|Year : 2015 | Volume
| Issue : 7 | Page : 164-167
Upregulated long noncoding RNA SPRY4-IT1 contributes to increased cell viability by activating zinc finger 703 expression in esophageal squamous cell carcinoma
J Xue-Liang1, W Ming-Dong2, Z Ya-Bi1, W Wang-Yue1
1 Department of Gastroenterology, Lishui People's Hospital, Lishui 323000, Zhejiang Province, PR China
2 Department of Clinical Pharmacy, Lishui People's Hospital, Lishui 323000, Zhejiang Province, PR China
|Date of Web Publication||20-Jul-2016|
Department of Gastroenterology, Lishui People's Hospital, Lishui 323000, Zhejiang Province
Source of Support: None, Conflict of Interest: None
OBJECTIVES: The function of long noncoding RNA SPRY4-IT1 in human esophageal squamous cell carcinoma (ESCC) has been showed in the former studies. The purpose of this study was to further analyze the underlined mechanisms responsible for its role in ESCC cells. MATERIALS AND METHODS: Quantitative reverse transcriptase polymerase chain reaction was firstly used to measure the expression of SPRY4-IT1 in 50 ESCC patients of different clinical stages. Loss of function approach was then applied to confirm the biological function, especially cell viabilities in cultured ESCC cells, by cell counting kit-8 and clonogenic assay. We further used western blot to reveal the activation of zinc finger 703 (ZNF703) by SPRY4-IT1. RESULTS: We validated that SPRY4-IT1 was upregulated in ESCC tissues of advanced clinical stages. In vitro function assays demonstrated that SPRY4-IT1 cause promotion of cell viability in ESCC cells. We further verified that SPRY4-IT1 could also activate the expression of ZNF703 in ESCC cells, which might contribute to the role of SPRY4-IT1 in ESCC cells. CONCLUSION: SPRY4-IT1 is a vital regulator in ESCC progression, and the SPRY4-IT1/ZNF703 axis might provide novel clues for future ESCC therapy.
Keywords: Cell viability, esophageal squamous cell carcinoma, SPRY4-IT1, zinc finger 703
|How to cite this article:|
Xue-Liang J, Ming-Dong W, Ya-Bi Z, Wang-Yue W. Upregulated long noncoding RNA SPRY4-IT1 contributes to increased cell viability by activating zinc finger 703 expression in esophageal squamous cell carcinoma. Indian J Cancer 2015;52, Suppl S3:164-7
|How to cite this URL:|
Xue-Liang J, Ming-Dong W, Ya-Bi Z, Wang-Yue W. Upregulated long noncoding RNA SPRY4-IT1 contributes to increased cell viability by activating zinc finger 703 expression in esophageal squamous cell carcinoma. Indian J Cancer [serial online] 2015 [cited 2020 Jan 28];52, Suppl S3:164-7. Available from: http://www.indianjcancer.com/text.asp?2015/52/7/164/186566
| » Introduction|| |
During the past few years, the development of genome-wide sequencing and the achievements from the encyclopedia of DNA elements project have facilitated the discovery of a new class of RNAs, named long noncoding RNA (lncRNA). ,, These findings dramatically altered our previous understandings of cell biology, especially cancer biology.  LncRNAs are more than 200 nt in length with limited or no protein-coding capacity and are often expressed in a disease-, tissue- or developmental stage-specific manner. ,, Recently, lncRNAs were found to be aberrantly expressed or dysregulated in a variety of tumors and acted as drivers of tumor suppressive or oncogenic functions. , In addition, it was showed that lncRNAs could regulate gene expression at the epigenetic, transcription, and posttranscription levels. 
The lncRNA named SPRY4-IT1 (Sprouty4-Intron 1; GenBank accession ID AK024556) is derived from the second intron of SPRY4 gene. It was originally identified in the adipose tissue and intensively studied in melanoma. , In melanoma cells, it was showed that SPRY4-IT1 was predominantly localized in the cytoplasm, and its knockdown resulted in defects in cell growth, differentiation, and higher rates of apoptosis.  High expression of SPRY4-IT1 predicts poor prognosis of clear cell renal cell carcinoma,  gastric cancer,  bladder cancer,  and esophageal squamous cell carcinoma (ESCC).  In addition, Xie et al. found that SPRY4-IT1 level was significantly upregulated in ESCC tissues and cell lines. They also showed that knockdown of SPRY4-IT1 reduced cell proliferation, invasiveness, and migration.  However, the underlying mechanism it utilized to take part in ESCC progression remains unknown.
ESCC is one of the most aggressive and lethal malignant digestive tract tumors.  Currently, surgical resection is a frequent strategy for early-stage ESCC patients. However, the prognosis for advanced-stage ESCC patients is unsatisfied, and recurrence often occurs after surgery.  Hence, it is of vital importance to understand its intrinsic pathology process of this disease.
| » Materials and Methods|| |
We obtained 50 paired ESCC and adjacent normal tissues from patients who underwent surgery at Lishui people's hospital between 2013 and 2015, and who were diagnosed with ESCC (Stages I, II, III, and IV) based on histopathological evaluation. All collected tissue samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until required. Written informed consent was obtained from all patients in our study.
Cell lines and cell culture
The human ESCC TE-13 cell line was purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). TE-13 cells were cultured in Dulbecco's modified Eagle's medium in humidified air at 37°C with 5% CO 2 . The media was supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin.
Small interfering RNAs and transfections
SPRY4-IT1 siRNAs (si-SPRY4-IT1) and negative control siRNA (si-NC) were purchased from Santa Cruz. TE-13 cells were transfected with si-SPRY4-IT1 or si-NC, using Lipofectamine 2000 transfection reagent. After 48 h, cells were harvested for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to detect the knockdown efficiency.
Quantitative real-time reverse transcriptase polymerase chain reaction
Total RNA was isolated from the tissues or cells using Trizol. The synthesis of cDNA was performed with a Takara reverse transcription kit. Glyceraldehyde-3-phosphate dehydrogenase was used for internal controls. The qRT-PCR was performed for 40 cycles. The results were expressed by the comparative 2−∆∆CT method.
Cell counting kit-8 assay
Cell proliferation was monitored using a cell counting kit (CCK-8). TE-13 cells transfected with si-NC or si-SPRY4-IT1 (3000 cells/well) were grown in 96-well plates. Cell proliferation was assessed every 24 h for the next 3 days following the manufacturer's protocol.
Colony formation assays
Transfected TE-13 cells (n = 1000) were placed in 6-well plates and maintained in media containing 10% FBS. The medium was replaced every 2 days. After 7 days, cells were fixed with methanol and stained with 0.1% crystal violet.
Western blot and antibodies
Western blot was performed as described below. Cells were lysed in radioimmunoprecipitation assay buffer. The total proteins were extracted by 10% sodium dodecyl sulfate-polyacrylamide gel, transferred onto polyvinylidene difluoride membranes and then incubated overnight with specific antibodies against zinc finger 703 (ZNF703) (Santa Cruz) followed by incubation with horseradish peroxidase-conjugated secondary antibodies. β-actin (Sigma) was used as loading control.
Statistical analysis was performed by SPSS software. For comparing SPRY4-IT1 expression in ESCC tissues with matched normal tissues, paired t-test was performed. For comparisons between two groups of samples, a grouped t-test was used. P < 0.05 was considered statistically significant.
| » Results|| |
Elevated levels of SPRY4-IT1 in esophageal squamous cell carcinoma tissues
QRT-PCR was used to validate the expression levels of SPRY4-IT1 in 50 clinical ESCC tissues matched with nontumor esophageal tissues. In comparison with the adjacent normal tissues, the ESCC tissues had a significantly elevated expression of SPRY4-IT1 at mRNA levels [Figure 1]a. We then evaluated whether the expression of SPRY4-IT1 correlated with the clinicopathological parameters of these ESCC patients, such as clinical stage. As shown in [Figure 1]b, the advanced tumors (III/IV) were associated with higher expression of SPRY4-IT1 (P < 0.05). These results validated the upregulation of this lncRNA in ESCC tissues, which supported the conclusion from another report.  Our observations suggested that upregulated SPRY4-IT1 expression was associated with ESCC progression.
|Figure 1: Expression of SPRY4-IT1 in esophageal squamous cell carcinoma tissues and cell lines. (a) Relative expression of SPRY4-IT1 in 50 paired esophageal squamous cell carcinoma tissues compared with corresponding adjacent normal tissues. Higher expression levels of SPRY4-IT1 were observed in esophageal squamous cell carcinoma tissues than in the adjacent normal tissues. (b) Relative expression of SPRY4-IT1 in esophageal squamous cell carcinoma tissues of early pathological Stages (I and II) compared with that of advanced pathological Stages (III and IV). SPRY4-IT1 expression was significantly higher in patients with a higher pathological stage, *P < 0.05|
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Effects of SPRY4-IT1 on esophageal squamous cell carcinoma cell viability in vitro
To explore the roles of SPRY4-IT1 in ESCC cell viability, we mainly focused its effects on cell proliferation by means of the small interfering RNA-mediated knockdown method. The ESCC cell line TE-13 was selected for functional analysis. We firstly constructed the SPRY4-IT1 knockdown TE-13 cells. As observed in [Figure 2]a, the mRNA expression of SPRY4-IT1 was significantly decreased in the siRNA treated group, comparing with the si-NC group. We next performed CCK-8 and clonogenic survival assays to evaluate the proliferation or cell viabilities of the two groups of TE-13 cells. As shown in [Figure 2]b and c, si-SPRY4-IT1 cells exhibited decreased cell proliferation and survival abilities in vitro. These results were consistent with the former study,  and also indicated an oncogenic role of SPRY4-IT1 in ESCC cells.
|Figure 2: Effects of SPRY4-IT1 expression on esophageal squamous cell carcinoma cell viability. (a) After transfection with si-SPRY4-IT1, quantitative reverse transcriptase polymerase chain reaction analysis was performed to detect the knockdown effect. (b) Cell counting kit-8 assay was performed after knockdown of SPRY4-IT1 to determine TE-13 cell proliferation. (c) Clonogenic survival assay was performed after knockdown of SPRY4-IT1 to determine TE-13 cell survival. The results were expressed as mean ± standard deviation of three individual experiments, **P < 0.01|
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SPRY4-IT1 regulated zinc finger 703 gene expression
Recently, one report in breast cancer  found that SPRY4-IT1 could increase cell proliferation by upregulating ZNF703, a genetic driver of the amplification at 8p12 in luminal B tumors. , However, whether this regulation might also existed in the ESCC cells remains unknown. To answer this question and test the effects of SPRY4-IT1 on the expression of ZNF703, we collect total cellular extracts from the above experiments for qRT-PCR and western blotting using ZNF703-specific primers and antibody, respectively. As shown in [Figure 3]a and 3b, we found that in our ESCC TE-13 cells, knockdown of SPRY4-IT1 could downregulated both the mRNA and protein expression of ZNF703. Moreover, we thus could suspect that, the growth-promotion ability of SPRY4-IT1 could partially interpreted by its regulation on ZNF703, which was already proved in breast cancer cells. 
|Figure 3: SPRY4-IT1 regulated zinc finger 703 gene expressions. (a) Quantitative reverse transcriptase polymerase chain reaction analysis of zinc finger 703 expression levels following TE - 13 cell treatment with si - SPRY4 - IT1. (b) Western blot analysis of zinc finger 703 expression levels following TE - 13 cell treatment with si - SPRY4 - IT1, **P < 0.01|
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| » Discussion|| |
Over the past decade, microRNAs served as the star RNAs among noncoding RNAs in cancer research. However, lncRNAs still remained as a novel and emerging field of noncoding RNAs in cancers, especially in ESCC cells. In this study, we confirmed that SPRY4-IT1 was increased expressed in ESCC tissues, which was consistent with the previous study.  Loss of function assays revealed that SPRY4-IT1 had the growth promotion activity. Furthermore, we also proved that SPRY4-IT1 positively regulated ZNF703 expression. Although evidence presented here was a bit limited, we still demonstrated that SPRY4-IT1 was a vital tumor-promoting lncRNA by enhancing cell viabilities, which might be interpreted by the regulation of ZNF703 expression.
The prognostic significance of SPRY4-IT1 had been addressed in a wide range of human cancers, including ESCC,  and clear cell renal cell carcinoma.  For ESCC, Xie et al. reported that higher SPRY4-IT1 expression had an advanced clinical stage and poorer prognosis than those with lower SPRY4-IT1 expression. Besides, the multivariate analysis revealed that SPRY4-IT1 expression level is an independent prognostic factor in ESCC patients.  We did not address the clinical and prognosis value of SPRY4-IT1 fully in our study. Instead, we found an elevated expression level of SPRY4-IT1 in advanced ESCC tissues, which also supported the previous conclusion from the former study. 
Regarding on the biological role of SPRY4-IT1 in cancer cells, it still remained paradoxical. Although most reports suggested that SPRY4-IT1 mainly impacted on cell proliferation, it could also regulate apoptosis, metastasis, and EMT processes. ,, However, on regulating metastasis, it was showed that overexpression of SPRY4-IT1 in gastric cancer cells reduced the migration and/or invasion capacity,  in which indicated a tumor-suppressing role of SPRY4-IT1 in these cells. Therefore, it was improper to absolutely describe if SPRY4-IT1 was an oncogene or not. We did not know if SPRY4-IT1 impacted on ESCC metastasis. Moreover, we believe that further investigation in this aspect will provide more insights and clues for ESCC treatment.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
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