IL-33 Promotes the Growth of Non-Small Cell Lung Cancer Cells Through Regulating miR-128-3p/CDIP1 Signalling Pathway
Authors Zhou X, Feng Y, Liu S, Li C, Teng Y, Li X, Lu J
Received 20 August 2020
Accepted for publication 7 January 2021
Published 12 March 2021 Volume 2021:13 Pages 2379—2388
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Seema Singh
Xiaorong Zhou, 1,* Yuxu Feng, 2,* Siwen Liu, 1 Chenchen Li, 1 Yue Teng, 1 Xiaoyou Li, 1 Jianwei Lu 1
1Department of Medical Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, 210009 Jiangsu Province, People’s Republic of China; 2Department of Orthopedics, The Pukou Centre Hospital, Nanjing, 210009 Jiangsu Province, People’s Republic of China
*These authors contributed equally to this work
Correspondence: Xiaoyou Li; Jianwei Lu
Department of Medical Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, The Affiliated Cancer Hospital of Nanjing Medical University, No. 42 Baiziting, Xuanwu District, Nanjing 210009, Jiangsu Province, People’s Republic of China
Email [email protected]; [email protected]
Background: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths, and it is also the most frequently diagnosed cancer. Previous studies indicate that IL-33 plays a crucial role in the development of NSCLC. In recent years, the role of miRNAs in cancer has become increasingly clear. However, reports focused on the relation between IL-33 and miRNAs in NSCLC have been limited.
Methods: The expression of IL-33 and miR-128-3p was detected by qPCR. MTT, EdU, and colony formation assays were used to detect the proliferation ability of NSCLC cells. Transwell assay was used to investigate the migration and invasion of NSCLC cells. The expression of bax, cyt-c, and caspase 3 was detected by Western blot. Finally, in vivo tumor xenograft was used to detect the effects of IL-33 and miR-128-3p on tumor growth capacity.
Results: IL-33 was notably increased in the serum and tumor tissue of NSCLC patients. The in vitro function study revealed that IL-33 significantly promotes the proliferation, migration, and invasion of the NSCLC cells. In vivo experiments further confirmed the pro-tumor effect of IL-33 on NSCLC. The study on the underlying mechanism elucidated that IL-33 regulates the expression of miR-128-3p, which can directly target and inhibit the expression of CDIP1. Furthermore, IL-33 regulates the expression of downstream apoptotic proteins such as bax, cyt-c, and caspase3. Rescue experiments demonstrated that miR-28-3p can reverse the effect of IL-33.
Conclusion: These findings indicated that IL-33 and miR-128-3p may play a potential role in the diagnosis and treatment of NSCLC.
Keywords: IL-33, NSCLC, miR-128-3p, CDIP1
Corrigendum for this paper has been published
Lung cancer is a leading cause of cancer death, accounting for approximately 26% of all female and 28% of all male cancer deaths in 2013. Non-small cell lung cancer (NSCLC), including squamous carcinoma, adenocarcinoma, and large cell carcinoma, is the most frequent type of lung cancer. NSCLC accounts for more than 80% of all lung cancers. Even with the most advanced therapeutic treatments, the five-year overall survival rate is less than 16%, and this rate has not changed appreciably over many decades. This poor prognosis emphasizes the urgent need for the development of novel strategies to prevent and effectively treat this deadly disease.
Interleukin 33 (IL-33) comes from the IL-1 family of cytokines, which induces the formation of pro-inflammatory cytokines complex and then regulates inflammatory responses.1,2 IL-33 is the ligand of the suppression of tumorigenicity 2 (ST2) receptor. The IL-33/ST2 signal pathway plays critical roles in immune response, inflammation, tumor growth, and metastasis.3,4 IL-33 attracts considerable attention as it is closely related to the initiation and development of cancer. Current research on IL-33 focuses mainly on tumor microenvironment, tumorigenesis, and tumor-associated inflammatory responses.5 IL-33-mediated mast cell activation promotes gastric cancer through macrophage mobilization.6 IL-33/regulatory T cell axis triggers the development of a tumor-promoting immune environment in chronic inflammation. Moreover, lower expression level of IL-33 is associated with the poor prognosis of lung cancer.7 IL-33 inhibition slows the tumor growth of lung cancer in immune-deficient mice.8 Nonetheless, further research is required for the better understanding of IL-33.
MicroRNAs (miRNAs/miRs) are small non-coding RNAs with 20–25 nucleotides that induce degradation or translational suppression of their target mRNAs by binding the seed sequences in the 3ʹ-untrans-lated regions (UTR). miRs participate in various biological processes, including cell proliferation, apoptosis, and differentiation. Dysregulated miR expression contributes to the development of cancers, including breast cancer, digestive tract cancers, and lung cancer among others. MiR-128-3p is indicated as an oncogene in ovarian cancer and hepatic cancer and serves a regulatory role in numerous physiological and pathological processes.9 MiR-128-3p participates in the antitumor process of dendritic cells (DCs), suggesting its potential value in tumor immune regulation. According to research reports, after the down-regulation of miR-128-3p expression in gastric cancer cells, activation of PDK1/Akt/NF-κB pathway promotes proliferation of gastric cancer cells. In addition, NF-κB activation can increase the expression of immunosuppressive factors such as IL-6, IL-8, and MCP-1 in tumor cells, thus promoting tumor growth. Therefore, we speculated that miR-128-3p may be involved in the release of immune-related cytokines by tumor cells or affect the infiltration of immune cells in the tumor microenvironment.
Cell death-inducing p53 target 1 is a regulator of stress-induced apoptosis. CDIP1 knockout mice also show reduced ER stress, as seen in embryonic stem cells (ESCs). In addition, specific activation of p53 by CDIP1 can trigger apoptosis. However, reports on the relationship between CDIP1 and the regulation of miRNA and the non-small cell immune microenvironment are few.
In this study, we aimed to discover the role of IL-33 in the initiation and progression of NSCLC. We also explored the effect of IL-33 on regulating miR-128-3p/CDIP1 signal pathway. We found that IL-33 significantly altered the expression level of miR-128-3p in NSCLC and further blocked the inhibitory effect of miR-128-3p on CDIP1 expression.
Methods and Materials
NSCLC tissues and adjacent normal tissues as well as blood sample were obtained from NSCLC patients at the Affiliated Cancer Hospital of Nanjing Medical University from November 2017 to March 2019. Blood sample from normal people was also obtained from the same hospital. The samples were snap frozen using liquid nitrogen and then stored at a temperature of −80°C. Informed consent to participate in the study was obtained from the research subjects prior to study commencement. All experiment procedures were performed in accordance with the Declaration of Helsinki of the World Medical Association and were approved by the Medical Ethics Committee of Jiangsu Cancer Hospital.
Cell Culture and Transfection
NSCLC cell lines A549 (TCHu150) and H1299 (TCHu160) were obtained from Chinese Academy of Science and were cultured in RPMI1640 (Gibco, Grand Island, NY, USA) medium supplemented with 10% FBS (Gibco, Grand Island, NY, USA) containing 100U/mL penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were cultured at 37°C in a 5% CO2 environment. IL-33 was obtained from Sigma Chemical Corporation (St. Louis, MO, USA). Adenovirus of sh-miR-128-3p and its negative control used in animal study were synthesized by Hanbio (Shanghai, China). The mimic and inhibitor of miR-128-3p along with their negative control were synthesized by Genepharma (Shanghai, China). The oligonucleotides were transfected into A549 and H1299 cells (200 nmol per well) using Lipo2000 (Invitrogen, Carlsbad, CA, USA). Ad-sh-miR-128-3p stably transfected A549 cells were derived from the parental cells by G418 (Sigma, St. Louis, MO) selection.
A total of 5000 cells/well were seeded onto 96-well plates and cultured for 12 h followed by transfection. Twenty-four hours after transfection,50 ng/mL IL-33 recombinant protein (Sigma, St. Louis, MO, USA) was added into each well. MTT assay was used to assess the cell viability 24 h later, and 0.5% MTT (Sigma, USA) was added to the culture medium at 37°C for 4 h. The supernatant was removed, and DMSO was added into each well. Afterward, the absorption at 490 nm was evaluated using a microplate reader (BioRad, CA, USA).
Colony Formation Assay
The cells were treated with indicated conditions and then seeded in 12-well plates (100/well). After two weeks of incubation, crystal violet (0.05%, Beyotime, Shanghai, China) was used to stain the colonies. Colonies containing more than 50 cells were counted.
The cell migration and invasion were detected by Transwell assay (Millipore, MA, USA). A total of 5000 cells were treated with indicated conditions and then seeded on the upper insert coated with 2% Matrigel (BD Biosciences, NY, USA) or not for the detection of invasion or migration, respectively. The upper insert was filled with medium lacking serum, and the lower chamber was filled with 600 μL DMEM supplemented with 10% FBS. After 24 h of incubation, cells invaded to the lower chambers were fixed with methanol, stained with crystal violet.
A549 and H1299 cells were supplemented with 45 μM 5-ethynyl-2ʹ-deoxyuridine (EdU, Beyotime, Shanghai, China) for 2 h at 37°C, with subsequent fixing in 4% paraformaldehyde for 30 min at room temperature. Then, 1% TritonX-100 was added for permeabilization, and the cells were reacted with an EdU reaction cocktail (Click-iT EdU microplate assay kit) for 30 min according to the instructions. Finally, the nuclei were stained with 1 mg/mL DAPI (Beyotime, Shanghai, China) for 15 min, and the samples were observed under a red fluorescence microscope (Leica, Wetzlar, Germany).
Quantitative Real-Time PCR
Total RNAs were extracted using Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instruction. One microgram of total RNAs was then reverse transcribed to cDNA using a RNA PCR Kit (Takara, Dalian, China) and was used as a PCR template. Quantitative real-time PCR (qRT-PCR) was performed in a FAST7500 System (ABI, USA) using the SYBR Green Super Mix (BioRad, CA, USA) according to manufacturer’s instructions. Small endogenous nuclear U6 snRNA was used as internal control for normalization of miRNA and GAPDH for mRNAs. The relative gene expression levels were calculated using (2−ΔΔCt) method. The specific primer sequences used in this study were as follows: IL-33 upstream primer: 5ʹ-TGGAGTCACAGAAGGAGTGGCTAAG-3ʹ and downstream primer: 5ʹ-TCTGACCACAGTGAGGAATGTCCAC-3ʹ. MiR-128-3p upstream primer: 5ʹ-AGCTAAGTATTAGAGCGGCGGCA-3ʹ and downstream primer: 5ʹ-GACATCAACACTCCCCTGACAAC-3ʹ. CDIP1 upstream primer: 5ʹ-ACACTCCAGCTGGGTCCCTGA-3ʹ and downstream primer: 5ʹ-CCCTGAACTCAACTGTGAAATA-3ʹ. U6 upstream primer: 5ʹ-GCCGCTGATTCTTTTGACAT-3ʹ and downstream primer: 5ʹ-AATCTTCTCCCCCCACCTTTGC-3ʹ. GAPDH upstream primer: 5ʹ-CGGAGTCAACGGATTTGGTC-3ʹ and downstream primer: 5ʹ-GACAAGCTTCCCGTTCTCAG-3ʹ.
Tumor tissue specimens were immobilized with 10% neutral formalin. Immunohistochemical staining was performed by SABC method in 4 μm continuous sections. All sections were routinely dewaxed, rinsed with PBS, and incubated at 3% H2O2 at room temperature for 10 min to inactivate endogenous peroxidase. Citric acid was heated for antigen repair. Normal goat serum was sealed. The primary antibody was added in drops and incubated at 4°C overnight. Then, biotinylated secondary antibodies and peroxidase-labeled antibodies were dropped and washed with PBS. DAB was used for color development and hematoxylin contrast dyeing. PBS was used as the negative control instead of primary antibody.
The total protein was extracted from the A549 and H1299 cells using a lysis buffer. Forty microgram protein was separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 5% skimmed milk for 2 h at room temperature to block non-specific binding, followed by incubation with primary antibodies (CD1P1#A14883, ABclonal; Bax#ab32503, abcam; Cytochrome C# ab133504, abcam; c-caspase3#ab13847, abcam; GAPDH#ab8245, abcam) overnight at 4°C. After three washes with TBST, the blots were incubated at room temperature for 2 h with horseradish peroxidase-conjugated goat anti-rabbit antibodies. GAPDH was used as an internal control. Finally, the blot was treated with ECL plus reagent (Pierce, Rockford, IL, USA) and visualized using charged-coupled device LAS 4000 (Fujifilm, Valhalla, NY, USA).
The wild-type and mutated 3ʹUTRs of CDIP1 were subcloned into the pGL3 vector (Promega, WI, USA). Cells were co-transfected with the plasmid containing pGL3-CDIP1-3ʹUTR or pGL3-CDIP1-3ʹUTR-mut along with miR-128-3p mimic or mimic control using Lipofectamine 2000 (Invitrogen, CA, USA). Subsequently, cells were transfected with 0.1 μg PRL-TK (TK-driven Renilla luciferase expression vector) as internal control. Luciferase activities were measured 48 h after transfection with a dual luciferase reporter assay kit (Promega, WI, USA).
Sections that are 5 µm thick were de-paraffinized in xylene and rehydrated in graded alcohol, and 3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity. Subsequently, the sections were incubated in 10 mM citrate buffer and heated for antigen repair. The slides were then incubated with primary antibody overnight at 4°C CDIP1 (Abcam, Cambridge, UK), followed by HRP-labeled secondary antibody incubation for 2 h. Afterward, DAB detection system (Dako, Glostrup, Denmark) was used for detection of primary antibodies.
In vivo Study
Thirty Balb/c nude male mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experiment was approved by the Animal Ethics Committee of Jiangsu Cancer Hospital as well as procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals. Mice were maintained under a specific pathogen-free condition and randomly divided into three groups. A549 and H1299 cells in the amount of 1×106 were subcutaneously injected into the flank region of the mice. IL-33 dissolved in normal saline was administered to these mice every day at the dose of 80 mg/kg for seven days before tumor inoculation until the end of the study. The mice in IL-33+sh-miR-128-3p group were tail injected with adi-sh-miR-128-3p after IL-33 administration. The tumor sizes were measured every two days using a caliper. The mice were sacrificed at day 30, and tumors were excised. Mice were placed in a plexiglass chamber with 5% isoflurane (VetOne, Shanghai, China) for 5 min and decapitated when fully sedated, as measured by a lack of active paw reflex. A part of the tissues was placed in 10% formalin for histological, and the remaining was frozen in −80°C.
All the data are presented as the mean±SD. One-way ANOVA was used to assess the difference between multiple groups. Differences between two groups were analyzed by the Student’s t-test. P<0.05 was considered as statistical significance.
IL-33 is Up-Regulated in the Serum of NSCLC Patient and the NSCLC Tissue
qPCR and IHC were used to evaluate the relative mRNA and protein expression of IL-33 in the NSCLC tissue, respectively. ELISA was used to detect the amount of IL-33 in the serum of NSCLC patient. As the results showed, IL-33 was up-regulated in the NSCLC tissue, and the level of IL-33 in the advanced phase of patients was higher than that in the early phase of NSCLC patients (Figure 1A–C). In addition, IL-33 level in serum of NSCLC patient was higher in that of normal individuals (Figure 1D).
MiR-128-3p is Up-Regulated by IL-33 Treatment
To verify the effect of IL-33, 50 ng/mL IL-33 stimulation was used. As shown in Figure 2, qPCR was used to screen several miRs after IL-33 treatment in H1299 cell, which potentially regulates IL-33 expression in TargetScan. We found that IL-33 remarkably elevated the expression level of miR-128-3p (Figure 2A). Moreover, after analyzing the data from StarBase dataset, we found an elevation of miR-128-3p in NSCLC (Figure 2B). Next, we evaluated the expression of miR-128-3p in NSCLC. The results demonstrated that miR-128-3p level was significantly increased in the serum and cancer tissues of NSCLC patient (Figure 2C and D). Moreover, the levels of miR-128-3p in the tissue of III–IV stage patients were remarkably higher than those in stage I–II patients (Figure 2E). Finally, we acquired survival analysis data from KM plotter dataset and found that high IL-33 exerted a poor prognosis in NSCLC (Figure 2F).
IL-33 Promotes the Proliferation and Clone Formation of A549 and H1299 Cell
We applied miR-128-3p inhibitor transfection to knock down miR-128-3p. The qPCR detection results indicated that IL-33 notably increased the level of miR-128-3p and that miR-128-3p inhibitor significantly reduced the level of miR-128-3p (Figure 3A). Next, we evaluated the effect of IL-33 in A549 and H1299 cell proliferation and clone formation. IL-33 promoted the proliferation and clone formation ability of A549 and H1299 cells. Moreover, knocking down miR-128-3p significantly reversed the effect of IL-33 on cell proliferation and clone formation (Figure 3B–E). The survival rate of BEAS-2B cells treated with 0, 25, 50, 100 ug/mL IL-33 treatment for 48 h did not obviously decrease, but significantly reduced for A549 and H1299 cells (Figure 3F). It is disclosed that appropriate IL-33 can selectively suppress lung cancer cells, but has no obvious inhibitory effect on normal lung epithelial cells.
IL-33 Promotes the Migration and Invasion of A549 and H1299 Cell
We further evaluated the effect of IL-33 in A549 and H1299 cell migration and invasion using Transwell assay. IL-33 promoted the migration and invasion of A549 and H1299 cells. Moreover, knocking down miR-128-3p significantly reversed the effect of IL-33 on cell migration and invasion (Figure 4A and B).
IL-33 Regulates the miR-128-3p/CDIP1 Signal Pathway
To investigate the mechanism through which miR-128-3p reversed the effect of IL-33, potential target genes of miR-128-3p were predicted by bioinformatic algorithms Targetscan7.2, and CDIP1 was selected as the potential target (Figure 5A). To further confirm whether miR-128-3p directly targeted IL-33 and suppressed its expression, a firefly luciferase reporter was constructed containing a wild type or mutated type fragment of the 3ʹ-UTR of IL-33’s mRNA. The wild type or mutated type luciferase reporters were co-transfected into A549 and H1299 cells along with miR-128-3p mimics or negative control. The data showed that the co-transfection of miR-128-3p mimics with wild-type 3ʹUTR but not with mutant 3ʹUTR significantly reduced the luciferase activity (Figure 5B). mRNA and protein expression of CDIP1 were remarkably inhibited by miR-128-3p overexpression and promoted by miR-128-3p knock down (Figure 5C). To further understand the mechanism underlying the effect of IL33 and miR-128-3p, we detected the expression of downstream proteins, including bax, cyt-c, and cleaved caspase-3. The Western blot assay results revealed that IL-33 significantly inhibited the expression of CDIP1 and the downstream proteins such as bax, cleaved caspase-3, and cytoplasm cyt-c as well as PARP, whereas Bcl-2 was enhanced. In addition, miR-128-3p knocked down by infection of miR-128-3p inhibitor significantly reversed this effect of IL-33 (Figure 5D and E).
IL-33 Promotes the Tumor Growth Which Could Be Reversed by miR-128-3p Knock Down
As shown in Figure 6A and B, IL-33 treatment significantly inhibited the tumor growth of A549 and H1299 cell in nude mice. However, sh-miR-128-3p adenovirus treatment notably reversed this effect of IL-33. Moreover, IL-33 treatment increased the tumor weight, and, accordingly, this effect was reversed by knocking down miR-128-3p (Figure 6C and D).
The role of IL-33 in malignancies is complicated. Numerous studies focused on the function of IL-33 in human cancers indicate its dual functions as a damage-associated molecular or nuclear factors mediating gene expression.10 As IL-33 is a cytokine implicated in the regulation of not only immunity response but also tumor growth, it exhibits different or even opposite functions under varying circumstances. For instance, IL-33 promoted the growth and metastasis of solid cancers, such as gastric cancer, colorectal cancer, ovarian cancer, and breast cancer. On the contrary, other studies reveal an anti-cancer function of IL-33. For example, recombinant IL-33 dramatically suppress the growth of leukemia via elevating the IFN-γ release from leukemia-reactive CD8+ T cells.11 We speculated that, depending on the environmental conditions, IL-33 may orchestrate antitumor immunity, activating CD8+ T cells.
Previous studies demonstrate an elevated IL-33 level in NSCLC, and a high amount of serum IL-33 indicates a poor prognosis in NSCLC patients.12,13 This was confirmed by another study further indicating the correlation between IL-33 and tumor stages of NSCLC.14–16 These findings may indicate the oncogenic effect of IL-33, which was verified by an in vivo study demonstrating that IL-33 blocking restricted tumor growth of NSCLC xenografts.8 However, the metastatic potential in the model of Lewis lung carcinoma is significantly attenuated by transgenic expression of IL-33.17 When depleting CD8(+) T cells and NK cells, pulmonary metastasis is significantly increased, indicating that IL-33 can mediate the anti-tumor immunity of CD8(+) T cells and NK cells. IL-33 inhibits NSCLC progression through various mechanisms, including diminishing regulatory T cells (Treg) cells in tumor tissues, educating immune surveillance in tumor microenvironments, abrogating polarization of M2 tumor-associated macrophages (TAMs), and reducing accumulations of Treg cells in tumor tissues.8,18 These findings are mainly involved in the immune response in tumor environment. However, we speculated that the role of IL-33 on immune regulation is not dominant here. In this study, we found that IL-33 was notably upregulated in the tumor tissues and serum of NSCLC patients, which is in accordance with the previous studies. Moreover, function studies indicate that IL-33 directly promotes the proliferation and migration of NSCLC cells.
We focused on miRs, which serve a regulatory role in numerous physiological and pathological processes. In our work, we first speculated whether an interaction exists between IL-33 and miRs in NSCLC, which has been studied in other diseases. For instance, IL-33 promotes recovery from acute colitis by inducing miR-320 to stimulate epithelial restitution and repair. Here, we screened miRs that are commonly expressed in lung cancer cells and found miR-128-3p, which is significantly up-regulated after IL-33 treatment. MiR-128-3p can induce Drosha depletion, which promotes lung cancer cell migration.19 It also confers chemoresistance-associated metastasis in NSCLC by activating Wnt/β-catenin and TGFβ signaling.20 We expanded our understanding of miR-128-3p in the progression of NSCLC.
MiRs negatively regulate gene expression post-transcriptionally by pairing to the 3ʹ-UTR of target mRNAs. We predicted the potential target gene of miR-128-3p and interestingly found that CDIP1 was one of the potential target genes of miR-128-3p. The reason we chose CDIP1 is that it is identified as a novel p53 target, and TNF-α-induced apoptosis is dependent upon CDIP.21 In our study, we indicated that miR-128-3p directly targeted CDIP1 and regulated its expression. The question whether IL-33 regulate CDIP1 expression via miR-128-3p naturally emerged. Further study confirmed our suspicion and indicated that IL-33 might exert its oncogenic function by inhibiting CDIP1 expression and thus reducing the apoptosis of NSCLC. For the first time, we found the interaction between IL-33 and miR in NSCLC and a novel underlying mechanism of IL-33 regulating CDIP1. However, the mechanism can be better explained with detection of apoptosis of NSCLC cells under IL-33 treatment.
In summary, our findings provide important insights into the mechanisms through which IL-33 promotes the proliferation of NSCLC cells by regulating miR-128-3p/CDIP1 signal pathway. The results extended our understanding of the function of IL-33 in preventing tumor progression and, in the meantime, expanded the regulation mechanism on CDIP1 and its downstream genes. Our findings may provide novel insights into the therapy methods in NSCLC.
The project was supported by Cadre Health Research Project of Jiangsu Province (BJI18033) and Youth Project of Nanjing Medical University Affiliated Cancer Hospital (ZN201603).
The authors declare that they have no conflicts of interest for this work.
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