Lnc RNA HOTAIR functions as a competing endogenous RNA to regulate HER2 expression by sponging miR-331-3p in gastric cancer

Abstract

Background: Accumulating evidence indicates that the long non-coding RNA HOTAIR plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of HOTAIR in gastric carcinogenesis remains largely unknown.

Methods: HOTAIR expression was measured in 78 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of HOTAIR on gastric cancer cells were studied by overexpression and RNA interference approaches in vitro and in vivo. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation (RIP). The positive HOTAIR/HER2 interaction was identified and verified by immunohistochemistry assay and bivariate correlation analysis.

Results: HOTAIR upregulation was associated with larger tumor size, advanced pathological stage and extensive metastasis, and also correlated with shorter overall survival of gastric cancer patients. Furthermore, HOTAIR overexpression promoted the proliferation, migration and invasion of gastric carcinoma cells, while HOTAIR depletion inhibited both cell invasion and cell viability, and induced growth arrest in vitro and in vivo. In particular, HOTAIR may act as a ceRNA, effectively becoming a sink for miR-331-3p, thereby modulating the derepression of HER2 and imposing an additional level of post-transcriptional regulation. Finally, the positive HOTAIR/HER2 correlation was significantly associated with advanced gastric cancers.

Conclusions: HOTAIR overexpression represents a biomarker of poor prognosis in gastric cancer, and may confer malignant phenotype to tumor cells. The ceRNA regulatory network involving HOTAIR and the positive interaction between HOTAIR and HER2 may contribute to a better understanding of gastric cancer pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics against this disease.

Figure 1

Relative HOTAIR expression in gastric cancer tissues and its clinical significance. (A) Relative expression of HOTAIR in gastric cancer tissues (n = 78) in comparison with corresponding non-tumor normal tissues (n = 78). HOTAIR expression was examined by qRT-PCR and normalized to GAPDH expression. Data was presented as fold-change in tumor tissues relative to normal tissues. (B) Examination of the correlation between HOTAIR expression and clinical pathological features showed that HOTAIR upregulation correlated with larger tumor size and advanced pathological stage. (C) HOTAIR expression was significantly higher in patients with distant metastasis than in those with non-distant metastasis. (D) Kaplan–Meier overall survival curves according to HOTAIR expression level. The overall survival of the High-HOTAIR group (n = 39: HOTAIR expression ratio ≥ median ratio) was significantly higher than that of Low-HOTAIR group (n = 39; HOTAIR expression ratio ≤ median ratio; P < 0.001, log-rank test). *P < 0.05, **P < 0.01.

Figure 2

The level of HOTAIR expression in gastric cancer cells and its effect on cell proliferation in vitro. (A) Results from qRT-PCR demonstrating HOTAIR expression levels of gastric cancer cell lines (MGC-803, SGC-7901, BGC-823, and AGS) compared with normal gastric epithelium cell line (GES-1), NSCLC cell line (SPC-A1) compared with normal human bronchial epithelial cell line (16HBE), and breast cancer cell lines MDA-MB-231 compared with MCF-7. (B) qRT-PCR analyses of HOTAIR expression level following treatment BGC-823 cells with siRNAs targeting HOTAIR and treatment SGC-7901 cells with pCDNA/HOTAIR vector. (C) MTT assay was performed to determine the proliferation of si-HOTAIR-transfected BGC-823 cells or pCDNA/HOTAIR-transfected SGC-7901 cells. Data represent the mean ± s.d. from three independent experiments. (D) Colony-forming growth assay was performed to determine the proliferation of si-HOTAIR transfected BGC-823 cells. Colonies were counted and captured. *P < 0.05 and **P < 0.01.

Figure 3

The effect of HOTAIR on gastric cancer cell apoptosis, migration and invasion in vitro. BGC-823 cells were transfected with si-HOTAIR or si-NC, and SGC-7901 cells were transfected with pCDNA/HOTAIR vector or empty vector control. (A) Hoechst staining assay of cell apoptosis; the percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was determined. (B) The apoptotic rates of cells were detected by flow cytometry. UL, necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. (C and D) Transwell assays were performed to investigate changes in cell migration and invasiveness. *P < 0.05 and **P < 0.01.

Figure 4

Effect of HOTAIR knockdown on tumor growth in vivo. (A) Tumor growth curves were measured after injection of BGC-823 cells transfected with sh-HOTAIR or pENTR vector. Tumor volume was calculated every 3 days. Data are presented as mean ± s.d. (n = 5). (B) Tumor weight. Values are means of tumor weight ± s.d. (C) qRT-PCR analysis of HOTAIR expression in tissues of resected tumors. (D) Tumors developed from BGC-823/sh-HOTAIR cells showed a lower level of PCNA protein than tumors developed from BGC-823/pENTR vector cells. Upper: H & E staining; Lower: immunostaining. *P < 0.05 and **P < 0.01.

Figure 5

HOTAIR is a target of miR-331-3P or miR-124 and controls miR-331-3p target. (A) The 11 putative miRNA binding sites in the HOTAIR sequence. The HOTAIR cDNA containing the putative miRNAs recognition sites was cloned downstream of the luciferase gene and named RLuc-HOTAIR. (B) Left: The luciferase reporter plasmid (RLuc-HOTAIR) was co-transfected into HEK-293 T cells with the 11 various miRNA-coding plasmids. Right: the luciferase reporter plasmid containing wild-type or mutant HOTAIR was co-transfected into HEK-293 T cells with miR-331-3p in parallel with an empty plasmid vector. Luciferase activity was determined using the dual luciferase assay and shown as the relative luciferase activity normalized to renilla activity. Histogram indicates the values of luciferase measured 48 h after transfection. (C) The 3’-UTR of HER2 was fused to the luciferase coding region (RLuc-HER2 3’-UTR) and transfected in HEK293T cells with miR-331-3p to confirm HER2 is the target of miR-331-3p. RLuc-HER2 3’-UTR and miR-331-3p constructs were co-transfected into HEK293T cells with plasmids expressing HOTAIR (pCDNA/HOTAIR) or with a control vector to verify the ceRNA activity of HOTAIR. rno-miRNA-344 was used as a negative control. Histogram indicates the values of luciferase measured 48 h after transfection. (D) RIP with mouse monoclonal anti-Ago2, preimmune IgG or 10% input from BGC 823-cell extracts. RNA levels in immunoprecipitates were determined by qRT-PCR. Top: levels of HOTAIR, miRNA331-3P and FOS RNA were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates. Bottom: relative RNA levels of U1 snRNA in SNRNP70 relative to IgG immunoprecipitates. Numbers are mean ± s.d. (n = 3). (E) Western blot analysis of HER2 protein level following treatment of BGC-823 cells with si-HOTAIR or pCDNA/HOTAIR, and SGC-7901 cells with pCDNA/HOTAIR. GAPDH was used as control. *P < 0.05, **P < 0.01 and N.S. not significant.

Figure 6

Positive correlation between HOTAIR and HER2 expression and the relevance to miR-331-3p/miR-124 expression levels. (A) Bivariate correlation analysis of the relationship between HOTAIR expression and miR-331-3p, or miR-124 level. (B) BGC-823 cells were transfected with plasmids encoding miR-331-3p or miR-124, and qRT-PCR was used to detect the miRNA levels compared with controls (miR-NC). (C) MTT assay and colony-forming growth assay were performed to determine the proliferation of miR-331-3p or miR-124 transfected BGC-823 cells. (D) Left: immunostaining of HER2 protein in advanced gastric cancer tissue samples. Upper: immunostaining of HER2 was negative in gastric cancer tissues with relatively low HOTAIR expression. Lower: immunostaining of HER2 was positive in gastric cancer tissues with relatively high HOTAIR expression. Right: bivariate correlation analysis of the relationship between HOTAIR and HER2 expression level. *P < 0.05, **P < 0.01.