SNHG5 Promotes Breast Cancer Proliferation by Sponging the miR-154-5p/PCNA Axis

Abstract

Breast cancer is the most common malignant tumor and the main cause of cancer-associated mortality in females worldwide. Long non-coding RNAs (lncRNAs) have been reported to play vital roles in breast cancer development and progression; however, our understanding of most lncRNAs in breast cancer is still limited. In this study, we demonstrated that small nucleolar RNA host gene 5 (SNHG5) promotes breast cancer cell proliferation both in vitro and in vivo, and depletion of SNHG5 significantly led to cell-cycle arrest at G1 phase. Accumulating evidence has shown that many lncRNA transcripts could function as competing endogenous RNAs (ceRNAs) by competitively binding common microRNAs (miRNAs). We found that SNHG5 acts as a sponge for miR-154-5p, reducing its ability to repress proliferating cell nuclear antigen (PCNA). SNHG5 promoted breast cancer proliferation and cell-cycle progression by upregulation of PCNA expression. Clinically, we observed an increased SNHG5 expression in breast cancer, whereas miR-154-5p was decreased in breast cancer tissues compared with the adjacent normal breast tissues. Furthermore, the SNHG5 expression was significantly negatively correlated with miR-154-5p expression. Taken together, our data uncover the SNHG5-miR-154-5p-PCNA axis and provide a novel mechanism to explain breast cancer proliferation.

Keywords: PCNA; SNHG5; breast cancer; cell cycle; miR-154-5p; proliferation.

Graphical abstract

Figure 1

Knockdown of SNHG5 Inhibits Cell Proliferation and Induces Apoptosis in Breast Cancer Both In Vitro and In Vivo (A) SNHG5 expression in breast cancer cell lines as determined by qRT-PCR. (B) SNHG5 expression in MDA-MB-231 cells transfected with small interfering RNAs targeting SNHG5 as determined by qRT-PCR. (C–E) Cell growth inhibition was determined by MTT (C), colony formation (D), and EdU (E) assays in cells as in (B). (F) Apoptosis analysis of cells as in (B). (G) SNHG5 expression in stable SNHG5-depleted MDA-MB-231 and control cells as determined by qRT-PCR. (H) Representative photos and weights of tumors formed by cells as in (G). (I) Tumor volume of xenograft mice injected with cells as in (G) at the indicated times. The data were presented as the mean ± SD obtained from at least three independent experiments. Significance was determined by Student’s t test; ***p < 0.001, **p < 0.01, *p < 0.05 versus negative control (NC).

Figure 2

Overexpression of SNHG5 Promotes Breast Cancer Proliferation Both In Vitro and In Vivo (A) The SNHG5 expression in stably transfected T47D with a SNHG5 expression vector or empty vector as determined by qRT-PCR. (B–D) Cell growth inhibition was determined by MTT (B), colony formation (C), and EdU (D) assays in cells as in (A). (E) Representative photos and weights of tumors formed by cells as in (A). (F) Tumor volume of xenograft mice injected with cells as in (A) at the indicated times. (G) H&E staining or Ki-67 expression in primary tumors harvested from mice bearing the indicated xenograft tumors. The data were presented as the mean ± SD obtained from at least three independent experiments. Significance was determined by Student’s t test; ***p < 0.001, **p < 0.01, *p < 0.05 versus empty vector.

Figure 3

Knockdown of SNHG5 Leads to Cell-Cycle Arrest at the G1 Phase (A) The cell-cycle distribution of 231-siSNHG5 and 231-siControl cells by flow cytometry analysis. (B) The cell-cycle distribution of 231-siSNHG5 and 231-siControl cells by flow cytometry analysis. These cells synchronized at the G1/S transition by a double-thymidine block and release for the indicated times. (C) The expression of Cyclin B1 in cells treated in (B) was detected by western blotting. (D) The cell-cycle distribution of 231-siSNHG5 and 231-siControl cells by flow cytometry analysis. The cells were synchronized at mitotic pre-metaphase by a thymidine-nocodazole block and were released for the indicated times. (E) The expression of Cyclin D1 in cells treated in (D) was detected by western blotting. (F) EdU analysis of cells as in (D) after released for 12 h. The data were presented as the mean ± SD obtained from at least three independent experiments. Significance was determined by Student’s t test; **p < 0.01 versus NC.

Figure 4

SNHG5 Acts as a Sponge for miR-154-5p (A) Representative fluorescence in situ hybridization indicated subcellular location of SNHG5 in MDA-MB-231 cells (green). Nuclei were stained by DAPI (blue). (B) Relative SNHG5 expression levels in nuclear and cytoplasmic fractions of MDA-MB-231 cells. (C) The predicted binding of miR-154-5p with SNHG5 3ʹ UTR. (D) Dual-luciferase reporter assay was performed to validate the interaction between miR-154-5p and SNHG5. (E) An RNA immunoprecipitation analysis of endogenous AGO2 binding to RNA in MDA-MB-231 cells; IgG was used as the control. SNHG5 and miR-154-5p levels were determined by qRT-PCR and presented as fold enrichment in AGO2 relative to input. (F) miR-154-5p expression in SNHG5-depleted MDA-MB-231 cells as determined by qRT-PCR. (G) miR-154-5p expression in stably transfected T47D with a SNHG5 expression vector or empty vector as determined by qRT-PCR. (H) miR-154-5p expression in T47D-SNHG5 cells transfected with miR-154-5p mimics as determined by qRT-PCR. (I–K) Cell growth inhibition was determined by MTT (I), colony formation (J), and EdU (K) assays in cells as in (H). The data were presented as the mean ± SD obtained from at least three independent experiments. Significance was determined by Student’s t test; ***p < 0.001, **p < 0.01 versus NC or empty vector. Mut, contains 7-base mutation at the miR-154-5p target seed region.

Figure 5

PCNA Is a Direct Target of miR-154-5p (A) The potential target genes of miR-154-5p analyzed by starBase v3.0. (B) The predicted binding of miR-154-5p with PCNA 3ʹ UTR. (C) Dual-luciferase reporter assay was performed to validate the miR-154-5p target, PCNA. (D) The expression of miR-154-5p and PCNA in miR-154-5p-overexpressed MDA-MB-231 and control cells determined by qRT-PCR. (E) The expression of miR-154-5p and PCNA in miR-154-5p-depleted T47D and control cells determined by qRT-PCR. (F) The expression of miR-154-5p and PCNA in indicated cells as determined by western blotting. The data were presented as the mean ± SD obtained from at least three independent experiments. Significance was determined by Student’s t test; ***p < 0.001, **p < 0.01 versus NC. Mut, contains 6-base mutation at the miR-154-5p target seed region.

Figure 6

SNHG5 Promotes Breast Cancer Proliferation by Regulating PCNA (A) SNHG5 expression in indicated T47D cells as determined by qRT-PCR. (B) The expression of PCNA, Cyclin D1, and p16 in indicated T47D cells as determined by western blotting. (C–E) Cell growth inhibition was determined by MTT (C), colony formation (D), and EdU (E) assays in indicated T47D cells. (F) The cell-cycle distribution of cells as in (A) by flow cytometry analysis. (G) The expression of PCNA in T47D-SNHG5 and T47D-vector xenograft tumors was examined by immunohistochemical staining. The data were presented as the mean ± SD obtained from at least three independent experiments. Significance was determined by Student’s t test; ***p < 0.001, **p < 0.01 versus empty vector.

Figure 7

SNHG5 Predicts Poor Prognosis and Inversely Correlates with miR-154-5p in Clinical Samples (A) SNHG5 expression in primary breast cancer tissues and paired normal breast tissues as determined by qRT-PCR. (B) miR-154-5p expression in primary breast cancer tissues and paired normal breast tissues as determined by qRT-PCR. (C) Kaplan-Meier analysis of the overall survival in patients with different SNHG5 expression levels by KM-plotter. (D) Kaplan-Meier analysis of the overall survival in patients with different miR-154-5p expression levels by KM-plotter. (E) The relationship between SNHG5 and miR-154-5p expression as determined by qRT-PCR. (F) A model for the role of SNHG5 in breast cancer proliferation.