Bioinformatic Identification of miR-622 Key Target Genes and Experimental Validation of the miR-622-RNF8 Axis in Breast Cancer

Abstract

Breast cancer is the leading cause of cancer-associated deaths among females. In recent decades, microRNAs (miRNAs), a type of short non-coding RNA that regulates gene expression at the post-transcription level, have been reported to participate in the regulation of many hub genes associated with tumorigenesis, tumor progression, and metastasis. However, the precise mechanism by which miRNAs regulate breast cancer metastasis remains poorly discussed, which limits the opportunity for the development of novel, effective therapeutic targets. Here, we aimed to determine the miR-622-related principal regulatory mechanism in cancer. First, we found that miR-622 was significantly related to a poor prognosis in various cancers. By utilizing an integrated miRNA prediction process, we identified 77 promising targets and constructed a protein-protein interaction network. Furthermore, enrichment analyses, including GO and KEGG pathway analyses, were performed to determine the potential function of miR-622, which revealed regulation networks and potential functions of miR-622. Then, we identified a key cluster comprised of six hub genes in the protein-protein interaction network. These genes were further chosen for pan-cancer expression, prognostic and predictive marker analyses based on the TCGA and GEO datasets to mine the potential clinical values of these hub genes. To further validate our bioinformatic results, the regulatory axis of miR-622 and RNF8, one of the hub genes recently reported to promote breast cancer cell EMT process and breast cancer metastasis, was selected as in vitro proof of concept. In vitro, we demonstrated the direct regulation of RNF8 by miR-622 and found that the predicted miR-622-RNF8 axis could regulate RNF8-induced epithelial-mesenchymal transition, cell migration, and cell viability. These results were further demonstrated with rescue experiments. We established a closed-loop miRNA-target-phenotype research model that integrated the bioinformatic analysis of the miRNA target genes and experimental validation of the identified key miRNA-target-phenotype axis. We not only identified the hub target genes of miR-622 in silico but also revealed the regulatory mechanism of miR-622 in breast cancer cell EMT process, viability, and migration in vitro for the first time.

miR-622 is a novel potential biomarker of breast carcinoma and impairs motility of breast cancer cells through targeting NUAK1 kinase

“…Recently, Liu reported, from in silico analysis, that in all breast cancer patients of the TCGA dataset, survival rate was negatively correlated with high level of miR-622. 21 However, we found that by analysing specific subtypes of breast cancers, high levels of miR-622 positively correlated with overall survival in the TNBC, ER-neg, HER2-pos and luminal B subtypes. These different results could be explained by the fact that breast cancer is an extremely heterogenous disease.…”

“… 20 In glioma miR-622 targets YAP 11 and in breast cancer targets RNF8. 21 Finally, a recent paper reported that in renal cell carcinoma, miR-622 suppressed cancer progression by targeting CCL18. 22 …”

Comprehensive analysis of hsa-miR-654-5p’s tumor-suppressing functions

“…MiRNAs are a class of endogenous noncoding small RNAs that play an important role in post-transcriptional gene silencing via partially or completely complementary binding to target mRNAs in their 3’ UTRs 23,36 . Currently, multiple researchers have confirmed that miRNAs are involved in the mechanisms of most biological processes including excessive growth, resistance to apoptosis, angiogenesis, invasion, and metastasis 35,37 .…”

miRNA Targets: From Prediction Tools to Experimental Validation

“…The intersection of the results obtained from these tools allowed identifying 77 promising targets and constructing a protein-protein interaction network. The results were experimentally validated in vitro, revealing the regulatory mechanisms of miR-622 in breast cancer cells [ 61 ].…”