LncRNA GATA6-AS Promotes Cancer Cell Proliferation and Inhibits Apoptosis in Glioma by Downregulating lncRNA TUG1

Research subjects

This study included 58 patients who were diagnosed in PLA Rocket Force General Hospital from May 2015 to April 2018. All patients were subjected to pathological examinations, which were performed by three experienced pathologists. Inclusion criteria: (1) glioma patients with the first diagnosis; (2) patients with no treatment performed before admission; (3) patients willing to participate in this study and signed informed consent. Exclusion criteria: (1) patients who received any treatments before admission; (2) patients complicated with other diseases. There are 18 cases of grade I, 16 cases of grade II, 14 cases of grade III, and 10 cases of grade IV. The 58 patients included 32 males and 26 females, and age ranged from 36 to 68 years, with a mean age of 50.5 ± 5.6 years. This study was approved by the Ethics Committee of the aforementioned hospital before the admission of patients.

Specimens and cell lines

Fasting blood (5 mL) was extracted from each participant before treatments. Blood was transferred to ethylenediamine tetraacetic acid (EDTA) tubes and centrifuged at 1200 g for 10 min to collect plasma samples.

Tumor tissues and tumor adjacent normal tissues were collected from each participant through biopsy. All tissues were confirmed by three experienced pathologists.

Our study included 2 human glioma cell lines, Hs 683 and CCD-25Lu (ATCC). Eagle's Minimum Essential Medium (10% fetal bovine serum [FBS]) was used as cell culture medium and cell culture conditions were 37°C and 5% CO₂.

RT-quantitative polymerase chain reaction

TRIzol reagent (Invitrogen) was mixed with tissue powders (ground in liquid nitrogen), plasma, and in vitro cultivated cells to extract total RNAs. Following DNase I digestion, SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Inc.) was used to synthesize cDNA through reverse transcription. To detect the expression of GATA6-AS and TUG1, polymerase chain reaction (PCR) systems were prepared using the SYBR^® Green Quantitative RT-qPCR Kit (Sigma-Aldrich, St. Louis, MO), and all PCRs were performed with 18S as endogenous control. All data normalizations were performed according to 2^−ΔΔCT method.

Cell transfection

GATA6-AS and TUG1 expression pcDNA3.1 vectors were constructed by Sangon (Shanghai, China). Cells of Hs 683 and CCD-25Lu cell lines were cultivated at confluence of 70%–80%, followed by cell transfections performed using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Inc.) with vectors at a concentration of 10 nM. Control and negative control cells were untransfected cells and cells transfected with empty vectors, respectively. Cells were harvested at 24 h after transfection for subsequent experiments (see Figure 1).

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FIG. 1.

GATA6-AS was significantly upregulated in glioma tissues. RT-quantitative polymerase chain reaction results showed that GATA6-AS expression was significantly upregulated in glioma tissues than in normal healthy tissues (*p < 0.05).

In vitro cell proliferation assay

Cells were harvested at 24 h after transfection. Cells were mixed with Eagle's Minimum Essential Medium (10% FBS) at a ratio of 4 × 10⁴ cells per 1 mL cell culture medium to prepare a single cell suspension, and each well of a 96-well plate was filled with 100 μL cell suspension. Cells were cultivated in an incubator (37°C, 5% CO₂), and CCK-8 solution (10 μL) was added into each well every 24 h until 94 h. After that, cell culture was performed for additional 4 h, followed by the addition of 10 μL dimethyl sulfoxide (DMSO). OD values at 450 nM were measured.

Cell apoptosis assay

Cells were harvested at 24 h after transfection, and 6 × 10⁴ cells were mixed with Eagle's Minimum Essential Medium (1% FBS) to prepare single cell suspensions. Each well of a six-well plate was filled with 2 mL cell suspension, followed by cell culture for 48 h. After that, cells were subjected to 0.25% trypsin digestion. Finally, propidium iodide (Dojindo, Japan) and Annexin V-FITC (Dojindo) staining was performed and flow cytometry was carried out to detect apoptotic cells.

Statistical analyses

Mean ± standard deviation was calculated using data from three biological replicates. All statistical analyses were performed using GraphPad Prism 6 software. Comparisons between normal and tumor tissues were performed by paired t-test. Differences among multiple cell treatment groups were explored by analysis of variance (ANOVA) (one-way) and Tukey's test. Correlation analysis was performed using linear regression. Univariate analysis was performed to analyze the correlations between clinical factors and GATA6 expression. p < 0.05 indicated a difference with statistical significance.

GATA6-AS was significantly upregulated in glioma tissues

RT-quantitative PCR (RT-qPCR) was performed to detect the differential expression of GATA6-AS in glioma and adjacent normal tissues of 58 glioma patients. Compared with normal tissues, significantly upregulated expression of GATA6-AS was observed in glioma tissues, indicating the involvement of this lncRNA in glioma. Univariate analysis found that age (> or ≤50 years), gender, and tumor stages have no significant effects on GATA6-AS expression. Risk ratios were 0.812, 1.428, and 1.232, respectively; 95% confidence intervals were 0.555–1.768, 0.711–2.020, and 0.508–2.545, respectively; p-values were 0.343, 0.888, and 0.596, respectively.

Plasma levels of GATA6-AS were correlated with its expression levels in glioma tissues

RT-qPCR was also performed to detect GATA6-AS in plasma. Linear regression was performed to investigate the correlations between plasma levels of GATA6-AS and its expression in glioma or normal tissues. It was observed that plasma levels of GATA6-AS were significantly and positively correlated with its expression levels in glioma tissues (Fig. 2A), but not in adjacent normal tissues (Fig. 2B).

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FIG. 2.

Plasma levels of GATA6-AS were correlated with its expression levels in glioma tissues. Linear regression analysis showed that plasma levels of GATA6-AS were significantly and positively correlated with its expression levels in glioma tissues (A), but not in adjacent normal tissues (B).

TUG1 was downregulated in glioma and inversely correlated with GATA6-AS

Differential expression of TUG1 in glioma and adjacent normal tissues of 58 glioma patients was detected by RT-qPCR. Compared with normal tissues, TUG1 was significantly downregulated in glioma tissues (Fig. 3A, p < 0.05). Linear regression was performed to investigate the correlations between expression levels of GATA6-AS and TUG1. It was observed that expression levels of TUG1 were inversely correlated with expression levels of GATA6-AS only in glioma tissues (Fig. 3B), but not in adjacent healthy tissues (Fig. 3C).

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FIG. 3.

TUG1 was downregulated in glioma and inversely correlated with GATA6-AS. TUG1 was significantly downregulated in glioma tissues than in adjacent normal tissues (A). Linear regression showed that expression levels of TUG1 were inversely correlated with expression levels of GATA6-AS only in glioma tissues (B), but not in adjacent healthy tissues (C).

GATA6-AS is an upstream inhibitor of TUG1 in glioma cells

Cells of Hs 683 and CCD-25Lu cell lines were transfected with GATA6-AS and TUG1 expression vectors to explore the possible interactions between GATA6-AS and TUG1. Overexpression of GATA6-AS and TUG1 was reached in cells of both cell lines at 24 h after transfection (Fig. 4A, p < 0.05). Compared with untransfection (control [C]) cells and cell transfected with empty vectors (negative control [NC]), GATA6-AS overexpression led to the downregulation of TUG1 (Fig. 4B, p < 0.05), whereas TUG1 overexpression failed to significantly affect GATA6-AS (Fig. 4C).

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FIG. 4.

GATA6-AS is an upstream inhibitor of TUG1 in glioma cells. Overexpression of GATA6-AS and TUG1 was reached in cells of both cell lines at 24 h after transfection (A). GATA6-AS overexpression led to TUG1 downregulation (B), whereas TUG1 overexpression failed to significantly affect GATA6-AS (C) (*p < 0.05).

GATA6-AS regulated glioma cell proliferation and apoptosis through TUG1

Results of in vitro cell proliferation and apoptosis assay showed that, compared with control (C) and negative control (NC) groups, GATA6-AS overexpression led to promoted proliferation (Fig. 5A) and inhibited apoptosis (Fig. 5B) of cells of both Hs 683 and CCD-25Lu cell lines (p < 0.05), TUG1 overexpression played an opposite role, and attenuated the effects of GATA6-AS overexpression.

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FIG. 5.

GATA6-AS regulated glioma cell proliferation and apoptosis through TUG1. GATA6-AS overexpression led to promoted proliferation (A) and inhibited apoptosis (B) of cells of both Hs 683 and CCD-25Lu cell lines. TUG1 overexpression played an opposite role and attenuated the effects of GATA6-AS overexpression (*p < 0.05).