Lysophosphatidic acid receptor 6 regulated by miR-27a-3p attenuates tumor proliferation in breast cancer

Abstract

Purpose: Lysophosphatidic acid (LPA) is a bioactive molecule which participates in many physical and pathological processes. Although LPA receptor 6 (LPAR6), the last identified LPA receptor, has been reported to have diverse effects in multiple cancers, including breast cancer, its effects and functioning mechanisms are not fully known.

Methods: Multiple public databases were used to investigate the mRNA expression of LPAR6, its prognostic value, and potential mechanisms in breast cancer. Western blotting was performed to validate the differential expression of LPAR6 in breast cancer tissues and their adjacent tissues. Furthermore, in vitro experiments were used to explore the effects of LPAR6 on breast cancer. Additionally, TargetScan and miRWalk were used to identify potential upstream regulating miRNAs and validated the relationship between miR-27a-3p and LPAR6 via real-time polymerase chain reaction and an in vitro rescue assay.

Results: LPAR6 was significantly downregulated in breast cancer at transcriptional and translational levels. Decreased LPAR6 expression in breast cancer is significantly correlated with poor overall survival, disease-free survival, and distal metastasis-free survival, particularly for hormone receptor-positive patients, regardless of lymph node metastatic status. In vitro gain and loss-of-function assays indicated that LPAR6 attenuated breast cancer cell proliferation. The analyses of TCGA and METABRIC datasets revealed that LPAR6 may regulate the cell cycle signal pathway. Furthermore, the expression of LPAR6 could be positively regulated by miR-27a-3p. The knockdown of miR-27a-3p increased cell proliferation, and ectopic expression of LPAR6 could partly rescue this phenotype.

Conclusion: LPAR6 acts as a tumor suppressor in breast cancer and is positively regulated by miR-27a-3p.

Keywords: Breast cancer; Cell proliferation; Lysophosphatidic acid receptor 6; miR-27a-3p.

Fig. 1

LPAR6 is downregulated in breast cancer, and decreased LPAR6 expression is correlated with poor clinicopathological features. a Different LPAR6 mRNA expression levels were determined from pan-cancer data (data from TCGA). b Differential LPAR6 mRNA expression in normal tissue and tumors from METABRIC dataset. ****p < 0.0001 (t test). c LPAR6 mRNA expression in normal tissues and tissues of subtypes of breast cancer (UALCAN). ****p < 0.0001 (t test). d LPAR6 mRNA expression in ER-positive and -negative breast cancer. **p < 0.01 (t test). e LPAR6 mRNA expression in HER2-positive and -negative breast cancer. ns: not significant (t test). f LPAR6 mRNA expression in three pathological grades of breast cancer. *p < 0.05, ****p < 0.0001 (Kruskal–Wallis test). g LPAR6 mRNA expression in clinical stages of breast cancer. h LPAR6 expression in tissues of four subtypes of breast cancer assessed using western blotting. GAPDH was used as an internal control. Abbreviations: ACC adrenocortical carcinoma, BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL cholangiocarcinoma, COAD colon adenocarcinoma, ER estrogen receptor, ESCA esophageal carcinoma, GAPDH glyceraldehyde-3-phosphate dehydrogenase, GBM glioblastoma multiforme, HER2 human epidermal growth factor receptor 2, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, LAML acute myeloid leukemia, LGG brain lower grade glioma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, METABRIC molecular taxonomy of breast cancer international consortium, OV ovarian serous cystadenocarcinoma, P para-cancer tissues, PAAD pancreatic adenocarcinoma, PRAD prostate adenocarcinoma, READ rectum adenocarcinoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, T paired tumor tissues, TCGA The Cancer Genome Atlas, TGCT testicular germ cell tumors, THCA thyroid carcinoma, TNBC triple-negative breast cancer, UCEC uterine corpus endometrial carcinoma, UCS uterine carcinosarcoma

Fig. 2

Decreased LPAR6 expression in breast cancer is significantly correlated with poor survival especially for hormone receptor-positive (HR +) patients. a–c Decreased LPAR6 expression significantly predicted poor overall survival (OS), disease-free survival (DFS), and distal metastasis-free survival (DMFS) in all patients. d–f Decreased LPAR6 expression significantly predicted poor OS, DFS, and DMFS in (HR +) patients. g–i Decreased LPAR6 expression could not predict OS and DMFS in HR- patients well but could predict DFS. j–o Decreased LPAR6 expression significantly predicted poor OS, DFS, and DMFS in patients with positive or negative lymph node metastasis

Fig. 3

LPAR6 inhibits breast cancer cell proliferation in vitro. a Efficient knockdown (KD) of LPAR6 with siLPAR6 in MCF-7 cells. *p < 0.05; ****p < 0.001; ns not significant (t test). b Efficient overexpression (OE) of LPAR6 with adenovirus in SK-BR-3 cells. ****p < 0.001 (t test). c and d Cell counting kit-8 assay was performed in MCF-7 and SK-BR-3 cells. **p < 0.01, ****p < 0.0001 (t test). e and f Plate colony formation assay was performed in MCF-7 and SK-BR-3 cells. *p < 0.05; ***p < 0.001 (t test)

Fig. 4

TCGA and METABRIC data analyses show that LPAR6 may be involved in the cell cycle arrest pathway. a and b GSEA analyses using TCGA and METABRIC datasets (red word indicating the most enriched pathway in both datasets). c Visualization of LPAR6 and RB1 genomic location using UCSC genome browser. d and e Pearson correlation between LPAR6 and RB1 expression in normal tissues and tumor groups of TCGA and METABRIC datasets, respectively. f–m Pearson correlation between LPAR6 expression and expression of E2F family members in tumors of TCGA dataset

Fig. 5

LPAR6 is positively regulated by miR-27a-3p in breast cancer. a Predicted binding site of miR-27a-3p on LPAR6 3′ untranslated region (3′ UTR) via TargetScan tool. The purple square indicates miR-27a-3p. b Pearson correlation of LPAR6 and miR-27a-3p precursor MIR27A expression. Data were from TCGA dataset. c Pearson correlation of LPAR6 and mature miR-27a-3p expression. Data were from TCGA dataset. d miR-27a-3p expression in a healthy mammary epithelial cell line (MCF-10A) and breast cancer cell lines. **p < 0.01, ****p < 0.0001 (compared to MCF-10A, t test). e Validation of miR-27a-3p manipulation with miRNA mimics and inhibitor. ****p < 0.0001 (t test). f LPAR6 mRNA level was regulated by miR-27a-3p expression. **p < 0.01 (t test). g Cell counting kit-8 assay showed deregulation of miR-27a-3p can increase cell proliferation, and LPAR6 ectopic expression can partly rescue this phenotype. **p < 0.01, ****p < 0.0001 (compared to negative control, t test)