Background
Oct4, encoded by
POU5F1 (POU domain, class 5, transcription factor 1), is a homeodomain transcription factor of the POU family. Oct4, Sox2 and Nanog are well-known pluripotency-associated transcription factors which maintain embryonic stem cells state [
1]. Metaplastic transformation, a precancerous condition, has been reported to recapitulate embryonic development. Therefore, the key factors involved in embryonic development may play critical roles in carcinogenesis. Studies have shown that Oct4 is overexpressed in human cancers such as bladder [
2], breast [
3], cervical cancer [
4], oral squamous cell carcinoma [
5], hepatocellular carcinoma [
6] and lung cancer [
7,
8]. In embryonic stem cells, Oct4 has been identified to regulate transcriptions of other transcription factors, chromatin modifiers, long non-coding RNAs (lncRNAs) and microRNAs [
9,
10]. For instance, Oct4 regulates lncRNAs expression, such as linc-RoR, which is a key reprogramming factor associated with pluripotency [
11]. Oct4 can also interact with Pontin, a chromatin remodeling factor, to regulate the transcription of lncRNAs, including linc1253, a lineage programme repressing lincRNA [
12]. However, transcription regulation of lncRNAs by Oct4 in tumorigenesis remains elusive.
LncRNAs is a subset of non-coding RNAs with length ranging from 200 nucleotides to 100,000 nucleotides. According to data obtained using next generation RNA-sequencing, the number of total human lncRNAs is approximately 20,000 transcripts and over 200 lncRNAs are confirmed to be functional [
13,
14]. Some lncRNAs are dysregulated in cancers and may serve as potential prognostic markers for specific cancer types [
15,
16]. Some lncRNAs have been characterized to possess oncogene-like or tumor suppressor-like function. For instance,
Hox Antisense Intergenic RNA (HOTAIR), acts as a bridge between PRC2 chromatin repressive and LSD1/CoREST/REST corepressor complexes to further modulate the metastasis-related gene expressions through changing chromatin states in breast cancer [
15,
17]. Another lncRNA,
HOXA transcript at the distal tip (HOTTIP), not only promotes pancreatic cancer progression but also confers chemoresistance to gemcitabine, which may be mediated by HOXA13 [
18,
19]. Accumulating evidence indicates that lncRNAs play critical roles in cancer biology.
Up to date, most of the studies on lncRNAs focus on the outcome and underlying mechanisms of dysregulated lncRNAs and their potential as prognosis markers. However, little is known about the upstream regulations responsible for aberrant expression of lncRNAs in cancers, especially at the transcriptional level. Our previous study using chromatin-immunoprecipitation sequencing (ChIP-seq) and functional analyses revealed a critical Oct4-driven transcriptional program [
8]. Genome-wide analysis of Oct4 targeting of this program suggests a novel role of Oct4-mediated transcriptional regulation of lncRNAs. In the current study, we have shown that Oct4 transcriptionally activated oncogenic lncRNAs expression through promoter- or enhancer-binding regulation. Moreover, Oct4-mediated high expression of lncRNAs such as
nuclear paraspeckle assembly transcript 1 (
NEAT1) and
metastasis-associated lung adenocarcinoma transcript 1 (
MALAT1) promoted lung cancer cell proliferation, migration and invasion abilities. Clinical studies further validated the importance of Oct4/
NEAT1/
MALAT1 signaling axis in lung cancer progression.
Methods
Cell lines and culture conditions
Human lung adenocarcinoma cell line A549 and normal bronchial epithelial cell line BEAS-2B was purchased from American Tissue Culture Company (ATCC). Human lung adenocarcinoma cell line CL1–0 was obtained from Dr. Pan-Chyr Yang (Department of Internal Medicine Medical College, National Taiwan University, Taiwan). All media were supplemented with 10% Fetal Bovine Serum (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco). Stable cell line expressing Oct4 or empty vector was established by ectopic transfection of Flag-Oct4 or empty vector plasmid into A549 and CL1–0 cells, and selected with puromycin. Transient transfections of Oct4 in BEAS-2B were carried out with lipofetamine 2000 (Invitrogen, Carslbad, CA, USA).
Transfection of plasmids and RNAi
The plasmids used in the study are listed in Additional file
1: Table S1. The interference RNA (RNAi) for Oct4 was obtained from Invitrogen (# Oct4-HSS143403, Invitrogen). Depletion of
NEAT1 or
MALAT1 was performed by transfection of smart-pool siRNAs (Dharmacon, Lafayette, CO, USA) at final concentration of 10 nM. Transfections of expression plasmids and RNAi were performed using lipofectamine 2000 (Invitrogen, Carslbad, CA, USA) according to the manufacturer’s protocol.
Chromatin-immunoprecipitation-polymerase chain reaction (ChIP-PCR) assay
Empty vector control and Oct4 stably-overexpressed A549 cells (1 × 10
7 cells) were cross-linked with 1% formaldehyde for 10 min at 37 °C, followed by preparation of nuclear lysates using Magna ChIP™ protein G Kit (Millipore Co., Billerica, MA, USA). Nuclear lysates were sonicated to shear crosslinked DNA to around 300 ~ 500 bps using Covaris-S2 machine. Chromatin was immunoprecipitated with Oct4 antibody (1:100, # ab-19857, Abcam, Cambridge, UK). Purified chromatin-immunoprecipitated DNA was subjected to PCR analysis using primers for the lncRNA promoter and enhancer regions listed in Additional file
1: Table S2.
RNA extraction and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays
Four μg of total RNA was reverse transcribed to cDNA using MultiScribe™ reverse transcriptase (Applied Biosystems, Foster City, CA, USA). cDNA was amplified using the Fast SYBR® Green Master Mix (Applied Biosystems). qRT-PCR was used to measure
Oct4 mRNA and lncRNA expression using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). The primer sequences and annealing temperature are listed in Additional file
1: Table S3.
Site-directed mutagenesis and luciferase promoter/enhancer activity assays
Mutations (Mut) of Oct4 binding elements within the
NEAT1 promoter,
MALAT1 enhancer or
urothelial carcinoma-associated 1 (UCA1) enhancer were generated by site-directed mutagenesis using wild-type (WT)
NEAT1 promoter,
MALAT1 enhancer or
UCA1 enhancer vectors as templates. The primers used are described in Additional file
1: Table S4.
For luciferase activity assays, cells were seeded the day before transfection. The pGL4-Renilla construct was included as an internal control. After 16 h of co-transfection with empty vector or gene promoter/enhancer vector, and pGL3-Basic or pGL4-Renilla, the dual luciferase reporter assay kit (Promega, Madison, WI, USA) was used to determine gene promoter or enhancer activity. The luminescence was measured with a Turner BioSystems luminometer (Promega). The data are represented as the means of ratio of firefly luciferase to Renilla luciferase activity by triplicate experiments.
RealTime-Glo viability assay
Cell viability was assayed using RealTime-Glo assay (Promega). Briefly, cells were transfected for 24 h and then reseeded at 2 × 103 cells/well in 96-well plates. MT Cell Viability Substrate and NanoLuc Enzyme were diluted and added to each well. The luminescence was measured with a Turner BioSystems luminometer (Promega) at 24, 48 and 72 h.
Transwell migration and invasion assay
The transwell insert with millipore membrane (pore size of 8 μm, Falcon, BD Franklin Lakes, NJ, USA) was used. For transwell migration assay, 2 × 105 A549 cells and 5 × 105 CL1–0 cells were seeded onto the upper chamber with 1 ml serum-free medium. For transwell invasion assay, the transwell inserted membranes were pre-coated with Matri-gel (2.5 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) 1 day before seeding cells. Complete medium with 20% FBS was supplemented into the lower chamber as chemoattractants. The cells were incubated for 16 ~ 24 h and then the cells attached on the reverse side of the membrane were then fixed and stained. Six random views were photographed and quantified under an upright microscope (Nikon E400, Yurakucho, Tokyo, Japan).
Study population
We recruited 124 lung cancer patients from National Cheng Kung University Hospital after obtaining appropriate institutional review board permission and informed consent from the patients. Surgically resected tumor tissue and corresponding normal tissue samples were collected. Total RNA of patient samples were prepared using Trizol reagent (Invitrogen) and reverse transcribed into cDNA as described above. qRT-PCR was conducted to measure the expressions of
Oct4,
NEAT1 and
MALAT1 using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). The expression of the target genes was normalized based on the levels of internal control gene,
GAPDH. The primers used for qRT-PCR analyses are described in Additional file
1: Table S3.
Statistical analysis
Pearson χ2 test was used to compare the correlation of Oct4 and lncRNAs expression and clinicopathological parameters in lung cancer patients. Overall survival curves were calculated according to the Kaplan-Meier method, and comparison was performed using the log-rank test. Two-way ANOVA and two-tailed Student’s t-test was used in cell and animal studies. Data represent mean ± SEM. P < 0.05 was considered to be statistically significant.
Discussion
In the present study, we have revealed that Oct4 binds to the genomic loci of lncRNAs through ChIP-seq and bioinformatic analysis (Fig.
1). We then validated that Oct4 bound on the promoter or enhancer regions of lncRNAs (Fig.
1). Dual luciferase activity assay further confirmed that Oct4 potentiated promoter activity of
NEAT1 and enhancer activities of
MALAT1 and
UCA1 lncRNAs (Fig.
2). Moreover,
NEAT1 and
MALAT1 acted as downstream effectors of Oct4 to promote proliferation, migration and invasion abilities of A549 lung cancer cells (Figs.
3 and
4). Of note, positive correlations between
Oct4 mRNA and
NEAT1/
MALAT1 lncRNAs were evident in lung cancer patient specimens (Fig.
5). Our study provides new evidence that Oct4 transcriptionally regulates lncRNAs expression by targeting their promoter or enhancer regions.
NEAT1 and
MALAT1 function as Oct4 downstream mediators to promote lung cancer proliferation, migration and invasion (Fig.
5e).
Recently, the roles of
NEAT1 in cancer have been uncovered. Studies have reported that
NEAT1 is upregulated in prostate cancer, colorectal cancer and lung cancer, and thus associated with poor prognosis in these cancer patients [
16,
26‐
28]. Rubin and associates demonstrated that estrogen receptor transcriptionally activates
NEAT1 expression to promote prostate tumorigenesis under the treatment of oestrogen [
16].
NEAT1 has also been shown to modulate prostate cancer-specific gene expression through chromatin modifications and thus contributes to cancer progression [
16]. However, the upstream mechanisms of
NEAT1 overexpression in cancers await to be uncovered. It is until recently that HIF-2α is demonstrated to transactivate
NEAT1 transcription under hypoxia, which promotes the formation of paraspeckles, accelerates tumor proliferation and cancer cell survival leading to poor prognosis in breast cancer patient [
29]. In our study, we have shown that a well-known stemness transcription factor Oct4 transcriptionally upregulates
NEAT1 expression through binding to Oct4 consensus binding element on promoter region (Figs.
1 and
2a), and therefore promoting lung cancer proliferation and motility (Figs.
3 and
4). Notably,
NEAT1 is found overexpressed in BRCA1-deficient breast cancer and promotes self-renewal abilities in breast cancer cells through epigenetically suppressing miR-129-5p, which targets to Wnt4 [
30]. The last-mentioned study together with our results revealed a potential role of
NEAT1 in maintaining stemness properties and suggested that Oct4-mediated
NEAT1 upregulation may play critical roles in embryonic or cancer stemness maintenance.
MALAT1 was first identified as a prognosis marker in early-stage metastasizing lung cancer [
31].
MALAT1 knockdown in lung cancer cells decreases cell migration abilities [
32]. In addition,
MALAT1 suppresses expression of anti-metastasis genes such as
MIA2 (melanoma inhibitory activity 2) and
ROBO1 (roundabout 1), while induces pro-metastasis genes including
LPHN2 (latrophilin 2) and
ABCA1 (ATP-binding cassette, sub-family A, member 1) to accelerate metastasis [
33]. However,
MALAT1-promoting lung cancer cell proliferation in different studies are contradictory. For example,
MALAT1 has no effect on cell proliferation in vitro and slightly promotes tumor growth in vivo [
33]. In contrast, knockdown of
MALAT1 in A549 lung cancer cells decreased proliferation [
34], which is consistent with our results that
MALAT1 plays a role in lung cancer cell proliferation (Fig.
3b and d) and this provides new insight into the role of
MALAT1 in various cancer types.
MALAT1 has been demonstrated to promote lung, bladder, colorectal, liver, oral and prostate cancer cells proliferation and migration [
32,
33,
35‐
40]. However, the upstream regulatory mechanisms of
MALAT1 expression remain unclear, especially at the transcription level. Recently, Sp1 is found to transcriptionally activate
MALAT1 expression through targeting the promoter region and the Sp1-
MALAT1 axis may play a critical role in cancers [
34]. In addition, Wnt signaling pathway acts upstream of
MALAT1 transcription, which is mediated by TCF4 binding on
MALAT1 promoter in endometrioid endometrial cancer [
41]. Importantly, our results provide the first evidence of Oct4-mediated
MALAT1 upregulation through enhancer regions.
Acknowledgements
We are grateful to Professors Ying Jin, Tetsuro Hirose and Kannanganattu V. Prasanth for generous help with the Oct4, NEAT1 and MALAT1 expression vectors. We thank the Human Biobank, Research Center of Clinical Medicine, National Cheng Kung University Hospital for providing the lung cancer specimens.