Background
Lung cancer is the primary cause of cancer-related death worldwide; NSCLC is the main type, accounting for approximately 85% of lung cancers [
1‐
3]. Despite the in-depth approaches to considerable innovations in targeted therapy, the survival of NSCLC patients is still not ideal, and the 5-year survival rate is less than 15% [
4]. Thus, the need to identify potential molecular targets for the treatment of NSCLC is urgent. In this paper, we demonstrated that EVI5 functions as an oncogene in the pathogenesis of NSCLC.
EVI5 belongs to a small subfamily of Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins [
5], which play enigmatically divergent roles as modulators of cell cycle progression, cytokinesis, and cellular membrane trafficking [
6,
7]. EVI5 contains a centrosomal targeting domain with homology to the structural maintenance of chromosomes (SMC) family of ATPases in the C-terminal half and a TBC domain in the N-terminal region [
8]; TBC domains are commonly associated with a functional class of proteins that act as GTPase-activating proteins (GAPs) for the Rab family GTPases and regulate membrane trafficking [
9].
EVI5 functions as a stabilizing factor which maintains Emi1 level in S/G2 phase of cell cycle, which resulted in the accumulation of Cyclins [
10]. Furthermore, a study showed that the expression of Emi1 is upregulated in several solid tumors, including NSCLC [
11]. Thus, we hypothesize that EVI5 could also promote the cell cycle of NSCLC by combining with Emi1 to induce Cyclins accumulation.
Noteworthy, tumor metastatic invasiveness is linked with the increased migration capacity of epithelial cells. This process is known as the epithelial-mesenchymal transition (EMT) [
12]. EMT is essential for morphogenesis during embryonic development and early tumour transformation into invasive malignancies [
13‐
15]. In particular, accumulating evidence indicates that TGF-β/Smad signaling is a potent inducer of EMT in various cancers, including NSCLC [
16,
17]. TGF-β/Smad signaling pathway can regulate cell proliferation, migration, invasion, and other functions through a complex pathway network comprising multiple pathways [
18,
19]. EVI5 could promote collective cell migration through its Rab-GAP activity [
20], the mechanism by which it regulates tumor metastasis remains unclear. Data extracted from GEPIA2 database (
http://gepia.cancer-pku.cn/) showed that the level of EVI5 is positively correlated with that of TGF-β receptors in NSCLC, especially TGF-β receptor II, which is an initiator of TGF-β/Smad signaling [
21]. Thus, we sought to investigate whether EVI5 could interact with TGF-β/Smad signaling pathway, which may promote the EMT progression of NSCLC, which would be a particularly important clinical significance.
In present study, the interaction between Emi1 and EVI5 suggested that EVI5 does play a protumoral role in NSCLC, in which the binding of EVI5 and Emi1 is the key to the accumulation of Cyclins. More important, we found a significant phenotype that EVI5 influence the migration and invasion of NSCLC by interacting with TGF-β receptors to activate the downstream TGF-β/Smad signaling pathway. In addition, we found that miR-486-5p represses EVI5 expression, and the low expression level of miR-486-5p may be one of the reasons for the elevated expression of EVI5 in NSCLC. Here, we aimed to evaluate the role of EVI5 in the tumorigenesis of NSCLC, and to explore the possible role of miR-486-5p in EVI5 dysregulation in lung carcinogenesis.
Methods
Tissue samples
Paired NSCLC and adjacent noncancerous lung tissue samples (60 of each) were collected with the informed consent of the patients from the First Affiliated Hospital of Soochow University between 2015 and 2018. The patients had been diagnosed with NSCLC based on their histological and pathological characteristics according to the Revised International System for Staging Lung Cancer. No patient had received chemotherapy or radiotherapy before tissue sampling. The tissue samples were snap frozen and stored in a cryofreezer at − 80 °C. This study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.
Cell lines and cultures
The human NSCLC cell lines A549, H226, H1299, H1650, SPC-A1 and H460 and the human immortalized normal epithelial cell line 16HBE were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% foetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and L-glutamine (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere containing 5% CO2.
RNA interference
Two pre-designed small interfering RNA (siRNA) sequences targeting different coding regions of EVI5 were directly synthesized (GenePharma). The target sequences of the siRNAs were as follows: siRNA-EVI5–1: 5′-GAG UCU CAG UGU GCA UUA ATT-3′; siRNA-EVI5–2: 5′-GGA CUC CUU ACU CAA UUA ATT-3′; and siRNA-Emi1: 5′-GCA CUA GAG ACC AGU AGA CTT-3′. Scrambled siRNA was used as a negative control. Cells were transiently transfected with 50 nM siRNA sequences using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). After 72 h of transfection, cells were harvested for further experiments.
Establishment of stable EVI5-overexpressing cell lines
To generate NSCLC cells in which EVI5 was stably overexpressed, a 2493-bp fragment of the EVI5 coding sequence was synthesized (Genewiz, Suzhou, China) and subcloned into the PLVX-IRES-Neo vector (PLVX) using the endonucleases EcoRI and XbaI for expression in a Lenti-X lentiviral expression system (Clontech, Mountain View, CA, USA). Empty vector was used as a negative control. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS at 37 °C in a humidified 5% CO2 incubator for 48 h. The EVI5 expression construct was co-transfected with packaging plasmids into HEK293T cells using Lipofectamine 3000. After incubation, the packaged lentiviruses were collected and used to infect A549 and H226 cells. After 48 h, stable cells were selected with 400 μg/ml G418 (Amresco, Solon, OH, USA).
Establishment of stable EVI5-knockout cell lines
To establish stable cell lines with silenced EVI5 expression, guide RNA (gRNA) sequences were synthesized (Genewiz). The target sequences of the EVI5-gRNA were as follows: Forward: 5′-CACCGAAAGGCAGCAGTCATTTTGT-3′ and Reverse: 5′-AAACACAAAATGACTGCTGCCTTTC-3′. Then, we subcloned the EVI5-gRNA into the lentiviral vector Lenti-CRISPR v2 (Cas-9, GenePharma, Shanghai, China) digested with BsmBI after phosphorylation and annealing. The correctness of the Lenti-CRISPR-sgEVI5 (EVI5-KO) plasmid was confirmed by sequencing. Empty vector was used as a negative control. Then, the EVI5 silencing construct or the negative control was co-transfected with packaging plasmids into HEK293T cells using Lipofectamine 3000 (Invitrogen). After incubation, the packaged lentiviruses were collected and used to infect A549 and H226 cells. After 48 h, stable cells were selected with 0.4 μg/ml or 2 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA).
Total RNA was extracted from cells by the addition of 1 ml of RNAiso Plus (Takara, Osaka, Japan) according to the manufacturer’s protocol. The RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was carried out with M-MLV reverse transcriptase (Takara). The primers used for reverse transcription and amplification of miR-486-5p and U6 were designed and synthesized by Guangzhou RiboBioCorp (Guangzhou, China). The primers for EVI5 and β-actin used for qRT-PCR analysis were as follows: EVI5, Forward: 5′-GCATCATCCTGGTTTCTGAC-3′ and Reverse: 5′-AGCTTGTCTGGG ACACCATC-3′; and β-actin, Forward: 5′-CACAGAGCCTCGCCTTTGCC-3′ and Reverse: 5′-ACCCATGCCCACCATCACG-3′. The primers specific for U6 were purchased from RiboBioCo., Ltd. (Guangzhou, China). qRT-PCR was performed using SYBR Premix ExTaq™ (Takara) according to the manufacturer’s instructions with an ABI Step One Plus Real-Time PCR system (Applied Biosystems). The PCR program was as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The expression values of EVI5 mRNA and miR-486-5p were normalized to those of the internal controls β-actin and U6, respectively. Relative expression was calculated using the
ΔΔCt method [
22].
Western blotting assay
Western blot analysis was performed as previously described by us [
23]. The following antibodies were used in the analysis: anti-EVI5 (Millipore, Billerica, MA, USA); anti-Emi1 and anti-TGF-β receptor II (Santa Cruz, CA, USA); anti-CyclinA2 (Proteintech, IL, USA); anti-pAkt (Ser473), anti-Akt, anti-Erk1/2, anti-pErk (Thr202/Tyr204), anti-CyclinD1, anti-MMP2, anti-p-Smad3, anti-Snail and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); anti-TGF-β receptor I (Abcam, London, UK); anti-N-cadherin and anti-Vimentin (BD Biosciences, USA); Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).
Co-immunoprecipitation (co-ip) assay
NSCLC cells were cultured in a 100 mm plate to 95–100% confluence. Then, the cells in each dish were washed twice with cold phosphate-buffered saline (PBS), collected by scraping, and lysed with 1 ml of modified RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Cell lysates were collected by centrifugation at 10,000×g at 4 °C for 30 min. Clear lysates were pre-cleared by the addition of 50 μl of protein G bead slurry and incubated at 4 °C overnight with rotation. Supernatants were transferred to a new Eppendorf tube and incubated with 1 μg of rabbit anti-EVI5 antibody (Abcam, ab70790) with rotation overnight in a cold room; this step was followed by an additional incubation for 3–4 h with protein G beads. The beads were washed three times with RIPA buffer and then boiled in 2× SDS protein loading buffer for 5 min. Samples (20 μl) were loaded on SDS-PAGE gels for western blot analysis.
Cell viability assay
Cell proliferation was examined using a Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China). Tumour cells were seeded in 96-well plates at a density of 3 × 103 cells per well and further grown under normal culture conditions for 24, 48 and 72 h. Cell viability was measured according to the manufacturer’s instructions at several time points (24, 48 and 72 h). We also assessed cell proliferation using a clonogenic assay. Briefly, tumour cells were diluted in complete culture medium, and 3000 cells were reseeded in a 60-mm plate. After incubation for 7–10 days, depending on the cell growth rate, colonies formed by at least 50 cells were stained with Giemsa and counted. Each experiment was performed in triplicate.
Wound healing assay
A wound healing assay was performed as described previously [
23]. Briefly, tumour cells were seeded into 6-well cell culture plates after 24 h of transfection and cultured in a monolayer to 70–80% confluence. The monolayer was gently and slowly scratched using a fresh 10-μl pipette tip across the centre of the well, aiming for a resulting gap distance equal to the outer diameter of the end of the tip. Another scratch was made perpendicular to the first to create a cross in each well. Detached cells were then removed by two gentle washes with 1 × PBS. The well was replenished with fresh medium, and cells were cultured for an additional 24 h. Cells were observed and imaged under a microscope (CKX41, Olympus) at the same magnification and settings. The width of the gap was evaluated quantitatively using Photoshop.
Migration and invasion assays
Transwell migration and invasion assays were performed as described previously [
23]. For the migration assay, 3 × 10
4 tumour cells in medium containing 1% FBS were seeded onto the upper chamber of a transwell insert, and 800 μl of medium containing 10% FBS was added to the lower chamber. For the invasion assay, 5 × 10
4 tumour cells in medium containing 1% FBS were seeded onto the upper chamber of a transwell insert coated with Matrigel matrix (BD Science, Sparks, MD, USA), and 800 μl of medium containing 10% FBS was added to the lower chamber. At 6 h later, if necessary, TGF-β1 (5 ng/ul) was added to the lower chambers, and the plates were incubated at 37 °C for 24 h. After 24 h of incubation, the cells that had migrated onto the lower surface of the chamber were fixed with 100% methanol and stained with 1% crystal violet. Finally, the cells were counted in at least three random fields under a light microscope.
Cell cycle analysis
According to the protocol of the Cell Cycle Analysis Kit (Beyotime, Shanghai, China), cells were cultured in 6-well plates and transfected with negative control miRNA (miR-NC), miR-486-5p, si-NC or si-EVI5 for 48 h. Cells were then harvested, washed with cold PBS, fixed with 70% ethanol at 4 °C for 24 h, washed with cold PBS again and stained with a propidium iodide (PI)/RNase A mixture. Next, cells were incubated in the dark at 37 °C for 30 min and analysed using a fluorescence-activated cell sorting (FACS) Calibur system (Beckman Coulter, Brea, CA, USA).
Cell apoptosis analysis
According to the protocol of the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China), cells were transfected with miR-NC, miR-486-5p, si-NC or si-EVI5. After 48 h, cells were harvested, washed with cold PBS, and resuspended in binding buffer containing Annexin V/FITC and PI (Beyotime). Stained cells were then detected using the FACS Calibur system (Beckman Coulter).
Xenografts
BALB/c athymic nude mice (female, 4–6 weeks old, weighing 16–20 g) were purchased from the Experimental Animal Center of Soochow University and bred under pathogen-free conditions. All animal experiments were carried out in accordance with the Guide for the Care and Use of Experimental Animals of the Experimental Animal Center of Soochow University. Two million stable EVI5 cells (A549-Cas-9 cells and A549-EVI5-KO cells) were suspended in 150 μl of FBS-free medium and subcutaneously injected into the nude mice, which were randomly divided into two groups (8 mice per group). Tumour growth was analysed by measuring the tumour length (L) and width (W) and calculating the volume (V) with the formula V = LW2/2.
Statistical analysis
All numerical data are presented as the mean ± SD. Statistical analysis was performed with an unpaired t test (two-tailed). A paired t test (two-tailed) was performed to determine the significance of the data from patient samples. Differences for which P was < 0.05 were considered significant. Statistical analyses were conducted using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA).
Discussion
Malignant tumor is essentially a disease involving unlimited cell proliferation [
30] and metastasis [
31]. In recent years, EVI5, an important protein regulating cell cycle [
32] and migration [
20], has been increasingly reported to be associated with HCC [
24], bladder cancer [
33], melanoma [
34], leukemia [
35], lymphoma [
25] and many other tumors. Here, we identified EVI5 as a novel prognostic biomarker for NSCLC.
In the present study, we first analyzed the expression of EVI5 in NSCLC tissues, its expression was upregulated in NSCLC tissues compared with that of matched paracancerous tissues. Moreover, we identified the higher expression of EVI5 in the NSCLC cell lines, which shows that EVI5 is frequently overexpressed in NSCLC. Our findings indicated that EVI5 significantly promotes NSCLC cell proliferation by accelerating the NSCLC cell cycle. The co-expression of EVI5 and Emi1 has been reported in other types of cancers [
10]. Consistent with the previous findings, EVI5 was found to promote Emi1 and Cyclins expression in our study. Therefore, our findings demonstrated that EVI5 may play a protumoral role in NSCLC via it effects on Emi1 accumulation. However, the interactions and underlying mechanisms need to be elucidated.
The public dataset from Kaplan-Meier Plotter indicated that higher expression of EVI5 was significantly associated with poor survival of patients with NSCLC. Metastasis is a critical issue leading to poor survival, as lymph node metastasis is a fundamental factor in the determination of the clinical staging and prognosis of NSCLC. The TGF-β/Smad signaling pathway plays an important role in EMT progression in various epithelial cell types [
36] and thus, ongoing research has focused on investigating ways to reverse or delay the EMT to prevent carcinogenic progression. Furthermore, as a GTPase that regulates intracellular transport, membrane trafficking, cytokinesis, and cell migration [
37], the tumor promoting metastasis role of EVI5 is poorly understood. When EVI5 knockdown in NSCLC, the TGF-β receptor II, TGF-β receptor I, p-Smad3, Snail, Vimentin, MMP2 and N-Cadherin levels were significantly decreased and E-Cadherin was significantly increased. The administration of TGF-β1 increased the migration and invasion of EVI5-knockout NSCLC cells, and increased the levels of p-Smad3 and its downstream signaling molecules, indicating a therapeutic role for EVI5 in inhibition of cancer metastasis. In the present study, we have reported for the first time that EVI5 could promote TGF-β/Smad induced cell migration and invasion by interacting with TGF-β receptors in NSCLC. Next, We sought to investigate the in-depth mechanism in which EVI5 may involved to be a binding partner and GTPase-activating protein domain for Rab11, and may influence the ability of Rab11 to recycling of endocytosed TGF-β receptors to the plasma membrane [
21]. This requires further exploration to reveal how EVI5 interacts with TGF-β receptors, thus affecting the metastasis of NSCLC.
As an important gene, EVI5 has only been reported to be regulated by miR-135b in HCC [
38]. Although there is ample evidence for the upregulation of EVI5 expression in NSCLC, the underlying mechanisms are poorly understood. The malignant progression of NSCLC is considered to be a comprehensive event that includes a gene expression network and alterations in the tumor microenvironment, in which microRNAs play critical roles [
39]. MiR-486-5p, widely documented to be a tumor-suppressive microRNA, inhibits proliferation and invasion in many types of cancers [
39,
40], In the present study, we observed an interaction between EVI5 and miR-486-5p. To address this question, a dual-luciferase reporter assay was performed, and it showed that miR-486-5p significantly inhibited luciferase activity in cells transfected with the wild-type EVI5 3′-UTR, Moreover, overexpression of miR-486-5p prevented EVI5-induced cell proliferation, migration and invasion in NSCLC cells, which further confirms the tumor-promoting function of EVI5 and provides more clues to the regulation network of EVI5.
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