DNA methyltransferase 1 inhibits microRNA-497 and elevates GPRC5A expression to promote chemotherapy resistance and metastasis in breast cancer.

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.

HINT3 suppresses AKT/mTOR signaling pathway activity during breast cancer tumorigenesis through PTEN transcriptional activation.

Histidine triad nucleotide­binding protein (HINT) belongs to the histidine triad protein family. Recent studies have demonstrated that HINT1 and HINT2 both play a pivotal role in cancer growth. However, the functions of HINT3 in various types of cancer, including breast cancer (BRCA), have not yet been fully elucidated. In the present study, the role of HINT3 in BRCA was investigated. Based on The Cancer Genome Atlas and reverse transcription­quantitative PCR analyses, HINT3 was found to be decreased in BRCA tissues. In vitro, HINT3 knockdown promoted the proliferation and colony formation of, and 5­ethynyl­2'­deoxyuridine incorporation in MCF­7 and MDA­MB­231 BRCA cells. By contrast, HINT3 overexpression suppressed DNA synthesis and the proliferation of both cell lines. Apoptosis was also found to be modulated by HINT3. In vivo, HINT3 ectopic expression attenuated the tumorigenesis of MDA­MB­231 and MCF­7 cells in a mouse tumor xenograft model. Furthermore, HINT3 silencing or overexpression also enhanced or inhibited, respectively, the migratory capacity of the MCF­7 and MDA­MB­231 cells. Finally, HINT3 upregulated phosphatase and tensin homolog (PTEN) at the transcriptional level, which resulted in the inactivation of AKT/mammalian target of rapamycin (mTOR) signaling both in vitro and in vivo. Taken together, the present study demonstrates that HINT3 inhibits the activation of the PTEN/AKT/mTOR signaling pathway, and suppresses the proliferation, growth, migration and tumor development of MCF­7 and MDA­MB­231 BRCA cells.

Knockdown of LINC00702 inhibits the growth and induces apoptosis of breast cancer through the Wnt/ß-catenin pathway.

Long non-coding RNAs (lncRNAs) are essential in many biological areas like cell growth and apoptosis. The role of recently discovered LINC00702 is yet to be explored. Therefore, we wanted to elucidate its role in breast cancer (BC) with bioinformatic and various methods. LINC00702 expression was predicted using bioinformatic analysis and confirmed by RT-qPCR. Furthermore, the impact of LINC00702 knockdown on BC progression was evaluated. High LINC00702 level could lead to a worse outcome in BC patients. Additionally, CCK-8, EdU,and Annexin V-APC7/AAD experiments showed that LINC00702 knockdown inhibited the growth of BT-474 and T-47D cells and promoted their apoptosis. Moreover, in vivo experiments showed that shLINC00702-2 significantly reduced tumor sizes and suppressed c-Myc and ß-catenin expressions. On the contrary, a rescue assay showed that HLY78, an activator of the Wnt/ß-catenin pathway, reversed the cell-inhibiting impact of LINC00702 knockdown. LINC00702 is an oncogenic lncRNA that promotes BC progression by stimulating the Wnt/ß-catenin pathway and downstream proteins, making it a promising target for further research on BC treatment.

Expression and promoter methylation status of OPCML and its functions in the inhibition of cell proliferation, migration, and invasion in breast cancer.

BACKGROUND: Opioid binding protein/cell adhesion molecule-like (OPCML) has been demonstrated to be a tumor suppressor gene, as it has been shown in previous studies to play a tumor-suppressive role in a variety of cancers. However, the role of OPCML in breast tumorigenesis remains unclear. METHODS: In this study, we analyzed OPCML expression in breast cancers and adjacent non-tumor tissue samples and examined its molecular function in the breast cancer-derived cell lines MDA-MB-231 and MCF7. RESULTS: We found that OPCML was downregulated in most breast cancer samples but that this protein was expressed in most adjacent non-tumor samples. The loss or downregulation of OPCML is associated with hypermethylation of its promoter. Methylation of the OPCML promoter was detected in all breast cancer cell lines and primary tumors but was not detected in surgical margin tissues and normal breast tissues. Furthermore, functional assays showed that ectopic OPCML expression could inhibit breast tumor cell proliferation in vivo and in vitro and further suppresses tumor cell migration and invasion. CONCLUSION: Our results show that OPCML exerts its tumor-suppressive functions in human breast cancer cells. Moreover, the promoter-specific hypermethylation of OPCML plays an important role in human breast cancer development.

MicroRNA-1297 downregulation inhibits breast cancer cell epithelial-mesenchymal transition and proliferation in a FA2H-dependent manner.

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.