PELI3 mediates pro-tumor actions of down-regulated miR-365a-5p in non-small cell lung cancer
To analyze the relative expression of PELI3 and its mechanistic involvement in the non-small cell lung cancer (NSCLC).
PELI3 expression in NSCLC tissue samples was determined by the immunohistochemistry. The transcripts abundance of PELI3 was measured with real-time PCR. The protein intensity was analyzed by western blot. The overall survival in respect to PELI3 or miR-365a-5p expression was plotted by the Kaplan–Meier’s analysis. Cell growth was determined by colony formation assay. Cell viability was measured by MTT assay. The migration and invasion were evaluated by wound healing and transwell assay respectively. The regulatory effect of miR-365a-5p on PELI3 was interrogated with luciferase reporter assay. The direct binding between miR-365a-5p and PELI3 was analyzed by pulldown assay.
PELI3 was aberrantly up-regulated in NSCLC both in vivo and in vitro. High level of PELI3 associated with poor prognosis. PELI3-deficiency significantly inhibited cell viability, colony formation, migration and invasion. We further identified that miR-365a-5p negatively regulated PELI3 in this disease. Ectopic expression of miR-365a-5p in both A549 and H1299 phenocopied PELI3-deficiency. Meanwhile, PELI3-silencing significantly abolished the pro-tumoral effect elicited by miR-365a-5p inhibition.
Our results highlighted the importance of dysregulated miR-365a-5p-PELI3 signaling axis in NSCLC.
KeywordsPELI3 miR-365a-5p Non-small cell lung cancer Cell viability
non-small cell lung cancer
epidermal growth factor receptor
receptor activator of nuclear factor kappa-B ligand
Pellino E3 ubiquitin protein ligase family member 3
Lung cancer is the most common human malignancy and the first cause of cancer-related death in men and second after breast cancer in women . In 2012, 1.82 million new cases were diagnosed worldwide and 1.56 million deaths were claimed which accounting for around 20% of all deaths from cancer . Lung cancer are conventionally classified by the histological types and two broad classes are distinguished including non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma . The most frequent risk factor related to the vast majority of lung cancer is long-term tobacco smoking . In the never smoker, the incidence of lung cancer is often due to the combinational influences of genetic abnormalities with occupational exposure to radon gas, asbestos, smoke and air pollution . Clinical options and end-point outcomes for this disease heavily depend on the type of cancer, progression and overall health conditions, while most cases are not curable. The mainstay therapeutics includes surgery, chemotherapy, radiotherapy, and more recently, the targeted therapies . However, the 5-year survival of NSCLC is relatively unfavorable and only 11.2% patients survive this disease. The clinical benefits of targeted therapies are greatly promising in expansion of the survival period, which are currently only applicable to those patients with specified genetic mutations in either epidermal growth factor receptor (EGFR) or receptor activator of nuclear factor kappa-B ligand (RANKL) . Despite of the tremendous advance in understanding the etiology of NSCLC, the molecular mechanism underlying the incidence and metastasis of some cases NSCLS are still elusive and to be precisely defined, which definitely would offer the chance for early diagnostic and therapeutic purpose.
Pellino E3 ubiquitin protein ligase family member 3 (PELI3) was initially identified as as a scaffolding protein which promotes activation of c-Jun and Elk-1  and an intermediate signaling protein in the innate immune response pathway. The subsequent investigations characterized the ligase activity and ubiquitin-protein transferase activity against IRAK1 . The PELI3 protein has also been reported to facilitate the transmission of the immune response signaling from Toll-like receptors to IRAK/TRAF6 complex . Autophagy-dependent PELI3 degradation inhibits proinflammatory IL1B expression . Notably, the potential involvement of PELI3 has been barely investigated until now.
microRNAs are defined as small oligonucleotides with average length between 18 and 25 base pairs and highly evolutional conservation . microRNAs exert their biological functions via recognition and binding to the 3′UTR region and post-transcriptionally modulated target gene expression. With advance in the assembling experimental evidences, microRNAs have been identified indispensable in multiple biological processing such as mRNA stability and translation. Dysregulation in micorRNAs therefore frequently links to variety of human disease including hereditary progressive hearing loss, skeletal and growth defects, cancer, kidney disease, alcoholism and obesity . Multiple dimensional effects of microRNAs have been increasingly uncovered in human malignancies as well involving in cell proliferation, cell cycle, apoptosis, angiogenesis, cell migration and invasion .
Here we set out to characterize the expression pattern of PELI3 in NSCLC and elucidate its mechanistic involvement in the initiation and progression of this disease. Our study will offer better understanding into the relevance of aberrantly regulated PELI3 with NSCLC.
Materials and methods
The non-small cell lung cancer patients were enrolled in the Second Hospital of Hebei Medical University in this study with written informed consents. This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University. All tumors were pathologically confirmed by at least three experienced experts.
The human non-small cell lung cancer cell lines A549, SPCA1, H1299, H358, PC9 and immortalized human bronchial epithelial cell 16HBE were obtained from the American Type Culture Collection (ATCC, VA, USA). The cell identities and mycoplasma-free were authenticated by DNA profiling. All cancer cells were maintained in RPMI modified medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HBEC cell was kept in Airway Epithelial Cell Basal Medium supplemented with the Bronchial Epithelial Cell Growth Kit (ATCC, VA, USA). Cell culture was performed in the humidified CO2 (5%) incubator. Exponentially growing cells were used for the experimental analysis. Lipofectamine 2000 (Invitrogen, MA, USA) was used for cell transfection purpose. Transfection efficacy was evaluated with GFP. For transient knockdown, siRNAs were synthesized by the Ribobio (Guangzhou, China).
PELI3 Forward primer: CTGGAAGGAAACCCTGAAGT
PELI3 Reverse primer: AGCGGCGTGGAGATGTG,
GAPDH Forward primer: ACAACTTTGGTATCGTGGAAGG;
GAPDH Reverse primer: GCCATCACGCCACAGTTTC.
miR-365a-5p reverse transcription primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACATC;
miR-365a-5p Forward primer: CTGAGGGACTTTTGGGGGCAG,
miR-365a-5p Reverse primer: GTGCAGGGTCCGAGGT;
U6 reverse transcription primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAA.
U6 Forward primer: TGCGGGTGCTCGCTTCGGCAGC,
U6 Reverse primer: GTGCAGGGTCCGAGGT.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cell viability was evaluated by the MTT method. The commercially available MTT Cell Proliferation Assay Kit (Cayman, Ann Arbor, MI, USA) was adopted for this assay. Briefly, transient PELI3-knockdown A549 and H1299 cells (1000 cells/well) were seeded into 96-well plate in triplicate and cultured for 24 h. 10 μL MTT solution was added into each well and incubated the plate in the CO2 chamber for 3 h to allow the formazan formation. Dissolution was conducted with 100 μL of dissolving solution and incubated for another 4 h. The absorption was measured at 570 nm with 490 nm as reference.
The indicated cells were lysed in ice-cold RIPA buffer (Beyotime, Nantong, China) for 30 min. The cell debris was discarded after refrigerated high-speed centrifugation (12,000 rpm by 15 min). The protein concentration was measured by the BCA method (Bicinchoninic Acid Kit, Sigma). The protein samples were first separated by the SDS-PAGE followed by the PVDF membrane transfer. The membranes were subjected to non-fat milk blocking, primary antibody incubation and secondary hybridization consecutively. The protein blots were visualized by the enhanced chemiluminescence kit (ECL Substrate Kit, Abcam, Cambridge, UK).
The fresh or flash-frozen in liquid nitrogen tissue samples were dehydrated first and followed by the fixation and paraffin embedding. Fixed and embedded in paraffin. The tissue blocks were cut into 5 μΜ sections and followed by the deparaffinization and rehydration. Antigen retrieval was performed with sodium citrate (pH 6.0) and boiled in microwave for 20 min. The endogenous peroxidase was blocked with 5% normal goat serum. The primary antibody (1:100) was incubated at 4 °C overnight. After rinsing with tris-buffered saline and Tween 20 buffer and H2O2 incubation, horseradish peroxidase-conjugated secondary antibody was applied for 1 h at room temperature. The protein was visualized with DAB (Boster, Wuhan, China).
Cell proliferation in response to PELI3 deficiency was determined by the colony formation assay. 1000 exponentially growing cells were seeded into 6-well plate in triplicate. Consecutive cell culture was performed in CO2 hood for 2 weeks. The formed colonies were fixed with methanol and stained with 0.25% crystal violet.
Cell invasive capacity was determined with Basal Membrane Extract (BD BioSciences, CA, USA)-precoated Transwell chambers (Corning, NY, USA). The single-cell suspension (20,000 cells/100 μL) was prepared in serum-free medium and laid on the upper insert. The complete culture medium (650 μL) was filled into the bottom compartment. After 12 h, the carbonate filter was cautiously removed and subjected to fixation and staining with crystal violet.
The migrative capacity of indicated cells was determined by the wound healing assay. The log phase single layer cells were cultured in 6-well plate overnight. The straight scratch of uniform width was created with sterile tips. The closure of scratch was continuously monitored under light microscope.
Luciferase reporter assay
Either wild-type or mutated luciferase reporter plasmids co-transfected into A549 and H1299 cells with miR-365a-5p. After 24 h, the relative luciferase activity was measured with Luciferase Assay System (Promega, WI, USA) in accordance with the manufacturer’s instruction.
The direct binding between miR-365a-5p and PELI3 transcript was interrogated by the pulldown assay. Biotin-labelled miR-365a-5p was purchase from Ribobio (Guangzhou, China). The cell lysate was prepared from both A549 and H1299 cell lines, which was subsequently incubated with biotin-miR-365a-5p at 4 °C for 2 h. The mixture was then pulled down by the Streptavidin-couple Dynabeads (ThermoFisher). After elution, the enriched PELI3 transcripts were quantified by the real-time PCR.
Xenograft tumor model
To establish xenograft tumor model, PELI3 was stably silenced in A549 by shRNAs, which was subjected to puromycin (2 μg/mL) selection and knockdown efficiency was confirmed by real-time PCR. BALB/c nude mice (4-week) were purchased from the Vital River (Beijing, China) and housed in the specific pathogen-free environment with free access to drinking water and food. 1 × 106 cells resuspended in serum-free medium was mixed with equal volume of Matrigel (BD BioSciences) and subcutaneously inoculated into right flank of nude mice. The tumor growth was monitored regularly until the size approaching 1500 mm3 and subjected to sacrifice. The xenograft tumor was resected and representative macroscopic images were captured. For lung metastasis evaluation, 5 × 105 cells were tail vein injected and lung nodules were determined by H&E staining.
All data were acquired from at least three independent experiments. GraphPad PRISM 6.0 software was used for data analysis and processing. Statistical comparison was performed with ANOVA followed the Student t-test. The p value was calculated and p < 0.05 was considered as significantly different.
The expression of PELI3 is up-regulated in NSCLC and increased PELI3 expression predicts poor prognosis in NSCLC patients
Correlation of PELI3 expression with tumor stage in NCSLC
Expression of PELI3
Knockdown of PELI3 inhibits proliferation, migration and invasion of NSCLC cell lines
Knockdown of PELI3 inhibits tumor growth and metastasis in vivo
PELI3 is a direct target of miR-365a-5p in NSCLC cells
MiR-365a-5p is frequently decreased in NSCLC and suppresses proliferation, migration and invasion of NSCLC cell lines
Correlation of miR-365a-5p expression with tumor stage in NCSLC
Expression of miR-365a-5p
PELI3 mediates the effects of miR-365a-5p on NSCLC cells proliferation, migration and invasion
PELI3 was an important member in the Pellino E3 ubiquitin protein ligase family and recently received attentions in the inflammatory response and insulin resistance . However, its involvement in human malignancies has been barely investigated so far. Here we first analyzed PELI3 expression both in vivo and in vitro. Our data for the first time uncovered the aberrant over-expression of PELI3 in NSCLC at both transcript and protein level. More importantly, high level of PELI3 significantly associated with poorer prognosis of NSCLC patients, which indicated the oncogenic properties of PELI3 in this disease. SiRNA-mediated transient knockdown of PELI3 in both A549 and H1299 cells greatly suppressed cell viability and colony formation capacity. Furthermore, the metastasis-related malignant behaviors such as migration and invasion were compromised by PELI3-silencing as well. We further pursued to address the regulatory mechanisms underlying the aberrant over-expression of PELI3 in NSCLC. The putative candidate miR-365a-5p was identified with bioinformatics online tool. We experimentally demonstrated that miR-365a-5p directly modulated the expression of PELI3 3′UTR-driven luciferase. Our pulldown assay confirmed the direct binding between miR-365a-5p and PELI3 transcript. The endogenous expression of PELI3 was greatly suppressed in response to ectopic introduction of miR-365a-5p, which was subsequently released by miR-365a-5p-inhibitor. Furthermore, the inverse correlation between endogenous PELI3 and miR-365a-5p was observed in NSCLC patients, which underlined the direct regulatory relationship in this scenario. The universal down-regulation of miR-365a-5p in NSCLC was further characterized both in vivo and in vitro. High miR-365a-50 significantly stratified the better survivor from the patients with poor prognosis. Exogenous miR-365a-5p greatly suppressed cell viability and colony formation in A549 and H1299 cells, as well as the wound healing and transwell invasion. More importantly, PELI3-deficiency significantly inhibited cell proliferation, migration and invasion provoked by the miR-365a-5p inhibition, which highlighted the dominant role of PELI3 in mediating the anti-tumor activity of miR-365a-5p in NSCLC.
miR-365a-5p has been increasingly recognized involving in range of human malignancies. For instance, Zhu et al.  demonstrated that miR-365 inhibited cell growth and promoted apoptosis in melanoma by targeting Bcl2 and Cyclin D1. Wang et al.  showed that miR-365 promoted lung carcinogenesis by downregulating the USP33/SLIT2/ROBO1 signaling pathway. In addition, Min et al.  verified that exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediated a horizontal transfer of drug-resistant trait by delivering miR-365. Study by Hamada et al.  also disclosed miR-365 induced gemcitabine resistance in pancreatic cancer by targeting the adaptor protein SHC1 and pro-apoptotic regulator BAX. On contrary, the anti-tumor properties of miR-365 were uncovered by vast investigations. Xu et al.  proposed that miR-365 functioned as a tumor suppressor by directly targeting CYR61 in osteosarcoma. Chen et al.  disclosed the prognostic significance and anti-proliferation effect of miR-365 in hepatocellular carcinoma. Bai et al.  provided evidences that miR-365 inhibited growth, invasion and metastasis of malignant melanoma by targeting NRP1 expression. In cutaneous squamous cell carcinoma, Zhou et al.  unraveled that miR-365-targeted nuclear factor I/B transcriptionally repressed cyclin-dependent kinase 6 and 4 to inhibit the tumor progression, which was further consolidated by the observation that loss of BAX by miR-365 promoted cutaneous squamous carcinoma progression by suppressing apoptosis . Wang et al.  displayed that miR-365 inhibited ovarian cancer progression by targeting Wnt5a as well. In line with the anti-tumoral notion of miR-365, here we provided novel evidences that down-regulation of miR-365a-5p in NSCLC, which further associated with unfavorable outcomes clinically.
Until now, multiple target genes of miR-365a-5p have been experimentally identified. For example, Mori et al.  described that HLA-G expression was regulated by miR-365 in trophoblasts under hypoxic conditions. Yang et al.  demonstrated that mechanical and IL-1β responsive miR-365 contributed to osteoarthritis development by targeting histone deacetylase 4. Zhang et al.  unraveled that miR-365 inhibited vascular smooth muscle cell proliferation through targeting cyclin D1. Wang et al.  showed that miR-365 promoted diabetic retinopathy through inhibiting Timp3 and increasing oxidative stress. Wu et al.  demonstrated that miR-365 accelerated cardiac hypertrophy by inhibiting autophagy via the modulation of Skp2 expression. Sa et al.  reported that post-transcriptional suppression of TIMP-1 in epithelial-differentiated adipose-derived stem cells seeded bladder acellular matrix grafts reduced urethral scar formation. Here our results for the first time identified that PELI3 as the direct target of miR-365a-5p, which consequently contributed to its anti-tumor properties.
In summary, here we characterized the aberrant down-regulation of miR-365a-5p and sequent up-regulation of PELI3 in NSCLC. Our results suggested the anti-tumoral activity of miR-365a-5p via suppression of PELI3 expression in this disease, which might hold great promise for diagnostic and therapeutic purpose.
YZH, YTS and RLL conceived and designed the experiments; ZCW and BHW performed the experiments; YZH, YTS and SJL analyzed and interpreted the data; YZH, YTS and SJL contributed reagents, materials, analysis tools or data; HLZ wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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This study was approved by the ethics committee of the Second Hospital of Hebei Medical University, and followed the ethical guidelines laid down in the 1975 Declaration of Helsinki.
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