MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to be involved in diverse biological processes by regulating gene expression. Numerous miRNAs have been identified in various species, and many more miRNAs remain to be detected. Generally, hundreds of mRNAs have been predicted to be potential targets of one miRNA, so it is a great challenge to identify the genuine miRNA targets. Here, we generated the cell lines depleted of Drosha protein and screened dozens of transcripts (including Cyclin D1) regulated potentially by miRNA-mediated RNA silencing pathway. On the basis of miRNA expressing library, we established a miRNA targets reverse screening method by using luciferase reporter assay. By this method, we found that the expression of Cyclin D1 (CCND1) was regulated by miR-16 family directly, and miR-16 induced G1 arrest in A549 cells partially by CCND1. Furthermore, several other cell cycle genes were revealed to be regulated by miR-16 family, including Cyclin D3 (CCND3), Cyclin E1 (CCNE1) and CDK6. Taken together, our data suggests that miR-16 family triggers an accumulation of cells in G0/G1 by silencing multiple cell cycle genes simultaneously, rather than the individual target.

Long non-coding RNAs (lncRNAs), which represent a new frontier in molecular biology, play important roles in regulating gene expression at epigenetic, transcriptional and post-transcriptional levels. More and more lncRNAs have been found to play important roles in normal cell physiological activities, and participate in the development of varieties of tumors and other diseases. Previously, we have only been able to determine the function of lncRNAs through multiple mechanisms, including genetic imprinting, chromatin remodeling, splicing regulation, mRNA decay, and translational regulation. Application of technological advances to research into the function of lncRNAs is extremely important. The major tools for exploring lncRNAs include microarrays, RNA sequencing (RNA-seq), Northern blotting, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), RNA interference (RNAi), RNA-binding protein immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), crosslinking-immunopurification (CLIP), and bioinformatic prediction. In this review, we highlight the functions of lncRNAs, and advanced methods to research lncRNA-protein interactions.

Maternally Expressed Gene 3 (MEG3) encodes a lncRNA which is suggested to function as a tumor suppressor. Previous studies suggested that MEG3 functioned through activation of p53, however, the functional properties of MEG3 remain obscure and their relevance to human diseases is under continuous investigation. Here, we try to illuminate the relationship of MEG3 and p53, and the consequence in hepatoma cells. We find that transfection of expression construct of MEG3 enhances stability and transcriptional activity of p53. Deletion analysis of MEG3 confirms that full length and intact structure of MEG3 are critical for it to activate p53-mediated transactivation. Interestingly, our results demonstrate for the first time that MEG3 can interact with p53 DNA binding domain and various p53 target genes are deregulated after overexpression of MEG3 in hepatoma cells. Furthermore, results of qRT-PCR have shown that MEG3 RNA is lost or reduced in the majority of HCC samples compared with adjacent non-tumorous samples. Ectopic expression of MEG3 in hepatoma cells significantly inhibits proliferation and induces apoptosis. In conclusion, our data demonstrates that MEG3 functions as a tumor suppressor in hepatoma cells through interacting with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes.

MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of various biological processes. Although there is emerging evidence that some microRNAs can function as oncogenes or tumor suppressors, the specific role of miRNA in human hepatocellular carcinoma (HCC) is unclear at this point. In this study, we examined the microRNA expression profiles in a set of 20 human HCC specimens by miRNA microarray and quantitative real-time polymerase chain reaction. The results showed that among the 20 HCC samples analyzed, microRNA-101 was significantly down-regulated twofold or more (twofold to 20-fold) in 16 samples compared with the matching nontumoral liver tissues. Using both a luciferase reporter assay and Western blot analysis, we showed that microRNA-101 repressed the expression of v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene, a key component of the activator protein-1 (AP-1) transcription factor. Moreover, using a luciferase expression vector (pAP-1-Luc) driven by seven copies of an AP-1 cis-element, we observed that microRNA-101 expression inhibited phorbol 12-myristate 13-acetate (PMA)–induced AP-1 activity. In in vitro Matrigel invasion and Transwell migration assays, enhanced microRNA-101 expression inhibited the invasion and migration of cultured HCC cells, respectively. These findings suggest that microRNA-101 may play an important role in HCC. Conclusion: MicroRNA-101, which is aberrantly expressed in HCC, could repress the expression of the FOS oncogene. (Hepatology 2009.)

Background: In recent years, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. In our previous studies, a set of miRNAs were revealed to be dysregulated in HCC tissues. However, the functions of these miRNAs in HCC remain largely undefined. Methods: The expression profiles of miR-183 were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was used to screen the potential target genes of miR-183. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-183 in hepatoma cells was examined. Results: Among the 25 HCC samples analyzed, microRNA-183 was significantly up-regulated (twofold to 367-fold) in 17 samples compared with the matching nontumoral liver tissues. Programmed cell death 4 (PDCD4) was identified as the target gene of miR-183. Moreover, PDCD4 is a proapoptotic molecule involved in TGF-β1-induced apoptosis in human HCC cells, we found that miR-183 transfectants were resistant to apoptosis induced by TGF-β1. Conclusions: We conclude that miR-183 can inhibit apoptosis in human HCC cells by repressing the PDCD4 expression, and miR-183 may play an important role in HCC development.