Long noncoding RNA UCA1 facilitates cell proliferation and inhibits apoptosis via activating PI3K/Akt pathway in retinoblastoma

Background Retinoblastoma (RB) is the most common intraocular malignancy of childhood. This study is aimed to explored the effect of lncRNA-UCA1 on RB and its potential molecular mechanisms. Methods In our study, the expression of lncRNA-UCA1 was measured by qRT-RCR in both RB tissues and RB HXO-RB44 and Y79 cells. The relationship between lncRNA-UCA1 expression and clinical parameters in RB patients were evaluated. Cell proliferation, cell clones, apoptosis and cell cycle of HXO-RB44 and Y79 cells were measured by cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry, respectively. In addition, the expressions of PCNA, Caspase-3, survivin, p16, p21, CDK2, PI3K, p-PI3K, Akt, p-Akt and S6k in HXO-RB44 and Y79 cells were measured by western blot. Results lncRNA-UCA1 was highly expressed in both RB tissues and RB HXO-RB44 and Y79 cells. Moreover, the expression of lncRNA-UCA1 in RB patients was remarkedly correlated with tumor size, optic nerve invasion and pathologic grade. lncRNA-UCA1 markedly facilitated cell proliferation and cell cycle procession, as well as inhibited cell apoptosis in HXO-RB44 and Y79 cells. lncRNA-UCA1 dramatically increased the expression of S6k and the phosphorylation of PI3K and Akt in RB cells. LY294002 (PI3K inhibitor) reversed the effects of lncRNA-UCA1 on RB cell proliferation, apoptosis and cell cycle procession. Conclusions Our study indicated that lncRNA-UCA1 could promote cell proliferation and cell cycle procession, as well as inhibit cell apoptosis in RB via activating PI3K/Akt pathway.


Background
Retinoblastoma (RB) is the most common intraocular cancer and mainly affects children under 5 years old [1,2]. The incidence of RB is about 1/15000 all over the world and has no gender predilection [3]. Although the therapeutic techniques have greatly improved, the survival rate of RB patients still remains poor, mainly due to its insensitivity to chemotherapy and radiotherapy [4]. Therefore, it is urgent to investigate new diagnostic markers and therapeutic targets for RB treatment.
Long noncoding RNAs (lncRNAs) are defined as RNA transcripts longer than 200 nts that have no protein-encoding ability [5]. Accumulating evidence has demonstrated that lncRNAs play important roles in the development and progression of cancers including RB [6]. More and more researches have confirmed that lncRNAs participate in numerous biological activities, such as cell proliferation, migration, invasion and cell cycle arrest [7,8]. For example, lncRNA BDNF-AS has been reported to suppress RB cell proliferation, migration and cell cycle transition [9]. Su et al. [10] have indicated that lncRNA BANCR could suppress cell proliferation, migration and invasion in RB. Dong et al. [11] have confirmed that lncRNA-HOTAIR could promote cell proliferation and invasion via regulating Notch pathway in RB. It is reported that lncRNA-H19 inhibited RB cell proliferation, migration and invasion, and promoted cell apoptosis . [12] However, the effect of lncRNA-UCA1 was still fully unclear in RB.
In this research, we explored the functions of lncRNA-UCA1 on cell proliferation and 4 apoptosis in RB, and its related molecular mechanisms. Our results indicated that lncRNA-UCA1 could facilitate cell proliferation and inhibit apoptosis through activating PI3K/Akt pathway in RB. Our results indicate that lncRNA-UCA1 may serve as a new therapeutic target for RB.

Clinical samples
Total 42 fresh RB specimens and the paired adjacent normal retina specimens from the patients of RB were provided by the Ophthalmology Department of our hospital. None of the patients received anti-tumor treatment, chemotherapy and radiotherapy before surgery. In addition, the characteristics of RB patients were presented in Table 1 Cell counting kit-8 assay

Colony formation assay
The transfected HXO-RB44 and Y79 cells were respectively seeded into 6-well plates at a density of 300 cells per well and cultivated for 14 d. After fixed in 4% cold formaldehyde for 30 min, the colonies were stained with 0.1% crystal violet (Beyotime, China) for 20 min. Finally, the colonies were photographed and counted under an optical microscope (Olympus, Tokyo, Japan).

Cell cycle analyses
The transfected HXO-RB44 and Y79 cells were harvested, washed with PBS, fixed in pre-

Statistical analysis
All statistical analyses were performed using SPSS 22.0 Statistical Software (Chicago, IL).
The results were presented as the mean ± SD. Statistical significance was tested using the one-way ANOVA or Student's t-test. P < 0.05 was considered to be statistically significant.

Results
The expression of lncRNA-UCA1 is upregulated and associated with clinical parameters in RB The results of qRT-PCR showed that lncRNA-UCA1 expression in RB tissue was markedly higher than that in paracancerous tissue (P < 0.01) (Fig 1A).. Similarly, lncRNA-UCA1 expression was remarkedly increased in HXO-RB44 and Y79 cells compared with ACBRI-181 cells (P < 0.01) (Fig 1B).. All the results suggested that lncRNA-UCA1 was highly expressed in RB. We also found that the expression of lncRNA-UCA1 was remarkedly correlated with tumor size (P < 0.01), optic nerve invasion (P < 0.01) and pathologic grade (P < 0.01) ( Table 1).. Meanwhile, the expression of lncRNA-UCA1 had no significantly correlation with age and gender in the patients with RB (P > 0.05) ( Table 1)..
In addition, the expression of lncRNA-UCA1 in transfected HXO-RB44 and Y79 cells was detected by qRT-PCR. The results showed that lncRNA-UCA1 expression of HXO-RB44 and Y79 cells was significantly increased in pcDNA-UCA1 group compared with BLANK and pcDNA-Mock group (P < 0.01) (Fig 1C).. Meanwhile, when compared with BLANK and si-NC group, lncRNA-UCA1 expression of HXO-RB44 and Y79 cells was markedly decreased in si-UCA1 group (P < 0.01) (Fig 1C),, suggesting that the transfection was successful.
lncRNA-UCA1 promotes cell proliferation in HXO-RB44 and Y79 cells 8 The results of CCK-8 assay showed that the proliferation ability of HXO-RB44 cells was markedly decreased in si-UCA1 group compared with BLANK and si-NC group at 48 h (P < 0.05), 72 h and 96 h (P < 0.01) (Fig 2A).. When compared with BLANK and pcDNA-Mock group, the proliferation ability of Y79 cells was remarkedly increased in pcDNA-UCA1 group at 48 h (P < 0.05), 72 h and 96 h (P < 0.01) (Fig 2A).. Furthermore, colony formation assay also confirmed that silencing lncRNA-UCA1 expression markedly decreased the cell clones number of HXO-RB44 cells (P < 0.01) (Fig 2B),, while lncRNA-UCA1 overexpression significantly increased the cell clones number of Y79 cells (P < 0.01) ( Fig 2B).. To further confirm the function of lncRNA-UCA1 on RB cell proliferation, we also measured the expression of PCNA in both HXO-RB44 and Y79 cells. The expression of PCNA of HXO-RB44 cells was dramatically decreased in si-UCA1 group compared with BLANK and si-NC group (P < 0.01) (Fig 2C).. When compared with BLANK and pcDNA-Mock group, the expression of PCNA of Y79 cells was markedly increased in pcDNA-UCA1 group (P < 0.01) (Fig 2C).. All the results indicated that lncRNA-UCA1 could promote cell proliferation in RB cells.
lncRNA-UCA1 promotes cell proliferation and cell cycle procession and inhibits apoptosis via activating PI3K/Akt signaling pathway in RB To further confirm whether lncRNA-UCA1 exerted its function on RB through activating PI3K/Akt signaling pathway, we performed a variety of experiments in Y79 cells. The results of CCK-8 showed that Y79 cells proliferation was markedly promoted in pcDNA-UCA1 group compared with BLANK and pcDNA-Mock group at 48 (P < 0.05), 72 (P < 0.01) and 96 h (P < 0.01) Fig 6A).. LY294002 markedly reversed the function of lncRNA-UCA1 on cell proliferation (P < 0.01) Fig 6A).. Colony formation assay results also confirmed that the cell clones number of Y79 cells was dramatically increased in pcDNA-UCA1 group compared with BLANK and pcDNA-Mock group (P < 0.01) (Fig 6B).. Meanwhile, when compared with pcDNA-UCA1 group, the number of Y79 cells clones was notably decreased in pcDNA-UCA1 + LY294002 group (P < 0.01) (Fig 6B).. As shown in Fig 6C, Y79 cells apoptosis was dramatically lower in pcDNA-UCA1 group than that in BLANK and pcDNA-Mock group (P < 0.01). When compared with pcDNA-UCA1 group, the apoptosis of Y79 cells was remarkedly increased in pcDNA-UCA1 + LY294002 group (P < 0.01). The results of western blot showed that Caspase-3, p16 and p21 expressions of Y79 cells in pcDNA-UCA1 group were markedly lower than those in BLANK and pcDNA-Mock group (P < 0.01) ( Fig 6D and E),, while survivin and CDK2 expressions were significantly higher (P < 0.01) (Fig 6D and E).. When compared with pcDNA-UCA1 group, Caspase-3, p16 and p21 expressions of Y79 cells were remarkedly higher in pcDNA-UCA1 + LY294002 group (P < 0.01), while survivin and CDK2 expressions were significantly lower (P < 0.01) (Fig 6D and   E).. All the results indicated that lncRNA-UCA1 could promote cell proliferation and cell cycle procession, as well as inhibit apoptosis via activating PI3K/Akt pathway in RB. Discussion RB, the most common intraocular malignancy, is diagnosed in approximately 8000 children all over the world each year . [18] In developing countries, the survival rate of RB patients is still below 70% . [19] It is urgent to investigate new and effective molecular targets for the treatment of RB. [20,21] In this study, we confirmed that lncRNA-UCA1 could facilitate cell proliferation and inhibit apoptosis through activating PI3K/Akt pathway in RB.
More and more evidences have indicated that lncRNAs play a carcinogenic or anticancer role in various malignant tumors, such as RB . In addition, lncRNAs are reported to be abnormally expressed in tumors. Previous researches have confirmed that the expression of lncRNA-UCA1 is upregulated in gastric cancer [16], ovarian cancer [22]and lung cancer.
[23] Furthermore, Sun et al. [24] have reported that lncRNA-UCA1 is highly expressed in laryngeal squamous cell carcinoma. In the present study, we assessed the expression of lncRNA-UCA1 in both RB tissues and RB HXO-RB44 and Y79 cells, and found that lncRNA-UCA1 was highly expressed in both RB tissues and RB HXO-RB44 and Y79 cells.
Furthermore, we also found lncRNA-UCA1 expression was associated with some clinical parameters of RB patients, such as tumor size, optic nerve invasion and pathologic grade.
Accumulating evidences have confirmed that lncRNAs participate in the regulation of cell proliferation, apoptosis, migration and cell cycle in various diseases [7,25]. For example, lncRNA CCAT1 could promote cell proliferation, migration and invasion, as well as suppress cell apoptosis of RB cells by modulating miR-218-5p. [26] Li et al. [27] have indicated that lncRNA 00152 could promote cell proliferation and migtation, and suppress apoptosis in RB. lncRNA H19 could suppress RB cell proliferation and promote cell cycle arrest and apoptosis through counteracting miR-17-92 cluster . [28] In this study, lncRNA-UCA1 promoted cell proliferation and cell cycle procession, and inhibited cell apoptosis in RB, indicating that lncRNA-UCA1 may serve as a potential prognostic biomarker for RB.
More and more researches have suggested that PI3K/Akt signaling pathway affects various biological behaviour in the process of malignancies including cell proliferation, migration, invasion, apoptosis and cell cycle . [29] S6K is a downstream target molecule of PI3K/Akt pathway. Once PI3K/Akt pathway is activated, S6K may be activated and then regulate important process of cell biology . [17] Moreover, previous findings have indicated that lncRNAs could participate in cell biological behaviour by modulating PI3K/AKT pathway in many kinds of cancers. For instance, lncRNA-UCA1 has been reported to regulate cell cycle progression through PI3K signaling pathway in bladder carcinoma cells .
[30] A study of Li et al. [17] has indicated that lncRNA-UCA1 significantly promotes cell proliferation and suppresses apoptosis by activating PI3K-Akt-mTOR signaling pathway in gastric cancer. Ma et al. [16] have suggested that lncRNA-UCA1 could promote osteosarcoma metastasis through the activation of PI3K/AKT signaling pathway. In the present study, silencing lncRNA-UCA1 significantly decreased the phosphorylation of PI3K and Akt, as well as the expression of S6k in HXO-RB44 cells, while lncRNA-UCA1 overexpression dramatically promoted the phosphorylation of PI3K and Akt, and S6k expression in Y79 cells, suggesting that lncRNA-UCA1 could activate PI3K/Akt signaling pathway in RB cells. In addition, we also confirmed that LY294002 reversed the function of lncRNA-UCA1 on RB cell proliferation, apoptosis and cell cycle procession. All results indicated that lncRNA-UCA1 could promote cell proliferation and cell cycle procession, as well as inhibit apoptosis via activating PI3K/Akt pathway in RB.

Conclusions
In conclusion, our work confirmed that lncRNA-UCA1 expression was dramatically upregulated in both RB tissue and RB HXO-RB44 and Y79 cells. In addition, we found that lncRNA-UCA1 could promote cell proliferation and cell cycle procession, as well as inhibit apoptosis via activating PI3K/Akt pathway in RB. Our research provides an innovatively regulatory mechanism about lncRNA-UCA1 in RB and point a new way to RB treatment.