Sage Journals: Discover world-class research

Abstract

Accumulating evidence has shown that lncRNA GAS5 is a novel tumour-promoting RNA that contributes to tumour progression by sponging miRNAs. However, the detailed role of lncRNA GAS5 in B lymphocytic leukaemia is still unclear. A qRT-PCR assay was used to examine the levels of lncRNA GAS5 and miR-222 in leukomonocytes of patients with B lymphocytic leukaemia and in healthy donors. Raji cells were transfected with GAS5 overexpression or shRNA-GAS5 plasmids for $48h$ , and cell proliferation was assessed by the CCK-8 assay, while apoptosis and cell cycle progression were assessed using flow cytometry. The Transwell assay was applied to detect the invasion of Raji cells with GAS5 overexpression or knockdown. The dual luciferase reporter assay and regression curve were conducted to evaluate the binding interaction between lncRNA GAS5 and miR-222. The results showed that the expression of lncRNA GAS5 was decreased in B lymphocytic leukaemia patients compared with the healthy group, and the levels of lncRNA GAS5 in B lymphocytic leukaemia cell lines were significantly higher than those in the normal B cell line, whereas the levels of miR-222 were increased in B lymphocytic leukaemia patients compared with the healthy group. Moreover, cell culture experiments indicated that lncRNA GAS5 overexpression decreased B lymphocytic leukaemia cell proliferation, promoted B lymphocytic leukaemia cell apoptosis, arrested B lymphocytic leukaemia cells in the G1 phase of the cell cycle, and inhibited B lymphocytic leukaemia cell invasion. Finally, the luciferase reporter assay showed a direct target interaction between lncRNA GAS5 and miR-222. The regression analysis showed a negative correlation between the levels of lncRNA GAS5 and miR-222. Thus, our data suggested that lncRNA GAS5 could effectively sponge miR-222 to modulate human B lymphocytic leukaemia cell tumourigenesis and metastasis. This work advances our understanding of the clinical significance of lncRNA GAS5 from the perspective of lncRNA-miRNA regulation.

Keywords

B lymphocytic leukaemia lncRNA GAS5/miR-222 axis tumourigenesis and metastasis

1. Introduction

Chronic lymphocytic leukaemia, defined as a lymphoproliferative disorder with low immunoglobulin levels and composed of monomorphic round B lymphocytes in peripheral blood, bone marrow and lymphoid organs, is the most common type of adult leukaemia in western countries [1, 2]. Recent studies have indicated that there are approximately 15000 new cases of chronic lymphocytic leukaemia per year, with approximately 4500 deaths, thereby chronic lymphocytic leukaemia and its associated high rate of morbidity and mortality are still a major health problem [3, 4]. In the past few years, chronic lymphocytic leukaemia treatment has evolved from chemotherapy to the use of agents specifically targeting various signalling pathways, but these treatment effects are still very limited [5, 6]. Thus, a detailed understanding of the molecular mechanisms involved in chronic lymphocytic leukaemia requires further investigation.

Dysregulated expression of miRNAs has been found in different human cancers, and the functions of miRNAs in cancer are quite complex; it has been well investigated that miR-222 functions as an oncogene in multiple cancer types, including colorectal cancer [7], cervical cancer [8], glioblastoma [9], osteosarcoma [10], and breast cancer [11]. In chronic lymphocytic leukaemia, Frenquelli et al. reported that enforced expression of miR-222 can modulate G1/S phase cell cycle transition by suppressing p27 expression, which could promote tumour cell proliferation [12].

Long non-coding RNAs (lncRNAs), a type of highly conserved RNA transcripts composed of more than 200 nucleotides in length and lacking protein-coding potential, have been found to be involved in transcriptional and post-transcriptional regulation of gene expression [13]. Accumulating evidence has demonstrated that lncRNAs could play pivotal roles in almost all major physiological processes as well as multiple pathological processes, especially the pathogenesis of different types of malignant tumours [14, 15]. Previous studies have reported that lncRNA GAS5 could play a role as a tumour-suppressor gene in the development of several cancer types [16, 17]. For example, lncRNA GAS5 can suppress ovarian cancer by inducing the formation of the inflammasome [18]; lncRNA GAS5 binds to Y-box binding protein 1 (YBX1) to regulate p21 expression, which arrests the cells at the G1 phase of the cell cycle in stomach cancer [19]; and lncRNA GAS5 inhibits oesophageal squamous cell carcinoma cell proliferation and migration by inactivating the phosphatidylinositol 3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) signalling pathway [20]. In addition, intensive research in the past two decades has uncovered that lncRNAs can bind to microRNAs (miRNAs) to sequester and control them, such as lncRNA-BGL3 (to miR-17, miR-93, miR-20a, miR-20b, miR-106a and miR-106b) [21], MIAT (to miR-150-5p) [22], Linc-RoR (to miR-145) [23], and lncRNA GAS5 (to miR-222) [24]. Hence, it is necessary to explore whether lncRNA GAS5 could function as a sponge for miR-222 to exert its role during the development of human B lymphocytic leukaemia.

2. Materials and methods

2.1 Patients and cell lines

Blood specimens were obtained from 30 human B lymphocytic leukaemia cell patients from November 2017 to March 2018 in the Qingdao Hospital of Traditional Chinese Medicine. In addition, 30 normal blood specimens from healthy donors were collected as control samples. Moreover, all samples were frozen in liquid nitrogen and maintained at $-$ 80 ${}^{\circ}$ C until use. All participants enrolled in this study were informed about the study, and written consent was obtained prior to the experiments. The Human Research Ethics Committee of the Qingdao Hospital of Traditional Chinese Medicine granted ethics approval for this study. All the methods in the present study were in accordance with the ethical guidelines of the Helsinki Declaration. The human B lymphocytic leukaemia cell lines RAMOS, ST486, Raji, and Farage and the normal B lymphocytic cell line IM9 were purchased from American Type Culture Collection (ATCC). All cells were cultured at 37 ${}^{\circ}$ C in a humidified 5% CO ${}_{2}$ environment in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) or Roswell Park Memorial Institute 1640 medium (RPMI-1640; Thermo Fisher Scientific, USA) supplemented with 10% foetal bovine serum (FBS; Gibco, USA) plus 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 $\mu$ g/mL streptomycin.

2.2 RNA extraction and quantitative RT-PCR (qRT-PCR) assay

Total RNA from clinical blood samples or cultured cells was extracted using TRIzol reagent (Invitrogen, USA) according to the provided protocol. Briefly, 10 ${}^{7}$ cells separated on lymphocyte separation medium (GE, 17-1440-02, Shanghai) or 10 ${}^{7}$ cultured lymphocyte cells were collected for RNA extraction. RNA quantity and quality were determined by a NanoDrop 2000c (Thermo Scientific, USA) spectrophotometer. Reverse transcription was performed using the PrimeScript RT Reagent Kit (Takara, Japan) to synthesize complementary DNA (cDNA). Then, qRT-PCR was carried out using a SYBR Green qPCR SuperMix Kit (Invitrogen, USA) with an ABI $\text{PRISM}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ 7500 Sequence Detection System (Applied Biosystems, USA). The reactions were incubated in a 96-well optical plate at 95 ${}^{\circ}$ C for 2 min, followed by 40 cycles of 15 s at 95 ${}^{\circ}$ C and 32 s at 60 ${}^{\circ}$ C and dissociation at 95 ${}^{\circ}$ C for 60 s, 55 ${}^{\circ}$ C for 30 s and 95 ${}^{\circ}$ C for 30 s. The relative expression of lncRNA GAS5 and miR-222 was calculated using the $2^{\rm-\Delta\Delta Ct}$ method by normalizing to 18S rRNA and U6 snRNA levels, respectively. The primer sequences in this study are presented as follows: 5’-CTT CTG GGC TCA AGT GAT CCT-3’ and 5’-TTG TGC CAT GAG ACT CCA TCA G-3’ for lncRNA GAS5; 5’-ACA CTC CAG CTG GGA GCT ACA TCT GGC TAC TG-3’ and 5’-CTC AAC TGG TGT CGT GGA-3’ for miR-222; 5’-GTA ACC CGT TGA ACC CCA TT-3’ and 5’-CCA TCC AAT CGG TAG TAG CG-3’ for 18S rRNA; 5’-CGC TTC GGC AGC ACA TAT ACT AAA ATT GGA AC-3’ and 5’-GCT TCA CGA ATT TGC GTG TCA TCC TTG C-3’ for U6 snRNA.

2.3 Cell transfection

To overexpress and knock down lncRNA GAS5 in cell lines, lncRNA GAS5-overexpression (GAS5-pcDNA3.1), lncRNA GAS5-knockdown (GAS5 shR-NA) and negative control (NC) plasmids were synthesized and purchased from Genepharma Inc. (China). Then, Raji cells were seeded in 6-well plates at a density of 5 $\times$ 10 ${}^{6}$ cells/well and transfected with the above plasmids using $\text{FuGENE}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ HD Transfection Reagent (Promega, USA) following the manufacturer’s instructions. After 48 h of transfection, the cells were collected to further detect the expression of lncRNA GAS5 and miR-222 via qRT-PCR.

2.4 Cell proliferation assay

Cell proliferation was assessed using a cell counting kit-8 (CCK-8) assay (Dojindo, Japan). A total of 2 $\times$ 10 ${}^{4}$ cells per well were placed into 96-well plates and maintained for the indicated time periods (0 day, 1 day, 2 days and 3 days). At the end of the incubation period, the medium was renewed, and 10 $\mu$ l CCK-8 solution was supplemented into each well. Cells were cultured at 37 ${}^{\circ}$ C for another 4 h, and a Varioskan LUX microplate reader (Thermo Fisher Scientific, USA) was used to measure OD values at 450 nm wavelength.

2.5 Flow cytometry analysis

Flow cytometry for cell apoptosis detection was carried out using an Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, China) in accordance with the manufacturer’s recommendations. Cells were harvested during the logarithmic growth phase and then successively stained with 250 $\mu$ l Annexin V binding buffer and 10 $\mu$ l freshly prepared Annexin V-propidium iodide (PI) mixed reagent. After incubation for 15 min at room temperature in a dark room, the cells were analysed within 30 min by a FACSCalibur flow cytometer (BD Biosciences, USA).

To determine the cell cycle, cell suspensions with a density of 4 $\times$ 10 ${}^{4}$ cells/ml were fixed with chilled 70% ethanol and kept overnight at $-$ 20 ${}^{\circ}$ C. Next, the cells were labelled with PI staining solution (10 $\mu$ g/mL RNase A and 50 $\mu$ g/mL PI) at 37 ${}^{\circ}$ C for 30 min in the dark. The cell cycle distribution was analysed using a FACSCalibur flow cytometer with the Cell-Quest software.

2.6 In vitro invasion assays

The 24-well Transwell insert chambers coated with Matrigel (BD Biosciences, USA) were utilized for the cell invasion assay. The transfected Raji cells were resuspended in 100 $\mu$ l serum-free medium at a density of 5 $\times$ 10 ${}^{4}$ cells/ml and were added to the upper chamber. The lower chamber was filled with 500 $\mu$ l complete medium containing 10% FBS as a chemoattractant. The cells were incubated in a cell incubator with 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C in a humidified atmosphere for 24 h, and then the cells on the top surface of the insert were carefully removed with a cotton swab. Invaded cells on the lower side of the membrane were fixed with methanol and stained with 0.5% crystal violet at room temperature for 20 min. After washing with PBS once, the number of cells was imaged and counted in five randomly chosen fields under a light microscope (Olympus Corporation, Japan), and the average number was calculated.

2.7 Dual luciferase reporter assay

To identify the direct interaction between lncRNA GAS5 and miR-222, 293T cells seeded in 24-well plates (5 $\times$ 10 ${}^{3}$ per well) were co-transfected with 50 nmol/L miR-222 mimics or negative control oli- gonucleotides, 20 ng firefly luciferase reporter, and 10 ng pRL-TK (Promega, USA) using $\text{FuGENE}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ HD Transfection Reagent (Promega, USA) according to the manufacturer’s guidelines. The luciferase reporter plasmid was constructed by cloning the predicted binding sequence of miR-222 in the lncRNA GAS5 gene and the mutant binding site sequence into the pMIR-REPORT vector (Ambion, USA). Forty-eight hours after transfection, the luciferase activity was detected by the Dual-Luciferase Reporter Assay System (Promega, USA) following the manufacturer’s protocols. The relative luciferase activity was presented as the firefly luciferase activity normalized to that of Renilla.

Figure 1.

qRT-PCR analysis of lncRNA GAS5 and miR-222 expressions in human B lymphocytic leukemia cells. (A) The levels of lncRNA GAS5 in leukomonocytes of 30 patients with B lymphocytic leukemia and in 30 heathy donors. (B) The levels of miR-222 in leukomonocytes of 30 patients with B lymphocytic leukemia and in 30 heathy donors. (C) The levels of lncRNA GAS5 in B lymphocytic leukemia cell lines (RAMOS, ST486, Raji, Farage) and normal B lymphocytic cell line IM9. * indicates $p<$ 0.05 compared with IM9.

2.8 Statistical analysis

SPSS 16.0 statistical software was used to perform statistical analyses, and GraphPad Prism 7 was applied for graphical data representation. Data are shown as the mean $\pm$ standard deviation (SD). Student’s t-test or non-parametric Mann-Whitney U test was conducted to analyse the differences between 2 groups, and multiple comparisons were assessed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Values of $P<$ 0.05 were considered statistically significant.

3. Results

3.1 Expression pattern of lncRNA GAS5 and miR-222 in leukomonocytes of human B lymphocytic leukaemia patients

We first determined the expression of lncRNA GAS5 and miR-222 in leukomonocytes of 30 B lymphocytic leukaemia patients and 30 healthy donors using qRT-PCR assay. It was found that the expression of lncRNA GAS5 was significantly decreased in patients with B lymphocytic leukaemia compared with the healthy group ( $p<$ 0.05), while the expression of miR-222 was notably increased in patients with B lymphocytic leukaemia compared with the healthy group ( $p<$ 0.05, Fig. 1a). Then, the expression of lncRNA GAS5 was further tested in B lymphocytic leukaemia cell lines. It was discovered that the levels of lncRNA GAS5 were conspicuously decreased in the B lymphocytic leukaemia cell lines RAMOS, ST486, Raji, and Farage compared with the normal B lymphocytic cell line IM9 ( $p<$ 0.05, Fig. 1b). Moreover, based on the lowest expression of lncRNA GAS5 in Raji, this cell line was chosen for subsequent experiments. Therefore, these data suggested that there might be an important role for lncRNA GAS5 and miR-222 during the development of human B lymphocytic leukaemia.

Figure 2.

The role of overexpression and knockdown of lncRNA GAS5 on B lymphocytic leukemia cell proliferation was evaluated by CCK-8 assay at days 0, 1, 2, 3. * and # indicate $p<$ 0.05 compared with NC group. N.S. indicates no significance compared with blank group.

Figure 3.

The effect of overexpression and knockdown of lncRNA GAS5 on B lymphocytic leukemia cell apoptosis was examined by flow cytometry. *indicates $p<$ 0.05 vs NC group, # indicates $p<$ 0.05 vs GAS5 group.

Figure 4.

The regulation of overexpression and knockdown of lncRNA GAS5 on B lymphocytic leukemia cell cycle was assessed by flow cytometry. * indicates $p<$ 0.05 compared with NC group. # indicates $p<$ 0.05 compared with GAS5group. N.S. indicates no significance compared with blank group.

Figure 5.

Overexpression and knockdown of lncRNA GAS5 modulates the invasion of human B lymphocytic leukemia cells. * indicate $p<$ 0.05 compared with NC group. # indicates $p<$ 0.05 compared with GAS5 group. N.S. indicates no significance compared with blank group.

3.2 The effects of lncRNA GAS5 on cell proliferation, apoptosis, cell cycle and invasion in B lymphocytic leukaemia in vitro

To investigate the functions of lncRNA GAS5, lncRNA GAS5-overexpression and lncRNA GAS5-knockdown plasmids were transfected into Raji cells for multiple cell culture experiments As shown in Fig. 1c, lncRNA GAS5 expression was sharply upregulated in Raji cells transfected with the lncRNA GAS5-overexpression plasmid and dramatically downregulated in cells transfected with the lncRNA GAS5-knockdown plasmid ( $p<$ 0.05). Moreover, it was also revealed that miR-222 expression was remarkably reduced in lncRNA GAS5-overexpressing Raji cells and markedly increased in lncRNA GAS5-knockdown Raji cells ( $p<$ 0.05). Thus, these results directly indicated that the transfection of Raji cells with lncRNA GAS5-overexpression and lncRNA GAS5-knockdown plasmids was effective, and there may be an intrinsic connection between the expression of miR-222 and lncRNA GAS5 in B lymphocytic leukaemia cells.

Figure 6.

MiR-222 is a direct target of lncRNA GAS5. (A) The putative binding sites of lncRNA GAS5 and miR-222 are presented. (B) Dual-luciferase assay was applied to determine the targeted interaction of lncRNA GAS5 and miR-222. (C) Regression analyses of GAS5 expression level and miR-222 expression level in 30 patients with B lymphocytic leukemia. * indicates $p<$ 0.05 compared with NC group. # indicates $p<$ 0.05 compared with GAS5 group. N.S. indicates no significance compared with blank group.

Subsequently, a series of cell function experiments was performed. The CCK-8 assay revealed that the cells in the different groups gradually proliferated with time, but the rate of Raji cell proliferation in the lncRNA GAS5-overexpression group was obviously lower than that in the Raji or NC groups, while the rate of Raji cell proliferation in the lncRNA GAS5-knockdown group was obviously higher than that in the Raji or NC groups ( $p<$ 0.05, Fig. 2). The flow cytometry analysis revealed that lncRNA GAS5 overexpression promoted Raji cell apoptosis and arrested Raji cells at the G1 phase of the cell cycle, but lncRNA GAS5 knockdown suppressed apoptosis and increased cell cycle distribution in G1 phase (Figs 3 and 4). The Transwell invasion assay showed that the rate of invasion in the lncRNA GAS5-overexpression group was greatly decreased when compared with that in the Raji or NC groups, whereas the rate of invasion in the lncRNA GAS5-knockdown group was prominently increased when compared with that in the Raji or NC groups ( $p<$ 0.05, Fig. 5). Hence, these findings implied that lncRNA GAS5 plays a tumour suppressor role in B lymphocytic leukaemia and that loss of GAS5 might promote Raji cell tumourigenesis and metastatic potential in vitro.

3.3 The binding interaction between lncRNA GAS5 and miR-222

Previous studies have demonstrated that many lncRNAs function as ceRNAs that regulate target genes by competitively binding miRNAs. In this study, bioinformatics software analysis (StarBase 2.0 programme, http://starbase.sysu.edu.cn/) predicted a potential interaction of lncRNA GAS5 and miR-222, and the putative binding sites are shown in Fig. 6a. Next, the dual luciferase reporter assay also confirmed this prediction and showed that the luciferase activity of the lncRNA GAS5 Wt group cotransfected with the miR-222 mimic group declined more notably compared with that in the control group ( ${P}<0.05$ ). In addition, in the lncRNA GAS5 Mut group, there was no significant difference in the luciferase activity among the miR-222 mimic co-transfected group, psiCHECK2 group and control group ( $p<$ 0.05, Fig. 6b). Additionally, the correlation between lncRNA GAS5 and miR-222 expression in leukomonocytes of patients with human B lymphocytic leukaemia was further analysed. The results of regression analysis showed that there was a negative correlation between the levels of lncRNA GAS5 and miR-222 in leukomonocytes of B lymphocytic leukaemia (Pearson’s $r=-$ 0.8927, Fig. 6c). Thus, these data demonstrated that miR-222 should be a direct target of lncRNA GAS5.

4. Discussion

Over the past decades, a large number of innovative targeted therapies, including monoclonal antibodies or bispecific T-cell engagers, personalized vaccines, and immunocellular therapy, have been widely reported [25, 26]. Although the survival outcome of patients with B lymphocytic leukaemia has dramatically improved, the subgroup of patients with relapsed/refractory B lymphocytic leukaemia still has a dismal prognosis [27]. This may be because the molecular mechanism of B lymphocytic leukaemia has not been clearly understood. lncRNAs, a novel class of RNA transcripts longer than 200 nucleotides that lack protein-coding potential, are dysregulated in many disease states, particularly in tumours [14, 28]. For example, lncRNA FBXl19-AS1 regulates osteosarcoma cell progression by sponging miR-346 [29]; lncRNA H19 promotes colorectal cancer cell formation by regulating the miR-29b-3p/PGRN-mediated Wnt signalling pathway [30]; and lncRNA MEG3 inhibits the progression of prostate cancer by modulating the miR-9-5p/QKI-5 axis [31]. Thus, these studies have demonstrated that lncRNAs can function as either carcinogens or tumour suppressors in diverse cancers. Moreover, the complex regulation between lncRNAs and miRNAs is thought to be one of the critical features of lncRNAs [32]. Additionally, lncRNA GAS5 has typically been considered a tumour suppressor and presents a declining expression trend in many cancers, such as cervical cancer, breast cancer, and stomach cancer [17, 33]. In the current study, it was also found that lncRNA GAS5 expression was significantly reduced both in leukomonocytes of patients with B lymphocytic leukaemia and B lymphocytic leukaemia cell lines. Then, the role of lncRNA GAS5 in B lymphocytic leukaemia cells was explored. Our results revealed that lncRNA GAS5 overexpression decreased B lymphocytic leukaemia cell proliferation, promoted B lymphocytic leukaemia cell apoptosis, arrested B lymphocytic leukaemia cell cycle in G1 phase, and inhibited B lymphocytic leukaemia cell invasion. Moreover, when lncRNA GAS5 expression was knocked down in B lymphocytic leukaemia cells, the opposite cell phenotype changes were observed. Therefore, these findings implied that lncRNA GAS5 might play an important role in the development of B lymphocytic leukaemia. The lncRNA GAS5 gene, first reported by Coccia et al. in 1992, is located on the human chromosome 1q25. Previous studies have demonstrated that lncRNA GAS5 is involved in the development of various cancers [17, 33]. For instance, lncRNA GAS5 regulates the proliferation and invasion of hepatoma cells by regulating Vimentin [34]; lncRNA GAS5 expression is notably decreased in colorectal cancer tissue samples, and its expression level could be an independent risk factor or a predictor of prognosis for colorectal cancer [35]; and lncRNA GAS5 has also been discovered to impede the viability, migration and invasion of pancreatic cancer cells [36, 37]. The roles of the lncRNA GAS5/miR-222 regulatory axis in cancer development have been illustrated by several studies. Xihe Zhao et al. showed that lncRNA GAS5 suppresses glioma tumour malignancy by inhibiting miR-222 [38]. Yanhua Li et al. indicated that the lncRNA GAS5/miR-222 axis regulates gastric cancer cell proliferation through the PTEN/Akt/mTOR pathway [24]. XF Zhang et al. have reported that GAS5 induces the suppression of papillary thyroid carcinoma cell proliferation by regulating miR-222 [39]. Our results in the present study were consistent with these previous findings, which uncovered the characteristics of the lncRNA GAS5/miR-222 axis in tumours.

Furthermore, miR-222 exhibited markedly increased expression in lncRNA GAS5-knockdown B lymphocytic leukaemia cells and decreased expression in lncRNA-overexpressing B lymphocytic leukaemia cel-ls. To further pinpoint the relationship between lnc-RNA GAS5 and miR-222, the dual luciferase reporter assay was performed, and the results clearly indicated that lncRNA GAS5 is a direct target of miR-222. A previous study showed that miR-222 could regulate p27 and form a miR-222/p27 regulatory loop to maintain chronic lymphocytic leukaemia cells in a resting state [12]. Therefore, the upregulation of miR-222 due to the absence of lncRNA GAS5 in chronic lymphocytic leukaemia cells may promote the proliferation of tumour cells by inhibiting p27 expression.

In summary, we first identified the regulatory role of the lncRNA GAS5/miR-222 axis in the cell proliferation, cell apoptosis, cell cycle and cell invasion of B lymphocytic leukaemia. The loss of lncRNA GAS5 expression in B lymphocytic leukaemia cells might be likely to stimulate the progression of B lymphocytic leukaemia. Moreover, the direct target interaction of lncRNA GAS5 and miR-222 was also verified. Hence, these data demonstrated that lncRNA GAS5 could effectively sponge miR-222 to modulate human B lymphocytic leukaemia cell tumourigenesis and metastasis. These results could greatly aid in exploring novel diagnostic biomarkers and treatment targets for B lymphocytic leukaemia in the future.

References

Rai

K.R.

and Jain

, Chronic lymphocytic leukemia (CLL)-then and now, Am J Hematol 91(3) (2016), 330–340.

Gauthier

Comont

Vergez

et al., Minimal residual disease in chronic lymphocytic leukemia: A still current issue in 2018, Bull Cancer 105(11) (2018), 1042–1051.

Hallek

, Chronic lymphocytic leukemia: 2017 update on diagnosis, risk stratification, and treatment, Am J Hematol 92(9) (2017), 946–965.

Montresor

Toffali

Rigo

et al., CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated bruton’s tyrosine kinase, Oncotarget 9(80) (2018), 35123–35140.

Jamroziak

Pula

and Walewski

, Current treatment of chronic lymphocytic leukemia, Curr Treat Options Oncol 18(1) (2017), 5.

Jain

and O’Brien

, Targeted therapies for CLL: Practical issues with the changing treatment paradigm, Blood Rev 30(3) (2016), 233–244.

Luo

Zhou

Wang

Sun

Han

and Bai

, microRNA-222 promotes colorectal cancer cell migration and invasion by targeting MST3, FEBS Open Bio 9(5) (2019), 901–913.

Pan

Z.X.

Zhang

X.Y.

Chen

S.R.

and Li

C.Z.

, Upregulated exosomal miR-221/222 promotes cervical cancer via repressing methyl-CpG-binding domain protein 2, Eur Rev Med Pharmacol Sci 23(9) (2019), 3645–3653.

C.H.

Liu

Xiao

L.M.

Chen

L.K.

Zheng

S.Y.

Zeng

E.M.

et al., Silencing microRNA-221/222 cluster suppresses glioblastoma angiogenesis by suppressor of cytokine signaling-3-dependent JAK/STAT pathway, J Cell Physiol, (2019).

10.

Guo

Liu

Guo

Bai

and Wang

, miR-222-3p promotes osteosarcoma cell migration and invasion through targeting TIMP3, Onco Targets Ther 11 (2018), 8643–8653.

11.

Zong

Zhang

Sun

Cheng

and Qin

, miR-221/222 promote tumor growth and suppress apoptosis by targeting lncRNA GAS5 in breast cancer, Biosci Rep 39(1) (2019).

12.

Frenquelli

Muzio

Scielzo

Fazi

Scarfo

Rossi

et al., MicroRNA and proliferation control in chronic lymphocytic leukemia: Functional relationship between miR-221/222 cluster and p27, Blood 115(19) (2010), 3949–3959.

13.

Wang

Zhou

et al., The functional roles of exosomal long non-coding RNAs in cancer, Cell Mol Life Sci, (2019).

14.

Huarte

, The emerging role of lncRNAs in cancer, Nat Med 21(11) (2015), 1253–1261.

15.

Martens-Uzunova

E.S.

Bottcher

Croce

C.M.

et al., Long noncoding RNA in prostate, bladder, and kidney cancer, Eur Urol 65(6) (2014), 1140–1151.

16.

et al., Altered expression of long non-coding RNA GAS5 in digestive tumors, Biosci Rep 39(1) (2019).

17.

Shi

Zhu

et al., The growth arrest-specific transcript 5 (GAS5): A pivotal tumor suppressor long noncoding RNA in human cancers, Tumour Biol 37(2) (2016), 1437–1444.

18.

Yang

et al., LncRNA GAS5 suppresses ovarian cancer by inducing inflammasome formation, Biosci Rep, (2017).

19.

Liu

Zhao

Zhang

et al., lncRNA GAS5 enhances G1 cell cycle arrest via binding to YBX1 to regulate p21 expression in stomach cancer, Sci Rep 5 (2015), 10159.

20.

Wang

Sun

Zhao

et al., Long non-Coding RNA (lncRNA) growth arrest specific 5 (GAS5) suppresses esophageal squamous cell carcinoma cell proliferation and migration by inactivating phosphatidylinositol 3-kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) signaling pathway, Med Sci Monit 24 (2018), 7689–7696.

21.

Guo

Kang

Zhu

Chen

Wang

Chen

et al., A long noncoding RNA critically regulates bcr-Abl-mediated cellular transformation by acting as a competitive endogenous RNA, Oncogene 34(14) (2015), 1768–1779.

22.

Leucci

Patella

Waage

Holmstrom

Lindow

Porse

et al., microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus, Sci Rep 2535 (2013).

23.

Zhou

Gao

Wang

Zhang

Liu

and Duan

, Linc-RNA-RoR acts as a “sponge” against mediation of the differentiation of endometrial cancer stem cells by microRNA-145, Gynecol Oncol 133(2) (2014), 333–339.

24.

and Lu

, The GAS5/miR-222 axis regulates proliferation of gastric cancer cells through the PTEN/Akt/ mTOR pathway, Dig Dis Sci 62(12) (2017), 3426–3437.

25.

Burger

J.A.

and O’Brien

, Evolution of CLL treatment – from chemoimmunotherapy to targeted and individualized therapy, Nat Rev Clin Oncol 15(8) (2018), 510–527.

26.

Chowdhury

S.R.

and Banerji

, Targeting mitochondrial bioenergetics as a therapeutic strategy for chronic lymphocytic leukemia, Oxid Med Cell Longev 2018 (2018), 2426712.

27.

Qin

Song

Shen

et al., Safety and efficacy of obinutuzumab in chinese patients with B-cell lymphomas: A secondary analysis of the GERSHWIN trial, Cancer Commun (Lond) 38(1) (2018), 31.

28.

Zhang

and Tang

, The application of lncRNAs in cancer treatment and diagnosis, Recent Pat Anticancer Drug Discov 13(3) (2018), 292–301.

29.

Pan

Ruan

et al., lncRNA FBXL19-AS1 regulates osteosarcoma cell proliferation, migration and invasion by sponging miR-346, Onco Targets Ther 11 (2018), 8409–8420.

30.

Ding

Zhao

et al., LncRNA H19/miR-29b-3p/PGRN axis promoted epithelial-Mesenchymal transition of colorectal cancer cells by acting on wnt signaling, Mol Cells 41(5) (2018), 423–435.

31.

Huang

Chen

et al., LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR-9-5p/QKI-5 axis, J Cell Mol Med 23(1) (2019), 29–38.

32.

Huang

, The novel regulatory role of lncRNA-miRNA-mRNA axis in cardiovascular diseases, J Cell Mol Med 22(12) (2018), 5768–5775.

33.

Pickard

M.R.

and Williams

G.T.

, Molecular and cellular mechanisms of action of tumour suppressor GAS5 lncRNA, Genes (Basel) 6(3) (2015), 484–499.

34.

Chang

Lan

et al., Decreased expression of long non-coding RNA GAS5 indicates a poor prognosis and promotes cell proliferation and invasion in hepatocellular carcinoma by regulating vimentin, Mol Med Rep 13(2) (2016), 1541–1550.

35.

Yang

Shen

Yan

et al., Long non-coding RNA GAS5 inhibits cell proliferation, induces G0/G1 arrest and apoptosis, and functions as a prognostic marker in colorectal cancer, Oncol Lett 13(5) (2017), 3151–3158.

36.

Fang

Wang

et al., Downregulation of gas5 increases pancreatic cancer cell proliferation by regulating CDK6, Cell Tissue Res 354(3) (2013), 891–896.

37.

Gao

Z.Q.

Wang

J.F.

Chen

D.H.

et al., Long non-coding RNA GAS5 antagonizes the chemoresistance of pancreatic cancer cells through down-regulation of miR-181c-5p, Biomed Pharmacother 97 (2018), 809–817.

38.

Zhao

Wang

Liu

Zheng

Liu

Chen

et al., Gas5 exerts tumor-suppressive functions in human glioma cells by targeting miR-222, Mol Ther 23(12) (2015), 1899–1911.

39.

Zhang

X.F.

and Zhao

S.J.

, LncRNA gas5 acts as a ceRNA to regulate PTEN expression by sponging miR-222-3p in papillary thyroid carcinoma, Oncotarget 9(3) (2018), 3519–3530.

Long non-coding RNA GAS5 regulates human B lymphocytic leukaemia tumourigenesis and metastasis by sponging miR-222

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and cell lines

2.2 RNA extraction and quantitative RT-PCR (qRT-PCR) assay

2.3 Cell transfection

2.4 Cell proliferation assay

2.5 Flow cytometry analysis

2.6 In vitro invasion assays

2.7 Dual luciferase reporter assay

3. Results

3.1 Expression pattern of lncRNA GAS5 and miR-222 in leukomonocytes of human B lymphocytic leukaemia patients

4. Discussion

References