To date, a large number of miRNAs have been identified in several organisms, including vertebrates and plants. In human, 533 miRNAs were validated (16
), and some computational studies estimated that the number of human miRNAs is as many as 2- to 4-fold higher (33
). However, only a handful of comprehensive studies on miRNA function have been completed, whereas the function of most miRNAs remains unknown. One of the main challenges in identification of miRNA function is to identify the genuine miRNA target gene (or genes) among many predicted targets.
In this study, we generated the cell lines depleted of Drosha protein, and indicated that 53 transcripts were upregulated remarkably in HepG2 cells depleted of Drosha. Given the roles of Drosha in the miRNA pathway (2
), these upregulated transcripts may be regulated by miRNAs. Rehwinkel et al.
) profiled the expression of mRNAs in Drosophila S2 cells depleted of Drosha, the results revealed that Drosha knockdown triggered upregulation of many transcripts. Furthermore, they concluded that the majority of those transcripts were genuine miRNA targets. Among these transcripts upregulated in HepG2 cells depleted of Drosha, CCND1 is a key cell cycle-related gene, and regulates G1/S transition. On the basis of miRNA expressing library (20
), we established a miRNA targets reverse screening method by using luciferase reporter assay. By this method, multiple miRNAs including miR-16, miR-195 and miR-424 were identified to regulate the expression of CCND1. Furthermore, these miRNAs triggered G1 cell cycle arrest partially by repressing CCND1 in A549 cells (). As all know, not all miRNA targets are downregulated at mRNA levels, thus, not all of them can be identified by the strategy analyzing mRNA expression in Drosha knockdown cells. Actually, for any genes we are interested in, this miRNA target reverse screening method can be used to analyze whether they are regulated by miRNAs, and screen the miRNAs regulating these mRNA genes. Using the reverse screening method, we found that several genes could be regulated by certain miRNAs, even some target genes could be regulated by multiple miRNAs simultaneously (data not shown). All these data suggest that this miRNA targets reverse screening method is very useful for identifying miRNA targets, and may play an important role in the characterization of miRNA function.
Although the biological roles of only a small fraction of identified miRNAs have been elucidated to date, the available evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and that miRNAs can function as tumor suppressors and oncogenes to regulate key pathways involved in cellular growth control (35
). Some miRNAs are involved in tumor genesis by modulating cell cycle progression (36
). For example, aberrant expression of miR-221/222 was observed in diverse tumors, such as papillary thyroid carcinoma (37
), pancreatic adenocarcinoma and glioblastoma (39
). Further study revealed that ectopic overexpression of miR-221/222 repressed the expression of p27(Kip1) by targeting the binding sites in the 3′-UTR of p27 mRNA and strongly affected cellular growth potentially by inducing a G1 to S shift in the cell cycle. These results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) downregulation (40
). Recently, miR-34 family members have been shown to be directly regulated by the tumor suppressor, TP53 (42–44
). Overexpression of miR-34 causes cell cycle arrest at G1 phase by downregulation of a significantly great number of genes promoting cell cycle progression. Several downregulated cell cycle genes (including CCNE2, CDK4 and MET) have been validated as direct targets by showing miR-34 regulation of reporters engineered to contain the 3′-UTRs of the respective targets (44
). Recently, Schultz et al.
) have shown that let-7b regulates CCND1 and induces G1 arrest in malignant melanoma cells. Our studies have revealed that miR-34a (46
) and miR-16 family can regulate CCND1 and induce G1 arrest in A549 cells too. To compare their effects on cell cycle, these miRNAs were synthesized and transfected into A549 cells. As shown in Figure S4, all of let-7b, miR-16 and miR-34a reduced CCND1 protein level and induced G1 arrest compared with Luc-siRNA. miR-16 and miR-34a had stronger inhibition effectiveness on CCND1 than let-7b, and miR-16 had the best effect on triggering accumulation of cells in the G0/G1 phase. Furthermore, compared with siRNA against CCND1, all these miRNAs had weaker inhibition effectiveness on CCND1, but triggered more prominent G0/G1-cell accumulation. These results suggest that all these miRNAs induce G1 cell cycle arrest by regulating multiple target genes, rather than a single key target.
miR-16 family negatively regulates cellular growth and cell cycle progression. They trigger the G0/G1 accumulation phenotype in diverse cell lines, including HCT116, DLD-1, A549, MCF7 and Tov21G cells (31
). Here, we found that miR-16 and miR-424 could regulate the expression of CCND1 by targeting putative target site (). Further study revealed that several other cell cycle genes (such as CCND3, CCNE1 and CDK6) could also be regulated by miR-16 family (), and all these cell cycle genes were involved in G1/S transition. Flow cytometry assay revealed that depletion of these targets led to a substantial arrest in G1, partly phenocopy activation of miR-16 (B). All results above indicated that simultaneous silencing of these genes was more effective on blocking cell cycle progression than deletion of an individual gene. Taken together, both our results and findings of Linsley et al.
) argue that miR-16 targets act in concert, rather than individually, to regulate G1/S transition (). In other words, multiple targets regulated by an individual miRNA can act coordinately to regulate the same biological process (36
Figure 7. miR-16 family modulates G1/S transition by regulating Cdk4/6-cyclin D complexes and Cdk2-cyclin E complexes. The G1/S phase transition is regulated primarily by D-type Cyclins (D1, D2 or D3) in complex with CDK4/CDK6, and E-type Cyclins (E1 or E2) in (more ...)
Just like siRNA against CCND1, miR-16 family repressed the expression of CCND1, but led to no G0/G1 accumulation phenotype in HepG2 cells. Likewise, miR-16 could not trigger cell cycle arrest in megakaryocytic cell line MEG-01 (data not shown). Cimmino et al.
) reported that miR-16 induced apoptosis by targeting Bcl-2 in MEG-01 cells. However, we detected no significant apoptosis following transfection with miR-16 in A549 cells. These data suggest that the effect of miR-16 family on cell cycle arrest and apoptosis is cell type dependent, that is to say, miR-16 leads to cell cycle arrest in some types of cells, but induces apoptosis in others. It is possibly because some types of cells are sensitive to the downregulation of the targets regulated by miRNA, whereas others are not. Or the deletion, mutation or editing of binding sites in target mRNAs can also result in the inactivation of miRNA (47
All data above argue that miR-16 family may participate in cell growth control and play tumor-suppressor roles by regulating cell proliferation and/or apoptosis pathway in various tumor cells. Both CCND1 (25
) and Bcl-2 (48
) play important roles in tumorigenesis, and serve as specific tumor therapeutic targets. And siRNAs against them can inhibit tumor cell growth significantly. Now that miR-16 can regulate multiple target genes promoting cell growth, including Bcl-2, CCND1, CCND3, CCNE1 and CDK6, and have stronger inhibitory effectiveness on cell growth than siRNAs against an individual gene, exogenous expression of miR-16 may be more effective for tumor therapy than depletion of single oncogene.