lncRNA PVT1 Promotes Metastasis of Non-Small Cell Lung Cancer
Through EZH2-Mediated Activation of Hippo/NOTCH1 Signaling
Although growing evidences have showed that long non-coding RNA (lncRNAs) plasmacytoma variant
translocation 1 (
Materials and Methods:
In this experimental study, Quantitative reverse transcription polymerase chain reaction (qRTPCR) was used to profile the expression of
These results showed that
These results suggested that lncRNA
Lung cancer is the leading cause of cancer-related death worldwide (1). The most common type of that is non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer new cases (2). The average 5-year survival rate of NSCLC cancer patients is still very low, because of the limited therapeutic options, I addition to the higher rate of tumor metastasis and recurrence (2).
Yes-associated protein 1 (YAP1) is highly expressed in NSCLC tissues and cells. It can positively regulate expression of NOTCH1, affecting proliferation, invasion and metastasis ability as well as drug sensitivity in lung cancer cells (3). Our previous work proved that YAP, a core transcription co-activator in Hippo signaling pathway, was overexpressed in NSCLC tissues and cells, positively regulated expression of NOTCH1 and markedly promoted cell proliferation and invasion (4). These results indicated that Hippo and NOTCH signaling pathways played an important role in development of NSCLC. However, the specific molecular mechanisms of these two signaling pathways in NSCLC tumorigenesis and development are not fully understood yet.
Long non-coding RNAs (lncRNAs) are non-coding transcripts with longer than 200 nucleotides,
which exhibit various functions and regulate different processes by many molecular
mechanisms (5). Growing evidences suggest that lncRNAs participate in the development and
progression of NSCLC.
LATS2 plays a pivotal role in regulating Hippo growth inhibitory signaling (12). Recent
study showed that LATS2 inhibition decreased YAP1 phosphorylation. It promoted nuclear
accumulation of YAP1 and upregulated the association of YAP1/ TEA domain transcription
factor 2 (TEAD2), which led to transcriptional activation of YAP1/TEAD2 (12). These results
indicated that lncRNA
NOTCH signaling was reported to be altered in approximately one third of NSCLCs (14).
Numerous studies have also suggested that activation of NOTCH correlates with poor clinical
outcomes in NSCLC patients without
Materials and Methods
This experimental study was approved by the Ethics Committee (Code No.: 20180521) of the First Affiliated Hospital of Nanchang University (Nanchang, China). Written informed consents were obtained from all patients. Thirty paired primary tumor tissues and adjacent tissues from these NSCLC patients were obtained. Clinical-pathological characteristics were recorded. No local or systemic treatment was conducted in these patients before surgery. All samples were immediately snap-frozen in liquid nitrogen and stored at -80˚C, until required.
Cell lines and cell culture
Human NSCLC cell lines A549, H1299, Calu-3, H1975 and PC-9 as well as human bronchial epithelial cells BEAS-2B were obtained from the American Type Culture Collection (ATCC, USA), cultured in their corresponding medium containing 10% FBS (Gibco, USA), 100 μg/ml streptomycin (HyClone, USA) and 100 U/ml penicillin (HyClone) and incubated at 37˚C in the presence of 5% CO2 .
RNA extraction and quantitative reverse transcription PCR
Trizol regent (Invitrogen, USA) was used to extract total RNA from tissue specimens and
cell samples. First-strand cDNA was generated by ImProm-II Reverse Transcription System
(Promega, USA). Then, SYBR Green qPCR assay (Takara, Japan) and gene-specific primers
(Table 1,) were used for quantitative reverse transcription PCR (qRT-PCR) analysis.
Plasmid generation and cell transfection
were also designed and synthesized by GenePharma (Shanghai, China). si-NC (5´-UUCUCCGAACGUGUCACGUTT-3´) was used as a negative control. Plasmid vectors and siRNA oligonucleotides were transfected into H1299 or A549 cells with Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Forty-eight hours after transfection, the cells were harvested for qRT-PCR or western blot analysis
|Genes||Paired primers||Sequences (5´-3´)|
Methylation analysis of miR-497 promotor was examined by Methylation-specific PCR (MSP). MethPrimer 1.0 was used to design MSP primers. A pair of methylation-specific primers (M-F: 5´-TTTGATTTAGGGAGAGGAAGGAC-3´; M-R: 5´-TAAACAAACAACTAAAAAACGACGA-3´) and a pair of unmethylation-specific primers at the same site (UF: 5´-TTTGATTTAGGGAGAGGAAGGAT-3´; M-R: 5´-TAAACAAACAACTAAAAAACAACAAA-3´) were chosen. Briefly, the isolated genomic DNA was treated with sodium bisulfite using an EZ DNA Methylation Gold kit (Zymo Research, USA). They were then subjected to PCR assay using the specific primers. The PCR products were digested with a restriction endonuclease BstUI, recognizing sequences unique to the methylated alleles, but not unmethylated alleles. The digested products were next electrophoresed on 3% agarose gels and stained with ethidium bromide. The ratio of gray scale value of the methylated band was calculated as methylation levels.
In vitro transcription and RNA pull-down assay
Colony formation assay
Cells after transfection were collected at logarithmic growth phase. Then, they were placed in a 6-well plate (1×103 /well) for two weeks. 4% paraformaldehyde was used to fix the cells for 15 minutes after discarding the medium, and Giemsa solution was added to stain for 5 minutes. The cells were then quantified by photographing three independent visual fields under the microscope.
Chromatin immunoprecipitation assays
ChIP assays were conducted using the SimpleChIP® Plus Enzymatic Chromatin IP Kit, according to the manufacturer’s instructions (CST, USA). H3 trimethyl Lys 27 antibody was obtained from Millipore (USA). EZH2 (5246) antibody was obtained from CST. Quantification of immunoprecipitated DNA was performed by quantitative PCR (qPCR). ChIP data were calculated as a percentage relative to the input DNA.
Transwell migration assay
8 mm pore 24-well transwell chambers (Corning, USA) were used for migration assay. 2×104 A549 or H1299 cells were seeded into the chambers and cultured with DMEM for 48 hours. Then, took out membranes at the bottom of chambers, and removed the cells on the upper membrane surface using a cotton swab. The cells on the lower surface of membrane surface were fixed with methanol and glacial acetic acid, at the ratio of (3:1) and they were stained with 10% Giemsa solution. Finally, five fields were selected randomly and counted for statistical analysis in each groups.
In vitro Matrigel invasion assay
Before seeding cells, the poly-carbonate membranes of the transwell upper chambers (8 µm pore size; Corning, USA) was pre-coated with Matrigel (BD, USA). Then, 4×105 cells, re-suspended in 200 µl serum-free medium, were placed in the upper chamber, followed by adding 600 µl of the same medium to the lower chamber. Then, the cells on the upper membrane surface were removed after 48 hours incubation at 37˚C. Meanwhile, the cells on the lower membrane surface were fixed with methanol and glacial acetic acid (3:1). They were next stained with 10% Giemsa solution. Finally, five fields selected randomly and counted for statistical analysis in each groups.
Western blot analysis
The cells were harvested and protein was isolated by IP lysis buffer (Thermo Fisher Scientific, USA) containing protease inhibitors (Roche, Switzerland). Then, the BCA Assay Kit (Thermo Fisher Scientific) was used to assess the concentration of proteins in the supernatants of cell lysates. Next, 10% SDS-PAGE gel electrophoresis was applied for separation of equal amount of protein samples. Then, they were transferred to PVDF membranes, which was later incubated with a specific primary antibody followed by incubating with secondary antibody marked by horseradish peroxidase (goat anti-rabbit; Abcam) at room temperature for one hour. Optical density method was used for quantitative autoradiography with β-actin (1:3000; Proteintech, USA), as controls.
Prism 6.0 (GraphPad Software, USA) was used for statistical analysis of data. All experiments were performed at least three times in triplicate. Data were expressed as the mean ± standard deviation (SD). Student’s t test (two tailed) and one-way analysis of variance (ANOVA) were used to evaluate the significant difference. P<0.05 was considered to be significantly different.
PVT1 was upregulated in NSCLC tissues and cell lines,
promoting cell proliferation, migration and invasion
To investigate the role of
Hippo/YAP and NOTCH signaling pathways are associated with the occurrence and
development of NSCLC (21). Western blot analysis demonstrated that protein levels of LAST2
and relative phosphorylated YAP1 were significantly down-regulated in NSCLC tissues than
that of the adjacent tissues, while total YAP1, TEAD and NOTCH1 proteins were up-regulated
in NSCLC tissues than that of the adjacent tissues (Fig .2A,), indicating that the Hippo
pathway was suppressed, promoting NOTCH signaling pathway activation in NSCLC
tumorigenesis. Furthermore, we further investigated the effects of lncRNA
Previous report found that knockdown of
PVT1 directly interacted with EZH2 in NSCLC cells
EZH2 is the functional enzymatic component of the polycomb repressive complex 2 (PRC2)
and it has been linked to many forms of cancer. lncRNA
PVT1 regulated the expression of YAP1 through EZH2-mediated
miR-497 promoter methylation
PVT1 promoted NSCLC cells epithelial-mesenchymal transition and
migration through activation of NOTCH1 signaling pathway
To test whether
NSCLC accounts for almost 80% of lung cancer, as the leading cause of cancer mortality
(22). Even though great progresses have been made in surgical resection, chemoradiotherapy
or target drugs, its prognosis is still poor (2). Hence, it is of most importance to uncover
the molecular mechanism of carcinogenesis. Accumulating studies have shown that some lncRNAs
associate with NSCLC generation and they participate in different biological processes in
NSCLC (23). Previously,
Hippo pathway is involved in the development of NSCLC. LATS1, the core component of Hippo
pathway, was reported to suppress NSCLC cell proliferation and migration (27). While,
Tafazzin (TAZ) was reported to be overexpressed in 70% NSCLC cell lines and it could cause
transformation of non-tumorigenic lung epithelial cells (28). Besides, constitutively
activated YAP, a TAZ paralog, was reported to drive NSCLC progression and metastasis (29).
In this study, we found that
Increasing evidences showed that EZH2 contributed to malignant transformation (30).
EMT progression is of great importance for migration of NSCLC cells (33). Investigations on
EMT will be of great benefit to the bulk of solid malignant tumors, as most of human
malignancies arise from the epithelium tissues 33. However, the functional role of lncRNA in
modulating EMT in NSCLC is still poorly understood. Here, we identified
In summary, we demonstrated that
There is no financially supported and conflict of interest.
J.C.L., S.G.Z.; Contributed to conception and design. S.G.Z., J.H.X., Y.W.; Contributed to all experimental work, data and statistical analysis, and interpretation of data. J.C.L.; Were responsible for overall supervision. X.M.W.; Drafted the manuscript, which was revised by Q.Y.Z. and S.H.D. All authors read and approved the final manuscript.