Mol. Cells 2020; 43(12): 975-988
Published online December 3, 2020
https://doi.org/10.14348/molcells.2020.0126
© The Korean Society for Molecular and Cellular Biology
Correspondence to : khbaek@khu.ac.kr
Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1AAS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxiainducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxiainduced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.
Keywords Hypoxia, hypoxia-inducible factor 1α, microarray, uroplakin 1A, uroplakin 1A antisense RNA 1
Hypoxia-inducible factor 1α (HIF-1α) is a key transcription factor that regulates the transcription of target genes under hypoxic conditions, thereby inducing hypoxic responses (Ke and Costa, 2006). Under normoxic conditions, HIF-1α protein is rapidly degraded by the ubiquitin-proteasome system (Maxwell et al., 1999). When oxygen levels are low, HIF-1α is stabilized and binds to the hypoxia response element (HRE) within the promoter to induce the transcription of downstream target genes (Jiang et al., 1996). Because HIF-1α is a key regulator of hypoxia-induced cellular processes, HIF-1α is a promising therapeutic target in the treatment of cancer (Hu et al., 2013; Semenza, 2003; Yu et al., 2017).
Long noncoding RNAs (lncRNAs) are endogenous, non-protein-coding RNAs greater than 200 nucleotides in length (Mercer et al., 2009). LncRNAs play roles in various biological processes (Autuoro et al., 2014; Kanduri, 2016; Penny et al., 1996; Pollex and Heard, 2012) and have been implicated in pathological processes, such as carcinogenesis (Faghihi et al., 2008; Scheuermann and Boyer, 2013; Schmitt and Chang, 2016; Schonrock et al., 2012). In cancer, dysregulation of lncRNAs affects the expression of oncogenes and tumor suppressors via epigenetic silencing, transcriptional regulation, and post-transcriptional processing (Tang et al., 2017). In tumor hypoxia, the expression of many genes and microRNAs is altered to adapt to the low oxygen environment via hypoxia-specific cellular processes (Elvidge et al., 2006; Hong et al., 2004; Kulshreshtha et al., 2007). However, the identities, biological functions, and mechanisms of action of lncRNAs with altered expression under hypoxic conditions have not been thoroughly studied.
Genome-wide expression profiling approaches such as microarray analysis and RNA sequencing (RNA-seq), have been used to identify differentially expressed lncRNAs under hypoxic conditions (Fiedler et al., 2015; Lin et al., 2015; Mimura et al., 2017; Voellenkle et al., 2016; Zhu et al., 2017). Recently, lncRNA microarray analysis of oral squamous cell carcinoma (Zhu et al., 2017) showed that hyaluronan synthase 2 antisense 1 (
Uroplakins (UPKs) are urothelial-specific transmembrane proteins that form plaques on the asymmetric unit membrane in the umbrella cells of the urothelium (Wu et al., 2009). The plaques consist of four distinct UPKs, i.e., UPK1A, UPK1B, UPKII, and UPKIII, which are highly expressed in the urothelium. These proteins regulate the membrane permeability of umbrella cells and strengthen the urothelium through interaction between the asymmetric unit membrane and cytoskeleton structure (Hall et al., 2005). Because of their specific expression in the urothelium, UPKs have not been studied in other cell types. However,
In this study, we evaluated the roles of lncRNAs during hypoxia in lung carcinoma cells using an lncRNA microarray assay. Our results provided important insights into the altered expression of lncRNAs in lung cancer cells at a genome-wide scale and the mechanisms underlying hypoxia-induced expression of
Small interfering RNA (siRNA) against
A549 (human epithelial, lung carcinoma-derived; KCLB 10185), NCI-H460 (human epithelial, lung carcinoma-derived; KCLB 30177), and T24 (human epithelial, bladder carcinoma; KCLB 30004) cell lines were obtained from the Korean Cell Line Bank (Korea). Cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C. To mimic hypoxia, HIF-1α protein was induced by treatment with 200 μM cobalt chloride (CoCl2) for 24 h at 21% oxygen. Cell culture under hypoxic conditions was achieved by incubation in a hypoxia chamber (MIC-101; Billups-Rothenberg, USA) containing 1% O2, 5% CO2, and 94% N2 at 37°C. For transfection, Lipofectamine 2000 and RNAiMAX (Invitrogen, USA) were used following the manufacturer’s protocol, as previously described (Kim et al., 2008).
Total protein was isolated using Pro-Prep protein extraction reagent (iNtRON Biotechnology, Korea). Western blotting was performed using standard procedures. Briefly, total proteins (25 μg) were separated on 4% to 12% precast protein gels (Koma Biotech, Korea) and then transferred to nitrocellulose membranes. Membranes were briefly washed with Tris-buffered saline containing 0.05% Tween 20 (TTBS), blocked for 1 h in TTBS containing 5% nonfat dry milk, and incubated with antibodies overnight at 4°C. Primary antibodies specific for HIF-1α (cat. No. 610958) and β-actin (C-4) were purchased from BD Biosciences (USA) and Santa Cruz Biotechnology (USA), respectively. Blots were then washed, incubated with secondary antibodies, and visualized using the Enhanced Chemiluminescence Plus western blotting reagent (Amersham Biosciences, USA).
Total RNA was isolated from A549 and NCI-H460 cells cultured under normoxic or hypoxic conditions using an RNeasy Mini kit (Qiagen, Germany). Each group consisted of three replicates. Microarray experiments were performed using Arraystar Human LncRNA Microarray v4.0, which contained 40,173 lncRNA and 20,730 mRNA probes (Arraystar, USA). Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, USA), with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY Eukaryotic mRNA Isolation Kit; Epicentre, USA). Next, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit; Arraystar). The labeled cRNAs were purified using an RNeasy Mini Kit (Qiagen). One microgram of each labeled cRNA was fragmented by adding 5 μl of 10× Blocking Agent and 1 μl of 25× Fragmentation Buffer. The mixture was then heated at 60°C for 30 min, and 25 μl of 2× GE Hybridization buffer was added to dilute the labeled cRNA. Next, 50 μl hybridization solution was dispensed into the gasket slide and assembled to produce the lncRNA expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed, and scanned using an Agilent DNA Microarray Scanner (part No. G2505C).
For data analysis, Agilent Feature Extraction software (ver. 11.0.1.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using GeneSpring GX v12.1 software (Agilent Technologies). Differentially expressed lncRNAs and mRNAs with a fold-change of at least 1.5 and statistical significance were identified through Volcano Plot filtering between the two groups. Hierarchical clustering was performed using R software. Gene ontology (GO) analysis was performed using the topGO package in the R environment for statistical computing and graphics, and pathway analysis was performed using Fisher’s exact test. The microarray data is available in the Gene Expression Omnibus (GEO)/NCBI repository (accession No. GSE151120).
Total RNA was isolated using an RNeasy Mini kit (Qiagen). Total RNA from human bladder tissues was obtained from BioChain (USA). For cDNA synthesis, 1 μg total RNA was reverse transcribed using an iScript cDNA synthesis Kit (Bio-Rad, USA). To determine relative mRNA levels, qRT-PCR was performed in triplicate in 384-well plates on an ABI Prism 7900 Sequence Detection System (Applied Biosystems, USA) using 2× SYBR Green PCR Master Mix (Applied Biosystems). The thermal cycling conditions consisted of an initial 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The expression of each cDNA was normalized to that of β-actin, and the comparative Ct method was used to obtain relative expression levels. The primers used for qRT-PCR are shown in Supplementary Table S2.
Nuclear and cytoplasmic RNA fractions were isolated using a Cytoplasmic & Nuclear RNA purification kit (Norgen Biotek, Canada) according to the manufacturer’s instructions. After incubating NCI-H460 cells at 37°C for 24 h in a hypoxia chamber (MIC-101) under conditions of 1% O2, 5% CO2, and 94% N2, 2 × 106 cells were harvested. The process was carried out according to the manufacturer’s protocol, and lysis buffer J was diluted to 15% before use. The nuclear RNA and cytoplasmic RNA concentrations were measured using a NanoDrop ND-1000, after which, qRT-PCR was performed. The primer sequences used in qRT-PCR are as shown in Supplementary Table S2.
NCI-H460 cells were harvested after incubating in a hypoxia chamber at 37°C for 24 h. Subsequent to RNA extraction using an RNeasy Mini kit (Qiagen), 90 μl RNA was incubated at 37°C for 1 h. After adding 10 μl of 10× RPA buffer (100 mM Tris-HCl, 3 M NaCl, and 50 mM ethylenediaminetetraacetic acid), the mixture was divided into two tubes (50 μl each). In one tube, 1 μl RNase A + T1 (Thermo Fisher Scientific, USA) was added, and both tubes were incubated at 37°C for 30 min. Following treatment with 200 μg/ml proteinase K (Biosesang, Korea), the tubes were incubated at 50°C for 1 h. RNA was isolated using an RNeasy Mini kit (Qiagen), and the concentration was measured. Subsequently, cDNA was synthesized by adding UPK-ss-primer and UPK-ds-primer using an iScript Select cDNA Synthesis Kit (Bio-Rad). After using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) to perform qRT-PCR on 1.5 μl cDNA, the product was identified by agarose gel electrophoresis. The thermal cycling conditions were as follows: 95°C for 10 min; and 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The primers used were as follows: UPK1A forward/UPK1A reverse and UPK-ds forward/UPK-ds reverse.
After incubating NCI-H460 cells for 24 h under hypoxic conditions, the cells were treated with 4 μg/ml actinomycin D (Thermo Fisher Scientific) and transfected with 20 nM NC,
Genomic DNAs were isolated from A549, NCI-H460, and T24 cells using a Wizard Genomic DNA purification kit (Promega, USA). Genomic DNAs from human lung, liver, and bladder tissues were obtained from BioChain. Genomic DNA was treated with bisulfite using an EZ DNA Methylation-Gold kit (Zymo Research, USA). Bisulfite-modified genomic DNA was then amplified by PCR using EpiMark Hot Start Taq DNA Polymerase (New England BioLabs, USA). PCR was performed in a 25 μl reaction volume at 95°C for 60 s, followed by 40 cycles of 95°C for 25 s, 55°C for 45 s, and 68°C for 45 s. The primer sequences were as follows: UPK-296, 5′-GGTTTTGGGTTATTATTTTTGTATG-3′; UPK-479, 5′-CTTAACTTATAAATTTACCCATCTAC-3′ (bisulfite sequencing for a 184-bp fragment within a CpG island located approximately 1100 bp upstream of exon 1); UPK-40, 5′-GGGTGTTTTTTTGTGTAAAATGTTT-3′; UPK-287, 5′-ATATTACCCACAACTAACAAACCCA-3′ (bisulfite sequencing for a 248-bp fragment containing an ATG initiation codon within exon 2); UPKAS-202, 5′-TATTTTTTTAGTGATGTTTTTTTGA-3′; UPKAS-402, 5′-CCTCCCATACAAATCAAACC-3′ (bisulfite sequencing for a 201-bp fragment containing exon 1 of
Data are shown as mean ± SD. Differences between groups were analyzed by two-tailed Student’s
To identify lncRNAs whose expression was altered under hypoxic conditions, we performed microarray experiments using total RNA isolated from A549 and NCI-H460 lung carcinoma cells. The human microarray slides used in the experiment contained 40,173 lncRNA and 20,730 mRNA probes. Among 40,173 lncRNAs, 7,506 lncRNAs were labeled as “Gold Standard LncRNAs”; these lncRNAs are well annotated, have been functionally studied, and have been experimentally identified as full-length lncRNAs. The cells were cultured under hypoxic or normoxic conditions, and total RNA was subjected to microarray analysis. Figs. 1A and 1C show mRNAs and lncRNAs that were up- or downregulated by greater than 1.5-fold under hypoxic conditions compared with those under normoxic conditions in A549 and NCI-H460 cells. For mRNAs, 892 and 1,185 mRNAs were upregulated in A549 and NCI-H460 cells, respectively. As expected, the gene showing the greatest upregulation in both A549 and NCI-H460 cells was CA9, a known marker of hypoxia. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that genes upregulated in hypoxia were involved in the HIF-1 signaling pathway, glycolytic pathway, and pentose phosphate pathway (Fig. 1B); these genes were also known downstream target genes of HIF-1α, including
Antisense lncRNAs are a subgroup of lncRNAs that are transcribed in the opposite direction of an associated protein-coding gene. Increasing evidence indicates that antisense lncRNAs regulate the expression of nearby sense coding genes in
In addition to A549 and NCI-H460 cells, we examined the expression of
To examine whether HIF-1α was involved in hypoxia-induced expression of
To investigate the interactions among
Next, to investigate the potential of duplex formation between
To investigate whether
Because antisense lncRNAs often play important roles in the expression of their sense counterparts via epigenetic regulation, we examined the methylation statuses of
The expression of
LncRNAs regulate the expression of many genes at the transcriptional and post-transcriptional levels (Bach and Lee, 2018). One of the mechanisms involved in regulation at the post-transcriptional level is the formation of a duplex between antisense lncRNA and the neighboring sense gene RNA to regulate RNA stability (Huang et al., 2016; Kimura et al., 2013; Sun et al., 2016; Zhang et al., 2019). For example, the lncRNA
DNA methylation regulates gene expression by affecting the chromatin structure (Jones and Takai, 2001; Klose and Bird, 2006). In this study, we found that the promoter region of
Several studies have indicated
Overall, in this study, we found that
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (grant No. 2015R1D1A1A01057433).
Y.C.C., J.Y., and K.B. conceived and designed the study. Y.B. and Y.C.C. performed the experiments. Y.J. analyzed the data. Y.C.C. and K.B. wrote and edited the manuscript. All authors read and approved the final manuscript.
The authors have no potential conflicts of interest to disclose.
Top 20 upregulated lncRNAs under hypoxic condition in both A549 and NCI-H460 cells
Sequence name | Gene symbol | Fold change A549 | Fold change H460 | RNA length | Chromosome | Strand | Start | End | Source |
---|---|---|---|---|---|---|---|---|---|
NR_046420 | UPK1A-AS1 | 21.5191323 | 38.5241975 | 813 | chr19 | – | 36158849 | 36164193 | RefSeq |
ENST00000522547 | RP11-14I17.2 | 10.6497245 | 38.3353098 | 508 | chr8 | + | 26280108 | 26281445 | GENCODE |
ENST00000417355 | AC114803.3 | 8.5930288 | 46.8519287 | 311 | chr2 | + | 220163723 | 220168852 | GENCODE |
ENST00000546523 | RP5-1057I20.2 | 8.384609 | 8.9300753 | 353 | chr12 | + | 48223276 | 48228337 | GENCODE |
T054705 | G012608 | 7.9549952 | 26.5872803 | 1448 | chr11 | + | 10382151 | 10383599 | RNA-seq: Iyer et al., 2015 |
T203015 | G046886 | 6.8663933 | 5.958497 | 1343 | chr2 | – | 173461092 | 173462435 | RNA-seq: Iyer et al., 2015 |
T036689 | G008310 | 6.8583296 | 4.7583565 | 773 | chr10 | + | 6428518 | 6429558 | RNA-seq: Iyer et al., 2015 |
ENST00000458314 | AC078883.3 | 6.1392631 | 5.030675 | 512 | chr2 | – | 173328989 | 173330750 | GENCODE |
TCONS_00000467 | XLOC_000695 | 5.9628345 | 10.5276413 | 410 | chr1 | – | 8066073 | 8066784 | RNA-seq: Cabili et al., 2011 |
ENST00000564127 | RP11-480I12.10 | 5.6380716 | 5.7666391 | 592 | chr1 | – | 202779365 | 202779957 | GENCODE |
NR_015421 | LOC154761 | 5.626012 | 3.2081902 | 2579 | chr7 | – | 143509060 | 143533810 | RefSeq |
T085710 | G019879 | 4.5601655 | 9.3665869 | 998 | chr12 | – | 106740693 | 106743112 | RNA-seq: Iyer et al., 2015 |
NR_040079 | LOC399715 | 4.3338854 | 5.1864333 | 2967 | chr10 | + | 6368506 | 6377943 | RefSeq |
NR_026804 | KLF3-AS1 | 4.2354962 | 6.9351289 | 2368 | chr4 | – | 38614321 | 38666249 | RefSeq |
T336396 | G078889 | 4.1126362 | 9.7690188 | 3072 | chr7 | + | 153646863 | 153654276 | RNA-seq: Iyer et al., 2015 |
NR_024006 | LINC00950 | 3.8685642 | 7.478096 | 5245 | chr9 | + | 35860270 | 35865515 | RefSeq |
T036645 | G008294 | 3.8119865 | 3.0235726 | 590 | chr10 | + | 6296150 | 6296740 | RNA-seq: Iyer et al., 2015 |
NR_120598 | GACAT2 | 3.7435975 | 2.183135 | 835 | chr18 | – | 8695853 | 8707619 | RefSeq |
uc001gwt.1 | AX747377 | 3.7160007 | 2.6729957 | 2162 | chr1 | – | 201604354 | 201606516 | UCSC_knowngene |
ENST00000608142 | RP11-1399P15.1 | 3.6876088 | 19.1472439 | 540 | chr2 | – | 87777013 | 87777553 | GENCODE |
Mol. Cells 2020; 43(12): 975-988
Published online December 31, 2020 https://doi.org/10.14348/molcells.2020.0126
Copyright © The Korean Society for Molecular and Cellular Biology.
Yuree Byun1,2 , Young-Chul Choi1,2
, Yongsu Jeong1
, Jaeseung Yoon1
, and Kwanghee Baek1,*
1Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Korea, 2These authors contributed equally to this work.
Correspondence to:khbaek@khu.ac.kr
Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1AAS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxiainducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxiainduced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.
Keywords: Hypoxia, hypoxia-inducible factor 1α, microarray, uroplakin 1A, uroplakin 1A antisense RNA 1
Hypoxia-inducible factor 1α (HIF-1α) is a key transcription factor that regulates the transcription of target genes under hypoxic conditions, thereby inducing hypoxic responses (Ke and Costa, 2006). Under normoxic conditions, HIF-1α protein is rapidly degraded by the ubiquitin-proteasome system (Maxwell et al., 1999). When oxygen levels are low, HIF-1α is stabilized and binds to the hypoxia response element (HRE) within the promoter to induce the transcription of downstream target genes (Jiang et al., 1996). Because HIF-1α is a key regulator of hypoxia-induced cellular processes, HIF-1α is a promising therapeutic target in the treatment of cancer (Hu et al., 2013; Semenza, 2003; Yu et al., 2017).
Long noncoding RNAs (lncRNAs) are endogenous, non-protein-coding RNAs greater than 200 nucleotides in length (Mercer et al., 2009). LncRNAs play roles in various biological processes (Autuoro et al., 2014; Kanduri, 2016; Penny et al., 1996; Pollex and Heard, 2012) and have been implicated in pathological processes, such as carcinogenesis (Faghihi et al., 2008; Scheuermann and Boyer, 2013; Schmitt and Chang, 2016; Schonrock et al., 2012). In cancer, dysregulation of lncRNAs affects the expression of oncogenes and tumor suppressors via epigenetic silencing, transcriptional regulation, and post-transcriptional processing (Tang et al., 2017). In tumor hypoxia, the expression of many genes and microRNAs is altered to adapt to the low oxygen environment via hypoxia-specific cellular processes (Elvidge et al., 2006; Hong et al., 2004; Kulshreshtha et al., 2007). However, the identities, biological functions, and mechanisms of action of lncRNAs with altered expression under hypoxic conditions have not been thoroughly studied.
Genome-wide expression profiling approaches such as microarray analysis and RNA sequencing (RNA-seq), have been used to identify differentially expressed lncRNAs under hypoxic conditions (Fiedler et al., 2015; Lin et al., 2015; Mimura et al., 2017; Voellenkle et al., 2016; Zhu et al., 2017). Recently, lncRNA microarray analysis of oral squamous cell carcinoma (Zhu et al., 2017) showed that hyaluronan synthase 2 antisense 1 (
Uroplakins (UPKs) are urothelial-specific transmembrane proteins that form plaques on the asymmetric unit membrane in the umbrella cells of the urothelium (Wu et al., 2009). The plaques consist of four distinct UPKs, i.e., UPK1A, UPK1B, UPKII, and UPKIII, which are highly expressed in the urothelium. These proteins regulate the membrane permeability of umbrella cells and strengthen the urothelium through interaction between the asymmetric unit membrane and cytoskeleton structure (Hall et al., 2005). Because of their specific expression in the urothelium, UPKs have not been studied in other cell types. However,
In this study, we evaluated the roles of lncRNAs during hypoxia in lung carcinoma cells using an lncRNA microarray assay. Our results provided important insights into the altered expression of lncRNAs in lung cancer cells at a genome-wide scale and the mechanisms underlying hypoxia-induced expression of
Small interfering RNA (siRNA) against
A549 (human epithelial, lung carcinoma-derived; KCLB 10185), NCI-H460 (human epithelial, lung carcinoma-derived; KCLB 30177), and T24 (human epithelial, bladder carcinoma; KCLB 30004) cell lines were obtained from the Korean Cell Line Bank (Korea). Cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C. To mimic hypoxia, HIF-1α protein was induced by treatment with 200 μM cobalt chloride (CoCl2) for 24 h at 21% oxygen. Cell culture under hypoxic conditions was achieved by incubation in a hypoxia chamber (MIC-101; Billups-Rothenberg, USA) containing 1% O2, 5% CO2, and 94% N2 at 37°C. For transfection, Lipofectamine 2000 and RNAiMAX (Invitrogen, USA) were used following the manufacturer’s protocol, as previously described (Kim et al., 2008).
Total protein was isolated using Pro-Prep protein extraction reagent (iNtRON Biotechnology, Korea). Western blotting was performed using standard procedures. Briefly, total proteins (25 μg) were separated on 4% to 12% precast protein gels (Koma Biotech, Korea) and then transferred to nitrocellulose membranes. Membranes were briefly washed with Tris-buffered saline containing 0.05% Tween 20 (TTBS), blocked for 1 h in TTBS containing 5% nonfat dry milk, and incubated with antibodies overnight at 4°C. Primary antibodies specific for HIF-1α (cat. No. 610958) and β-actin (C-4) were purchased from BD Biosciences (USA) and Santa Cruz Biotechnology (USA), respectively. Blots were then washed, incubated with secondary antibodies, and visualized using the Enhanced Chemiluminescence Plus western blotting reagent (Amersham Biosciences, USA).
Total RNA was isolated from A549 and NCI-H460 cells cultured under normoxic or hypoxic conditions using an RNeasy Mini kit (Qiagen, Germany). Each group consisted of three replicates. Microarray experiments were performed using Arraystar Human LncRNA Microarray v4.0, which contained 40,173 lncRNA and 20,730 mRNA probes (Arraystar, USA). Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, USA), with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY Eukaryotic mRNA Isolation Kit; Epicentre, USA). Next, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit; Arraystar). The labeled cRNAs were purified using an RNeasy Mini Kit (Qiagen). One microgram of each labeled cRNA was fragmented by adding 5 μl of 10× Blocking Agent and 1 μl of 25× Fragmentation Buffer. The mixture was then heated at 60°C for 30 min, and 25 μl of 2× GE Hybridization buffer was added to dilute the labeled cRNA. Next, 50 μl hybridization solution was dispensed into the gasket slide and assembled to produce the lncRNA expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed, and scanned using an Agilent DNA Microarray Scanner (part No. G2505C).
For data analysis, Agilent Feature Extraction software (ver. 11.0.1.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using GeneSpring GX v12.1 software (Agilent Technologies). Differentially expressed lncRNAs and mRNAs with a fold-change of at least 1.5 and statistical significance were identified through Volcano Plot filtering between the two groups. Hierarchical clustering was performed using R software. Gene ontology (GO) analysis was performed using the topGO package in the R environment for statistical computing and graphics, and pathway analysis was performed using Fisher’s exact test. The microarray data is available in the Gene Expression Omnibus (GEO)/NCBI repository (accession No. GSE151120).
Total RNA was isolated using an RNeasy Mini kit (Qiagen). Total RNA from human bladder tissues was obtained from BioChain (USA). For cDNA synthesis, 1 μg total RNA was reverse transcribed using an iScript cDNA synthesis Kit (Bio-Rad, USA). To determine relative mRNA levels, qRT-PCR was performed in triplicate in 384-well plates on an ABI Prism 7900 Sequence Detection System (Applied Biosystems, USA) using 2× SYBR Green PCR Master Mix (Applied Biosystems). The thermal cycling conditions consisted of an initial 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The expression of each cDNA was normalized to that of β-actin, and the comparative Ct method was used to obtain relative expression levels. The primers used for qRT-PCR are shown in Supplementary Table S2.
Nuclear and cytoplasmic RNA fractions were isolated using a Cytoplasmic & Nuclear RNA purification kit (Norgen Biotek, Canada) according to the manufacturer’s instructions. After incubating NCI-H460 cells at 37°C for 24 h in a hypoxia chamber (MIC-101) under conditions of 1% O2, 5% CO2, and 94% N2, 2 × 106 cells were harvested. The process was carried out according to the manufacturer’s protocol, and lysis buffer J was diluted to 15% before use. The nuclear RNA and cytoplasmic RNA concentrations were measured using a NanoDrop ND-1000, after which, qRT-PCR was performed. The primer sequences used in qRT-PCR are as shown in Supplementary Table S2.
NCI-H460 cells were harvested after incubating in a hypoxia chamber at 37°C for 24 h. Subsequent to RNA extraction using an RNeasy Mini kit (Qiagen), 90 μl RNA was incubated at 37°C for 1 h. After adding 10 μl of 10× RPA buffer (100 mM Tris-HCl, 3 M NaCl, and 50 mM ethylenediaminetetraacetic acid), the mixture was divided into two tubes (50 μl each). In one tube, 1 μl RNase A + T1 (Thermo Fisher Scientific, USA) was added, and both tubes were incubated at 37°C for 30 min. Following treatment with 200 μg/ml proteinase K (Biosesang, Korea), the tubes were incubated at 50°C for 1 h. RNA was isolated using an RNeasy Mini kit (Qiagen), and the concentration was measured. Subsequently, cDNA was synthesized by adding UPK-ss-primer and UPK-ds-primer using an iScript Select cDNA Synthesis Kit (Bio-Rad). After using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) to perform qRT-PCR on 1.5 μl cDNA, the product was identified by agarose gel electrophoresis. The thermal cycling conditions were as follows: 95°C for 10 min; and 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The primers used were as follows: UPK1A forward/UPK1A reverse and UPK-ds forward/UPK-ds reverse.
After incubating NCI-H460 cells for 24 h under hypoxic conditions, the cells were treated with 4 μg/ml actinomycin D (Thermo Fisher Scientific) and transfected with 20 nM NC,
Genomic DNAs were isolated from A549, NCI-H460, and T24 cells using a Wizard Genomic DNA purification kit (Promega, USA). Genomic DNAs from human lung, liver, and bladder tissues were obtained from BioChain. Genomic DNA was treated with bisulfite using an EZ DNA Methylation-Gold kit (Zymo Research, USA). Bisulfite-modified genomic DNA was then amplified by PCR using EpiMark Hot Start Taq DNA Polymerase (New England BioLabs, USA). PCR was performed in a 25 μl reaction volume at 95°C for 60 s, followed by 40 cycles of 95°C for 25 s, 55°C for 45 s, and 68°C for 45 s. The primer sequences were as follows: UPK-296, 5′-GGTTTTGGGTTATTATTTTTGTATG-3′; UPK-479, 5′-CTTAACTTATAAATTTACCCATCTAC-3′ (bisulfite sequencing for a 184-bp fragment within a CpG island located approximately 1100 bp upstream of exon 1); UPK-40, 5′-GGGTGTTTTTTTGTGTAAAATGTTT-3′; UPK-287, 5′-ATATTACCCACAACTAACAAACCCA-3′ (bisulfite sequencing for a 248-bp fragment containing an ATG initiation codon within exon 2); UPKAS-202, 5′-TATTTTTTTAGTGATGTTTTTTTGA-3′; UPKAS-402, 5′-CCTCCCATACAAATCAAACC-3′ (bisulfite sequencing for a 201-bp fragment containing exon 1 of
Data are shown as mean ± SD. Differences between groups were analyzed by two-tailed Student’s
To identify lncRNAs whose expression was altered under hypoxic conditions, we performed microarray experiments using total RNA isolated from A549 and NCI-H460 lung carcinoma cells. The human microarray slides used in the experiment contained 40,173 lncRNA and 20,730 mRNA probes. Among 40,173 lncRNAs, 7,506 lncRNAs were labeled as “Gold Standard LncRNAs”; these lncRNAs are well annotated, have been functionally studied, and have been experimentally identified as full-length lncRNAs. The cells were cultured under hypoxic or normoxic conditions, and total RNA was subjected to microarray analysis. Figs. 1A and 1C show mRNAs and lncRNAs that were up- or downregulated by greater than 1.5-fold under hypoxic conditions compared with those under normoxic conditions in A549 and NCI-H460 cells. For mRNAs, 892 and 1,185 mRNAs were upregulated in A549 and NCI-H460 cells, respectively. As expected, the gene showing the greatest upregulation in both A549 and NCI-H460 cells was CA9, a known marker of hypoxia. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that genes upregulated in hypoxia were involved in the HIF-1 signaling pathway, glycolytic pathway, and pentose phosphate pathway (Fig. 1B); these genes were also known downstream target genes of HIF-1α, including
Antisense lncRNAs are a subgroup of lncRNAs that are transcribed in the opposite direction of an associated protein-coding gene. Increasing evidence indicates that antisense lncRNAs regulate the expression of nearby sense coding genes in
In addition to A549 and NCI-H460 cells, we examined the expression of
To examine whether HIF-1α was involved in hypoxia-induced expression of
To investigate the interactions among
Next, to investigate the potential of duplex formation between
To investigate whether
Because antisense lncRNAs often play important roles in the expression of their sense counterparts via epigenetic regulation, we examined the methylation statuses of
The expression of
LncRNAs regulate the expression of many genes at the transcriptional and post-transcriptional levels (Bach and Lee, 2018). One of the mechanisms involved in regulation at the post-transcriptional level is the formation of a duplex between antisense lncRNA and the neighboring sense gene RNA to regulate RNA stability (Huang et al., 2016; Kimura et al., 2013; Sun et al., 2016; Zhang et al., 2019). For example, the lncRNA
DNA methylation regulates gene expression by affecting the chromatin structure (Jones and Takai, 2001; Klose and Bird, 2006). In this study, we found that the promoter region of
Several studies have indicated
Overall, in this study, we found that
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (grant No. 2015R1D1A1A01057433).
Y.C.C., J.Y., and K.B. conceived and designed the study. Y.B. and Y.C.C. performed the experiments. Y.J. analyzed the data. Y.C.C. and K.B. wrote and edited the manuscript. All authors read and approved the final manuscript.
The authors have no potential conflicts of interest to disclose.
. Top 20 upregulated lncRNAs under hypoxic condition in both A549 and NCI-H460 cells.
Sequence name | Gene symbol | Fold change A549 | Fold change H460 | RNA length | Chromosome | Strand | Start | End | Source |
---|---|---|---|---|---|---|---|---|---|
NR_046420 | UPK1A-AS1 | 21.5191323 | 38.5241975 | 813 | chr19 | – | 36158849 | 36164193 | RefSeq |
ENST00000522547 | RP11-14I17.2 | 10.6497245 | 38.3353098 | 508 | chr8 | + | 26280108 | 26281445 | GENCODE |
ENST00000417355 | AC114803.3 | 8.5930288 | 46.8519287 | 311 | chr2 | + | 220163723 | 220168852 | GENCODE |
ENST00000546523 | RP5-1057I20.2 | 8.384609 | 8.9300753 | 353 | chr12 | + | 48223276 | 48228337 | GENCODE |
T054705 | G012608 | 7.9549952 | 26.5872803 | 1448 | chr11 | + | 10382151 | 10383599 | RNA-seq: Iyer et al., 2015 |
T203015 | G046886 | 6.8663933 | 5.958497 | 1343 | chr2 | – | 173461092 | 173462435 | RNA-seq: Iyer et al., 2015 |
T036689 | G008310 | 6.8583296 | 4.7583565 | 773 | chr10 | + | 6428518 | 6429558 | RNA-seq: Iyer et al., 2015 |
ENST00000458314 | AC078883.3 | 6.1392631 | 5.030675 | 512 | chr2 | – | 173328989 | 173330750 | GENCODE |
TCONS_00000467 | XLOC_000695 | 5.9628345 | 10.5276413 | 410 | chr1 | – | 8066073 | 8066784 | RNA-seq: Cabili et al., 2011 |
ENST00000564127 | RP11-480I12.10 | 5.6380716 | 5.7666391 | 592 | chr1 | – | 202779365 | 202779957 | GENCODE |
NR_015421 | LOC154761 | 5.626012 | 3.2081902 | 2579 | chr7 | – | 143509060 | 143533810 | RefSeq |
T085710 | G019879 | 4.5601655 | 9.3665869 | 998 | chr12 | – | 106740693 | 106743112 | RNA-seq: Iyer et al., 2015 |
NR_040079 | LOC399715 | 4.3338854 | 5.1864333 | 2967 | chr10 | + | 6368506 | 6377943 | RefSeq |
NR_026804 | KLF3-AS1 | 4.2354962 | 6.9351289 | 2368 | chr4 | – | 38614321 | 38666249 | RefSeq |
T336396 | G078889 | 4.1126362 | 9.7690188 | 3072 | chr7 | + | 153646863 | 153654276 | RNA-seq: Iyer et al., 2015 |
NR_024006 | LINC00950 | 3.8685642 | 7.478096 | 5245 | chr9 | + | 35860270 | 35865515 | RefSeq |
T036645 | G008294 | 3.8119865 | 3.0235726 | 590 | chr10 | + | 6296150 | 6296740 | RNA-seq: Iyer et al., 2015 |
NR_120598 | GACAT2 | 3.7435975 | 2.183135 | 835 | chr18 | – | 8695853 | 8707619 | RefSeq |
uc001gwt.1 | AX747377 | 3.7160007 | 2.6729957 | 2162 | chr1 | – | 201604354 | 201606516 | UCSC_knowngene |
ENST00000608142 | RP11-1399P15.1 | 3.6876088 | 19.1472439 | 540 | chr2 | – | 87777013 | 87777553 | GENCODE |
Su-Kyeong Jang, Byung-Ha Yoon, Seung Min Kang, Yeo-Gha Yoon, Seon-Young Kim, and Wankyu Kim
Mol. Cells 2019; 42(3): 237-244 https://doi.org/10.14348/molcells.2018.0413You-Sun Kim, Nurdan Kokturk, Ji-Young Kim, Sei Won Lee, Jaeyun Lim, Soo Jin Choi, Wonil Oh, and Yeon-Mok Oh
Mol. Cells 2016; 39(10): 728-733 https://doi.org/10.14348/molcells.2016.0095Chunggab Choi, Seung-Hun Oh, Jeong-Eun Noh, Yong-Woo Jeong, Soonhag Kim, Jung Jae Ko, Ok-Joon Kim, and Jihwan Song
Mol. Cells 2016; 39(4): 337-344 https://doi.org/10.14348/molcells.2016.2317