lncRNA Metastasis-Associated Lung Adenocarcinoma Transcript 1
Promotes Proliferation and Invasion of Non-Small Cell Lung
Cancer Cells via Down-Regulating miR-202 Expression
The first two authors equally contributed to this work.
Tiansheng G, Junming H, Xiaoyun W, Peixi Ch, Shaoshan D, Qianping Ch. lncRNA metastasis-associated lung adenocarcinoma transcript 1 promotes proliferation and invasion of non-small cell lung cancer cells via down-regulating miR-202 expression. Cell J. 2020; 22(3): 375-385. doi: 10.22074/ cellj.2020.6837.
Accumulating evidences indicate that long non-coding RNAs (lncRNAs) play key roles in cancer. This study
aims to clarify role of the metastasis-associated lung adenocarcinoma transcript 1 (
Materials and Methods
In this experimental study,
This study demonstrated that lncRNA-
Lung cancer is the first cause of cancer deaths worldwide, leading to about 1.6 million patients die per year (1). According to the pathological diagnosis, lung cancer is divided into small-cell lung cancer (SCLC, around 15%) and non-small cell lung cancer (NSCLC, around 85%). Although diagnostic techniques and therapy strategies (such as surgical techniques and targeted treatment) have progressed, the 5-year overall survival rate is still below 15%. Besides, this 15% of patients are accompanied with high recurrence rates (2). Thus, it is necessary to determine oncogenes involved in lung cancer development and progression and explore the underlying mechanism, facilitating development of more effective treatment methods.
Long noncoding RNAs (lncRNAs) are an emerging
class of transcripts, which is longer than 200 nucleotides
(nt). Although lncRNAs are coded by the genome, they
are hardly translated into proteins. Previous researches
revealed that lncRNAs serve as new regulators, controlling
gene expressions epigenetically and post-transcriptionally.
They also play crucial roles in modulating chromatin
dynamics, cell growth, differentiation and development
(3). Increasing evidences indicated that many lncRNAs
are observed to be abnormally expressed in many types
of cancer (4). For instance, Wei and Wang (5) found that
The metastasis-associated lung adenocarcinoma
transcript 1 (
Previous studies have identified
In this study, we observed that lncRNA-
Materials and Methods
Patients and tissue samples
Forthy NSCLC tissues as well as corresponding adjacent normal tissues specimens were collected from Guangzhou Panyu Hospital of Chinese Medicine between June 2015 and July 2018. Patients involved in this study had not received any preoperative radiotherapy or chemotherapy. All specimens were identified as NSCLC tissues or normal lung tissues via histopathological observation. After resection, all tissues were dipped in liquid nitrogen promptly and then were stored at -80˚C for further studies. All enrolled patients were informed to sign the written informed consent and this study was approved by the Ethics Committees of Guangzhou Panyu Hospital of Chinese Medicine (license number of ethics statement: 2015HW126).
In this experimental study, normal lung cell BEAS- 2B, NSCLC cell lines (A549, NCI-H23, NCI-H292, NCI-H1299 and NCI-H1975) and HEK293T cell were obtained from ATCC. BEAS-2B cell was cultured in BEBM medium (Lonza/Clonetics Corporation, Switzerland) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA). NSCLC cell lines and HEK293T cell were cultured in RPMI-1640 medium (Thermo Fisher Scientific, USA) supplemented with 10% (v/v) FBS. All cells were maintained in a humidified atmosphere with 5% CO2 at 37˚C.
RNA extraction and quantitative real time polymerase chain reaction assay
Total RNA was extracted from tissue specimens and cell
lines by using TRIzol reagent (Invitrogen, USA) according
to manufacturer’s protocol and treated with DNase I
(Thermo Fisher Scientific, USA) to remove genomic
DNA. cDNA was synthesized with the Transcriptor
First Strand cDNA Synthesis Kit (Roche, Switzerland).
For miRNAs, reverse transcription was conducted with
TaqMan Micro-RNA Reverse Transcription Kit (Applied
Biosystems, USA). Expression level of lncRNA-
siRNAs oligo targeting
si-NC: 5′-CGUACGCGGAAUA CUUCGAdTdT-3′.
At 24 hours post-transfection, 1×103 cells/well were seeded in 96-well plates and cultured overnight. The cell viability was measured with CCK-8 (Beyotime Biotechnology, China) at different time of culture (0, 24, 48 and 72 hours) following the manufacturer’s instruction.
Western blot assay
Total protein was extracted from cell pellet using RIPA lysis buffer (Thermo Fisher Scientific, USA) supplemented with protease inhibitors and phosphatase inhibitors (Roche) according to the manufacturer’s protocol. Concentration of total protein was determined by using BCA™ Protein Assay Kit (Thermo Fisher Scientific, USA). Then, 40 μg of protein per lane was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. 5% skim milk was used to block PVDF membranes for 1 hour at room temperature. Next, membranes were incubated with primary antibodies overnight at 4˚C, followed by incubation of secondary antibodies for 1 hour at room temperature. Next, protein bands were visualized using the enhanced chemiluminescence system (Bio-Rad Clarity Western ECL, USA). Primary antibodies, including MMP2 (1:1000), MMP9 (1:1000) and β-actin (1:1000) and HRP-conjugated secondary antibodies (1:5000) were obtained from Cell Signaling Technology (CST Inc., USA). β-actin was regarded as the internal control.
Transwell invasion assay
24-well Transwell chambers were purchased from Corning (USA). After 24 hours transfection, 1×104 suspended cells in 100 μl serum free medium were seeded in upper chambers smeared on Matrigel (BD Biosciences, USA). Bottom chambers were filled with 600 μl medium containing 10% FBS. After 48 hours culture, upper chambers were fixed with 4% formaldehyde and stained with 0.05% crystal violet. Then, a cotton swab was used to rub away cells on the above membrane. The invaded cells through membrane were counted using optical microscopy.
Dual-luciferase reporter gene assay
Firstly, the full-length 3′-UTR of
RNA-binding protein immunoprecipitation assay
A Magna RIP RNA binding protein immunoprecipitation kit was obtained from Millipore (Darmstadt, German) and the Ago2 antibody was purchased from Abcam (Cambridge, USA). RIP assay was conducted using the magna RIP RNA binding protein immunoprecipitation kit and Ago2 antibody in accordance with the instruction of manufacturer. qRT-PCR was used to determine expression level of co-precipitated RNAs.
RNA pull-down assay
Biotin-labeled miR-NC and biotin-labeled
Statistical analyses were processed with GraphPad Prism 6.0 software (GraphPad software, USA) and all data were expressed as mean ± standard deviation (SD). Student t test or one-way ANOVA was used to determine the differences between two groups or among multiple groups respectively. P<0.05 was considered as statistically significant.
Overexpression of lncRNA-
MALAT1 is observed in
NSCLC tissues and cell lines
To investigate the role of lncRNA-
Knocking-down of lncRNA-
MALAT1 inhibits cell
growth and invasion
To investigate biological function of MALAT1 in
NSCLC, A549 cells were transfected with siRNAs
oligo against MALAT1. As the knockdown efficiency
of si-MALAT1-1 was better than that of si-MALAT1-2
(data was not shown), we silenced lncRNA-
Ectopic expression of lncRNA-
MALAT1 promotes cell
growth and invasion
To further characterize the biological function of
lncRNA-MALAT1 binds to miR-202 and reduces its expression
As there is complementary sequence of miRNA in
lncRNA, they can act as a competing endogenous RNAs,
regulating miRNA expressions and biological function
(19). To explore the mechanism of lncRNA-
MALAT1 negatively regulates miR-202
expression in NSCLC tissues
MALAT1 promotes NSCLC cells proliferation
and invasion via decreasing miR-202
Next, we conducted rescue experiments via
More and more evidences have shown that the aberrant
expression of lncRNAs was observed in lung cancer
tissues, indicating that lncRNAs play multiple roles in
carcinogenesis of lung cancer (20). For instance, Nie et al.
(21) found that lncRNA urothelial carcinoma-associated
The ceRNA theory proposes that lncRNAs sharing
miRNA response elements (MREs) with mRNAs can act
as miRNA decoys. It has been reported that lncRNAs can
act as ceRNA by sponging miRNAs in cancer progression
(28). The underlying molecular mechanisms involved
in lncRNAs interacting with miRNAs are as follows: i.
lncRNA indirectly inhibits negative regulation of miRNAs
on target genes by competing with miRNAs to bind to the
3´-UTR of target gene mRNA, ii. Some lncRNAs form
miRNA precursors by intracellular cleavage, which is then
processed into specific miRNAs, regulating expression of
the target genes, iii. Some lncRNAs function as endogenous
miRNA sponges inhibiting miRNA expression (29). For
instance, in gastric cancer, lncRNA-HOTAIR was reported
to serve as a ceRNA to modulate HER2 expression via
sponging miR-331-3p (30). Huang et al. (31) found that
The molecular mechanism whereby
This study elucidated that
There is no financial support and conflict of interest in this study.
G.T., H.J.; Contributed to conception and design. W.X., C.P., D.S., C.Q.; Contributed to all experimental works, data and statistical analyses, and interpretation of data. G.T.; Were responsible for overall supervision. H.J.; Drafted the manuscript, which was revised by G.T. and W.X. All authors read and approved the final manuscript.