Upregulation of Small Nucleolar RNA 78 Promotes Cell Proliferation and Invasion of Breast Cancer


 OBJECTIVE: Recent studies suggest that abnormal snoRNA expression may play crucial role in tumor development and progression. In the study, we aim to explore the clinical significance and functional role of Small nucleolar RNA 78 (SNORD78) in breast cancer.PATIENTS AND METHODS: The expression of SNORD78 in 92 breast cancer and adjacent non tumor tissues was analyzed by quantitative real time polymerase chain reaction (qRT-PCR). The association between SNORD78 expression and clinicopathological factors was evaluated. The biological functions in vitro were examined by MTT cell proliferation, colony formation and transwell invasion assays. QRT-PCR and Western blot assays were used to analyze the mRNA and protein expression of WNT1, GSK3β and β-catenin.RESULTS: SNORD78 expression showed a significant increase in breast cancer tissues and cell lines. Higher SNORD78 expression positively was related to lymph node metastasis and TNM stage in patients. Furthermore, knockdown of SNORD78 inhibited the abilities of cell proliferation, colony formation, and cell invasion in MDA-MB-231 and SKBR-3 cells. Moreover, we demonstrated that knockdown of SNORD78 inhibited WNT/β-catenin signaling pathway in MDA-MB-231 and SKBR-3 cells via downregulating the relative protein expression of WNT1 and β-catenin and upregulating the GSK3β expression.CONCLUSION: Our results indicated that SNORD78 may be potential therapeutic target for breast cancer.


Introduction
Breast cancer is the most common malignancy in women worldwide. In 2015, 231,840 women were diagnosed with breast cancer, and approximately 40,290 died of the disease [1,2]. Tumor recurrence and metastasis cause larger cancer related morbidity and mortality [3]. In recent decades, large advances in the diagnosis and therapy for breast cancer have been improved, however, patients who are diagnosed at advanced stage present poor outcome [4]. Therefore, to investigate tumor makers of early detection or therapeutic target of breast cancer is important.
Small nucleolar RNAs (snoRNAs) are a class of RNA without protein coding capacity and about 60-300 nucleotides in length [5]. Recent studies have indicated that snoRNAs were involved in some disease process including tumor development [6]. For instance, small nucleolar RNA U91 is a new internal control for accurate microRNAs quanti cation in pancreatic cancer [7]. SNORD76, not its host gene, is selectively downregulated in glioblastoma (WHO grade IV) and acts as a tumor suppressor in glioblastoma [8].
SNORD113-1 suppresses tumorigenesis in hepatocellular carcinoma by inactivating the phosphorylation of ERK1/2 and SMAD2/3 in MAPK/ERK and TGF-β pathways [9]. However, the clinical role and potential functions of small nucleolar RNA 78 (SNORD78) in breast cancer remain unknown.
In the study, we demonstrated that SNORD78 expression was signi cantly upregulated in breast cancer.
SNORD78 silencing obviously inhibited breast cancer cells proliferation, cell invasion and Wnt/β-catenin signaling pathway. Thus, our results indicated that SNORD78 may be potential therapeutic target for breast cancer.

Patients And Methods
Human patient tissue specimens Freshly breast tissue specimens obtained from breast cancer patients who underwent curative surgical treatment between April 2010 and March 2012 were immediately frozen in liquid nitrogen for further RNA extraction. All of patients were selected at Department of Oncology, Peking University Shenzhen Hospital. The study was approved by Peking University Shenzhen Hospital and written informed consent was obtained from all of the patients.

MTT assay
Cell growth rate was investigated using a MTT assay. 3 × 10 3 transfected cells/well were seeded into 96well plates. At indicated time 0, 24, 48, and 72 h, cells were incubated with 20 µl MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and cultured for 4 h at 37 °C with 5% CO 2 . The cell proliferation was analyzed using an ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the absorbance at 490 nm.
Cell clonogenic assay 300 transfected cells were placed in 12-well plates cultured in medium added with 10% fetal bovine serum at 37 °C with 5% CO 2 for 14 days. Colonies were xed with 100% methanol, stained with 0.1% crystal violet, and then were counted under a microscope.

Cell invasion assays
Transwell chambers with an 8 µm pore polycarbonate membrane (Costar; Corning Incorporated, Corning, NY, USA) were used to assess cell invasion ability. 1 × 10 5 cells were added on the upper chamber supplemented with 300 µl culture medium without FBS and 500 µl culture medium supplemented with 20% FBS was added on the lower chamber. After cell cultured for 24 h, cells were xed with 100% methanol, and then stained with 0.1% crystal violet. Cells on the lower chamber were counted in ve random elds under a light microscope.

Western blot analysis
Cells were lysed using radioimmunoassay precipitation assay (RAPA) lysis buffer with a proteinase inhibitor. The quantity of approximately 40 µg proteins in the lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene di uoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with anti-WNT1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), GSK3β (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 12 h at 4 °C and then washed three times for 5 min.
Following horseradish peroxidase-conjugated secondary antibodies were incubated. The blot was visualized by an enhanced chemiluminescence kit (Thermo Fisher Scienti c, Inc., Rockford, IL, USA).

Statistical analysis
All of results are presented as means ± SD from at least three or more independent experiments. The association between SNORD78 expression and clinical characteristics was evaluated using the chisquare test. Differences between groups were compared using Student's t-test or an analysis of variance (ANOVA). A p < 0.05 were considered statistically signi cant.

SNORD78 expression is upregulated in breast cancer tissues and associates with clinicopathologic features
In the study, SNORD78 expression was determined by qRT-PCR in 92 breast cancer tissues compared with adjacent normal tissues. As shown in Fig. 1A, results analysis demonstrated that SNORD78 expression in breast cancer tissues was signi cantly higher compared with adjacent normal tissues (p < 0.05). According to the median expression rate of SNORD78 expression in breast cancer tissues, we divided patients into higher expression group and lower expression group. The association between the SNORD78 expression and the clinical characteristics of breast cancer patients were analyzed. We demonstrated that higher SNORD78 expression was signi cantly associated with the lymph node status (p = 0.001, Table I) and advanced TNM stage (p = 0.023, Table I), but was not associated with the other characteristics in breast cancer patients (p > 0.05, Table I).

Downregulation of SNORD78 expression suppresses cell growth and invasion in MDA-MB-231 and SKBR-3 cells in vitro
Additionally, we evaluated the expression of SNORD78 in three human breast cancer cell lines (MDA-MB-231, MCF-7, and SKBR-3) and one normal mammary epithelial cell line (MCF-10A). As shown in Fig. 1B, the expression of SNORD78 was higher in three human breast cancer cell lines than MCF-10A cells (p < 0.05). To further analyze the biological effects of on cell growth and invasion, two siRNAs targeting SNORD78 oligos were transfected into MDA-MB-231 and SKBR-3 cells ( Fig. 2A-2B). The si-SNORD78-1 was chosen to knock down SNORD78 expression in following experiments due to its higher silencing e ciency. MTT analysis results showed that knockdown of SNORD78 in MDA-MB-231 and SKBR-3 cells notably suppressed cell proliferation at 48 h and 72 h, compared with control group (Fig. 2C-2D

Discussion
Small nucleolar noncoding RNAs (snoRNAs) regulate function of ribosomes and increasing evidence indicates that dysregulation of small nucleolar RNAs (snoRNAs) were associated with tumorigenesis [11].
Such as, integrated analysis of the prostate cancer small-nucleolar transcriptome reveals SNORA55 as a driver of prostate cancer progression [12]. SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred signi cantly in multiple human tumor types [13]. Small nucleolar RNA 47 promotes tumorigenesis by regulating EMT markers in hepatocellular carcinoma [14]. Small nucleolar RNA ACA11 promotes cell proliferation, migration and invasion in hepatocellular carcinoma by targeting the PI3K/AKT signaling pathway [15]. In the study, we found that SNORD78 expression in breast cancer tissues was signi cantly higher compared with adjacent normal tissues. Higher SNORD78 expression was signi cantly associated with the lymph node status and advanced TNM stage. Moreover, knockdown of SNORD78 inhibited cell proliferation, colony formation and cell invasion capacities in breast cancer. Consistent with our ndings, SNORD78 was observed to be upregulated in NSCLC tissues compared to their adjacent normal tissues. SNORD78 promoted cell proliferation and invasion of NSCLC cells, which indicated SNORD78 function as an oncogene [16].
Furthermore, we demonstrated that WNT/β-catenin signaling pathway related protein expression levels of WNT1 and β-catenin was signi cantly downregulated, but GSK3β expression were upregulated when SNORD78 was knocked down in MDA-MB-231 and SKBR-3 cells. The WNT pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis [10]. Aberrantly activated Wnt/β-catenin signaling could affect diverse biological processes including cell motility, migration, differentiation, proliferation, and invasion [17]. Overexpression of Wnt/β-catenin protein Lrp6 or stabilized β-catenin, or loss of APC, resulted in mouse mammary hyperplasia or tumors [18]. To elucidate the underlying mechanisms of SNORD78 affecting cell proliferation and invasion, we demonstrated that knockdown of SNORD78 inhibited cell proliferation and invasion by regulating WNT/β-catenin signaling pathway.

Conclusions
In conclusion, our results found that SNORD78 expression was signi cantly upregulated in breast cancer.
Thus, our results indicated that SNORD78 may be potential therapeutic target for breast cancer.

Declarations
Ethics approval and consent to participate The study was approved by Peking University Shenzhen Hospital and written informed consent was obtained from all of the patients.

Consent for publication
All the authors agreed that the article should be published.

Availability of data and material
All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare no con ict of interest.