Triple-negative breast cancer (TNBC) is characterized by fast progression with high potential for metastasis, and poor prognosis. The dysregulation of microRNAs (miRNAs) occurring in the initiation or progression of cancers often leads to aberrant gene expression. The aim of the present study was to explore the function of miR-126 in TNBC cells. Expression levels of miR-126-3p were determined by quantitative real-time PCR. Then, the effects of miR-126-3p on migration, proliferation, invasion, and angiogenesis were assessed through in vitro experiments including Cell Counting Kit-8, colony formation, Transwell invasion and vasculogenic mimicry formation assays. One of the target genes for miR-126-3p predicted by TargetScan was confirmed by luciferase activity assay. Results indicated that miR-126-3p expression was reduced in TNBC cell lines. Functional assays revealed that miR-126-3p overexpression inhibited cell proliferation, migration, invasion, colony formation capacity and vasculogenesis by 1.2-, 1.8-, 2.3-, 2.0-and 3.3-fold, respectively, compared to the miRNA-negative control group of MDA-MB-231 cells (P<0.001, respectively). In addition, the regulator of G-protein signaling 3 (RGS3) was hypothesized and validated as a direct target of miR-126-3p in TNBC. The proliferation, migration, invasion, colony formation capacity and vasculogenesis of MDA-MB-231 cells were significantly increased by 1.4-, 2.0-, 1.8-, 1.4-and 3.2-fold, respectively, in cells transfected with pcDNA3.0-RGS3 compared to pcDNA3.0-negative control groups (P<0.001, respectively). The influence of miR-126-3p expression was reversed by RGS3 restoration. Collectively, the present study revealed that miR-126-3p plays a role as a tumor suppressor in regulating TNBC cell activities by targeting RGS3, indicating that the miR-126-3p/RGS3 axis may be a potential treatment target.

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Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.

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Background: Breast cancer (BC), the leading cause of cancer-related deaths among women, remains a serious threat to human health worldwide. The biological function and prognostic value of disulfidptosis as a novel strategy for BC treatment via induction of cell death remain unknown.Methods: Gene mutations and copy number variations (CNVs) in 10 disulfidptosis genes were evaluated. Differential expression, prognostic, and univariate Cox analyses were then performed for 10 genes, and BC-specific disulfidptosis-related genes (DRGs) were screened. Unsupervised consensus clustering was used to identify different expression clusters. In addition, we screened the differentially expressed genes (DEGs) among different expression clusters and identified hub genes. Moreover, the expression level of DEGs was detected by RT-qPCR in cellular level. Finally, we used the least absolute shrinkage and selection operator (LASSO) regression algorithm to establish a prognostic feature based on DEGs, and verified the accuracy and sensitivity of its prediction through prognostic analysis and subject operating characteristic curve analysis. The correlation of the signature with the tumor immune microenvironment and tumor stemness was analyzed.Results: Disulfidptosis genes showed significant CNVs. Two clusters were identified based on three DRGs (DNUFS1, LRPPRC, SLC7A11). Cluster A was found to be associated with better survival outcomes(p &lt; 0.05) and higher levels of immune cell infiltration(p &lt; 0.05). A prognostic signature of four disulfidptosis-related DEGs (KIF21A, APOD, ALOX15B, ELOVL2) was developed by LASSO regression analysis. The signature showed a good prediction ability. In addition, the prognostic signature in this study were strongly related to the tumor microenvironment (TME), tumor immune cell infiltration, tumor mutation burden (TMB), tumor stemness, and drug sensitivity.Conclusion: The prognostic signature we constructed based on disulfidptosis-DEGs is a good predictor of prognosis in patients with BC. This prognostic signature is closely related to TME, and its potential correlation provides clues for further studies.

Introduction Recently, the significant regulatory effects of lncRNAs on the oncogenesis and growth of tumor have been demonstrated by an increasing number of research projects. A previous study showed that LL22NC03-N64E9.1 could promote the development of colorectal cancer, especially via enhanced cell proliferation. Similarly, this lncRNA should have comparable functions in breast cancer (BC), which requires in-depth investigation. Therefore, this study was designed to explore the correlation of LL22NC03-N64E9.1 with BC. Methods qRT-PCR was used to assess the relative expression of LL22NC03-N64E9.1 in BC tissues. Cell viability examination and colony formation experiments were performed to investigate the role of LL22NC03-N64E9.1 in BC cell’s proliferation. Transwell assays were used to explore the effects of LL22NC03-N64E9.1 on BC cell’s migration. RNA immunoprecipitation, chromosome immunoprecipitation assay and rescue experiments were performed to analyze the association of LL22NC03-N64E9.1 with target proteins and genes in BC cells. Results We identified that LL22NC03-N64E9.1 is an oncogene, upregulated in BC, which was verified in a cohort of 48 pairs of BC tissues. Based on the loss-of-function experiments, silencing LL22NC03-N64E9.1 expression significantly inhibited malignancy progression. In terms of the mechanism, LL22NC03-N64E9.1 acted on the enhancer of zeste homolog 2 ( EZH2 ) by direct binding, which promoted BC cell growth. Furthermore, in the promoters of KLF2 , the trimethylation of H3K27 could be regulated by LL22NC03-N64E9.1 as the mediator. Conclusion Relying on the LL22NC03-N64E9.1 / EZH2 / KLF2 pathway, the lncRNA LL22NC03-N64E9.1 was significantly associated with BC development and could, therefore, be a potential therapeutic target to block BC growth.