1 Introduction
Lung cancer is the leading cause of cancer-related deaths, resulting in 1.8 million deaths in 2020 worldwide [
1]. The overall survival (OS) time of patients with driver gene mutations and advanced lung cancer is only 10–12 months in the absence of specific medical intervention. With targeted therapies using drugs that modulate the activity of genes or proteins involved in cancer cell growth and survival, the median OS can be extended to more than 3 years. Lung adenocarcinoma (LUAD) is a type of non-small-cell lung cancer (NSCLC) and accounts for about 40% of lung cancers [
2]. It is generally considered that LUAD shows distinct genomic alterations with respect to other NSCLC subtypes [
2,
3]. Prolonging the survival of patients and improving their quality of life via targeted therapies are undoubtedly beneficial in the treatment of lung cancer. Thus it is critical to understand the molecular mechanism regulating its occurrence and progression for the identification of novel therapeutic targets.
There is increasing evidence suggesting a key role of post-transcriptional control mediated by non-coding RNAs in the pathogenesis and development of various cancers [
4]. Long non-coding RNAs (lncRNAs) are critically involved in the regulation of gene expression in lung cancers by acting as both tumor suppressors and oncogenes [
5]. Thus, they are promising biomarkers and therapeutic targets for lung diseases [
6]. The IncRNA
LINC01279 was originally identified as a down-regulated non-coding gene during osteogenic differentiation [
7]. Subsequent works suggested that its function could be associated with the pathogenesis of endometriosis [
8,
9]. Interestingly, more recent studies indicated a correlation between
LINC01279 expression and progression of gastric cancer [
10], suggesting a possible function of this IncRNA in regulating tumor growth. However, whether it plays a role in other cancers is unknown. In particular, the molecular and cellular mechanisms by which it functions to regulate tumorigenesis remain unclear.
In this study, we report the function of LINC01279 in the progression of LUAD. Using clinical samples, xenografts and NSCLC cell lines, we show that the expression of LINC01279 is significantly up-regulated in LUAD tumor tissues and NSCLC cell lines. Functional analyses indicate that suppression of LINC01279 decreases the expression of focal adhesion kinase (FAK) and extracellular-regulated kinase (ERK) signaling. Moreover, we demonstrate that LINC01279 complexes with and stabilizes the transcriptional co-repressor SIN3A. Knockdown of LINC01279 or SIN3A activates autophagy and apoptosis in NSCLC cells. Importantly, inhibition of LINC01279 function reduces tumor growth in xenografts derived from NSCLC cells. These observations suggest that LINC01279 displays pro-tumor function and plays an important role in the development of lung cancer. Its expression may be considered as a predictive factor of LUAD. Our findings provide insights into the mechanism underlying LINC01279-mediated lung tumorigenesis. They may also help to identify potential therapeutic targets for cancer diagnosis and prognosis.
2 Materials and methods
2.1 Ethical statement
This project was approved by the Research Ethics Committee of the Affiliated Hospital of Guangdong Medical University. All experiments using animals were performed in accordance with the ARRIVE guidelines. Clinical tissues were collected with written informed consents of each patient.
2.2 Collection of clinical tissues
Lung carcinoma tissues and adjacent noncancerous tissues were collected from 90 consecutive patients with LUAD. These patients were subjected to curative resection between September 2014 and September 2016 at the Department of Thoracic Surgery of the Affiliated Hospital of Guangdong Medical University (Zhanjiang, China). Tissue blocks were selected from lung cancer tissue specimens and analyzed with respect to the clinicopathological and follow-up data of patients. The histopathological diagnosis was based on the standard of the World Health Organization.
2.3 Cell lines and transfection
H1299, H1650, H838 and PC-9 cells (human NSCLC cell lines with different metastatic potentials) were purchased from Kobio Biology (Nanjing, China) and tested routinely for mycoplasma contamination as described previously [
11]. They were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C with 5% CO
2 in a humidified atmosphere. Cell transfection was performed using the Lipofectamine
® RNAiMAX Reagent (Invitrogen) according to the manufacture’s instructions.
For OS analysis, HLugA180Su06 LUAD tissue microarray with clinical pathological data and survival information (Shanghai Outdo Biotech Biotechnology, China) were used to examine LINC01279 expression levels. This microarray contains 94 tumors and 86 paired adjacent normal tissues, which were collected from patients subjected to surgical resection from July 2004 to June 2009. The follow-up of these patients lasted 5 to 10 years until August 2014.
2.4 Knockdown by siRNA and shRNA
For siRNA-mediated knockdown, cells were plated at a desired density and further cultured for 12 h to 24 h. They were then transfected with gene-specific siRNA or a non-targeting siRNA (Supplementary Table S1) at a final concentration of 10 μM, using the Lipofectamine® RNAiMax Reagent (Invitrogen) in OptiMEM medium according to the manufacturer’s instructions. For shRNA-mediated knockdown, lentiviral LV10-U6/RFP&Puro shuttle vector (GenePharma, Shanghai, China) was used to allow the expression of LINC01279-specific shRNAs or a non-targeting shRNA (Supplementary Table S1). Cells were cultured and transfected as above.
2.5 Tumor xenografts
Five-week-old male BALB/C nude mice were maintained under specific pathogen-free conditions. Stably transfected H1299 cells (5 × 106 cells in 200 µL of PBS) were implanted into the armpit on both sides of the mouse. Xenografts were examined every 3 days with a digital caliper and tumor volumes were calculated using the formula (length x width2)/2. After 27 days, mice were sacrificed, and tumor samples were embedded in paraffin for immunohistochemistry labeling using Ki-67 antibody, followed by hematoxylin and eosin staining.
2.6 Cell proliferation, transwell migration and invasion assays
Cell proliferation test was performed using the WST-1 kit (Beyotime Biotechnology, Shanghai, China). NSCLC cells were plated in 96-well plates at a density of 1000 cells/well and cultured for 24 h. They were transfected with siRNA and further cultured for 96 h. The optical density was measured at 450 nm after adding 10 μL of WST-1 solution to 100 μL of RPMI-1640 medium.
Cell migration and invasion were determined by transwell migration assay and matrigel invasion assay (BD Falcon, San Jose, CA, USA), according to the manufacture’s instructions. Briefly, transwell migration assay was performed by suspending 5 × 104 cells in 200 µL of serum-free RPMI-1640 medium and placing them in the cell culture insert (8 µm pore size) of a plate containing pre-warmed RPMI-1640 medium with 20% fetal bovine serum. Cells were cultured for 12 h and fixed with 4% paraformaldehyde. For matrigel invasion assay, 1 × 105 cells were placed in the cell culture insert precoated with matrigel. Following addition of pre-warmed RPMI-1640 medium with 20% fetal bovine serum to the well, they were further cultured for 24 h and fixed with 4% paraformaldehyde. Cells were stained with 0.1% crystal violet and random regions were imaged using an optical microscope.
2.7 Reverse transcription and quantitative PCR (RT-qPCR)
Total RNAs were extracted from cells or tissues using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Cytoplasmic and nuclear RNAs were prepared using the RNA Subcellular Isolation Kit (Active Motif) as described previously [
7]. Reverse transcription of total RNAs (500 ng) was performed using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) in the presence of random primers. qPCR was performed using the Roche LightCycler
® 480 System and SYBR Premix Ex Taq (TaKaRa, Dalian, China), in the presence of gene-specific primers (Supplementary Table S2).
Cells were digested with trypsin and seeded in 6-well plates with a density of 200 cells/well and cultured at 37 °C. After 10 days, the culture medium was removed, and cells were washed twice with PBS. They were fixed with methanol for 20 min, and colonies were stained with crystal violet for 20 min. After washing excess of crystal purple with PBS, the plates were dried and imaged for colony counting.
2.9 Flow cytometry assays of apoptosis
Fluorescein Isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BD Biosciences) was used to detected apoptosis. After 48 h of transfection, cells were collected and washed with PBS. They were resuspended in the binding buffer and stained with 5 µL of Annexin V-FITC and propidium iodide in the dark for 15 min. Cells were sorted by flow cytometry and analyzed using the BD FACSDiva6.1 software (BD Biosciences).
2.10 In situ hybridization
Lung tissues were fixed in 4% formaldehyde overnight at 4 °C, and paraffin sections of 10 µm were prepared using a microtome (Leica, RM2125RTS). Digoxigenin-labeled LINC01279 probe was synthesized using the DIG RNA labeling Kit (Roche). After hybridization and incubation with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche), LINC01279 signals were visualized using NBT/BCIP as a substrate.
2.11 Western blot
Cells were lysed using the RIPA buffer in the presence of a mixture of protease inhibitors. Proteins were separated by SDS-PAGE and transferred to the polyvinylidene fluoride membrane. Non-specific antibody binding was blocked using 5% skimmed milk for 1 h, and incubation with primary antibodies (Supplementary Table S3) was performed at 4 °C overnight. After washing several times with TBST and incubation with secondary antibodies for 2 h, protein bands were detected using Luminate Western HRP substrates (Millipore) and Tanon 5200 chemiluminescence imaging system (Shanghai, China).
2.12 RNA immunoprecipitation (RIP) assay
This was performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). Cells were lysed in 100 µL of RIP lysis buffer containing protease inhibitor cocktail and DNase inhibitor. RNA–protein complexes were immunoprecipitated with SIN3A antibody (5 µg) or control IgG at 4 °C overnight. The magnetic beads were washed 6 times in washing buffer, and proteins were digested using protease K at 55 °C. Precipitated RNAs were analyzed by qPCR after reverse transcription in the presence of random primers.
2.13 Analysis of fluorescent-tagged LC3 punctae
A tandem fluorescent-tagged LC3 construct was used to monitor the formation of GFP-LC3 and RFP-LC3 punctae [
12]. Plasmids were transfected into H1299 and PC-9 cells using the Lipofectamine
® RNAiMAX Reagent (Invitrogen). After 48 h, cells were washed with PBS and incubated with Earle's balanced salt solution (E2888, Sigma) for an appropriate period. The formation of fluorescent punctae was visualized by confocal microscopy.
2.14 Statistical analysis
All data were collected from three independent experiments. They were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA) and expressed as mean ± SD. Survival data were calculated using the Kaplan–Meier method and analyzed using the log-rank test. Unless specified, Student’s t-test was performed to determine statistical significance. P value < 0.05 was considered as statistically significant.
4 Discussion
There is increasing evidence suggesting that dysregulation of lncRNAs occurs in various diseases and critically contributes to cancer development [
43,
44]. In this work, we have demonstrated that
LINC01279 is up-regulated in LUAD and is associated with cancer progression. It is involved in the migration and invasion of NSCLC cells through regulation of FAK and ERK signaling. Furthermore, we also found that it complexes with the tumor suppressor transcriptional co-repressor SIN3A and regulates the expression of this protein. Knockdown of
LINC01279 and SIN3A inhibited cell proliferation by inducing autophagy and apoptosis. Importantly, inhibiting the function of
LINC01279 prevented tumor progression in xenografts derived from NSCLC cell lines. These observations strongly implicate
LINC01279 in lung cancer development, making it a potential target for diagnosis and treatment of this cancer.
Based on the results from knockdown approaches using cultured NSCLC cell lines and xenografts derived from these cells, we conclude that increased expression of
LINC01279 promotes LUAD oncogenesis by regulating several proteins involved in cell migration and proliferation. Analyses of
LINC01279-depedent protein expression changes indicate that FAK and ERK signaling pathways are possible targets of
LINC01279 in LUAD cells. FAK is a non-receptor tyrosine kinase highly expressed in cancer. It acts as a multi-functional regulator of cell signaling in the tumor microenvironment and controls cell migration and invasion through kinase-dependent and -independent mechanisms [
29,
45]. Knockdown of
LINC01279 decreased FAK protein levels but had no effect on the stability of FAK mRNA, thus it is likely that
LINC01279 modulates FAK expression through translation of the mRNA, thereby influencing integrin-mediated cell adhesion. ERK/MAPK (mitogen-activated protein kinase) signaling pathway has been also implicated in multiple cellular processes such as proliferation, migration and apoptosis. There are many lines of evidence indicating that FAK can mediate phosphorylation of ERK and activation of the ERK pathway [
13‐
16]. We found that knockdown of
LINC01279 had little effect on the expression of total ERK protein but significantly reduced p-ERK level. However, knockdown of FAK led to reduced expression of ERK mRNA and protein in different NSCLC cell lines. These observations suggest that FAK should mediate the activity of
LINC01279 and functions upstream of ERK to regulate cancer cell proliferation and migration. Nevertheless, how does FAK regulate ERK expression requires further investigation.
SIN3A, a member of the SIN3 family, functions as a component of the histone deacetylase (HDAC) complex and regulates key cellular processes linked to cancer pathogenesis and progression [
33]. However, the exact role of SIN3A in tumorigenesis remains elusive. Previous studies suggested that SIN3A could function as a tumor suppressor. It regulates the expression of several genes involved in cell invasion [
30,
31]. Recent works showed that it cooperates with STAT3 to repress the transcription of tumor suppressor genes, thus interfering with its expression leads to transcriptional derepression, resulting in increased tumor cell death and reduced tumorigenic potential of anaplastic large-cell lymphoma [
32]. This oncogenic activity of SIN3A is consistent with our present observations. We made the interesting finding that SIN3A protein associates with and is stabilized by
LINC01279. Moreover, we found that knockdown of
LINC01279 or SIN3A in NSCLC cells similarly induced autophagy and led to apoptosis, suggesting that they normally function to inhibit these processes. Although future studies will be needed to determine how
LINC01279 regulates SIN3A protein level, the present results suggest that increased expression of
LINC01279 in LUAD likely prevents cell death-related autophagy through regulation of SIN3A protein stability.
It is well known that cancer cells exhibit autophagy-dependent cell death, which is linked to apoptosis [
46]. In the present study, we showed that suppression of
LINC01279 enhanced autophagy flux since LC3-II level further increased in the presence of chloroquine. Therefore, inhibiting the activity of
LINC01279 can induce autophagy and apoptosis, thereby preventing progression of LUAD. By contrast, increased expression of this IncRNA should lead to inhibition of autophagy-dependent cell death, thereby promoting proliferation of LUAD. Thus, our results imply that
LINC01279 may normally function to promote cancer development by inhibiting autophagy-dependent cell death. Whether
LINC01279 directly regulates autophagy-related proteins still needs further investigations. However, we found that SIN3A displays a similar activity as
LINC01279 in regulating the expression of autophagy-related proteins. It is of note that autophagy exhibits dual functions during tumorigenesis, often in a stage-dependent manner. It may act as a tumor suppressor that inhibits the expression of oncogenic proteins at early stages or may exert pro-tumor activity by promoting tumor cell survival at advanced stages [
47]. Since
LINC01279 interacts with SIN3A, its may exert pro-tumor activity through regulation of SIN3A expression. In this regard, it has been shown recently that the SIN3A transcriptional repressor complex interacts with STAT3 and is involved in silencing tumor repressor genes, thus promoting cell survival in different cancers [
32]. Our results showing that knockdown of SIN3A activates autophagy and attenuates cell proliferation supports a pro-tumor activity of this epigenetic regulator.
The relationship between
LINC01279-regulated autophagy and apoptosis remains unclear and needs further investigations. Knockdown of
LINC01279 increased p53 protein levels and cleavage of PARP, which are hallmarks of apoptosis. This may be both dependent and independent of autophagy-induced cell death. Indeed, there exists a close interplay between autophagy and apoptosis, which may be regulated by common signaling pathways [
46]. Results from the present study suggest that
LINC01279 may be an important regulator of both processes. We found that either high or low expression of
LINC01279 is correlated with a reduced OS rate in patients with LUAD. There is also evidence that upregulation of
LINC01279 may be related to tumor invasion in gastric cancer [
10]. Thus, it may represent a potential therapeutic target to prevent tumor growth. Indeed, we showed that suppression of
LINC01279 efficiently prevented tumor growth in xenografts derived from NSCLC cells.
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