Hewen Wu1,2, Junli Li1, En'en Guo1, Suxia Luo2, Guohui Wang2,3. 1. Department of Infectious Diseases, Henan Province People's Hospital, Zhengzhou, China. 2. Department of medicine, the Affiliated Tumor Hospital, Zhengzhou University, Zhengzhou, China. 3. Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA.
Abstract
BACKGROUND/AIMS: Endoplasmic reticulum lipid raft-associated 2 (ERLIN2) is reported to be overexpressed in human breast cancer cells and plays an important role in cell proliferation. MicroRNAs (miRNAs) act as post-transcriptional regulators of gene expression and are involved in the development of multiple malignancies, including breast cancer. However, the molecular mechanism of the aberrant ERLIN2 expression in human breast cancer remains poorly understood. METHODS: MiR-410 expression level was analyzed using Real-time PCR, and ERLIN2 expression was analyzed using Western blot, Real-time PCR and immunohistochemical staining. The effect of miR-410 on ERLIN2 3'UTR intensity was performed using a luciferase assay. Cell proliferation was analyzed using CCK-8 and colony formation assay, together with an Annexin V-PE/7-AAD kit for cell apoptosis assay. Cell migration and invasion was detected using a Transwell migration and invasion assay. Methylation specific PCR was used to examine whether miR-410 promoter was demethylated. RESULTS: In this study, we validated that ERLIN2 was a direct target of miR-410 and miR-410 suppressed ERLIN2 expression at the post-transcriptional level. Importantly, the regulation of ERLIN2 by miR-410 was estrogen receptor (ER) dependent. Functional studies demonstrated that miR-410 inhibited breast cancer cell proliferation, migration and invasion, but promoted cell apoptosis. However, inhibition of miR-410 resulted in opposite effects. A xenograft nude mouse model further confirmed that miR-410 suppressed breast tumor growth. In addition, miR-410 modulated the expression levels of epithelial-mesenchymal transition (EMT)-related genes. ERLIN2 knockdown suppressed cell proliferation, migration and invasion, as well as EMT. ERLIN2 overexpression can restore the cell proliferation, migration and invasion that were inhibited by miR-410. Furthermore, our data demonstrated that miR-410 inhibition suppressed the expression of endoplasmic reticulum-stress (ERS)-related genes, while ERLIN2 knockdown abrogated the effects of miR-410 inhibitor. Finally, we showed that miR-410 was downregulated in human ER-positive breast cancer tissues, inversely correlated with ERLIN2. We further demonstrated the downregulation of miR-410 in breast cancer might be due to the hypermethylation of its promoter. CONCLUSIONS: Our study indicates that miR-410 suppresses cell growth, migration and invasion by directly downregulating ERLIN2 in ER positive breast cancer, acting as a tumor suppressor. Our study also suggests that miR-410 may serve as a potential therapeutic target for patients with ER positive breast cancer.
BACKGROUND/AIMS: Endoplasmic reticulum lipid raft-associated 2 (ERLIN2) is reported to be overexpressed in humanbreast cancer cells and plays an important role in cell proliferation. MicroRNAs (miRNAs) act as post-transcriptional regulators of gene expression and are involved in the development of multiple malignancies, including breast cancer. However, the molecular mechanism of the aberrant ERLIN2 expression in humanbreast cancer remains poorly understood. METHODS:MiR-410 expression level was analyzed using Real-time PCR, and ERLIN2 expression was analyzed using Western blot, Real-time PCR and immunohistochemical staining. The effect of miR-410 on ERLIN2 3'UTR intensity was performed using a luciferase assay. Cell proliferation was analyzed using CCK-8 and colony formation assay, together with an Annexin V-PE/7-AAD kit for cell apoptosis assay. Cell migration and invasion was detected using a Transwell migration and invasion assay. Methylation specific PCR was used to examine whether miR-410 promoter was demethylated. RESULTS: In this study, we validated that ERLIN2 was a direct target of miR-410 and miR-410 suppressed ERLIN2 expression at the post-transcriptional level. Importantly, the regulation of ERLIN2 by miR-410 was estrogen receptor (ER) dependent. Functional studies demonstrated that miR-410 inhibited breast cancer cell proliferation, migration and invasion, but promoted cell apoptosis. However, inhibition of miR-410 resulted in opposite effects. A xenograft nude mouse model further confirmed that miR-410 suppressed breast tumor growth. In addition, miR-410 modulated the expression levels of epithelial-mesenchymal transition (EMT)-related genes. ERLIN2 knockdown suppressed cell proliferation, migration and invasion, as well as EMT. ERLIN2 overexpression can restore the cell proliferation, migration and invasion that were inhibited by miR-410. Furthermore, our data demonstrated that miR-410 inhibition suppressed the expression of endoplasmic reticulum-stress (ERS)-related genes, while ERLIN2 knockdown abrogated the effects of miR-410 inhibitor. Finally, we showed that miR-410 was downregulated in humanER-positive breast cancer tissues, inversely correlated with ERLIN2. We further demonstrated the downregulation of miR-410 in breast cancer might be due to the hypermethylation of its promoter. CONCLUSIONS: Our study indicates that miR-410 suppresses cell growth, migration and invasion by directly downregulating ERLIN2 in ER positive breast cancer, acting as a tumor suppressor. Our study also suggests that miR-410 may serve as a potential therapeutic target for patients with ER positive breast cancer.