A tumor suppressive role of lncRNA GAS5 in human colorectal cancer

Yan Lei; Li Jing-jing; Zhang Ke-nan; Tian Qing-zhong; Li Jin

doi:10.1515/biol-2016-0014

Article Open Access

A tumor suppressive role of lncRNA GAS5 in human colorectal cancer

Yan Lei , Li Jing-jing , Zhang Ke-nan , Tian Qing-zhong and Li Jin

Published/Copyright: July 26, 2016

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From the journal Open Life Sciences Volume 11 Issue 1

Abstract

Objective

It is already known that long non-coding RNA growth arrest-specific 5 (GAS5) is downregulated in human colorectal cancer (CRC) cells inhibiting cell proliferation. We further analyzed its involvement in cell cycle distribution and apoptosis induction.

Methods

We measured the expression level of GAS5 in CRC tissues and cell lines with the corresponding non-tumoral cells. We also analyzed the roles of GAS5 in modulation of cell growth, cell cycle distribution and apoptosis by the CCK-8 method and flow cytometry. Western blots were performed to evaluate the protein level of cyclin D1 and p21 after overexpression of GAS5

Results

GAS5 expression was significantly reduced in CRC samples and cell lines. Overexpression of GAS5 induced cell growth arrest and induced cell apoptosis in vitro. Meanwhile, we found that the growth suppressive role of GAS5 might be attributed to the inhibition of G1-S phase transition, reflected by the downregulation of cyclin D1 and upregulation of p21.

Conclusion

Our results demonstrate that GAS5 is a crucial tumor suppressor in human CRC cells.

Key words: GAS5; CRC; cell cycle; apoptosis; tumor suppressor

1 Introduction

Recent large-scale genome-wide projects have identified groups of non-coding RNAs (ncRNAs) ubiquitous throughout the mammalian genome. These ncRNAs are actively transcribed, but do not code for proteins [1,2]. Like many other emerging disciplines, the ncRNA field has received much attention in recent years, and the progress has been extensively viewed from the perspective of mechanistic research [3-5] and/or biological functions for these ncRNAs [6-8]. Interestingly, many of these ncRNAs emerge as tissue-specific in expression and the sequences are somewhat conserved across species. In general, ncRNAs have been classified based on an arbitrary size cut-off of 200 nt to separate small ncRNAs from long non-coding RNAs (lncRNAs). LncRNAs could interact with numerous biomolecules, including DNA, RNA and proteins, to regulate gene expression at transcriptional, post-transcriptional and epigenetic levels [4,9]. LncRNAs are known to be involved in many human diseases, particularly in the development and progression of human malignancies [10,11]. For instance, functional studies revealed a broad spectrum of mechanisms utilized by lncRNAs such as HOTAIR, MALAT1, ANRIL or lincRNA-p21 to fulfill their functions in human cancers [11-13].

Growth arrest-specific transcript 5 (GAS5), located at 1q25, is a growth suppressor [14] that is upregulated in T cells when cell growth is inhibited by starvation or rapamycin treatment [15,16]. In bladder cancer, pancreatic cancer and gastric cancer cells, GAS5 had been shown to similarly act as a tumor suppressor, in which the downregulation of GAS5 contributed to elevated cell proliferation capacity, and importantly for these cancers, GAS5 negatively regulated the expression of cyclindependent kinase 6 (CDK6) [17-19]. Besides proliferation, other reports have also implicated GAS5 in cell-cycle regulation [20] and apoptosis [21,22] in stomach cancer, non-small cell lung cancer, and breast cancer, respectively. Nevertheless, in human colorectal cancer (CRC) cells, Yin et al discovered that GAS5 was downregulated in clinical CRC tissues, and GAS5 expression served as an independent predictor for overall survivalin the CRC patients. Moreover, overexpression of GAS5 significantly repressed the proliferation both in vitro and in vivo [23]. These findings provide new insights regarding GAS5 as a novel tumor suppressor in CRC tumorigenesis.

2 Materials and methods

2.1 Clinical CRC tissues

Twenty-four pairs of clinical CRC tissues were collected from Jingzhou Central Hospital, Affiliated Hospital of Tongji Medical College hospital from 2015 August to 2015 December. According to the pathologist, the tumor tissues contained 80-90% CRC cancer cells. Informed written consent was obtained from all patients.

2.2 Cell lines

Human normal intestine epithelial cells CCD-841 were obtained from the American Type Culture Collection (USA). Human colorectal cancer cells HCT116, SW620, LoVo and HT29 cells were obtained from Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 2 mM L-glutamine (Invitrogen, USA) and were grown at 37°C in a 10% CO₂ atmosphere.

2.3 Construction of vector and plasmidtransfection

Based on the National Center for Biotechnology Information (NCBI) database, the full length of the homo GAS5 coding sequence was amplified and cloned into a pcDNA3.1 vector (Invitrogen). The control vector or the pcDNA3.1-GAS5 plasmid was transfected into HCT116 cells cultured in 6-well plates using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions.

2.4 RNA isolation and qRT-PCR analyses

Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The isolated RNA was reverse-transcribed into cDNA using a reverse transcription kit (Takara). The results were normalized against GAPDH expression. qRT-PCR data collection was performed on an ABI 7900 (Applied Biosystems). The relative quantification of GAS5 expression was calculated using the 2^-DDCT method relative to GAPDH. All of the qRT-PCR reactions were performed in triplicate.

2.5 Flow Cytometric Analysis of Apoptosisand Cell Cycle

The pcDNA3.1 control vector or pcDNA3.1-GAS5 plasmid-transfected HCT116 cells were cultured in 6-well plates for 48 h. The cells were harvested. Following double staining with FITC-annexin V and propidium iodide (PI), the cells were analyzed by flow cytometry (BD Biosciences). The percentage of cells in the G0/G1, S, and G2/M phases were counted and compared.

2.6 CCK-8 assay

HCT116 cells were transfected with pcDNA3.1 control vector or pcDNA3.1-GAS5 plasmid for 24 h. After which, cells were trypsinzed and seeded into 96-well plates at a density of 2,500 cells. The CCK-8 assay was carried out using the CCK-8 Kit for 4 days. Absorbance was detected at 450 nm using a microplate reader. Experiments were repeated in triplicate independently.

2.7 Western blot

HCT116 cells transfected with indicated plasmids were lysed in lysis RIPA buffer and centrifugated at 12,000 rpm for 30 min at 4°C. After concentrations were determined by BCA method, protein samples were denatured at 95°C for 5 min prior to loading onto a 10% SDS-PAGE gel. After gel separation, proteins were transferred to PVDF membranes and incubated with primary antibodies over night at 4°C. The membranes were washed five times, 8 min per wash, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1h at room temperature, followed by another five washes. Proteins were visualized by chemiluminescence detection imaging. Cyclin D1, p21 and GAPDH antibodies were purchased from Santa Cruz.

2.8 Statistical analysis

All data were analyzed using SPSS 16.0 software (SPSS, Chicago, IL). A two-tailed Student’s t-test was used when appropriate, and a P < 0.05 was considered statistically significant.

3 Results

3.1 Downregulation of GAS5 was observedinCRC tissues and cell lines

Since a previous study had shown the significant downregulation of GAS5 in CRC tissues [23], in our experiment, we firstly confirmed its expression on clinical CRC samples by quantitative RT-PCR (qRT-PCR). As shown in Figure 1A, GAS5 was decreased in 24 pairs of CRC tissues (P < 0.05). We then measured its expression in the established CRC cell lines. As shown in Figure 1B, the results from the qRT-PCR demonstrated notable decreased expression of GAS5 in HCT116 (P < 0.01), SW620 (P < 0.05) and LoVo (P < 0.05), when compared with the non-cancerous intestine epithelial cell line CCD-841 cells. The results confirmed that GAS5 was indeed downregulated in CRC tissues and cells, and our findings for these cell lines allowed us to choose the HCT116 cell line for further functional analysis to disclose the functions of GAS5 in HCT116 cells.

Figure 1

The GAS5 expression in human CRC tissues and cell line. qRT-PCR results demonstrating GAS5 expression in CRC tissues (A) and cell lines (B) (HCT116, SW620, LoVo, HT29) compared to human normal intestine epithelial cells (CCD-841). *: P < 0.05; **: P < 0.01.

3.2 GAS5 promoted G1 phase arrestinHCT116 cells

To study the effect of GAS5 overexpression on cellular functions, the pcDNA3.1-GAS5 plasmid was constructed. This plasmid or pcDNA3.1 alone was transfected into HCT116 cells. Upon transfection, GAS5 was significantly upregulated, as shown in Figure 2A. Additionally, the percentage of cells in G1 phase significantly increased after GAS5 overexpression (Figure 2B). Meanwhile, cell growth rate was monitored by the CCK-8 method. Consistent with the results in Figure 2B, GAS5 overexpression led to a significant decrease in the growth rate of HCT116 cells in culture (Figure 2C). Furthermore, we applied western blotting to analyze the protein expression of cyclin D1 and p21, and found that cylin D1was downregulated by GAS5 overexpression, while p21, the negative G1/S regulator, was increased by GAS5 overexpression (Figure 2D). These results indicate that GAS5 overexpression could cause cell cycle arrest at the G1 phase, thus leading to reduced cell growth rates.

Figure 2

The effect of overexpressing GAS5 on CRC cell cell cycle and proliferation in vitro. (A)GAS5 expression, measured by qRT-PCR, following the transfection of HCT116 cell line with pCDNA3.1 control vector or pcDNA3.1-GAS5 plasmid. (B) The percentage of cells arresting in G1 phase was increased in HCT116 when overexpression of GAS5. (C) Cell Counting assay was performed to measure the proliferation of HCT116 cells after transfected with pcDNA3.1 control vector or pcDNA3.1-GAS5 plasmid. *: P < 0.05; **: P < 0.01. (D) Western blot was carried out the analyze the expression of cyclin D1 and p21 in indicated cells. GAPDH was as the internal control.

3.3 Effects of GAS5 overexpression on cellapoptosis

Cell apoptosis rate was measured for transfected plasmids by double staining with FITC-annexin V and propidium iodide (PI) followed by flow cytometry. As shown in Figure 3, for the GAS5 overexpressed cells, the ratio of apoptotic cells substantially increased relative to the control vector-transfected cells (P < 0.01). It was notable that the apoptosis induced by GAS5 overexpression seemed more serious than the cell growth limitation, which indicated that apoptosis induction might be the major cellular consequence of GAS5 overexpression in HCT116 cells.

Figure 3

The effect of overexpressing GAS5 on CRC cell apoptosis in vitro. The proportion of apoptotic cells is increased after transfec-tion with pcDNA3.1-GAS5. **: P < 0.01.

4 Discussion

Our findings demonstrate that lncRNA GAS5 acts as a potential effective tumor suppressor in human CRC cells. Particularly, expression was significantly reduced in CRC cell lines when compared to normal CCD-841 cells. Functionally, lncRNA GAS5 not only inhibited cell growth, which can be explained by the cell cycle arrest, but also induced cell apoptosis in CRC cells. Our findings reveal that GAS5 is a multi-functional lncRNA in CRC cells.

As the current understanding of GAS5 in CRC cells had merely considered that it could regulate cell proliferation [23], it was unclear whether GAS5 could also impact other cellular processes. Our study enriched the knowledge reagrding the roles of GAS5 in CRC cells. In the previous study [23], the researchers analyzed the correlation of decreased expression of GAS5 with clinicalpathological features of the CRC patients. That study illustrated that downregulated GAS5 levels was significantly correlated with large tumor size, low histological grade and advanced TNM stage. They also performed multivariate analysis and found that GAS5 expression could be utilized as an independent predictor for overall survival of CRC patients. These findings strongly highlighted the possible usage of GAS5 as an efficient biomarker or therapeutic target in CRC. However, the researchers only illustrated the proliferation suppressing function of GAS5 in CRC cells. It might also be important that GAS5 takes part in other cellular processes. Indeed, we discovered here that GAS5 also induced cell apoptosis. But whether it is involved in other cellular processes, including metastasis and angiogenesis, still needs to be clarified in future studies.

In this study, we only demonstrated the phenotypes of GAS5 overexpression in CRC cells in vitro. Hence, in vivo data is still lacking to further clarify its function Meanwhile, in vitro studies can help address why GAS5 is decreased in CRC cells and how it impacts CRC cell growth and apoptosis. In other types of cancers, it has been illustrated that GAS5 could modulate the expression of CDK6 [1719], however, we did not observe significant changes in CDK6 when GAS5 was overexpressed (data not shown). We instead found that cyclin D1 was downregulated and p21 was upregulated by GAS5 overexpression. We next will pay more attention to the molecular mechanisms that GAS5 governs in CRC cells. Future substantial works thus might provide more information about this lncRNA.

Conflict of interest

Authors declare nothing to disclose.

Authors’ contributions

YL performed most of the experiments and wrote the manuscript. LJJ, ZKN and TQZ collected the clinical data. LJ designed the experiments. All authors read and approved experiment.

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Received: 2016-4-27

Accepted: 2016-6-15

Published Online: 2016-7-26

Published in Print: 2016-1-1

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