In this study, we demonstrated the functional effects of miR-34a on c-Myc transcriptional complexes in RCC. We showed that miR-34a inhibited c-Myc by binding to its 3′ UTR and subsequently suppresses the assembly and function of the c-Myc–Skp2–Miz1 complex that activates RhoA and the c-Myc–P-TEFb complex that elongates transcription of a variety of genes in RCC. Mutations of c-Myc are found in various cancers and its de-regulated expression causes the uncontrolled expression of many genes, some of which regulate cell proliferation and results in tumor development (33
). Although Myc alone is not able to transform cells and requires a co-operating oncogene, such as ras oncogene (35
), c-Myc is one of the major factors in tumor development. Thus, inhibition of c-Myc is thought to be a significant function of miR-34a. Our results also show that miR-34a strongly inhibits cell proliferation, in vivo
xenograft tumor growth and cell invasion, indicating that miR-34a functions as a tumor suppressor in A498 and 769P cells.
Rho GTPases, a family of small G proteins, regulate intracellular actin dynamics, including cell polarity, migration, vesicle trafficking, cell proliferation, apoptosis and gene expression (20
). RhoA, a member of the Rho family of GTPases, is implicated in transformation and metastasis. RhoA has been documented to be an important factor associated with various human cancers (36
Cross talk between c-Myc and RhoA has been documented. Proteomic experiments have revealed that c-Myc controls the activity of RhoA-dependent routes (37
). Transcriptomal profiling has revealed that c-Myc is important for Rho-mediated transformation (38
). Ras activates Rho, which leads to activation of c-Myc through phosphorylation (39
). c-Myc also inhibits the RhoA/Rock-dependent cytoskeleton (40
). Existence of unknown pathways in association between c-Myc and RhoA has been implicated. As a transcription factor, c-Myc activates a variety of genes with its partner protein, Max and with other proteins (12
). The c-Myc–Skp2–Miz1 transcriptional complex was shown to activate RhoA and to be essential for cell invasion and cancer metastasis (25
). We found that c-Myc is a target of miR-34a in A498 cells, and we studied the effect of miR-34a on RhoA activation through the c-Myc–Skp2–Miz1 transcriptional complex. miR-34a reduced RhoA expression and overexpression of c-Myc reversed the reduction of RhoA and inhibition of cell invasion. ChIP assay revealed that miR-34a suppresses the recruitment of c-Myc to the RhoA promoter. We demonstrated that miR-34a targets c-Myc and suppressed RhoA transcription by reducing the c-Myc–Skp2–Miz1 transcriptional complex. These data document that miR-34a suppresses RhoA activation at the initiation of transcription via targeting c-Myc. Our results also demonstrate that miR-34a suppresses the assembly and function of the c-Myc complex that activates RhoA transcription. A schematic model of the mechanisms is shown in . Although miR-34a reduced c-Myc protein expression in 769P cells, RhoA expression was not significantly suppressed, which suggests that other mechanisms prevent reduction of RhoA expression. However, this result supports the small effect of miR-34a on 769P cell invasion and RhoA dependence of miR-34a regulation in A498 cell invasion.
Fig. 6. Schematic model of miR-34a repression of c-Myc complexes. (A) The c-Myc–Skp2–Miz1 transcriptional complex activates RhoA. (B) miR-34a targets c-Myc and represses RhoA expression by suppressing the c-Myc–Skp2–Miz1 transcriptional (more ...)
The P-TEFb regulates the promoter-proximal pause release of the elongation phase of transcription by Pol II (26
). P-TEFb is composed of Cyc and Cdk9 (26
). c-Myc interacts with CycT1, the regulatory component of P-TEFb, and regulates the elongation phase of transcription of Pol II (28
). P-TEFb is globally involved in the generation of mRNAs and gene expression (30
) and is recruited to genes by a variety of transcription factors (26
). Our results demonstrate that miR-34a suppresses the assembly and function of the c-Myc complex that elongates transcription, which may affect global gene expression. The miR-34a-induced c-Myc immunocomplex may be heterogeneous since the ratios of the components were different from those in the control immunocomplexes. Since miR-34a affects wide variety of genes besides reducing c-Myc expression, the miR-34a-induced c-Myc immunocomplex may be heterogeneous.
P-TEFb has been shown to be involved in aberrant transcriptional elongation in mixed-lineage leukemia (43
) and various tumor cell lines overexpress P-TEFb (45
). We demonstrated here that miR-34a inhibits the assembly of the c-Myc–P-TEFb complex presumably by targeting c-Myc, which is of significance in cancer studies. Our results suggest that miR-34a suppresses by reducing c-Myc and affects Pol II pause release in tumor cells, which partly accounts for its dramatic tumor-suppressing effects. A schematic model of the mechanisms is shown in .
In this study, we show that miR-34a inhibits kidney cancer cell (A498 and 769P) cell invasion. We also show miR-34a inhibits c-Myc and its complexes, which we observed in prostate cancer cells (Yamamura,S, Saini,S, Majid,S, Hirata,H, Ueno,K, Deng,G and Dahiya,R, unpublished results). c-Myc activates a variety of genes through its protein complexes and also regulates transcriptional elongation. We demonstrate here that miR-34a suppresses assembly and function of the c-Myc–Skp2–Miz1 complex that activates RhoA and the c-Myc–P-TEFb complex that elongates transcription of various genes. Therefore, our study reveals a novel role of miR-34a in the regulation of transcription by c-Myc.