Background
Breast cancer (BC) is the most common malignancy occurring in women and it accounts for 30% of all diagnosed cancer in women around the world. BC remains the second leading cause of cancer-related mortality in women, especially between 20 and 59 years of age [
1]. BC is a complex heterogeneous disease and has been classified into several unique molecular subtypes based on different molecular and histological characteristics [
2,
3]. Although BC-related mortality has declined over the past two decades owing to great advances in cancer prevention, early diagnosis and treatment, there are still issues that need to be addressed, including micro-invasion, micro-metastasis and varied responses in patients undergoing similar surgery and adjuvant therapy [
4,
5]. Routine prognostic markers such as estrogen, progesterone, and human epidermal growth factor receptors have proven to be insufficient for estimating risk of recurrence and death. Therefore, there is an urgent need to explore the complex molecular mechanisms and identify better prognostic markers to improve the life quality for patients with BC.
In recent years, small nucleolar RNAs (snoRNAs), one of the less studied classes of small non-coding RNAs of approximately 60–300 nucleotides in length, have attracted a great deal of attention as regulatory RNAs [
6]. SnoRNAs originate within the intronic regions of protein-coding or non-protein coding genes and often function as housekeeping genes to guide the enzymatic modifications of other RNAs, mainly rRNA. There are two main groups of snoRNAs based on differences in structure—the box H/ACA snoRNAs (SNORAs), which are associated with pseudouridylation of rRNA, and box C/D snoRNAs (SNORDs), which are involved in 2′-
O-methylation of rRNA [
7‐
9]. Apart from the traditional function of modifying other RNAs, compelling evidence suggests that dysregulation of snoRNAs can also influence the development and progression of various human diseases such as Prader Willi syndrome, some metabolic stress disorders and several types of cancers [
10‐
14]. The first report that highlighted the pathological importance of snoRNAs showed that H5sn2 (a box H/ACA snoRNA) was distinctly down-regulated in meningiomas [
15]. Further, SNORD50 was reported to have a tumor suppressive role in breast and prostate cancer [
16,
17], while SNORA42 was reported to act as an oncogene in lung and colorectal cancer [
18,
19]. Su et al. have demonstrated the importance of snoRNAs in breast cancer [
20]. With the advance of high-throughput RNA-sequencing and microarray-based analysis, snoRNAs are beginning to be considered as plausible disease biomarkers.
In the present study, we used a series of bioinformatics analysis and identified SNORA7B as a potential oncogene in BC. In order to further investigate its potential role, we performed a series of gain- and loss-of-function experiments in vitro and evaluated the relationship between the expression level of SNORA7B and clinicopathological parameters. This is the first study to explore the role of SNORA7B in BC pathogenesis.
Materials and methods
Patients and samples
This study analyzed 1077 patients with breast cancer and 104 patients with non-cancerous tissues from The Cancer Genome Atlas (TCGA) database. In addition, 30 pairs of matched breast cancer and adjacent normal tissues, which came from BC patients enrolled at the First affiliated hospital of Wenzhou Medical University, were used to further verify the SNORA7B expression level in BC. The use of all tissues samples in this study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, and informed consent was obtained from each patient.
Cell culture
Breast cancer cell lines (MDA-MB-231, MDA-MB-468, MDA-MB-453, MDA-MB-436, MCF7, BT549, BT474, and SK-BR-3) and a non-neoplastic breast epithelial cell line (MCF10A) were purchased from Stem Cell Bank, Chinese Academy of Sciences. These cells were cultivated in RPMI1640 (Gibco, CA, USA) or DMEM (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, CA, US) and incubated in a humidified atmosphere with 5% CO2 at 37 °C. All cell lines were propagated following the standard protocols from ATCC.
RNA extraction and quantitative real-time RT-PCR
Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and then reverse transcribed into cDNA using a cDNA synthesis kit (Toyobo, Tokyo, JP). The quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the THUNDERBIRD SYBR qPCR Mix (Toyobo, Tokyo, JP) according to the manufacturer’s instructions. ALL qRT-PCR were performed on the Applied Biosystems 7500 Real-Time PCR System ( (Bio-Rad, Hercules, CA, USA). GAPDH was measured as an internal control. The following gene-specific primers were used: GAPDH (F: 5′-GGTCGGAGTCAACGGATTTG-3′; R: 5′-ATGAGCCCCAGCCTTCTCCAT-3′). SNORA7B (F: 5′-TCCTGGGATCGCATCTGGA-3′; R: 5′-GGAATGGAATGGGTGCCTCT-3′). RPL32P3 (F: 5′-CGGCACCAGTCAGACCGATA-3′; R: 5′-CCTGCACCCGTGGTATAAAG-3′). Each sample was run in triplicate.
Transfection of antisense oligonucleotides, small interfering RNAs, and plasmid DNAs
For functional studies, cells were transfected with the specific antisense oligonucleotides (ASO) flanked at both ends by locked nucleic acids or amido-bridged nucleic acids (AmNAs) targeting SNORA7B, siRNAs targeting RPL32P3, and SNORA7B-expression plasmids, which were synthesized and purchased from RiboBio. Cell transfection was performed with the Lipofectamine RNA iMAX (Life Technologies, Carlsbad, CA, USA) or Lipofectamine 3000 Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The efficiency of transfection was confirmed by qRT-PCR. The sequence of each antisense oligonucleotides and siRNA is given in Additional file
1.
Cell proliferation assay and colony formation assay
We utilized the colony formation and Cell Counting Kit-8 (CCK-8, Sigma, St Louis, MO, USA) assays to determine proliferative ability. For the colony formation assay, transfected MDA-MB-231 and BT-549 cells (2 × 103 cells/well) were seeded in 6-well plates. After 10 days, cells were fixated with 4% paraformaldehyde (PFA) for 30 min and stained with 0.1% crystal violet for 30 min. Colonies were counted only if they included at least 50 cells. For the proliferation assay, the transfected cells (2 × 103) were plated in 96-well plates and measured every 24 h using the CCK-8 reagent following the manufacturer’s instruction. The absorption was measured at 450 nm after adding the reagent and incubating for 2 h in a 37 °C incubator. All experiments were performed in triplicate.
Cell migration and invasion ability analyses
Cellular migration and invasion assays were performed in a transwell cell culture chamber system and Matrigel invasion chamber system, respectively; both had a pore size of 8 mm according to the manufacturer’s instruction (Corning Costar, Cambridge, MA, USA). For migration assays, the transfected cells (8 × 105 cells for MDA-MB-231 and 6 × 105 cells for BT-549) were seeded in the upper chamber, which was placed into a 24-well plate filled with medium containing 10% FBS. Cells were incubated for 26 h (MDA-MB-231 cells) or 28 h (BT-549 cells) at 37 °C and 5% CO2 in an incubator. Then, after washing off cells that did not traverse the filter, cells adhering to the lower surface of the membrane were fixed with 4% PFA for 30 min, stained with 0.01% crystal violet for 30 min and photographed by a light microscope. For invasion assays, inserts coated with Matrigel matrix were used. The transfected cells (12 × 105 cells for MDA-MB-231 and 10 × 105 cells for BT-549) were added to the upper chamber, the medium in the lower chamber was supplemented with 20% FBS, and the chamber system was incubated for 24 h. The subsequent steps were similar to the migration assay. All experiments were performed at least three times.
Cell apoptosis assay
Two days after infection, cells were harvested and double stained with annexin V conjugated to phycoerythrin and 7-aminoactinomycin (7-AAD) (Apoptosis Detection Kit-1, BD Pharmingen, San Diego, CA, USA). Apoptotic events were analyzed using FlowJo software. These experiments were repeated in triplicate.
Statistical analysis
Data are expressed as the mean ± SD. The different gene expression levels of SNORA7B in tumor and healthy samples were analyzed using Wilcoxon signed-rank test, Mann–Whitney U test, and paired sample t-test. Spearman’s correlation analysis was used to determine correlation between SNORA7B and RPL32P3 expression level. Chi square test was used to access the relationship between SNORA7B expression and clinical characteristics. Survival curves were plotted by the Kaplan–Meier method and the log-rank tests. Both univariate and multivariate Cox proportional hazard models were applied to assess the relationship between effect of SNORA7B expression and survival. We used the Student’s t-test or one-way ANOVA to test the differences in expression between cell lines, the expression changes after transfection and all cell function assays. All p-values were two sided, and a p-value of 0.05 and less was considered statistically significant. Statistical analysis was performed with SPSS software version 19.0 (SPSS, Chicago, IL, USA). GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) was used for plotting graphs.
Discussion
SnoRNAs are a type of noncoding RNA reported to play important regulatory roles in several physiological and pathological events [
21‐
23]. A growing volume of evidence indicates that multiple snoRNAs are involved in various cancers [
24‐
28]. In light of the potential value of snoRNAs as biomarkers and therapeutic targets, as well as to further explore the potential molecular mechanisms underlying cancer formation and development, we identified and investigated potential snoRNAs involved in BC. In this study, we firstly screened for specific snoRNAs using the publicly available TCGA database. Our analysis of the database indicated that the expression of SNORA7B was prominently up-regulated in BC compared with normal tissues, which was also verified in our breast cancer clinical specimens and cell lines. Intriguingly, we also confirmed that SNORA7B expression was positively associated with some key clinicopathological parameters, such as age, tumor size, and lymph node metastasis. Moreover, patients with high expression of SNORA7B showed much worse prognosis of BC. These results suggested that the SNORA7B might play an oncogenic role in breast cancer progression. Thus, SNORA7B, which is positioned on the intron of RPL32P3 gene and belongs to the H/ACA snoRNAs for sequence-specific pseudouridylation of other RNAs, was selected for further functional examination.
In most cases, the transcription of the host gene determines the levels of intron encoded snoRNA genes [
29]. Thus, considering the possibility that RPL32P3 also might play a role in BC, we examined the expression of RPL32P3 in TCGA database and BC cell lines. Interestingly, RPL32P3 exhibited only little difference in RNA expression level between BC cell lines and normal breast cell line, and no correlation was found between transcriptional levels of SNORA7B and RPL32P3. Moreover, we also evaluated the expression of SNORA7B following the knockdown of RPL32P3 and found that its expression was only slightly and insignificantly altered. These results suggested that the function of SNORA7B might be independent of its host gene.
We then performed a series of gain- and loss-of- function analyses in vitro to further determine the functional significance of SNORA7B in BC cell lines. By using specific anti-SNORA7B-ASOs to suppress SNORA7B expression, we elucidated that SNORA7B knockdown exhibited a strong capacity to promote cell apoptosis, which resulted in the inhibition of cell growth, proliferation, migration, and tumor invasion abilities of the BC cells. The oncogenic effect of SNORA7B in BC was subsequently confirmed with SNORA7B overexpression, which showed consistent results with that from the loss-of-function experiments.
Previous study and computational analysis indicated that there are several isoforms of SNORA7 [
23]. Among these paralogues, SNORA7B and SNORA7A are over 98% identical and target the same 28S rRNA pseudouridylation sites [
30,
31]. Zhang et al. [
32] demonstrated that both SNORA7B and SNORA7A could promote the self-renewal of human umbilical cord blood-derived mesenchymal stem cells (uMSCs). The SNORA7B gene is located on chromosome 3q21 as an intron in the RPL32P3 gene. Interestingly, genomic alterations in the 3q21 locus have been observed in leukemia [
33,
34], colorectal cancer [
35], prostate cancer [
36], and breast cancer [
37‐
39]. Each class of snoRNAs interacts with a specific set of highly conserved proteins to form the well-defined C/D box and H/ACA box small nucleolar ribonucleoproteins (snoRNPs). The H/ACA box snoRNAs are associated with four proteins including DKC1, GAR1, NHP2, and NOP10 to catalyze a certain pseudouridylation site of 18S or 28S rRNA [
8,
40]. Considering that cancer cells often show perturbation at the translation level, snoRNAs and snoRNPs are likely to contribute to tumorigenesis through effects on ribosomes and protein translation. Zhang et al. [
32] also showed that SNORA7A functioned by inducing snoRNP formation by binding DKC1 and subsequently catalyzing pseudouridines in 28S rRNA. Since SNORA7B and SNORA7A target the same 28S rRNA pseudouridylation sites, it is likely that SNORA7B may also act through snoRNP to regulate the tumor behavior in BC.
The present study still had several limitations. First, this study only conducted in vitro experiments. Therefore, the effects of SNORA7B in BC need to be verified in vivo experiments. Second, the mechanism of SNORA7B oncogenic role needs to be further explored. Despite these limitations, this study is the first to identify the oncogenic role of SNORA7B and show that it promotes BC cell growth, proliferation, invasion, and migration through decreased apoptosis of BC cells in vitro. Furthermore, the expression of SNORA7B, which was significantly up-regulated in BC, may have diagnostic potential and present a useful prognostic molecular marker of BC.
Conclusions
In summary, we found that expression level of SNORA7B increased in BC tissues compared to that in non-tumor tissues. Further, SNORA7B expression level was positively correlated with poor survival time and worse clinicopathologic parameters. SNORA7B impaired apoptosis to promote BC cell growth, proliferation, migration, and invasion. Here, for the first time, we identified that SNORA7B functions as an oncogene in BC and may have diagnostic potential and sever as a potential prognostic biomarker for BC.
Authors’ contributions
XHZ and EDC conceived of the study and designed experiments. YHS, YFL and DRY performed experiments and statistical analysis. YHS write the manuscript. All authors read and approved the final manuscript.