microRNAs (miRNAs) dysregulation is involved in the development and progression of various human cancers, including hepatocellular carcinoma (HCC). However, how to identify the miRNAs targeting specific mRNA in cells is a significant challenge because of the interaction complexity and the limited knowledge of rules governing these processes. Some miRNAs are not predictable by current computer algorithms available. Here, using p21 mRNA as target, we established a new method, named as miRNA in vivo precipitation (miRIP), to identify which kind of miRNAs can actually bind to the specific mRNA in cells. Several unpredictable miRNAs which bound p21 mRNA in HepG2 and PC-3 cells were identified by miRIP method. Among these miRNAs identified by miRIP, miR-92a was found and confirmed to interact robustly with p21 mRNA both in HepG2 and PC-3 cells. miR-92a was found to be remarkably increased in HCC tissues, and higher expression of miR-92a significantly correlated with lower expression of p21 which is related with poor survival of HCC patients. Moreover, inhibition of miR-92a could significantly suppress HCC growth in vitro and in vivo by up-regulating p21. Together, miR-92a, which is identified by miRIP, is functionally shown to be associated with HCC growth as an oncogenic miRNA by inhibiting expression of targeting gene p21. In addition, several unpredictable miRNAs which target STAT3 mRNA were also identified by miRIP method in HepG2 cells. Our results demonstrated that miRIP approach can effectively identify the unpredictable but intracellular existing miRNAs which target specific mRNA in vivo.


