Background Abnormal DNA methylation of tumor suppressor gene promoter has been found in breast cancer. Therefore, the current study set out to explore how DNA methyltransferase 1 (DNMT1) affects breast cancer through mediating miR-497/GPRC5A axis. Methods After loss and gain-of-function approaches were conducted in MCF-7/ADR and MCF-7 cells, cell viability, IC50 value, invasion, migration and apoptosis were measured, respectively. In addition, drug resistance, metastasis and apoptosis-related protein expression were examined using immunoblotting. ChIP and dual-luciferase reporter gene assays were carried out to validate relationship among DNMT1, miR-497, and GPRC5RA. Subcutaneous xenograft tumor model in nude mice was established to detect effects of DNMT1 on growth and metastasis of breast cancer in vivo. Results It was found that DNMT1 was notably increased, while miR-497 was poorly-expressed in breast cancer. Highly-expressed DNMT1 could promote chemotherapy resistance and metastasis of breast cancer. Meanwhile, DNMT1 modified methylation of CpG island in miR-497 promoter region, thereby repressing miR-497 level. In addition, miR-497 targeted GPRC5A expression to curb chemotherapy resistance and metastasis of breast cancer cells. Lastly, in vivo experiments showed that knockdown of DNMT1 could suppress breast cancer growth and metastasis. Conclusions Collectively, our findings indicated that DNMT1 may inhibit miR-497 and boost the expression of GPRC5A through methylation, thus augmenting breast cancer chemotherapy resistance and metastasis, which provides novel mechanistic insight into the unrecognized roles of DNMT1 in breast cancer.

No abstract

Breast cancer (BC) is one of the most common malignant tumors among women worldwide. MicroRNAs (miRs) may be involved in several types of human cancer, including gastric, liver, lung and breast cancer. The aim of the present study was to investigate the effect of miR-1297 on MDA-MB-231 cell epithelial-mesenchymal transition (EMT) and proliferation, and the underlying molecular mechanisms. MDA-MB-231 cells were transfected with miR-1297 inhibitor or inhibitor control for 48 h. Subsequently, MTT and flow cytometry assays indicated that miR-1297 inhibitor significantly decreased cell proliferation and induced apoptosis compared with the inhibitor control group. In addition, reverse transcription-quantitative PCR and western blotting suggested that miR-1297 inhibitor suppressed EMT in MDA-MB-231 cells compared with the inhibitor control group. TargetScan bioinformatics analysis and a dual-luciferase reporter gene assay were performed, which predicted that miR-1297 directly targeted fatty acid 2-hydroxylase (FA2H). Furthermore, MDA-MB-231 cells were transfected with control-plasmid or FA2H-plasmid for 48 h. The results demonstrated that FA2H overexpression decreased MDA-MB-231 cell proliferation and increased apoptosis compared with the control-plasmid group. Additionally, FA2H-plasmid increased E-cadherin expression levels, and reduced N-cadherin and matrix metalloproteinase 9 expression levels at both the protein and mRNA level compared with control-plasmid. Finally, MDA-MB-231 cells were transfected with control-small interfering (si) RNA, FA2H-siRNA, inhibitor control, miR-1297 inhibitor, miR-1297 inhibitor + control siRNA or miR-1297 inhibitor + FA2H-siRNA, and the results suggested that the biological effects of miR-1297 inhibitor were reversed by co-transfection with FA2H siRNA. In conclusion, the present study indicated that miR-1297/FA2H might serve as a novel potential biomarker and therapeutic target for BC.

No abstract

Background Histidine triad nucleotide-binding (HINT) protein belongs to the histidine triad proteins family. Recent studies have shown that HINT1 and HINT2 play pivotal roles in cancer growth. However, the function of HINT3 in cancers, including breast cancer (BRCA) remains to be determined. In the present study, we investigated the role of HINT3 in BRCA. Methods The clinical relevance of HINT3 in BRCA was analyzed using the datasets from The Cancer Genome Atlas. HINT3 was over-expressed and knocked down using lentivirus system. qRT-PCR and Western blot assays were performed to detect mRNA and protein expression. CCK-8 and colony formation assays were used to assess cell proliferation. Migration was analyzed using Transwell assay. Luciferase reporter activity assay was performed using pGL3.Basic/TK system. Xenogrfted tumorigenesis was performed to evaluate the effect of HINT3 on tumor development. Results HINT3 was down-regulated in BRCA tissues based on TCGA analysis and our qPCR analysis. TCGA database also showed that HINT3 transcript was much lower in BRCA tissues with higher stage. In vitro, HINT3 knockdown promoted the cell proliferation, colony growth and EDU cooperation in MCF7 and MDA-MB-231 cells. Oppositely, HINT3 overexpression suppressed the DNA synthesis and proliferation in both cells. In vivo, HINT3 ectopic expression blunted the xenografted tumorigenesis of MDA-MB-231 cells. Furthermore, HINT3 silencing or overexpression enhanced and inhibited the migration capacity of MCF7 and MDA-MB-231 cells, respectively. Lastly, HINT3 upregulated PTEN at transcription level, which resulted in inactivation of AKT/mTOR signaling in vitro and in vivo. Conclusions Taken together, HINT3 inhibits PTEN/AKT/mTOR signaling pathway and suppresses the proliferation, growth, migration and tumor development of BRCA cells.