Lung cancer is one of the most frequently diagnosed malignant tumors and remains the leading cause of cancer-related deaths worldwide [1
]. According to the biological characteristics, there are two main types of lung cancer: non-small cell lung cancer (NSCLC; approximately 85% of lung cancer cases) and small cell lung cancer (SCLC) [3
]. Since diagnosis is usually made at advanced stages of the disease, the five year overall survival rate among NSCLC patients is under 20% [5
]. In addition, the overall survival rate is two-fold higher among women than among men. Nonetheless, there is no conclusive evidence to explain the difference in the survival rate between the genders.
The protein-coding genes of the X and Y chromosomes have been relatively well characterized. Some studies have shown that the dysregulation of X inactivation and the loss of the Y chromosome are involved in several types of cancers. Human Y chromosome deletions and rearrangements are associated with various cancers, including prostate cancer [7
], bladder cancer [9
], male sex cord stromal tumors, lung cancer [10
], and esophageal carcinoma [12
], indicating the presence of oncogenes and tumor suppressor genes on this chromosome. Nevertheless, the chromosomal regions responsible for the production of noncoding RNAs are not well known.
Long noncoding RNAs (lncRNAs) are transcribed from thousands of loci in the mammalian genome and play a wide range of roles in gene regulation and other cellular processes, including mRNA splicing, RNA decay, translation, and chromatin remodeling. In addition to contributing to a normal physiology, lncRNA expression and function have been linked to the initiation and progression of cancer. Differences in cancer susceptibility are consistently associated with gender in cancer epidemiology [13
]. Men have worse prognoses and a higher mortality rate than women do. Nevertheless, so far, the sex-related lncRNAs are poorly characterized and the potential link between these lncRNAs and cancer has not been studied.
The human Y chromosome can be divided into three regions: (a) male-specific regions of the Y chromosome (MSY), (b) pseudoautosomal regions (PAR1 and PAR2), and (c) heterochromatin in the Yq area [14
]. The Y chromosome contains more than 200 specific genes that are important for male sex determination, germ cell differentiation, and masculinization of various tissues [15
]. On the other hand, the cellular function of many lncRNAs encoded in MSY has yet to be elucidated. Recently, a study revealed that an intergenic Y-linked lncRNA named lnc-KDM5D-4 (lysine demethylase 5D) is associated with fatty liver, atherosclerosis, and coronary artery disease in men [16
]. There is currently only a limited amount of data available regarding the association between Y chromosome-linked lncRNAs and human phenotypes. Moreover, the regulatory mechanisms of these lncRNAs remain to be studied.
Testis-specific transcript Y-linked 15 (TTTY15
, 5263 bp) is encoded in the chromosome region Yq11.21 of MSY. Some studies have shown that a fusion of the TTTY15
gene with USP9Y (TTTY15
–USP9Y) predicts prostate biopsy results [17
] in multiple cases of hepatocellular carcinoma, lung cancer, and prostate cancer, suggesting that TTTY15
-USP9Y fusion is a potential driver of carcinogenesis. Nevertheless, the function of TTTY15
itself in cells remains unknown. In this study, we investigated the mechanism of action of TTTY15
in the progression of NSCLC. We found that TTTY15
was significantly downregulated in NSCLC tissue samples compared to adjacent noncancerous tissues. Furthermore, decreased expression of TTTY15
was associated with poor prognosis among patients with NSCLC. Our data also indicate that a knockdown of TTTY15
promotes cell proliferation, cell cycle progression, migration, and invasion. In addition, investigation of the mechanism revealed that TTTY15
can target and affect T-box transcription factor 4 (TBX4) expression via DNA (cytosine-5)-methyltransferase 3A (DNMT3A)-mediated regulation. Our results show for the first time that TTTY15
participates in NSCLC progression.
Recent studies suggest that gender affects the risk, incidence, and progression of various human diseases, including cancers [20
]. The most important genetic difference between men and women is the genes on the sex chromosomes. At present, there is a limited number of studies on Y chromosome-linked lncRNAs in human diseases and cancers because the Y chromosome is often excluded from genomic analysis studies [25
]. Our study provides the first evidence of the expression of a Y chromosome-linked lncRNA called TTTY15
in NSCLC. Some authors have reported that ubiquitin specific peptidase 9, Y-linked (USP9Y)–TTTY15
is expressed in both tumor and nonmalignant samples and can be used to predict the outcome of a prostate biopsy [17
]. The TTTY15
gene can get fused to >zinc finger DHHC-type containing 2 (ZDHHC2–TTTY15
), and this fusion is found in patients with acute myeloid leukemia [28
]. In addition to forming fusion genes, TTTY15
(its product) can protect cardiomyocytes from hypoxia-induced cell injury by targeting miR-455-5p and thus regulating Jun dimerization protein 2 (JDP2) expression [29
has also been reported to mainly localize in the cytoplasm and promote prostate cancer progression by functioning as an RNA sponge and binding to let-7 [30
]. These studies show that TTTY15
plays important roles in cells. In our study, we found that TTTY15
was downregulated in lung cancer and was mainly located in the nucleus, upregulating TBX4 expression by targeting DNMT3A. These different findings suggest that TTTY15
may play a dual part in the cytoplasm and nucleus in various types of cancer. This study is the first to determine the clinical significance and biological function of TTTY15
in NSCLC, and the results indicate that TTTY15
is a tumor suppressor in NSCLC.
LncRNAs are long and can form complex secondary and tertiary structures to participate in gene regulation. A growing amount of research is suggesting that lncRNA genes can function as genes in certain situations and can regulate elements located on different chromosomes through processes involving the physical associations between different chromosomes (“trans-interactions”) [31
]. Chromosome interactions can promote the silencing and/or activation of genes within the three-dimensional structure of the nuclear matrix [35
]. To elucidate the regulatory mechanism of action of TTTY15
in the NSCLC, we employed the 4C technique to identify the target regions of TTTY15
in the genome. The 4C data showed that TTTY15
(chromosome 10) and LINC00674
(chromosome 17). After confirming the knockdown, we found that the knockdown of TTTY15
dramatically decreased the expression of T-box transcription factor 4 (TBX4). TBX4 is located in chromosomal region 17q23. Its protein product is reported to act as a transcription factor in hind limb growth and to regulate some processes during embryonic development [38
]. TBX4- and TBX5-deficient mice show severely reduced lung branching during the second trimester, suggesting that TBX4 performs an important function in the regulation of proliferation, migration, and differentiation of mesenchymal cells, in addition to supporting and helping to produce peripheral epithelial cells [40
]. In the present study, 4C was used in combination with next-generation sequencing and bioinformatic analysis to investigate the possible interactions of TTTY15
with other targets in the genome. This approach was effective at identifying the regulation targets of the lncRNA genes.
TBX4 is a mesenchymal transcription factor that drives the accumulation of myofibroblasts and the development of pulmonary fibrosis [41
]. Low expression levels of TBX4 indicate a worse prognosis in patients with stage II pancreatic ductal adenocarcinoma (PDAC) [19
]. Our results revealed that TBX4 is downregulated in NSCLC and the knockdown of TBX4 increases the NSCLC cell migration and invasion. MMPs are known to degrade proteins in the extracellular matrix and basement membrane, and to promote tumor metastasis [42
]. Although the inhibition of the T-box transcription factor Brachyury is shown to downregulate MMP2 and MMP24 in cancer, to date [43
], there are no reports on the direct participation of TBX4 in MMP gene expression. Therefore, we conducted experiments here to determine whether there is any correlation between MMP and TBX4. Our data showed that TBX4 silencing increases MMP9 expression in A549 and H441 cells. These results suggest that TBX4 is a tumor suppressor in NSCLC and inhibits the migration and invasiveness of NSCLC by decreasing the activity of MMP9.
In addition to epigenetic control via chromosome modification, lncRNA can also affect gene regulation by its association with DNA methylation. Promoter methylation status is known to be involved in the initiation and progression of tumors. TBX4 is reported to be highly methylated in PDAC and human salivary adenoid cystic carcinoma [19
]. In mammalian cells, DNMT3A and DNMT3B are known to establish a DNA methylation pattern de novo, while DNMT1 takes part in the maintenance of methylation status during DNA replication [45
]. One study showed that lncRNA Dum interacts with three DNMTs to mediate and maintain local DNA methylation in the Dppa2 promoter [47
]. In the present study, TTTY15
was found to regulate TBX4 expression via DNA methylation status. Our RNA immunoprecipitation and ChIP data revealed that TTTY15
can interact with DNMT3A and that the binding of DNMT3A to the promoter of TBX4 was blocked by TTTY15
. Furthermore, the overexpression of a DNMT in a variety of tumors results in highly methylated and carcinogenically activated genes [48
]. Moreover, either DNMT3A or DNMT3B is found in many clinical tumor samples, and the increased expression of DNMT3A was previously reported to participate in the progression of hepatocellular carcinoma [49
]. Our results also showed that DNMT3A is overexpressed in NSCLC. These findings suggest that TTTY15
may interact with DNMT3A and decrease the ability of DNMT3A to bind to the TBX4 promoter.
4. Materials and Methods
4.1. Patients and Sample Collection
Thirty-seven paired NSCLC tissue samples and adjacent nontumor tissue samples from male patients (mean age 65.0, SD ± 9.6) were obtained from the Biobank of China Medical University Hospital (CMUH) between 2006 and 2014. This study was conducted with the approval of the CMUH’s Institutional Research Ethics Committee on 11 December 2014 (CMUH103-REC2-140), according to the Declaration of Helsinki’s guidelines. None of the patients had received radiotherapy or chemotherapy prior to surgical resection. All tumor specimens were snap-frozen and stored in liquid nitrogen until analysis. The clinical and histopathological characteristics of each patient were also recorded. Informed written consent was obtained from all the patients.
4.2. Cell Culture and Stable Transfection
Cell lines A549, H441, H2170, and H520 were cultured in in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (10% FBS, Gibco, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in humidified air at 37 °C with 5% of CO2. The medium was refreshed every day, and cell passaging was performed once every 3 days using 0.25% trypsin. The synthesis of shRNA (short hairpin RNA) downregulating TTTY15 was based on the pSUPER RNAi system expression vector (OligoEngine, Seattle, WA, USA). The oligo sequences designed for targeting TTTY15 were as follows: TTTY15-forward: 5′-GATCCCCTTTACAAAGAATTCCAGCTCTGTGGTTCAAGAGACCACAGAGCTGGAATTCTTT GTAAATTTTTA-3′ and TTTY15-reverse: 5′-AGCTTAAAAATTTACAAAGAATTCCAGCTCTGTG GTCTCTTGAAGCCACAGAGCTGGAATTCTTTGTAAAGGG-3′. The annealing and ligation procedures were carried out according to the manufacturer’s instructions. The A549 and H441 cells were transfected with the shTTTY15 plasmid and selected by means of G418. The efficiency of the TTTY15 knockdown was determined by qRT-PCR.
4.3. SiRNA Transfection
The siRNAs for targeting TBX4 and DNMT3A mRNA were constructed by MDBio Inc. (Taipei, Taiwan). The targeting sequences for TBX4 and DNMT3A were 5′-CCGAUGACCAUCGCUACAA TT-3′ and 5′-CAGUGGUGUGUGUUGAGAATT-3′, respectively. The scrambled ribooligonucleotide served as the negative control (a scrambled %GC matched oligonucleotide). A549 and H441 cells were transfected with various doses of siRNA or a scramble ribooligonucleotide by means of RNAimax Lipofectamine (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.
4.4. Subcellular Fractionation, Total RNA Extraction, and qRT-PCR Analysis
The nuclear and cytosolic fractions of the A549 and H441 cell lines were fractionated using the PARIS Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Total RNA was extracted with the TRIzol reagent (Life Technologies, Scotland, UK), according to the manufacturer’s instructions. The RNA was quantitated on a nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA was reverse-transcribed into cDNA using the cDNA Reverse Transcriptase Kit (Thermo Fisher Scientific-Applied Biosystems, Waltham, MA, USA). qRT-PCR was performed via a TaqMan assay, where glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control for mRNA levels. The relative expression levels of the target genes were calculated as ratios normalized to GAPDH. The primers were synthesized by Genomics BioSci and Tech (Taipei, Taiwan). The following primer sequences were used (5′–3′): TTTY15-forward: tgagggagggatgtagctttt; TTTY15-reverse: gaagtcaagcaggcaactga; CCDC3-forward: tttctagccttttccagttttca; CCDC3-reverse: caaacaaggccttctgcac; LINC00674-forward: aagctgggctcaagagatcc; LINC00674-reverse: tggctgtggtggcttgta; KPNA2-forward: tgggccgtgaccaactatac; KPNA2-reverse: tgccacagtgaacaaggtaca; TBX4-forward: ccatcgctacaagttctgtgac; TBX4-reverse: gaatccgggtggacatacag; TIMP2-forward: gtgggtccaaggtcctcat; TIMP2-reverse: cgaagccccagacacatagt; DNMT3A-forward: cctgaagcctcaagagcagt; DNMT3A-reverse: tggtctccttctgttctttgc; MMP2-forward: ccccaaaacggacaaagag; MMP2-reverse: cttcagcacaaacaggttgc; MMP9-forward: cgcagacatcgtcatccagt; MMP9-reverse: cgcagacatcgtcatccagt. The average value of genes was measured using the 2−ΔΔCt method.
4.5. A Cell Proliferation Assay
This assay was conducted to determine whether the TTTY15 knockdown affected the viability of NSCLC cells. Briefly, TTTY15 stable knockdown A549 and H441 cells were seeded in 96-well plates (at 5000 cell/well). After 24, 48, and 72 h, cell proliferation and viability were examined in an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All the experiments were conducted in triplicate.
4.6. Wound Healing Scratch and Transwell Invasion Assays
A wound healing assay was performed to assess the migration ability of cells. A549 and H441 cells were seeded in a 24-well plate as a monolayer at a density of 95–100%. The cell monolayer was gently scratched across the center of the well with a 200 μL plastic pipette tip. The rate of wound closure was observed by imaging by means of an inverted microscope (DMi1; Leica, Wetzlar, Germany). We then used the free software Tscratch to verify and calculate the migration movement of the entire wound area. A cell invasion assay was performed with transwell chambers (8 μm pore size), which were coated with 0.1% gelatin (50 μL/well), in 24-well plates. Approximately 105 either scramble cells or shTTTY15 cells were seeded into the upper chamber of the insert. After incubation for 24 h at 37 °C with 5% CO2, any cells that had invaded the membrane were stained with a 0.1% crystal violet solution. The cells were counted in three randomly selected visual fields under the inverted microscope (DMi1; Leica). For each experimental group, the assay was performed in triplicate.
4.7. Flow Cytometry for Cell Cycle Analysis
Transfected A549 and H441 cells were harvested 48 h after transfection. The cells were fixed in 70% ethanol at −20 °C overnight. The fixed cells were then washed once with phosphate-buffer-saline (PBS) and labeled with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) in the presence of RNase A (Sigma-Aldrich, St. Louis, MO, USA) and Triton X-100 for 30 min in the dark. The samples were run on a FACSCanto flow cytometer (BD Biosciences, San Diego, CA, USA) and the percentages of the cells within each phase of the cell cycle were analyzed in the ModFit LT software.
4.8. Circular Chromosome Conformation Capture
As previously described, 4C was performed [51
]. In brief, A549 cells were crosslinked with 1% formaldehyde for 10 min at room temperature to preserve the three-dimensional nuclear architecture. Sac
I was used to digest the crosslinked chromatin (primary digestion). The digested chromatin was then ligated with the T4 DNA ligase. The digested and ligated chromatin was then de-crosslinked and subjected to the second restriction digestion using CviQ
I to reduce the size of the fragments. Inverse PCRs were carried out with TTTY15
-specific primers containing Illumina adapter sequences to amplify the genomic DNA fragments ligated to TTTY15
(first PCR: forward 5′-tggtgcgatcttgatttactgc-3′, reverse 5′-tgccttttgtctgtatgt gca-3′; nested PCR: forward 5′-tcattcttgttgcccagtctg-3′, reverse 5′-ttcaccttttgttggctccc-3′).
4.9. Next-Generation Sequencing and Bioinformatic Analysis of TTTY15 4C-Sequencing Data
The PCR products were purified with the Qiagen Mini-Elute kit (Qiagen, Hilden, Germany). The amplicon was then prepared for sequencing using the TruSeq DNA library preparation kit (Illumina, San Diego, California). After that, 100 ng purified amplicon pools were repaired to generate blunt-ended ligations, according to the TruSeq DNA Sample Preparation protocol. 5′-Phosphorylated DNA and an A-tailing reaction compatible with the adapter ligation strategy were performed. The ligation product was then purified using sample purification beads. In order to enrich the library, an enhanced PCR mix was used for PCR amplification. The size distribution of the library was verified using the High-Sensitivity DNA Kit (Agilent, Technologies, Waldbronn, Germany), and the concentration of the library was quantified with the GeneRead Library Quant Kit (Qiagen, Hilden, Germany). The library was diluted and sequenced with 500 paired-end cycles on the Illumina MiSeq platform by following the standard protocol. For bioinformatic analysis, any known fragments were removed from the sequencing reads as follows. First, the forward and reverse sequencing reads were merged into one sequence, such that the length of the overlapping region was over 20 nucleotides. Next, the bioinformatic software of sequence BLAST was used to identify the location of the known fragments and primers [52
]. The known fragments were located in the regions between the primers and the cutting site and the alignment similarity for BLAST was set to 95%. Finally, these primers and known fragments of sequencing reads were removed from the sequences. The remaining region of the sequences was labeled as “unknown fragments”. The Bowtie2 software is an efficient tool for the identification of potential TTTY15
interaction regions, therefore by aligning sequencing reads against reference sequences [53
], we aligned unknown fragments against human genome sequences (Grch38.p2 was employed in the present study) in this software. Subsequently, fourSig was used to identify the potential TTTY15
-interacting genome regions [54
]. After scanning for the interacting regions, fourSig provided two categories of regions that were defined as potential TTTY15
interaction regions. The regions and their associated genes were listed and annotated according to the known human genomic location. These two interaction regions were used as the targeting candidates for TTTY15
4.10. The Western Blot Assay
Whole-cell extracts were prepared from A549 and H441 cells by adding radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) (Sigma-Aldrich, St. Louis, MO, USA) with complete protease inhibitor cocktails (Sigma). Equal quantities of total protein samples were separated on 10% SDS-PAGE gels and transferred onto poly vinylidene fluoride (PVDF) membranes. The blots were incubated with primary antibody against DNMT3A (ab13888, Abcam, Cambridge, MA, USA) overnight at 4 °C. After second antibody incubation, the electrochemiluminescence (ECL) kit (EMD Millipore, St. Charles, MO, USA) was utilized to visualize the protein signals. β-Actin (Proteintec, Rosemont, IL, USA) served as the internal control.
4.11. RNA Immunoprecipitation
This procedure was carried out using ChIP-IT Kit (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s instructions. Briefly, endogenous DNMT1 and DNMT3A complexes from the whole-cell extract were pulled down using anti-DNMT1 (ab13537) or anti-DNMT3A (ab13888, Abcam, Cambridge, MA, USA) antibody-coated beads. The beads were then washed with wash buffer and eluted with elution buffer. The eluted samples were incubated with 0.5 mg/mL protease K to remove proteins. The isolate from the immunoprecipitation product was further validated by qRT-PCR.
4.12. Chromatin Immunoprecipitation
The DNA ChIP assay was performed using the ChIP-IT Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, anti-DNMT1 and anti-DNMT3A antibody-coated beads were used to pull down the DNMT1 and DNMT3A complexes from A549 and H441 cells. The beads were washed three times with washing buffer. The beads were then eluted and subjected to reverse crosslinking. The isolate from the IP product was then validated by qRT-PCR. The following primers were designed to amplify the TBX4 promoter region: TBX4 promoter (−749 to −672 bp)-forward: 5′-cagagcctggatcagtcacc-3′, reverse: 5′-tctggcacagacatcctcac-3′ and TBX4 promoter (−1734 to −1626 bp)-forward: 5′-TGAACCAGCTCCTCACAGG-3′, reverse: 5′-CTCTGCTGGGCTCTTG TCAC-3′.
4.13. Methylation-Specific PCR
The methylation status of the promoter region on the TBX4 gene (from −1842 to −1626 bp) was analyzed by methylation-specific PCR. Briefly, the genomic DNA of A549 cells was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) and was then modified with the Zymo EZ DNA Methylation Kit (Zymo Research, Tustin, CA, USA), according to the manufacturer’s instructions. The modified DNA was amplified using a 20 μL mixture including HotStarTag buffer (2.0 mM Mg2+
, 0.2 mM deoxy-ribonucleoside triphosphate (dNTP), 1 U HotStarTag polymerase (Qiagen, Germany)), 0.2 μM of each primer and 1 μL of template DNA. The amplification program for methylation-specific PCR was as follows: an initial incubation for 10 min at 95 °C, followed by 35 cycles of 95 °C for 30 s, 50 °C for 1 min, 72 °C for 1 s, and a final 5 min incubation at 72 °C. The primer information for methylation-specific PCR was shown in Table S3
4.14. Statistical Analysis
All experimental data from the three independent experiments were analyzed in GraphPad Prism version 5 (GraphPad Software Inc., La Jolla, CA, USA). The results were expressed as the mean ± SD (standard deviation). The associations between the relative TTTY15 RNA expression levels and the clinical parameters (age, tumor size, lymph node metastasis, and TNM stage) were analyzed by Fisher’s exact test. A Student’s t-test was conducted to analyze the differences, where p < 0.05 was assumed to indicate a statistically significant difference.