谷氨酸代谢型受体 4（GRM4）可抑制乳腺癌细胞的增殖、迁移和侵袭，其表达受 miR-328-3p 和 miR-370-3p 的调控。
Glutamate metabotropic receptor 4 (GRM4) inhibits cell proliferation, migration and invasion in breast cancer and is regulated by miR-328-3p and miR-370-3p.
机构信息
Department of Laboratory Medicine, General Hospital of Southern Theatre Command of PLA, Guangzhou, 510010, China.
Department of Laboratory Medicine, Dermatology Hospital, Southern Medical University, Guangzhou, China.
出版信息
BMC Cancer. 2019 Sep 6;19(1):891. doi: 10.1186/s12885-019-6068-4.
BACKGROUND
Glutamate metabotropic receptors (GRM) play a variety of roles in neuronal cells. However, their clinical significance and biological functions in breast cancer remain unknown.
METHODS
RNA sequencing data of breast cancer was obtained from the TCGA dataset (v2) and mined for the expression profiles of GRM family according to cancer subtypes. mRNA expression of GRM family in breast cancer tissues and para-cancerous tissue samples as well as breast cancer cell lines were measured by qPCR. The effects of over- and under-expression of GRM4 on cell capabilities to survive, migrate and invade were determined by colony formation, transwell migration and invasion assays. To explore the upstream regulation pattern of GRM4, miRNAs that target GRM4 were predicted and validated by dual luciferase reporter assay. In addition, the mRNA and protein expression of GRM4 regulated by these miRNAs were further measured by qPCR and western blot assay.
RESULTS
GRM4 was the only GRM member that expressed in breast cancer tissues. Ectopic expression of GRM4 was correlated with better prognosis of breast cancer patients. Overexpression of GRM4 could significantly inhibit cell proliferation, migration and invasion capacity in MDA-MB-231, while knockdown of GRM4 could promote these processes. miR-328-3p and miR-370-3p were predicted to regulate the expression of GRM4 and dual luciferase reporter assay demonstrated that miR-328-3p and miR-370-3p directly bound to the 3' UTR of GRM4 and mutations on the binding regions on GRM4 significantly decreased the luciferase activity. qPCR demonstrated that expression of miR-328-3p and miR-370-3p was significantly decreased in breast cancer tissues and cells compared with that in control samples. However, there were no correlations between the expression of miR-328-3p and GRM4, as well as the expression of miR-370-3p and GRM4. Moreover, overexpression of miR-328-3p and miR-370-3p counteracted the inhibitory effect of GRM4-induced cell proliferation, migration and invasion.
CONCLUSIONS
Our results suggest that GRM4 might be a tumor suppressor gene in breast cancer under the direct regulation of miR-328-3p and miR-370-3p.
背景
谷氨酸代谢型受体（GRM）在神经元细胞中发挥多种作用。然而，它们在乳腺癌中的临床意义和生物学功能尚不清楚。
方法
从 TCGA 数据集（v2）中获取乳腺癌的 RNA 测序数据，并根据癌症亚型挖掘 GRM 家族的表达谱。通过 qPCR 测量乳腺癌组织和癌旁组织样本以及乳腺癌细胞系中 GRM 家族的 mRNA 表达。通过集落形成、Transwell 迁移和侵袭实验确定 GRM4 过表达和低表达对细胞存活、迁移和侵袭能力的影响。通过双荧光素酶报告实验预测和验证靶向 GRM4 的 miRNAs，以探索 GRM4 的上游调控模式。此外，通过 qPCR 和 Western blot 实验进一步测量这些 miRNA 调控的 GRM4 的 mRNA 和蛋白表达。
结果
GRM4 是唯一在乳腺癌组织中表达的 GRM 成员。GRM4 的异位表达与乳腺癌患者的更好预后相关。GRM4 的过表达可显著抑制 MDA-MB-231 中的细胞增殖、迁移和侵袭能力，而 GRM4 的敲低可促进这些过程。miR-328-3p 和 miR-370-3p 被预测可调节 GRM4 的表达，双荧光素酶报告实验表明 miR-328-3p 和 miR-370-3p 可直接结合到 GRM4 的 3'UTR 上，而 GRM4 结合区域的突变则显著降低了荧光素酶活性。qPCR 表明，与对照样本相比，miR-328-3p 和 miR-370-3p 在乳腺癌组织和细胞中的表达明显降低。然而，miR-328-3p 和 GRM4 的表达之间以及 miR-370-3p 和 GRM4 的表达之间没有相关性。此外，miR-328-3p 和 miR-370-3p 的过表达可拮抗 GRM4 诱导的细胞增殖、迁移和侵袭的抑制作用。
结论
我们的结果表明，在 miR-328-3p 和 miR-370-3p 的直接调控下，GRM4 可能是乳腺癌中的肿瘤抑制基因。
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