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

Abstract

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.

Keywords: EMT; NSCLC; PI3K/AKT; SNORA47; cell growth.

Figure 1

Knockdown of SNORA47 significantly inhibited the proliferation of NSCLC cells. (A) A549 or NCI-H23 cells were transfected with SNORA47 shRNA1 or shRNA2. Then, the expression of SNORA47 in NSCLC cells was detected by RT-qPCR. (B) The viability of NSCLC cells was tested by CCK-8 assay. (C) The proliferation of A549 cells was measured by Ki67 staining. Red fluorescence indicates Ki67. Blue fluorescence indicates DAPI. (D) The proliferation of NCI-H23 cells was measured by Ki67 staining. **P < 0.01 compared to Blank.

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-β. (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.

Figure 3

SNORA47 shRNA significantly suppressed the migration and invasion of NSCLC cells. (A) The migration of A549 cells was detected by transwell assay. (B) The migration of NCI-H23 cells was detected by transwell assay. (C) The invasion of A549 cells was detected by transwell assay. (D) The invasion of NCI-H23 cells was detected by transwell assay. **P < 0.01 compared to Blank.

Figure 4

SNORA47 knockdown inhibited PI3K/Akt, MAPK/ERK and the EMT process in NSCLC cells. (A) The protein expressions of E-cadherin, N-cadherin, Akt, p-Akt, ERK, p-ERK, and cleaved caspase 3 in NSCLC cells were detected by western blot. (B–F) The relative expressions were quantified by normalizing to β-actin. **P < 0.01 compared to Blank.

Figure 5

SNORA47 knockdown notably induced G1 arrest in NSCLC cells. (A) The cell cycle distribution in NSCLC was detected by flow cytometry. (B) The protein expressions of p27 Kip1, Cyclin D1, and CDK2 in NSCLC cells were detected by western blot. (C–E) The relative expressions were quantified by normalizing to β-actin. **P < 0.01 compared to Blank.

Figure 6

Knockdown of SNORA47 significantly inhibited the tumor growth of NSCLC in vivo. A549 cells were subcutaneously injected into nude mice to mimic NSCLC in vivo. SNORA47 shRNA1 or vector control was directly injected into the tumors. (A) Tumor volumes were assessed once a week. (B) Tumor tissues of mice were collected at the end of the study. (C) Tumor weights of mice were investigated. (D) The level of SNORA47 in mice was investigated by RT-qPCR. (E) The proliferation of NSCLC cells was detected by Ki67 staining. (F) The protein levels of p-Akt and p-ERK in tissues of mice were measured by IHC staining. **P < 0.01 compared to control.