Open Life Sci. 2016; 11: 105–109

Topical Issue on Cancer Signaling, Metastasis and Target Therapy Open Access Research Article

Yan Lei, Li Jing-jing, Zhang Ke-nan, Tian Qing-zhong, Li Jin* A tumor suppressive role of lncRNA GAS5 in human colorectal cancer

DOI 10.1515/biol-2016-0014 Received April 27, 2016; accepted June 15, 2016 1 Introduction

Abstract: Objective: It is already known that long non- Recent large-scale genome-wide projects have identified coding RNA growth arrest-specific 5 (GAS5) is downregulated groups of non-coding RNAs (ncRNAs) ubiquitous in human colorectal cancer (CRC) cells inhibiting cell throughout the mammalian genome. These ncRNAs are proliferation. We further analyzed its involvement in cell actively transcribed, but do not code for proteins [1,2]. cycle distribution and apoptosis induction. Methods: We Like many other emerging disciplines, the ncRNA field has measured the expression level of GAS5 in CRC tissues and received much attention in recent years, and the progress cell lines with the corresponding non-tumoral cells. We also has been extensively viewed from the perspective of analyzed the roles of GAS5 in modulation of cell growth, cell mechanistic research [3-5] and/or biological functions for cycle distribution and apoptosis by the CCK-8 method and these ncRNAs [6-8]. Interestingly, many of these ncRNAs flow cytometry. Western blots were performed to evaluate emerge as tissue-specific in expression and the sequences the protein level of cyclin D1 and p21 after overexpression are somewhat conserved across species. In general, of GAS5 Results: GAS5 expression was significantly reduced ncRNAs have been classified based on an arbitrary size in CRC samples and cell lines. Overexpression of GAS5 cut-off of 200 nt to separate small ncRNAs from long induced cell growth arrest and induced cell apoptosis in non-coding RNAs (lncRNAs). LncRNAs could interact vitro. Meanwhile, we found that the growth suppressive role with numerous biomolecules, including DNA, RNA and of GAS5 might be attributed to the inhibition of G1-S phase proteins, to regulate gene expression at transcriptional, transition, reflected by the downregulation of cyclin D1 and post-transcriptional and epigenetic levels [4,9]. LncRNAs upregulation of p21. Conclusion: Our results demonstrate are known to be involved in many human diseases, that GAS5 is a crucial tumor suppressor in human CRC cells. particularly in the development and progression of human malignancies [10,11]. For instance, functional studies Keywords: GAS5, CRC, cell cycle, apoptosis, tumor revealed a broad spectrum of mechanisms utilized by suppressor 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 *Corresponding author: Li Jin, Department of Oncology Surgery, in T cells when cell growth is inhibited by starvation or Xuzhou Central Hospital, Affiliated Hospital of Medical College of Southeast University, Xuzhou, 221009, People’s Republic of China, rapamycin treatment [15,16]. In bladder cancer, pancreatic E-mail: [email protected] cancer and gastric cancer cells, GAS5 had been shown Yan Lei, Zhang Ke-nan, Department of Gastrointestinal surgery. Jin- to similarly act as a tumor suppressor, in which the gzhou Central Hospital, Affiliated Hospital of Tongji Medical College, downregulation of GAS5 contributed to elevated cell Huazhong University of Science and Technology, Jingzhou, 434020, proliferation capacity, and importantly for these cancers, People‘s Republic of China Li Jing-jing, Department of Hepatobiliary Surgery.Jingzhou Central GAS5 negatively regulated the expression of cyclin- Hospital, Affiliated Hospital of Tongji Medical College, Huazhong dependent kinase 6 (CDK6) [17-19]. Besides proliferation, University of Science and Technology, Jingzhou, 434020, People‘s other reports have also implicated GAS5 in cell-cycle Republic of China regulation [20] and apoptosis [21,22] in stomach cancer, Tian Qing-zhong, Department of Oncology Surgery, Xuzhou Central non-small cell lung cancer, and breast cancer, respectively. Hospital, Affiliated Hospital of Medical College of Southeast Univer- Nevertheless, in human colorectal cancer (CRC) cells, sity, Xuzhou, 221009, People‘s Republic of China Lei Yan and Jingjing Li contribute equally to this work Yin et al discovered that GAS5 was downregulated in

© 2016 Yan Lei et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. 106 Y. Lei, et al. clinical CRC tissues, and GAS5 expression served as an performed on an ABI 7900 (Applied Biosystems). The independent predictor for overall survival in the CRC relative quantification of GAS5 expression was calculated patients. Moreover, overexpression of GAS5 significantly using the 2−DDCT method relative to GAPDH. All of the repressed the proliferation both in vitro and in vivo [23]. qRT-PCR reactions were performed in triplicate. These findings provide new insights regarding GAS5 as a novel tumor suppressor in CRC tumorigenesis. 2.5 Flow Cytometric Analysis of Apoptosis 2 Materials and methods and Cell Cycle The pcDNA3.1 control vector or pcDNA3.1-GAS5 plasmid- 2.1 Clinical CRC tissues transfected HCT116 cells were cultured in 6-well plates for 48 h. The cells were harvested. Following double staining Twenty-four pairs of clinical CRC tissues were collected with FITC-annexin V and propidium iodide (PI), the cells from Jingzhou Central Hospital, Affiliated Hospital of were analyzed by flow cytometry (BD Biosciences). The Tongji Medical College hospital from 2015 August to 2015 percentage of cells in the G0/G1, S, and G2/M phases were December. According to the pathologist, the tumor tissues counted and compared. contained 80-90% CRC cancer cells. Informed written consent was obtained from all patients. 2.6 CCK-8 assay

2.2 Cell lines HCT116 cells were transfected with pcDNA3.1 control vector or pcDNA3.1-GAS5 plasmid for 24 h. After which, Human normal intestine epithelial cells CCD-841 were cells were trypsinzed and seeded into 96-well plates at a obtained from the American Type Culture Collection (USA). density of 2,500 cells. The CCK-8 assay was carried out Human colorectal cancer cells HCT116, SW620, LoVo and using the CCK-8 Kit for 4 days. Absorbance was detected HT29 cells were obtained from Chinese Academy of Sciences at 450 nm using a microplate reader. Experiments were (Shanghai, China). Cells were cultured in Dulbecco’s repeated in triplicate independently. 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. 2.7 Western blot

HCT116 cells transfected with indicated plasmids were 2.3 Construction of vector and plasmid lysed in lysis RIPA buffer and centrifugated­ at 12,000 rpm transfection for 30 min at 4°C. After concentra­tions were determined by BCA method, protein samples were denatured at 95°C Based on the National Center for Biotechnology for 5 min prior to loading onto a 10% SDS-PAGE gel. Information (NCBI) database, the full length of the homo After gel separation, proteins were transferred to PVDF GAS5 coding sequence was amplified and cloned into a membranes and incubated­ with primary antibodies pcDNA3.1 vector (Invitrogen). The control vector or the over night at 4°C. The membranes were washed five pcDNA3.1-GAS5 plasmid was transfected into HCT116 times, 8 min per wash, and incubated with horseradish cells cultured in 6-well plates using Lipofectamine 2000 peroxidase (HRP)-conjugated sec­ondary antibodies for (Invitrogen), according to the manufacturer’s instructions. 1h at room temperature, followed by another five washes. Proteins were visualized by chemiluminescence detection imaging. Cyclin D1, p21 and GAPDH antibodies were 2.4 RNA isolation and qRT-PCR analyses purchased from Santa Cruz.

Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The isolated 2.8 Statistical analysis RNA was reverse-transcribed into cDNA using a reverse transcription kit (Takara). The results were normalized All data were analyzed using SPSS 16.0 software (SPSS, against GAPDH expression. qRT-PCR data collection was Chicago, IL). A two-tailed Student’s t-test was used when A tumor suppressive role of lncRNA GAS5 in human colorectal cancer 107 appropriate, and a P < 0.05 was considered statistically 3.2 GAS5 promoted G1 phase arrest significant. in HCT116 cells

To study the effect of GAS5 overexpression on cellular 3 Results functions, the pcDNA3.1-GAS5 plasmid was constructed. This plasmid or pcDNA3.1 alone was transfected into 3.1 Downregulation of GAS5 was observed HCT116 cells. Upon transfection, GAS5 was significantly in CRC tissues and cell lines upregulated, as shown in Figure 2A. Additionally, the percentage of cells in G1 phase significantly increased after Since a previous study had shown the significant GAS5 overexpression (Figure 2B). Meanwhile, cell growth downregulation of GAS5 in CRC tissues [23], in our rate was monitored by the CCK-8 method. Consistent experiment, we firstly confirmed its expression on with the results in Figure 2B, GAS5 overexpression led to clinical CRC samples by quantitative RT-PCR (qRT-PCR). a significant decrease in the growth rate of HCT116 cells As shown in Figure 1A, GAS5 was decreased in 24 pairs of in culture (Figure 2C). Furthermore, we applied western CRC tissues (P < 0.05). We then measured its expression blotting to analyze the protein expression of cyclin D1 and in the established CRC cell lines. As shown in Figure 1B, p21, and found that cylin D1was downregulated by GAS5 the results from the qRT-PCR demonstrated notable overexpression, while p21, the negative G1/S regulator, decreased expression of GAS5 in HCT116 (P < 0.01), SW620 was increased by GAS5 overexpression (Figure 2D). These (P < 0.05) and LoVo (P < 0.05), when compared with the non- results indicate that GAS5 overexpression could cause cell cancerous intestine epithelial cell line CCD-841 cells. The cycle arrest at the G1 phase, thus leading to reduced cell results confirmed that GAS5 was indeed downregulated growth rates. 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 3.3 Effects of GAS5 overexpression on cell HCT116 cells. apoptosis

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.

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 Figure 1 The GAS5 expression in human CRC tissues and cell line. arrest, but also induced cell apoptosis in CRC cells. Our qRT-PCR results demonstrating GAS5 expression in CRC tissues (A) and cell lines (B) (HCT116, SW620, LoVo, HT29) compared to human findings reveal that GAS5 is a multi-functional lncRNA normal intestine epithelial cells (CCD-841). *: P < 0.05; **: P < 0.01. in CRC cells. 108 Y. Lei, et al.

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.

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 Figure 3 The effect of overexpressing GAS5 on CRC cell apoptosis in in CRC. However, the researchers only illustrated the vitro. The proportion of apoptotic cells is increased after transfec- proliferation suppressing function of GAS5 in CRC cells. tion with pcDNA3.1-GAS5. **: P < 0.01. A tumor suppressive role of lncRNA GAS5 in human colorectal cancer 109

[8] Fatica A., Bozzoni I., Long non-coding RNAs: new players in cell It might also be important that GAS5 takes part in other differentiation and development, Nat. Rev. Genet., 2014 ,15 (1), cellular processes. Indeed, we discovered here that GAS5 7-21. also induced cell apoptosis. But whether it is involved [9] Lai F., Orom U.A., Cesaroni M., Beringer M., Taatjes D.J., Blobel in other cellular processes, including metastasis and G.A., et al., Activating RNAs associate with Mediator to enhance angiogenesis, still needs to be clarified in future studies. chromatin architecture and transcription, Nature, 2013, 494 In this study, we only demonstrated the phenotypes of (7438), 497-501. [10] Harries LW., Long non-coding RNAs and human disease, GAS5 overexpression in CRC cells in vitro. Hence, in vivo Biochem. Soc. Trans., 2012, 40 (4), 902-6. data is still lacking to further clarify its function Meanwhile, [11] Gutschner T., Diederichs S., The hallmarks of cancer: a long in vitro studies can help address why GAS5 is decreased non-coding RNA point of view, RNA Biol., 2012, 9 (6), 703-19. in CRC cells and how it impacts CRC cell growth and [12] Han D., Wang M., Ma N., Xu Y., Jiang Y., Gao X., Long noncoding apoptosis. In other types of cancers, it has been illustrated RNAs: novel players in colorectal cancer, Cancer Lett., 2015, 361 (1), 13-21. that GAS5 could modulate the expression of CDK6 [17- [13] Wang G.Y., Zhu Y.Y., Zhang Y.Q., The functional role of long 19], however, we did not observe significant changes in non-coding RNA in digestive system carcinomas, Bull. Cancer, CDK6 when GAS5 was overexpressed (data not shown). 2014, 101 (9), E27-31. We instead found that cyclin D1 was downregulated and [14] Coccia E.M., Cicala C., Charlesworth A., Ciccarelli C., Rossi p21 was upregulated by GAS5 overexpression. We next G.B., Philipson L., et al., Regulation and expression of a growth will pay more attention to the molecular mechanisms that arrest-specific gene (gas5) during growth, differentiation, and development, Mol. Cell. Biol., 12 (8), 3514-21. GAS5 governs in CRC cells. Future substantial works thus [15] Mourtada-Maarabouni M1., Hedge V.L., Kirkham L., Farzaneh F., might provide more information about this lncRNA. Williams GT., Growth arrest in human T-cells is controlled by the non-coding RNA growth-arrest-specific transcript 5 (GAS5), J. Cell Conflict of interest: Authors declare nothing to disclose. Sci., 121 (7), 939-46. [16] Mourtada-Maarabouni M., Hasan A.M., Farzaneh F., Williams G.T., Inhibition of human T-cell proliferation by mammalian Authors’ contributions: YL performed most of the target of rapamycin (mTOR) antagonists requires noncoding RNA experiments and wrote the manuscript. LJJ, ZKN and TQZ growth-arrest-specific transcript 5 (GAS5), Mol. Pharmacol.,. collected the clinical data. LJ designed the experiments. 2010, 78 (1), 19-28. All authors read and approved experiment. [17] Liu Z., Wang W., Jiang J., Bao E., Xu D., Zeng Y., et al. Downre- gulation of GAS5 promotes bladder cancer cell proliferation, partly by regulating CDK6, PLoS One., 2013, 8 (9), e73991. References [18] Lu X., Fang Y., Wang Z., Xie J., Zhan Q., Deng X., et al., Downre- gulation of gas5 increases pancreatic cancer cell proliferation by [1] Zaratiegui M., Irvine D.V., Martienssen R.A., Noncoding RNAs regulating CDK6, Cell Tissue Res., 2013, 354 (3), 891-6. and gene silencing, Cell, 2007, 128 (4), 763-76. [19] Guo X., Deng K., Wang H., Xia J., Shan T., Liang Z., et al., GAS5 [2] Fu X.D., Non-coding RNA: a new frontier in regulatory biology, Inhibits Gastric Cancer Cell Proliferation Partly by Modulating Natl. Sci. Rev., 2014, 1 (2), 190-204. CDK6, Oncol. Res. Treat., 2015, 38 (7-8), 362-6. [3] Wang X., Song X., Glass C.K., Rosenfeld M.G., The long arm [20] Liu Y., Zhao J., Zhang W., Gan J., Hu C., Huang G., et al., lncRNA of long noncoding RNAs: roles as sensors regulating gene GAS5 enhances G1 cell cycle arrest via binding to YBX1 to transcriptional programs, Cold. Spring. Harb Perspect Biol., regulate p21 expression in stomach cancer, Sci. Rep., 2015, 5, 2011, 3 (1), a003756. 10159. [4] Rinn J.L., Chang H.Y., Genome regulation by long noncoding [21] Shi X., Sun M., Liu H., Yao Y., Kong R., Chen F., et al., A critical RNAs, Annu. Rev. Biochem., 2012, 81, 145-66. role for the long non-coding RNA GAS5 in proliferation and [5] Yang L., Froberg J.E., Lee J.T., Long noncoding RNAs: fresh apoptosis in non-small-cell lung cancer, Mol. Carcinog., 2015, perspectives into the RNA world, Trends. Biochem. Sci., 2014, 54, Suppl 1, E1-E12. 39 (1), 35-43. [22] Pickard M.R., Williams G.T., Regulation of apoptosis by long [6] Esteller M., Non-coding RNAs in human disease, Nat. Rev. non-coding RNA GAS5 in breast cancer cells: implications for Genet., 2011, 12 (12), 861-74. chemotherapy, Breast. Cancer. Res. Treat., 2014, 145 (2), 359-70. [7] Batista P.J., Chang H.Y., Long noncoding RNAs: cellular [23] Yin D., He X., Zhang E., Kong R., De W., Zhang Z., Long noncoding address codes in development and disease, Cell, 2013 ,152 (6), RNA GAS5 affects cell proliferation and predicts a poor prognosis 1298-307. in patients with colorectal cancer, Med., Oncol., 2014, 31 (11), 253.