Impact of novel oncogenic pathways regulated by antitumor miR‐451a in renal cell carcinoma

Abstract

Recent analyses of our microRNA (miRNA) expression signatures obtained from several types of cancer have provided novel information on their molecular pathology. In renal cell carcinoma (RCC), expression of microRNA‐451a (miR‐451a) was significantly downregulated in patient specimens and low expression of miR‐451a was significantly associated with poor prognosis of RCC patients (P = .00305) based on data in The Cancer Genome Atlas. The aims of the present study were to investigate the antitumor roles of miR‐451a and to identify novel oncogenic networks it regulated in RCC cells. Ectopic expression of miR‐451a significantly inhibited cancer cell migration and invasion by RCC cell lines, suggesting that miR‐451a had antitumor roles. To identify oncogenes regulated by miR‐451a in RCC cells, we analyzed genome‐wide gene expression data and examined information in in silico databases. A total of 16 oncogenes and were found to be possible targets of miR‐451a regulation. Interestingly, high expression of 9 genes (PMM2,CRELD2,CLEC2D,SPC25,BST2,EVL,TBX15,DPYSL3, and NAMPT) was significantly associated with poor prognosis. In this study, we focused on phosphomannomutase 2 (PMM2), which was the most strongly associated with prognosis. Overexpression of PMM2 was detected in clinical specimens and Spearman's rank test indicated a negative correlation between the expression levels of miR‐451a and PMM2 (P = .0409). Knockdown of PMM2 in RCC cells inhibited cancer cell migration and invasion, indicating overexpression of PMM2 could promote malignancy. Analytic strategies based on antitumor miRNAs is an effective tool for identification of novel pathways of cancer.

Molecular pathogenesis of renal cell carcinoma: Impact of the anti‐tumor miR‐29 family on gene regulation

“…In the present study, we used two human RCC cell lines, 786‐O and A498. Both cell lines were obtained from the American Type Culture Collection . The cells stocks were immediately made and were used within 3 months.…”

“…The following molecular reagents were used: mature miRNAs, Pre‐miR miRNA precursors ( hsa‐miR‐29a‐3p , assay ID: ; hsa‐miR‐29b‐3p , assay ID: ; hsa‐miR‐29c‐3p , assay ID: MC10518; Applied Biosystems, Foster City, CA, USA), negative control miRNA (assay ID: ; Applied Biosystems) and siRNA (Stealth Select RNAi siRNA; si‐ SERPINH1 P/N: and ; Invitrogen, Carlsbad, CA, USA). The transfection procedures were described previously …”

“…Tissue samples were incubated overnight at 4°C with anti‐SERPINH1 antibodies diluted 1:50 (sc‐5293; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The procedures for western blot analysis were described …”

“…Anti‐GAPDH antibodies (1:1000 dilution; ; Abcam, Cambridge, UK) were used as an internal control. The procedures for western blot analysis have been previously described …”

“…Renilla luciferase intensity ( Renilla /firefly) was used as normalized data. The luciferase reporter assay was carried out as previously described …”