lncRNA TPT1-AS1 promotes cell migration and invasion in esophageal squamous-cell carcinomas by regulating the miR-26a/HMGA1 axis

2.1 Research subjects

60 ESCC patients from Shanxi Cancer Hospital (Shanxi, China) between January 2017 and January 2019 were involved. Patients who had a history of malignancies or ESCC treatment or had other clinical diseases were excluded. The correlation between TPT1-AS1/miR-26a/HMGA1 expressions and clinicopathological factors of ESCC patients are listed in Tables 1–3.

1. Ethics approval and consent to participate: The present study was approved by the Ethics Committee of Shanxi Cancer Hospital. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. All patients and healthy volunteers provided written informed consent prior to their inclusion within the study.

2.2 RNA interaction prediction

The interaction between TPT1-AS1 and miR-26a was predicted using IntaRNA software (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp).

2.3 ESCC tissue samples and cells

Fine needle biopsies under the guidance of MRI were performed to collect ESCC tissues and their matched adjacent normal-appearing tissues. The tissue sections were stained by hematoxylin and eosin, and their histopathological features were examined by three professional pathologists. The ESCC cancer tissues were diagnosed according to the World Health Organization’s classification for esophageal cancer [28]. The matched adjacent normal-appearing tissues were diagnosed with the criteria that there were no detectable cancer cells in the samples.

KYSE510 and KYSE-30 ESCC cell lines were from BFB Biotechnology Co., Ltd (China). They were cultured in RPMI-1640 medium with 10% FBS under 5% CO₂ at 37°C. The cells were harvested from passages 3–6 for subsequent experiments. All experiments were performed with mycoplasma-free cells, which were authenticated by STR analysis.

2.4 Cell transfection

Short hairpin RNAs targeting TPT1-AS1 (shTPT1-AS1) and scrambled vector (shNC) from Invitrogen (Carlsbad, CA, USA) were used to knockdown TPT1-AS1. The expression vectors of TPT1-AS1 (NCBI accession: NR_024458.1) and HMGA1 (NCBI accession: KJ891364.1) were constructed by Sangon Biotech (China). The miR-26a mimic (5′-UUCAAGUAAUCCAGGAUAGGCU-3′) and negative control (5′-GUACGUAGCUAGUACGGUCCCA-3′) were designed by Invitrogen (USA). The HMGA1 overexpression plasmid was purchased from Gikai Gene Company (GV492, Shanghai, China) (Table 4). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for cell transfection.

2.5 Dual luciferase reporter assay

Dual luciferase reporter assay was performed following a previous work [29]. Briefly, pGL3 Promoter Luciferase Reporter Vector (Promega Corporation) was used to construct TPT1-AS1 vector. KYSE510 cells were transfected with pGL3-TPT1-AS1-promoter + pRL-TK + NC miRNA (NC group) or pGL3-TPT1-AS1-promoter + pRL-TK + miR-26a mimic (miR-26a group) using Lipofectamine 2000. The luciferase activity was determined using LucPair™ Duo-Luciferase Assay Kit (GeneCopoeia), and the firefly/Renilla activity ratio was calculated.

HMGA1 wild-type and mutant-type luciferase reporter vector targeting the miR-26a binding site were constructed. The vectors and miR-26a mimics were co-transfected into cells using Lipofectamine 2000 reagent, and luciferase activities were detected 48 h later using the dual luciferase reporter system (Promega, USA).

2.6 RNA preparations and RT-qPCR

Total RNAs and miRNAs were extracted from KYSE510 cells and tissue samples using Direct-zol RNA Kit (R2061, Zymo Research) and PureLink miRNA Isolation Kit (K157001, Thermo Fisher Scientific), respectively. The cDNA samples were obtained by using QuantiTect Reverse Transcription Kit (205311, QIAGEN). RT-qPCR reactions were performed with KAPA SYBR® FAST qPCR Master Mix (2X) Kit (KR0390, Kapa Biosystems) or All-in-One™ miRNA qRT-PCR Detection Kit (QP015/AOMD-Q020, GeneCopoeia). GAPDH and U6 were selected as the internal controls for regular genes and miRNA, respectively. The corresponding primer sequences were 5′-CGTTTGGACCCCTGTCTTGGAC-3′ (forward) and 5′-CAGAAAGAAAGCAGGTCATT-3′ (reverse) for TPT1-AS1; 5′-TGAGTCGAGCTCGAAGTCCAG-3′ (forward) and 5′-CTTAGGTGTTGGCACTTCGC-3′ (reverse) for HMGA1; and 5′-GTCTCCTCTGACTTCAACAGC-3′ (forward) and 5′-CCACCCTGTTGCTGTAGCCAA-3′ (reverse) for GAPDH. The forward miR-26a primer was 5′-UUCAAGUAAUCCAGGAUAG-3′. The reverse miR-26a primer and U6 primers were from the kit. The PCR reaction conditions were 1 min at 95°C followed by 40 cycles of 95°C for 10 s and 58°C for 50 s. Each experiment was performed with 3 replicates, and the relative expression level was calculated using the 2^−ΔΔCt method.

2.7 Subcellular fractionation

The nuclear and cytoplasm extracts of KYSE510 cells were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (pierce-78835, Pierce, USA). The TPT1-AS1, U6, and GAPDH levels in the nuclear and cytoplasmic extracts were detected by RT-qPCR.

2.8 Western blotting

Total proteins were isolated from in vitro cultivated cells and quantified using bicinchoninic acid assay method. 50 μg (10 μL) of total proteins were loaded to each sample well and separated by 10% SDS-PAGE. The separated proteins were transferred onto PVDF membranes, which were blocked with 5% nonfat dry milk for 2 h and incubated with antibodies against GAPDH (ab9845, Abcam) and HMGA1 (ab226850, Abcam) at 4°C for 18 h, followed by incubation with secondary antibody (ab6721, Abcam) at room temperature for 2 h. The target protein signals were detected using the enhanced chemiluminescence system (Amersham).

2.9 Transwell assays

Transwell assays were performed using KYSE510 and KYSE-30 cells collected at 48 h post-transfection using Transwell inserts (8 µm, 3415, Corning). For invasion assay, the Transwell inserts were pre-treated with 50 μL/well Matrigel (dilution rate = 1:3) at 37°C for 6 h. For migration assay, the uncoated Transwell inserts were used. 4,000 cells were seeded into the upper chamber, and RPMI-1640 media with 20% FBS were added into the lower chamber. 12 h later, the lower surface of membranes was stained using crystal violet (0.1%, Sigma-Aldrich) for 20 min. Images were analyzed using ImageJ software to measure the percentage of the area of cells present in each insert.

2.10 Statistics

The experimental data (three replicates) were analyzed using SPSS 17.0 software. Data from ESCC tissue samples were analyzed using paired t test and Pearson’s correlation coefficient. One-tailed t-test was used in the study. Unpaired t test was applied to compare the difference between two groups. ANOVA was used to compare the difference among multiple groups. The threshold of p-value was set to 0.05.

3.1 TPT1-AS1 interacted with miR-26a

TPT1-AS1 was predicted to interact with miR-26a by IntaRNA (Figure 1a). As shown in Figure 1b, miR-26a overexpression markedly inhibited the luciferase activity in KYSE510 cells transfected with pGL3-TPT1-AS1-promoter and pRL-TK, indicating that TPT1-AS1 interacted with miR-26a. Moreover, we isolated the nuclear and cytoplasmic extracts from ESCC cell line KYSE510. Subsequent RT-qPCR assay indicated that TPT1-AS1 was mainly localized in the cytoplasm of KYSE510 cells (Figure 1c). MiR-26a overexpression significantly reduced the luciferase activity of the HMGA1-wt vector but failed to decrease that of the HMGA1-mut (Figure A1).

Figure 1

TPT1-AS1 interacted with miR-26a. (a) TPT1-AS1 was predicted to interact with miR-26a. (b) KYSE510 cells were transfected with pGL3-TPT1-AS1-promoter + pRL-TK + NC miRNA or pGL3-TPT1-AS1-promoter + pRL-TK + miR-26a mimic. Luciferase activity was assessed by using the corresponding assay kit. (c) TPT1-AS1 level in the nucleus and cytoplasm of ESCC cells. *, p < 0.05.

3.2 TPT1-AS1 level was increased while miR-26a level was reduced in ESCC tissues

TPT1-AS1 and miR-26a levels in ESCC and matched non-tumor tissues were assessed. The results indicated that TPT1-AS1 level was markedly increased in ESCC tissues (Figure 2a), while miR-26a level was apparently reduced in ESCC tissues (Figure 2b). The expression levels of TPT1-AS1 and miR-26a/HMGA1 in these two cells were compared with KYSE180 cells and normal cells. The results indicated that TPT1-AS1 level was increased in KYSE180 cells and KYSE510 cells (Figure A3a), while miR-26a level was reduced in KYSE180 cells and KYSE510 cells (Figure A3b). In addition, HMGA level was increased in KYSE180 cells and KYSE510 cells (Figure A3c). Moreover, TPT1-AS1 level was increased in KYSE180 cells and KYSE-30 cells (Figure A3d), while miR-26a level was reduced in KYSE180 cells and KYSE-30 cells (Figure A3e). Moreover, HMGA level was increased in KYSE180 cells and KYSE-30 cells (Figure A3f).

Figure 2

TPT1-AS1 level was increased, while miR-26a level was reduced in ESCC tissues. TPT1-AS1 (a) and miR-26a (b) levels in ESCC and matched non-tumor tissues were measured by RT-qPCR. ***, p < 0.001.

3.3 TPT1-AS1 enhanced HMGA1 expression in ESCC cells by sponging miR-26a

The correlation among TPT1-AS1, miR-26a, and HMGA1 levels were assessed. As shown in Figure 3a, TPT1-AS1 level in ESCC tissues was not obviously correlated with miR-26a level. Interestingly, TPT1-AS1 level in ESCC tissues was positively correlated with HMGA1 mRNA, a target of miR-26a (Figure 3b). We then transfected KYSE510 cells with TPT1-AS1 expression vector or miR-26a mimic. As shown in Figure 4a, TPT1-AS1 overexpression and miR-26a mimic remarkably upregulated TPT1-AS1 and miR-26a levels in KYSE510 cells, respectively. As shown in Figure 4b, TPT1-AS1 and miR-26a overexpression did not affect the expression levels of each other. In addition, we also assessed the effects of TPT1-AS1 and miR-26a overexpression on HMGA1. The results indicated that TPT1-AS1 overexpression promoted HMGA1 mRNA and protein levels, while miR-26a overexpression inhibited HMGA1 mRNA and protein levels (Figure 4c and d). Moreover, miR-26a overexpression attenuated the effects of TPT1-AS1 overexpression on HMGA1 mRNA and protein expression (Figure 4c and d). Furthermore, TPT1-AS1 knockdown had no effect on miR-26a expression (Figure 4e) but decreased HMGA1 expression (Figure 4f). Collectively, TPT1-AS1 might enhance HMGA1 expression by sponging miR-26a.

Figure 3

TPT1-AS1 was positively correlated with HMGA1 mRNA. (a) The correlation between TPT1-AS1 and miR-26a across ESCC tissues. (b) The correlation between TPT1-AS1 and HMGA1 mRNA across ESCC tissues.

Figure 4

TPT1-AS1 enhanced HMGA1 expression by sponging miR-26a. TPT1-AS1 expression vector and miR-26a mimic were transfected into KYSE510 cells. (a and b) TPT1-AS1 and miR-26a levels were assessed by RT-qPCR. (c and d) HMGA1 expression level was assessed by RT-qPCR and western blotting. (e and f) The expressions of miR-26a and HMGA1 were assessed by RT-qPCR in KYSE510 cells transacted with shTPT1-AS1. *, p < 0.05.

3.4 TPT1-AS1 enhanced ESCC cell migration and invasion by regulating the miR-26a/HMGA1 axis

The function of TPT1-AS1 in ESCC cells was explored. TPT1-AS1 and HMGA1 overexpression markedly promoted migration and invasion of ESCC cells, while miR-26a overexpression and TPT1-AS1 knockdown obviously suppressed migration and invasion of ESCC cells. Moreover, the effects of TPT1-AS1 and HMGA1 overexpression on migration and invasion were alleviated by miR-26a overexpression (Figure 5a and b).

Figure 5

TPT1-AS1 regulated the miR-26a/HMGA1 axis to enhance KYSE510 and KYSE-30 cell migration and invasion. ESCC cells migration (a) and invasion (b) abilities were determined by Transwell assays. *, p < 0.05.