Knockdown of SNORA47 Inhibits the Tumorigenesis of NSCLC via Mediation of PI3K/Akt Signaling Pathway

Background Non-small cell lung cancer (NSCLC) is a frequently diagnosed aggressive cancer all over the world. Small nucleolar RNAs (snoRNAs) are a group of non-coding mediatory RNAs. A previous report indicated that small nucleolar RNA 47 (SNORA47) is upregulated in NSCLC. However, the role of SNORA47 in NSCLC is unclear. Material and Methods Cell proliferation was measured by immunofluorescence staining. Cell apoptosis and cycle of NSCLC were tested by flow cytometry and the protein expressions were investigated by Western-blot. Meanwhile, cell migration and invasion were detected by transwell assay. Xenograft mice model was established to detect the effect of SNORA47 knockdown on tumor growth of NSLC in vivo. Results Knockdown of SNORA47 significantly inhibited the proliferation of NSCLC cells via inducing cell apoptosis. Moreover, migration and invasion of NSCLC cells were notably decreased by SNORA47 shRNA. SNORA47 knockdown significantly induced G1 arrest in NSCLC cells via regulation of p27 Kip1, CDK2, and cyclin D1. Meanwhile, SNORA47 shRNA inhibited EMT process and PI3K/Akt signaling in NSCLC cells. Finally, silencing of SNORA47 significantly inhibited the tumor growth of NSCLC in vivo. Conclusion Knockdown of SNORA47 significantly inhibited the tumorigenesis of NSCLC via inhibition of PI3K/Akt signaling and EMT process. Thereby, our finding might shed a new light on exploring the strategies for the treatment of NSCLC.

INTRODUCTION Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer which is known as the leading cause of cancer-associated death all over the world (1). Nowadays, surgery, radiation and chemotherapy are the main treatments of NSCLC (2). Although many efforts have been made to treat NSCLC in the past twenty years, 5-year survival rates of patients with NSCLC remain low due to metastasis and recurrence of lung cancer (3). Therefore, it is necessary to explore new strategies for the treatment of NSCLC.
Small nucleolar RNAs (snoRNAs) are a group of non-coding mediatory RNAs about the length of 60-300 nucleotides which are located in nucleolus of cells (4). It has been previously reported that more than 100 snoRNAs throughout the human body are closely correlated with cellular processes, genesis of miRNA and stress response (5). Since snoRNAs are participated in many physiological processes, dysregulation of snoRNAs may lead to the progression of diseases (6). In addition, it has been confirmed that snoRNAs can act as important mediators in NSCLC. For example, Zheng D et al. found that SNORA78 could promote the tumorigenesis of NSCLC in vitro and in vivo (7). In addition, Mei YP et al. indicated that SNORA42 could act as an oncogene in NSCLC (8). Meanwhile, SNORA47 is known to be upregulated in NSCLC (9). However, the role of SNORA47 in NSCLC remains unclear.
In the current study, we sought to detect the function of SNORA47 in NSCLC. We hope our research would shed new lights on exploring the new methods for the treatment of NSCLC.

Immunofluorescence Staining
Cells were plated onto a 96-well plate at the density of 5.0 × 10 3 cells/well. After incubation, cells were transfected with NC or SNORA47 shRNA1 for 72 h. After that, cells were fixed with 4% paraformaldehyde and incubated with rabbit monoclonal antibody anti-Ki67 (1:100, Abcam Cambridge, MA, USA) at 4°C overnight. Then, cells were incubated with anti-rabbit IgG secondary antibody (1:1000, Abcam) for 1 h at room temperature. Finally, the results were observed under a fluorescence microscope (Olympus BX53, Tokyo, Japan).

Flow Cytometry
Cells were trypsinized and resuspended in binding buffer. After that, cells were stained with 5 µl annexin V-FITC and PI in the dark at 37°C for 30 min. Later on, flow cytometry (FACScan ™ ; BD Biosciences, Franklin Lake, NJ, USA) was applied to analyze the apoptosis rate using CellQuest ™ software.

Cell Cycle Detection
Cell cycle distribution was investigated according to a recent report (10). NSCLC cells were harvested, fixed, permeabilized, and stained with Pharmingen PI/RNase (BD). After incubation for 15 min, cells were analyzed using ModFit and the data were quantified by FlowJo software (BD).

Transwell Assay
The upper chamber is pre-treated with 100 ml matrigel (BD Biosciences, Franklin Lake, NJ, USA). Cell migration was tested without Matrigel. Subsequently, transfected NSCLC cells were seeded in upper chambers with serum-free media, while the media in lower chambers was supplemented with 10% serum. After 24 h of incubation, transwell chambers were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet. The migrated and invaded cells were calculated under a microscope.

Xenograft Mice Model
All animal experiments were approved by the research ethics committee of Chongqing University Cancer Hospital. NSCLC cells were infected for 24 h with a control or SNORA47 shRNAexpressing lentivirus (Genepharma), and then injected subcutaneously (2 × 10 6 cells/per mouse) into 5-week-old BALB/c nude mice (Chinese Academy of Sciences, Shanghai, China). Tumor volume was calculated every week for 5 weeks according to the equation: (length×width×width)/2. At the end of the study, mice were sacrificed for collection of tumor tissues.

Immunohistochemical (IHC) Staining
Tissues of mice were fixed, paraffin-embedded, and cut into 5mm-thick sections. Paraffin sections were deparaffinized and rehydrated. The sections were heated in sodium citrate buffer in a microwave for antigen retrieval, washed with phosphatebuffered saline (PBS) for 5 min (three times) at room temperature, incubated in 3% H 2 O 2 at room temperature for 25 min. The sections were, washed with PBS for 5 min (three times) and blocked and incubated in goat serum for 30 min. Then, samples were incubated with primary antibody (anti-p-Akt and anti-p-ERK, Abcam) overnight at 4°C. After that, samples were incubated with secondary antibody (HRPlabeled) for 30 min at 37°C. Finally, freshly prepared diaminobenzidine (DAB) was added for color development. The tissues were observed under a microscope.

Statistical Analysis
Data are presented as the mean ± standard deviation. CCK-8 assay was performed in quintuplicate. RT-qPCR, flow cytometry, Ki67 staining, transwell assay, and western blot were repeated three times. Comparisons were analyzed by the Student's t-test (between two groups) or one-way ANOVA followed by Tukey's post hoc test (among multiple groups, GraphPad Prism; version 7; GraphPad Software, Inc.). P < 0.05 indicates a statistically significant difference.

RESULTS
Knockdown of SNORA47 significantly inhibited the proliferation of NSCLC or BEAS-2B cells To detect the efficiency of cell transfection, RT-qPCR was used. As revealed in Figure 1A and Supplementary Figure 1A, the expression of SNORA47 in NSCLC or BEAS-2B cells was downregulated by SNORA47 shRNA1. Moreover, cell viability of NSCLC or BEAS-2B was notably decreased by SNORA47 knockdown ( Figure 1B Figure 1C). Meanwhile, the viability and proliferation of BEAS-2B cells were limitedly affected by SNORA47 shRNA2 (Supplementary Figures 1B, C). Altogether, knockdown of SNORA47 significantly inhibited the proliferation of NSCLC cells.

Silencing of SNORA47 Notably Induced the Apoptosis in NSCLC Cells
In order to investigate the effect of SNORA47 knockdown on cell apoptosis, flow cytometry was used. As indicated in Figures 2A,  B and Supplementary Figure 1D, SNORA47 shRNA1 notably induced the apoptosis of NSCLC or BEAS-2B cells, while SNORA47 shRNA2 had very limited effect on BEAS-2B cell apoptosis. Meanwhile, NSCLC cells were more sensitive to SNORA47 shRNA1, compared to BEAS-2B cells ( Figures 1A-D,  2A, B and Supplementary Figures 1A-D). Furthermore, SNOTRA47 knockdown-induced NSCLC cells apoptosis was partially reversed by TGF-b (Figures 2A, B). Thus, silencing of SNORA47 significantly inhibited the proliferation of NSCLC cells via inducing cell apoptosis. Moreover, NSCLC cells were more sensitive to SNORA47 shRNA1, compared to SNORA47 shRNA2. Thus, SNORA47 shRNA1 were selected of use in subsequent experiments.

SNORA47 shRNA1 Significantly Suppressed the Migration and Invasion of NSCLC Cells
In order to detect the migration and invasion of NSCLC cells, transwell was performed. The data showed that migration of NSCLC cells was significantly inhibited by silencing of SNORA47 ( Figures 3A, B). Consistently, SNORA47 knockdown obviously alleviated the invasion of NSCLC cells (Figures 3C, D). Since A549 cells were more susceptible to SNORA47 shRNA treatment, A549 cells were selected for further analysis. Taken together, SNORA47 shRNA significantly suppressed the migration and invasion of NSCLC cells.

SNORA47 Knockdown Inhibited PI3K/Akt, MAPK/ERK and the EMT Process in NSCLC Cells
In order to investigate the mechanism by which SNORA47 mediated the progression of NSCLC, western blot was used. As revealed in Figures 4A-F, knockdown of SNORA47 significantly upregulated the expressions of E-cadherin and cleaved caspase 3 and downregulated the expressions of N-cadherin, p-Akt, and p-ERK. In addition, the effect of SNORA47 shRNA1 on E-cadherin and N-cadherin was further increased by LY294002 or PD98059 (Supplementary Figure 2A). Altogether, SNORA47 knockdown inhibited PI3K/Akt signaling, MAPK/ERK and the EMT process in NSCLC cells.

SNORA47 Silencing Notably Induced G1 Arrest in NSCLC Cells
For the purpose of detecting the effect of SNORA47 knockdown on cell cycle distribution, flow cytometry was performed. As shown in Figure 5A, silencing of SNORA47 significantly induced G1 arrest in NSCLC cells. Moreover, the effect of SNORA shRNA1 on cell cycle distribution was further enhanced by LY294002 or PD98059 (Supplementary Figure  2B). Meanwhile, the expression of p27 Kip1 in NSCLC cells was notably upregulated by knockdown of SNORA47 (Figures 5B,  C). In contrast, silencing of SNORA47 significantly inhibited the protein levels of cyclin D1 and CDK2 ( Figures 5B, D, E). To sum up, SNORA47 knockdown notably induced G1 arrest in NSCLC cells via mediation of p27 Kip1, cyclin D1, and CDK2.

Knockdown of SNORA47 Significantly Inhibited the Tumor Growth of NSCLC In Vivo
Finally, xenograft mice model was established to investigate the function of SNORA47 in NSCLC. As revealed in Figures 6A, B, the tumor sizes of mice were significantly decreased by knockdown of SNORA47. Consistently, SNORA47 shRNA1  notably reduced the tumor weight of mice ( Figure 6C). Meanwhile, the level of SNORA47 in mice was significantly decreased when injected with SNORA47 shRNA1 (Figure 6D), and SNORA47 shRNA1 notably increased Ki67 positive cell rate in tumor tissue ( Figure 6E). In addition, the protein levels of p-Akt and p-ERK in tissues of mice were notably inhibited by SNORA47 shRNA1 (Figure 6F). In summary, knockdown of SNORA47 significantly inhibited the tumor growth of NSCLC in vivo.

DISCUSSION
In this study, we found that silencing of SNORA47 could lead to the inhibition of cell growth, migration, and invasion in NSCLC. In addition, our research firstly indicated that SNORA47 could play a key role in tumorigenesis of NSCLC via mediation of PI3K/Akt/EMT axis. Therefore, our findings would be meaningful for the improvement of new targeted therapy.
It has been reported that snoRNAs are involved in tumorigenesis of NSCLC. For instance, Tang G et al. found that SNORA71A could promote the growth, migration, and invasion of NSCLC cells via mediation of MAPK/ERK pathway (11). In addition, Zheng D et al. found SNORA78 could promote the tumorigenesis of NSCLC (7). Moreover, Gao et al. reported that SNORA68 and SNORA21 were notably upregulated in NSCLC tissues, compared with adjacent normal tissues (12). Since these snoRNAs have been confirmed to be participated in the progression of NSCLC, it is necessary to compare the underlying mechanisms of these snoRNAs in NSCLC with SNORA47 for identifying a potential therapeutic target.
PI3K/Akt signaling pathway is frequently upregulated in malignant tumors (13,14). In the current research, SNORA47 shRNA notably inhibit PI3K/Akt signaling in NSCLC. A recent study found that inactivation of PI3K/Akt signaling contributes to cell apoptosis in NSCLC (15). Thus, our study was consistent to this previous research. Moreover, our data also showed that silencinhg of SNORA47 inactivated p-ERK in NSCLC cells, and ERK inhibitor further increased the apoptotic effect of SNORA47 A B FIGURE 2 | Silencing of SNORA47 notably induced the apoptosis in NSCLC cells. A549 or NCI-H23 cells were treated with NC, SNORA47 shRNA1, SNORA47 shRNA2, or SNORA47 shRNA1 + TGF-b. (A) The apoptosis of A549 cells was tested by flow cytometry. (B) The apoptosis of NCI-H23 cells was tested by flow cytometry. **P < 0.01 compared to Blank. ## P < 0.01 compared to SNORA47 shRNA1.
shRNA. Since ERK is the downstream protein of MAPK pathway (16), our study firstly found the function of SNORA47 in MAPK/ ERK signaling. Meanwhile, our findings suggested that knockdown of SNORA47 downregulated EMT process in NSCLC cells. A previous report indicated that activation of PI3K/Akt signaling was correlated with the upregulation of EMT process (17). Taken together with these data, our findings indicated that SNORA47 knockdown could inhibit the progression of NSCLC via inhibition of EMT process and PI3K/ Akt signaling pathway.
In this research, we found that p27 Kip1, CDK2, and cyclin D1 were the downstream targets of SNORA47 by which SNORA47 shRNA regulated cell cycle distribution of NSCLC. P27 Kip1 is known to be a cell cycle regulator which plays a pivotal role in the G1-to-S transition of the cell cycle (18)(19)(20). Consistently, our data demonstrated that SNORA47 knockdown could result in G1 phase arrest in NSCLC cells through upregulation of p27 Kip1. Meanwhile, CDK2 and Cyclin D1 are known to be cell cycle regulators in NSCLC (21)(22)(23). SNORA78 could induce cell cycle arrest in NSCLC cells via upregulation of CDK2 (7). In addition, SNORA42 could act as an oncogene in lung cancer via upregulation of Cyclin D1 (8). Our data was in line with the previous findings of CDK2 and Cyclin D1 as cell-cycle mediators.
According to Li G et al. (24), SNORA47 could promote the tumorigenesis of hepatocellular carcinoma by regulation of EMT process. Similarly, our data also revealed that SNORA47 shRNA could inhibit EMT process in NSCLC cells. In this previous research, SNORA47 significantly inhibited the expression of Ecadherin and increased the level of N-cadherin. Consistently, our study also indicated that SNORA47 silencing inhibited the EMT process via regulation of E-cadherin and N-cadherin. Thus, the function of SNORA47 on these two proteins might result in this similarity. Meanwhile, our data further explored the function of SNORA47 in PI3K/Akt signaling.
Of course, there are some limitations in this research as follows: (1) the correlation between SNORA47 and PI3K/Akt signaling is still unclear; (2) other molecular mechanisms by which SNORA47 mediates the tumorigenesis of NSCLC remain to be further explored. Thereby, more investigations are needed in the future.  In conclusion, knockdown of SNORA47 significantly inhibited the tumorigenesis of NSCLC via mediation of PI3K/ Akt and MAPK/ERK signaling. Thus, SNORA47 might serve as a new target for the treatment of NSCLC.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by all animal experiments were approved by the research ethics committee of Chongqing University Cancer Hospital.  The cell cycle distribution was tested by flow cytometry. **P < 0.01 compared to Blank. ## P < 0.01 compared to SNORA47 shRNA1.