Role of LncRNA MIR99AHG in breast cancer: Bioinformatic analysis and preliminary verification

This research was aimed to preliminarily explore the clinical roles and potential molecular mechanisms of MIR99AHG and its significant transcripts in breast cancer (BRCA).

Public databases were utilized to analyze the expression and prognostic roles of MIR99AHG and its transcripts. Relationships between MIR99AHG expression and immune cells infiltration were analyzed in Xiantao platform. In addition, co-expressed genes and interacting proteins of MIR99AHG were predicted. CancerSEA analyzed its relationship with functional states. Next, CNV status, DNA methylation, interacting transcription factors (TFs) and ceRNA network were analyzed to explore its possible mechanisms. Then, RNA ISH and FISH assays were used to detect its expression and location in BRCA tissues and cell lines, respectively. Finally, qRT-PCR was utilized to investigate MIR99AHG expression in cell lines.

Compared with the corresponding normal tissues, MIR99AHG expression levels were lower in all BRCA subtypes, and luminal B's was the lowest one. And MIR99AHG expression was negatively related to the tumor stage. In addition, 4 transcripts (ENST00000619222.4, ENST00000418813.6, ENST00000602901.5 and ENST00000453910.5) of MIR99AHG showed significant differences in the expression. Databases also suggested that the high MIR99AHG expression levels indicated good prognosis, especially in patients without lymph node metastasis. Xiantao found that MIR99AHG was positively related to 17 immune cells and negatively linked with 2 immune cells. CancerSEA analysis showed no relationships between MIR99AHG and functional states. From GEPIA and BCIP databases, 19 co-expressed genes were highly related to these four significant transcripts of MIR99AHG. StarBase, RNAct and HDOCK showed that several tumor-associated proteins, including U2AF65, hnRNPC, AEBP2, CHIC1 and so on, might interact with MIR99AHG. Genetically, BRCA had a higher proportion of MIR99AHG CNV loss than CNV gain, and the high level of DNA methylation indicated a good prognosis. Furthermore, 19 TFs were predicted to combine with the promoter of MIR99AHG. Then, we screened out 10 miRNAs potentially interacting with the significant transcripts of MIR99AHG, and five were significantly increased in breast tumors compared to normal tissues, including miR-194-5p, miR-320 b and so on, which could combine 14 mRNAs. Through ISH and FISH assays, we verified that MIR99AHG was down-regulated in BRCA samples and cell lines in comparison to non-tumor tissues and mammary epithelial cell line (MCF10A), and MIR99AHG was located both in cytoplasm and nucleus. qRT-PCR assay also showed the lower expression of MIR99AHG in breast cancer cells than that in MCF10A.

These results indicate that MIR99AHG can be a favorable prognostic indicator for BRCA. ENST00000619222.4, ENST00000418813.6, ENST00000602901.5 and ENST00000453910.5 are significant transcripts and their down-regulation may play crucial roles in the progression of BRCA.