Schematic view of miRNA biogenesis and functioning pathways. Genes encoding miRNA are transcribed into pri-miRNA by RNA polymerase II (RNA Pol II), the pri-miRNA is first processed by the type III RNA endonuclease, Drosha, into pre-miRNAs that are 60–70 nucleotides in length and have a stem-loop structure. The pre-miRNA is exported out of nucleus by exportin-5 and is further processed by another type III RNA endonuclease, Dicer, to generate a mature miRNA duplex 22-nucleotides in length. The sense strand of the miRNA duplex is then loaded into RISC, while the complementary strand, “star” form (*) of the miRNA duplex is degraded. The RISC complex regulates gene expression through inhibition of RNA translation or degradation of target mRNA by base-pairing the “seed region” of a miRNA to the 3′ UTR of target mRNA.

Strategies for identifying miR-210 targets. Schematic view of four major strategies utilized to identify miRNA targets. By immunoprecipitating a major component of the RISC complex, Argonaute 2 protein (AGO2), miRNP-IP pulls down the mRNA targets associated with miRNA within RISC. The mRNA targets are identified by next-generation sequencing or microarray. Because miRNA regulates target gene expression through the inhibition of RNA translation or degradation of target mRNA, two strategies can be used to identify miRNA targets, proteomics analysis or mRNA microarray profiling. When miRNA targets identified through experimental approaches are combined with computational prediction, which is mainly based on the homology of the short miRNA “seed region” to the 3′ UTR sequence of target gene, a list of candidate miRNA target genes are generated. 3′ UTR sequences of the putative targets are cloned into a reporter construct and a luciferase reporter assay is then performed to measure inhibition of protein production. Western blotting is also performed to verify decreased protein levels. The verified miRNA target gene is then subject to appropriate functional assays for validation.

Genes regulated by HIF1α. HIF1α regulates a broad spectrum of genes under hypoxia, including genes involved in glucose transport (glucose transport 1 (GLUT1)), glycolysis (phosphoglycerate kinase 1 (PGK1)), angiogenesis (VEGF), tumor metastasis (lysyl oxidase (LOX), chemokine (C-X-C motif) receptor 4 (CXCR4), osteopontin (OPN)), and pH control (CA9). Recently, miR-210 was identified as a HIF1α target under hypoxia. MiR-210 suppresses the expression of a number of genes involved in a variety of cellular functions. By up-regulating miR-210 expression under hypoxia, HIF1α can indirectly inhibit the expression of a subset of genes so cells can adjust and adapt to the hypoxic environment.