).
miPEP133 knockdown with siRNAs and CRISPR/Cas9-mediated miPEP133 deletion
siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s instruction. miPEP133 siRNAs were synthesized by Ribo Biotechnology (Guangzhou, China). The sequences of siRNAs and the method of CRISPR/Cas9-mediated miPEP133 deletion are listed in Additional file
2.
Cell fractionation and RNA/protein extraction
The cytoplasmic and nuclear RNA was extracted from cells using Cytoplasmic and Nuclear RNA Purification kit (Norgen BioTeck, Thorold, ON, Canada). Cell fractionation and protein extraction were performed using Qproteome Mitochondria Isolation kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instruction.
Cell proliferation assay
Five thousand cells/well were plated in 96-well plates. Cell viability reagent WST-8 (Abcam, Branford, CT) was added to each well and incubated for 3 h at 37 °C at different time points. Optical density at 450 nm was measured using plate reader.
Flow cytometry analysis of apoptosis, cell cycle, and mitochondrial membrane potential
Cells were stained according to the instruction of the Annexin V-APC/7-AAD kit (Keygen Biotech, Nanjing, China) and analyzed using FACSLyric Flow cytometry system (BD, Franklin Lakes, NJ) to assess the apoptotic cell populations. Cell cycle status was analyzed by propidium bromide (PI) staining and flow cytometry as detailed in Additional file
2. JC-1 mitochondrial membrane potential assay kit (abcam) was used to stain the cells and assess mitochondrial membrane potential by flow cytometer. A potent mitochondrial uncoupler, carbonyl cyanide chlorophenylhydrazone (CCCP) was used as a positive control.
Migration assay and invasion assay
For wound healing assay, cells were seeded in 6-well plates, grown to 90% confluence. A scratch wound was made using a pipet tip. Wound width was measured under microscope at 24 h. For the transwell migration assay, cells were seeded into the transwells directly (Corning, Corning, NY, USA) with 0.2 mL serum-free medium. For the invasion assay, cells were seeded into the Matrigel-coated transwells (Corning). The bottom wells were filled with normal growth medium. After 24 h, cells in the upper wells were removed using a cotton swab. The migrating/invading cells at the bottom of the transwells were fixed in 4% polyoxymethylene, stained with crystal violet, and imaged in randomly chosen fields. The stained cells were counted.
p53 response element luciferase reporter assay
PG13-luc (wt p53 binding sites) was a gift from Bert Vogelstein (Addgene plasmid # 16442;
http://n2t.net/addgene:16442; RRID:Addgene_16,442). Renilla luciferase control reporter pRL vector was purchased from Promega (Madison, WI, USA). miPEP-133 or control vector was co-transfected with PG13-luc plasmid. p53 transcriptional activation was evaluated as previously described [
24].
Immunofluorescence (IF) staining and confocal microscopy
Formalin-fixed paraffin-embedded tissue sections were deparaffinized and hydrated. Slides were submersed in 1X citrate unmasking solution and heated in a microwave to incubate at 95–98 °C for 10 min. Cells were cultured on chamber slides, fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, and then washed with PBS. The slides were incubated in blocking buffer (5% normal serum and 0.3% Triton X-100 in PBS) for 60 min at room temperature. Primary antibodies were diluted in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in 1X PBS) and incubated with the slides overnight at 4 °C. Fluorochrome-conjugated secondary antibody was incubated for 1 h at room temperature in the dark. Mount medium with DAPI was used to mount the slides (Cell Signaling, Danvers, MA, USA). The stained slides were imaged using Leica SP8 Laser Scanning Confocal microscope. Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining was performed using In Situ Cell Death Detection kit (Roche, Branford, CT, USA).
Luminescence-based ATP detection assay
Cells were counted using a hemocytometer. Cell suspension was mixed with the same volume of ATP assay reagent (Promega) to incubate for 10 min. The luminescent signal was measured using GloMax Navigator plate reader (Promega).
Mouse model
Six-week-old female BALB/c-nude mice (Shanghai Laboratory Animal, Shanghai, China) were subcutaneously injected with 107 C666–1 cells that expressed miPEP133 or control lentivirus vector. Tumor width (W) and length (L) were measured daily. Tumor volume was calculated using formula V = π/6*L*W2. Three weeks after injection, mice were euthanized to collect the tumors.
Statistics
Data were presented as the mean ± standard deviation. All experiments included at least 3 biological repeats. Student’s t-test, two-way ANOVA or chi-square test was used in statistical analysis as specified for each experiment. Kaplan–Meier analysis was employed for the survival analysis of two groups (miPEP133 low and high groups). Survival was defined as time from diagnosis until death or until time last followed. The differences in the survival probabilities were estimated using the log-rank test. P values less than 0.05 were considered to be statistically significant.
Discussion
In this study, we have identified a novel microprotein that is encoded by an ORF in the precursor of miR-34a. This microprotein, namely miPEP133, mainly localizes in the mitochondria and plays a role as a tumor suppressor with the activity to induce cell cycle arrest, cell growth inhibition, and apoptosis in cancer cells in addition to slowing down cancer cell migration and invasion. We revealed the mechanisms of its function that involves interacting with mitochondrial chaperon HSP9A and disrupting the interaction of HSP9A with other proteins. Our findings demonstrated the tumor suppressor activity of miPEP133 and its potential value as a prognostic marker and therapeutic target.
miPEPs are more common in plant, but rare in animals. miPEP133 is the first miPEP that is identified in the pri-miRNA of tumor suppressor miRNA in human. It shares the same promoter as its associated miRNA, miR-34a. Therefore, the expression patterns of miPEP133 are similar to miR-34a. miPEP133 and miR-34a are both downregulated in tumor tissues and both regulated by p53. The other inducers of miR-34a, such as ELK1 and TAp73 [
29], may also induce miPEP133 expression, which is yet to be determined in the future studies. The target genes of miR-34a are involved in oncogenic signaling pathways, such as cell proliferation (e.g. cyclins, cyclin-dependent kinases, MYCN, NOTCH1, and MDMX), anti-apoptosis (e.g. BCL2 and SIRT1), cancer stem-like cell properties (e.g. CD44, NANOG, and SOX2), metastasis (e.g. SNAI1 and MET), and immune evasion (e.g. PD-L1). miPEP133 can promote the transcription of miR-34a to enhance the silencing of these miR-34a-targeted genes. We have observed the miPEP133-induced downregulation of ATF3, AXIN2, CDKN1A, DLL1, E2F1, E2F2, FOXP1, JAG1, MYCN, SIRT1, and SOX2, which are miR-34a-targeted genes (data not shown).
The mechanisms underlying miPEP133-induced expression of miR-34a may be explained by different co-existing mechanisms under different cellular contexts. In cells with wild-type functional p53, such as HEK293 cells, miPEP133 can disrupt mitochondrial functions, which activates wild-type p53 transcriptional activity consequently inducing miR34a expression. In the meantime, the miPEP133-induced mitochondrial dysfunction can activate other transcriptional regulators of miR-34a, such as TAp73, to directly bind to the promoter of miR34a [
29,
30]. The latter mechanism may be responsible for the miPEP133-induced upregulation of miR-34a in p53-null cancer cells like ovarian cancer cell line SKOV3 [
31] and cancer cells without functional wild-type p53. p53 mutation is very common in ovarian cancer, particularly in high-grade serous ovarian carcinoma. NPC cells usually have wild-type p53, however, NPC is highly associated with Epstein-Bar virus that can inactivate p53 [
32]. Cervical cancer Hela cell line is also known for having p53 inactivated by human papillomavirus proteins [
33]. Our findings demonstrated the p53-dependent and p53-independent tumor suppressor roles of miPEP133 in different cellular contexts with diverse p53 status.
HSPA9 is involved in stress response, antigen processing, control of cell proliferation, differentiation and tumorigenesis [
34]. HSPA9 has anti-apoptotic and pro-proliferative activities [
35,
36]. Increasing levels of HSPA9 permit cells to survive lethal conditions [
37‐
39]. HSPA9 influences the function, dynamics, morphology, and homeostasis of mitochondria as the only ATPase component of the mitochondrial protein import machinery [
35,
40,
41]. Mitochondrial protein precursors are chaperoned by HSPA9 into the mitochondrial matrix with the assistance of co-chaperones [
42]. HSPA9 forms a complex with HSP60 and floats freely in the mitochondrial matrix to regulate protein folding [
43]. HSPA9 also binds the translocase of the mitochondrial inner and outer membranes [
44,
45]. Their complexes control the translocation of precursor proteins and their distribution in the matrix and across the mitochondrial membranes [
46‐
48]. HSPA9-VDAC1 complex modulates voltage-dependent anion-selective channel properties [
49]. The interaction with miPEP133 prevents HSPA9 from acting as a chaperon and interacting with its partner proteins, which demonstrates a new mechanism for regulating mitochondrial morphology and function.
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