lncRNA JPX/miR-33a-5p/Twist1 axis regulates tumorigenesis and metastasis of lung cancer by activating Wnt/β-catenin signaling
MicroRNAs (miRNAs) and Twist1-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination are well established, but the involvement of long noncoding RNAs (lncRNAs) in Twist1-mediated signaling remains largely unknown.
RT-qPCR and western blotting were conducted to detect the expression levels of lncRNA JPX and Twist1 in lung cancer cell lines and tissues. The impact of JPX on Twist1 expression, cell growth, invasion, apoptosis, and in vivo tumor growth were investigated in lung cancer cells by western blotting, rescue experiments, colony formation assay, flow cytometry, and xenograft animal experiment.
We observed that lncRNA JPX was upregulated in lung cancer metastatic tissues and was closely correlated with tumor size and an advanced stage. Functionally, JPX promoted lung cancer cell proliferation in vitro and facilitated lung tumor growth in vivo. Additionally, JPX upregulated Twist1 by competitively sponging miR-33a-5p and subsequently induced EMT and lung cancer cell invasion. Interestingly, JPX and Twist1 were coordinately upregulated in lung cancer tissues and cells. Mechanically, the JPX/miR-33a-5p/Twist1 axis participated in EMT progression by activating Wnt/β-catenin signaling.
These findings suggest that lncRNA JPX, a mediator of Twist1 signaling, could predispose lung cancer cells to metastasis and may serve as a potential target for targeted therapy.
KeywordsEpithelial-mesenchymal transition Twist1 Long noncoding RNA Wnt/β-catenin signaling Lung cancer
Competitive endogenous RNA
Dulbecco’s modified Eagle’s medium
Long noncoding RNA
miRNA response elements
Non-small cell lung cancer
Quantitative polymerase chain reaction
Serine hydroxymethyl transferase 2
Small interfering RNA
Lung cancer is one of the most malignant of all cancer, and the 5-year survival rates vary from 4 to 17% depending on stage and regional differences [1, 2]. Although many advances have been made in the diagnosis and treatment of lung cancer in recent years, metastasis still remains the main challenge posed by advanced lung cancer leading to high mortality . Thus, the elucidation of a new oncogenic pathway is required to precisely target lung cancer and to serve as a prognostic factor. Long noncoding RNAs (lncRNAs) are a class of RNA molecules longer than 200 nucleotides in length with considerable potential to drive cancer development [4, 5, 6, 7].
Aberrantly expressed lncRNAs have been found to be associated with the occurrence and development of various types of cancers [8, 9, 10]. In addition, lncRNAs affect gene expression through various mechanisms in which lncRNAs regulate their target genes by acting as microRNA (miRNA) sponges, thereby affecting the growth, proliferation, migration, and invasion of cancer cells [11, 12]. In the lncRNA-miRNA-mRNA regulatory network, lncRNAs act as competitive endogenous RNAs (ceRNAs) of specific mRNAs . Specifically, lncRNA SMAD5-AS1 could upregulate adenomatous polyposis coli expression by sponging miR-135b-5p and inactivate the canonical Wnt/β-catenin pathway to inhibit diffuse large B cell lymphoma proliferation . Furthermore, lncRNA PVT1 was found to regulate hexokinase 2 (HK2) expression by competitively binding to endogenous miR-143 in gallbladder cancer (GBC) cells, suggesting an important role of the PVT1/miR-143/HK2 axis in cell proliferation and metastasis by modulating aerobic glucose metabolism in GBC cells . However, the function and mechanism of most aberrantly expressed lncRNAs as ceRNAs in lung cancer remain unclear.
Our previous work showed that miR-33a-5p negatively regulated Twist1, thus inhibiting the invasion and metastasis of non-small cell lung cancer (NSCLC) , and served as a potential biomarker for early lung cancer diagnosis . Twist1 is an important transcription factor that mediates epithelial-mesenchymal transition (EMT) progression and tumor metastasis [18, 19]. In addition, Wnt/β-catenin signaling is a critical driver in EMT and cancer metastasis . It has been found that miRNAs can participate in the EMT process by regulating the Wnt/β-catenin pathway in a variety of cancers [21, 22]. Therefore, we considered whether an lncRNA could form a ceRNA network with miR-33a-5p and Twist1 to participate in EMT and malignant processes in lung cancer. In the present study, we identified that JPX, an upregulated lncRNA in lung cancer, acted as a ceRNA for Twist1 through binding with miR-33a-5p. As an oncogene, JPX affected the tumor size, TNM staging, and metastasis of lung cancer. Functionally, JPX promoted cell proliferation, migration, and invasion and facilitated tumor growth in xenograft mouse model. Further assays revealed that JPX participated in the activation of Wnt/β-catenin signaling by regulating miR-33a-5p/Twist1, which in turn promoted the EMT process, ultimately influencing the of lung cancer process. The results indicate that the JPX/miR-33a-5p/Twist1 axis regulates lung carcinoma by activating Wnt/β-catenin signaling, suggesting a therapeutic potential for lung cancer treatment.
Clinical subjects and specimens
Total of 116 lung cancer tissues and corresponding adjacent tissues were collected from the Affiliated Hospital of Medical School of Ningbo University (Ningbo, China) and Ningbo Medical Center Lihuili Eastern Hospital (Ningbo, China). All of the patients were diagnosed with primary lung cancer and did not receive preoperative radiotherapy, chemotherapy, targeted therapy and immunotherapy. At the same time, general clinical information and detailed pathology records were collected. All patients received written informed consent and the study protocol was approved by the Clinical Research Ethics Committee of the Medical School of Ningbo University (Approval No.: NBUSM20171006). All experimental protocols were implemented in accordance with relevant regulations.
RNA extraction and quantitative real-time PCR (RT-qPCR)
Total RNA was isolated from lung cancer tissue and cells using Trizol reagent (Invitrogen, USA) following the manufacturer’s protocol. DeNovix DS-11 Spectrophotometer (DeNovix, USA) detects the purity and concentration of RNA. We synthesized cDNA by reverse transcription reaction using a ReverTra Ace qPCR RT Master Mix with gDNA Remove kit (Toyobo, Japan) or a commercial miRNA reverse transcription PCR kit (GenePharma, China). RT-qPCR was conducted using a SYBR Premix Ex Taq II (Takara, Japan) on a Mx3005P real-time PCR System (Stratagene, USA) according to manufacturer’s instructions. Results were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 as an internal control. To account for the assessment of technical variability, the assays were performed in triplicate for each case. Primer sequences are shown in Additional file 1: Table S1.
Cell culture and transfection
All cell lines were obtained from the Chinese Academy of Sciences Cell Bank (CASCB, China), including 1 human normal bronchial epithelial cell (BEAS-2B) and 4 human lung adenocarcinoma cells (SPC-A-1, LTEP-a-2, A549, NCI-H1299). All human lung cancer cell lines were cultured in RPMI-1640 (Hyclone, USA), with 10% fetal bovine serum (PAN, Germany). BEAS-2B was maintained in Dulbecco’s modified Eagle’s medium (DMEM) that was supplemented with 10% FBS. All cell lines were placed in a cell culture incubator (Thermo Fisher, USA) containing 5% CO2 at 37 °C. JPX small interfering RNA (siRNA, GenePharma, China) and Twist1 siRNA with the corresponding control RNA (siRNA NC), or recombinant plasmid overexpressing JPX with the empty pcDNA3.1 vector (Tiandz, China), or miR-33a-5p mimics (GenePharma, China) with corresponding control RNA (mimics NC) were transfected into cells in logarithmic growth phase. The transfection was performed using the Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. The transfected sequences of the miR-33a-5p mimics and siRNA oligonucleotides are shown in Additional file 1, Table S2.
Recombinant plasmid construction
The sequences of JPX was amplified by PCR from the genomic DNA of SPC-A1 cell line, and sub-cloned into the pcDNA3.1 vector or pGL3-control vector (Promega, USA) as described in our previous work . The primer sequences are shown in Additional file 1, Table S1.
Cell counting Kit-8 (CCK-8) assay
The transfected cells were seeded in 96-well plates at a concentration of 5 × 103 per well at different time points (24, 48, 72, and 96 h), and 10 ml CCK-8 reagent (Dojindo, Japan) was added to each well after cell attachment, and cells were incubated at 37 °C for 2 h. We determined the cell growth rate by measuring their optical density (OD) value at 450 nm using a microplate reader (Labsystems, Finland).
Colony formation assay
The transfected cell suspension was collected, and 500 cells were seeded ito a 6-well plate and cultured in a cell culture incubator. After 2 weeks, the cell colonies were washed 3 times with 1 × PBS. Colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Solarbio, China) for 30 min.
Wound healing assay
The confluent cell monolayer was manually damaged by scraping the cells with a 200 μl pipette tip. Photographs were taken using an optical microscope (Olympus, Japan) at 0, 24, and 48 h, respectively. The distances were measured by Image-Pro Plus 6.0 software.
Transwell invasion assay
The transfected cells were collected and resuspended in serum-free medium. Then, 1 × 105 cells were seeded into a pre-packed Matrigel (BD Bioscience, USA) chamber (Corning, USA), and the chamber was inserted into a well containing 20% serum from 24-well plate. After 24 h incubation, the cells remaining on the upper membrane surface were removed using a cotton swab, and the cells adhering to the lower membrane surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were then counted under an optical microscope.
Nuclear and cytoplasmic RNA fractionation analysis
Nuclear and cytosolic fractions were separated using a PARIS kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. The expression levels of GAPDH, U6 and JPX in the nuclear and cytoplasm of lung cancer cells were detected by RT-qPCR assays.
Cell lysates and western blotting
We extracted the protein (including total, nuclear and cytoplasmic protein) of the cells using RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1%Triton X-100, and 1 protease inhibitor cocktail tablet/10 ml) and detected the protein concentration with a BCA kit (Beyotime, China). The western blotting was conducted as previously described . The primary antibodies were anti-E-cadherin (Bioss, USA), anti-N-cadherin (Santa Cruz, USA), anti-Vimentin (CST, USA), anti-GSK-3-β (Bioss, USA), anti-β-Catenin (CST, USA), anti-Twist1 (Sigma, USA), anti-GAPDH (Santa Cruz, USA), and anti-Lamin B (Bioss, USA).
The putative miRNA binding sites on JPX sequences were predicted using StarBase V3.0 (http://starbase.sysu.edu.cn/).
Luciferase reporter assay
JPX wild-type and mutant-type luciferase reporter vector targeting the miR-33a-5p binding site were constructed. The vectors and miR-33a-5p mimics were co-transfected into cells by Lipofectamine 2000 reagent, and luciferase activities were measured 24 h later using the dual luciferase reporter system (Promega, USA). Renilla luciferase activity was used as a standardized control.
In vivo tumorigenesis assay
Four-week-old BALB/c male nude mice were purchased from Shanghai SLAC Laboratory Animals Co., Ltd. (Shanghai, China). We screened lung cancer cells stably expressing JPX and empty plasmids using G418 and stably expressed miR-33a-5p by transfecting AgomiR-33a-5p (GenePharma, China). AgomiRNA is a specially labeled and chemically modified double-stranded small RNA that mimics the endogenous miRNA to regulate the biological function of the target gene. The subcutaneous xenograft mouse model was used to assess the tumor formation ability. First, 5 × 106 lung cancer cells (SPC-A-1 and NCI-H1299) stably expressing miR-NC/miR-33a-5p, and empty plasmid/JPX were suspended in 200 μL phosphate-buffered saline (PBS) and then were injected subcutaneously into the right flanks of BALB/c nude mice (single-factor experiment, n = 7 per group; rescue experiment n = 3 per group). The tumor dimensions were measured every two days via digital caliper measurements; after 4 weeks, the mice were sacrificed by cervical dislocation and the tumors were excised for weighing. The tumor volume was calculated with the formula V = (length × width2)/2. To determine lung cancer cell metastasis, SPC-A-1 and NCI-H1299 cells stably expressing miR-NC/miR-33a-5p, and empty plasmid/JPX were used to construct a tail vein metastasis animal model. Each BALB/c nude mouse was injected with 200 μL PBS containing 5 × 106 cells (n = 3 each group). All nude mice were sacrificed humanely after 30 days, and intact lung tissues were obtained f and imaged. The tissue sections were used for subsequent experiments, such as H&E staining. To prevent AgomiR-33a-5p degradation in vivo, we injected a dose of 1 nmol AgomiR-33a-5p (in 20 μL PBS) into the subcutaneous tumors and tail veins of nude mice weekly. The experiments were performed in accordance with the approved guidelines of the Laboratory Animal Ethical Committee at Ningbo University (Approval No.: NBULA20180902/ NBULA20191109).
Immunohistochemistry assay (IHC)
The paraffin-embedded tumor tissue sections were deparaffinized and rehydrated for IHC, and the antigen was retrieved with high pressure in 0.01 M sodium citrate buffer solution. After incubating with the primary and secondary antibodies, the sections were incubated with diaminobenzidine and counterstained with hematoxylin (Solarbio, China). Images were taken by a microscope with 200× magnification (Olympus, Japan). Primary antibody for IHC: anti-Twist1 (Bioss, USA), anti-β-catenin (Bioss, USA).
The statistical analyses were carried out with using GraphPad Prism 8 software. Data are presented as the mean ± SD, and all experiments were performed in triplicate. The relationship between JPX expression and the clinical characteristics of patients with lung cancer were evaluated using the chi-squared test. Analysis of differences between the two groups were performed using Student’s t test, one-way ANOVA, and Pearson’s correlation analysis. For all analyses, a P-value less than 0.05 from a two-tailed test was considered statistically significant.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
JPX was upregulated in lung cancer tissues and cells
Correlations between JPX and clinical characteristics of 116 lung cancer patients
lncRNA JPX level†
High and moderate
I + II
III + IV
JPX promoted lung cancer cell proliferation in vitro and facilitated lung tumor growth in vivo
JPX promoted migration and invasion of lung cancer cells
JPX acted as a sponge for miR-33a-5p
JPX promoted cell proliferation, migration, and invasion of lung cancer cells by regulating miR-33a-5p
JPX promoted lung tumor growth and metastasis in vivo by regulating miR-33a-5p
JPX and Twist1 were coordinately upregulated in lung cancer tissues and cells
JPX/miR-33a-5p/Twist1 axis participated in lung cancer cell EMT progression via the Wnt/β-catenin signaling
Together, the data demonstrated that JPX regulated miR-33a-5p/Twist1-mediated EMT progression by activating the Wnt/β-catenin signaling (Fig. 8j).
Although encouraging progress has been made in understanding the molecular mechanisms of lung cancer development, the prognosis of patients with advanced lung cancer remains unfavorable . Recent studies have shown that abnormally expressed lncRNAs are closely related to lung cancer occurrence and development [28, 29, 30]. Specifically, lncRNA HCP5 was found to be upregulated in lung adenocarcinoma (LUAD), resulting in increases of Snail and Slug to promote EMT progression by adsorbing miR-203 . As an early specific antisense lncRNA, SBF2-AS1 promoted LUAD tumorigenesis through substantially decreased miR-338-3p and miR-362-3p and substantially increased E2F1, and served as a prognostic marker and potential therapeutic target for LUAD . Based on our previous work, we have found that miR-33a-5p negatively regulated the target gene of Twist1 and participated in the EMT process of lung cancer cells. We combined an lncRNA microarray and bioinformatic prediction to screen out lncRNA JPX, which has potential binding sites with miR-33a-5p and is associated with lung cancer tumorigenesis. In this study, we showed that JPX was significantly upregulated in lung cancer tissues and cells. Importantly, JPX promoted lung cancer malignant processes and tumor growth in vivo. The results indicate that JPX plays an oncogenic role in lung cancer.
JPX is a molecular switch that inactivates the X chromosome [33, 34]. Recent study has shown that exosomal JPX from hepatocellular carcinoma (HCC) cells promotes XIST expression by inhibiting the function of CCCTC-binding factor (CTCF) in blood cells . Relevant research shows that human JPX and its mouse homolog of lncRNA Jpx have great differences in their nucleotide sequences and RNA secondary structures, but both lncRNAs show strong binding to CTCF, and human JPX can functionally compensate for the loss of Jpx in mouse embryonic stem cells . It has also been shown that JPX is lowly expressed in HCC and inhibits HepG2 cell growth or tumorigenesis in a XIST-dependent manner, revealing that JPX has a tumor-suppressing effect in HCC . In addition, highly expressed JPX shows poor prognosis; promotes the proliferation, invasion, and migration of human ovarian cancer cells, and inhibits cell apoptosis by activating the PI3K/Akt/mTOR signaling . Recently, there have been reports on JPX in lung cancer. As an oncogene, JPX is significantly upregulated in NSCLC tissues and is associated with poor prognosis; JPX upregulates cyclin D2 expression in the ceRNA mechanism by interacting with miR-145-5p, which stimulates NSCLC development and progression . These studies show that JPX plays different roles in different types of human cancers. On the one hand, JPX acts as an oncogene to promote the development of ovarian and lung cancer. On the other hand, JPX acts as a tumor suppressor gene to inhibit HCC development. In our study, we found for the first time that JPX was highly expressed in lung cancer patients and was significantly linked to tumor size and TNM stage. Interestingly, JPX expression was higher in patients with advanced lung cancer than in those with early lung cancer. Consistently, JPX expression was also higher in patients with metastatic lung cancer than in those without metastasis. We further revealed a negative correlation between JPX and miR-33a-5p in lung cancer patients. The miR-33a-5p-induced inhibition of cell growth and metastasis could be restored by JPX overexpression. Additionally, Twist1,a target of miR-33a-5p, was found to be coordinately upregulated with JPX in lung cancer. These findings indicate that JPX plays an oncogenic role via its interaction with miR-33a-5p and Twist1.
In the past decade, ceRNAs have become a very important class of post-transcriptional regulators that affect tumor occurrence and development by altering the corresponding gene expression through miRNA-mediated mechanism . As a type of ceRNA, lncRNAs can act as molecular sponges to adsorb miRNAs through the same miRNA response elements (MREs), thereby regulating their target genes and ultimately affecting tumor progression. For instance, lncRNA CA7–4 regulated the autophagy and apoptosis of vascular endothelial cells by inducing miR-877-3p and miR-5680 under high-glucose condition . Similarly, LINC01234 acted as a ceRNA of miR-642a-5p, resulting in the suppression of the endogenous serine hydroxymethyl transferase 2 (SHMT2), suggesting that the LINC01234-miR642a-5p-SHMT2 axis plays a key role in colon cancer . There are also other lncRNAs that play crucial roles as ceRNAs in cancers development [43, 44]. The present work demonstrated that lncRNA JPX, miRNA-33a-5p, and Twist1 constituted a ceRNA network to regulate lung cancer growth and metastasis. However, the ceRNA hypothesis is still in the verification stage, and much research is needed to identify the abundance of the three components and verify the functional activities of ceRNAs.
It has been reported that EMT and the associated Wnt/β-catenin pathway can be important drivers of tumor growth and metastasis . Twist1 is a member of the basic helix-loop-helix transcription factor family and is an important transcription factor that induces EMT, migration and invasion in cancer cells [18, 19]. Furthermore, Twist1 is highly expressed and acts as an oncogene in many invasive types of cancers, such as lung cancer , breast cancer , and HCC . Additionally, studies have shown that Twist1 affects the cancerous behavior of tumor cells via the Wnt/β-catenin pathway . In the current study, we found that JPX could increase Twist1 expression by adsorbing miR-33a-5p, thereby activating Wnt/β-catenin signaling pathway to promote EMT progression in lung cancer cells. However, whether Twist1 participates in the Wnt/β-catenin pathway directly or indirectly, and whether JPX affects Twist1 expression or regulates other pathways through RNA-binding proteins to promote lung cancer development remain unclear. The above speculation needs further investigation. Importantly, recent studies have shown that lncRNA is closely related to the stemness of cancer cells . For example, lncRNA CCAT1 which is notably upregulated in breast cancer stem cells (BCSCs) and contributes to the stemness of BCSCs ; lncRNA SPRY4-IT1 increases TCF7L2 expression by targeting miR-6882-3p, thereby promoting breast cancer cell proliferation and stemness as well as BCSC renewal and maintenance ; and lncRNA LOXL1-AS1 facilitates the stemness of gastric carcinoma through regulating the miR-708-5p/USF1 axis . Although we have not tested the function of JPX in lung cancer stem cells at present, our results provide a deeper understanding of the role of JPX in lung cancer cells as well as a new direction for future research.
In summary, our results demonstrate that the lncRNA JPX/miRNA-33a-5p/Twist1 axis may act as a new ceRNA regulatory network, participating in the EMT process by activating the Wnt/β-catenin signaling pathway, thus accelerating the malignant processes of lung cancer. These findings suggest that JPX may serve as a potential therapeutic target and a novel biomarker for the precise treatment of lung cancer.
We thank Dr. Hui-Kuan Lin (Department of Cancer Biology, Wake Forest University School of Medicine, USA) for helpful discussion on the manuscript.
Z.G. and J.P. developed the concepts for the manuscript and designed the study. J.P., H.T., and S.F. performed the experiments on RT-qPCR, Western blot, cell phenotype and in vivo experiments. C.Z., H.T., J.H., and W.S. collected the clinical samples and performed the clinicopathologic analyses. J.P., Z.G., X.M., and X.J. performed the data analysis and discussed results. J.P. prepared the manuscript and all authors contributed to editing the paper. All authors read and approved the final manuscript.
This work was supported in part by research grants from the Non-profit Technology Research Program of Zhejiang (LGF18H160006), the Natural Science Foundation of Zhejiang (LQ18H200001), the Non-profit Technology Research Program of Ningbo (2019C50040), the Natural Science Foundation of Ningbo (2018A610204), the Scientific Innovation Team Project of Ningbo (2017C110019) and the K.C. Wong Magna Fund in Ningbo University.
Ethics approval and consent to participate
Human lung primary cancer tissue specimens and cancer-adjacent tissues were collected from the patients underwent the radical surgery of lung cancer or palliative resection of lung cancer at the Affiliated Hospital of Medical School of Ningbo University and Ningbo Medical Center Lihuili Eastern Hospital. Written informed consent was obtained from all patients and the study protocol was approved by the Clinical Research Ethics Committee of Medical School of Ningbo University (Approval No.: NBUSM20171006). All methods were performed in accordance with relevant guidelines and local regulations.
Consent for publication
The authors have no conflicts of interest to disclose.
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