Characterization Of Molecular Mechanisms Underlying G1/S Transition Of Cell Cycle

It is well known that cell cycle progression is regulated by elaborate gene regulatory networks. Deregulation of cell cycle, especially abnormality in G1/S transition, is a key step during carcinogenesis. A large number of proteins have been characterized as cell cycle regulators. Recently, microRNAs (miRNAs), a class of small noncoding RNAs, have been identified. miRNAs play powerful roles in repressing protein expression. However, the biological function of these small RNAs in the regulatory networks of cell cycle is still largely unknown. In the previous study, we identified differentially expressed miRNAs in Hepatocellular carcinoma (HCC) tissues using microarray technique. We further predicted the targets of these differentially expressed miRNAs using bioinformatic tools. Interestingly, significant enrichment of G1/S transition-related genes was observed among the predicted targets of miR-195 and miR-26. In this study, we therefore aimed to elucidate the function of miR-195 and miR-26 in the cell cycle control and its underlying mechanisms. Our results are summarized as follows:1) miR-195 blocks G1/S transition by repressing multiple molecules both upstream (cyclin D1, CDK6) and downstream (E2F3) of pRb. We found that ectopic expression of miR-195 blocked G1/S transition, whereas inhibition of miR-195 promoted cell cycle progression. Subsequent investigation using luciferase reporter assay and Western blot characterized three G1/S transition-related molecules, including cyclin D1, CDK6 and E2F3, as direct targets of miR-195. Moreover, introduction of miR-195 significantly repressed the phosphorylation of pRb as well as the transactivation of downstream target genes of E2F3. These results indicate that miR-195 may block G1/S transition by repressing cyclin D1, CDK6 and E2F3, and provide new insight into a fail-safe mechanism, which may guard the cell from runaway proliferation in the case of abnormal activation of E2F3 or aberrant inactivation of pRb.2) Intronic miR-26a/b and their host genes CTDSP1/2/L display synergistic blocking effect on the G1/S transition by activating pRb protein. miR-26 family composes of miR-26a and miR-26b. They are transcribed from three genomic loci, miR-26a-1, miR-26a-2 and miR-26b, which reside in the introns of genes coding for CTDSPL, CTDSP2 and CTDSP1 proteins, respectively. The serum starvation-stimulation assay in primary fibroblasts and the two-thirds partial-hepatectomy in mice, two classical models which are often used to study cell cycle, revealed that miR-26a/b and CTDSP1/2/L displayed similar expression pattern, that was, increased expression of miR-26a/b and CTDSP1/2/L in quiescent cells and down-regulation of both families during cell proliferation. We also found that both miR-26 and CTDSP family members were down-regulated in HCC tissues, and similar expression pattern was observed between miR-26a/b and their respective host genes. These data implicate that miR-26a/b may express concomitantly with their host genes in both physiological and pathological processes. Moreover, gain- and loss-of-function studies demonstrated that miR-26a/b can act in concert with CTDSP1/2/L to block G1/S transition. Subsequent investigation showed that miR-26a/b repressed CDK6 and G1 cyclins, and CTDSP1/2/L dephosphorylated the ppRb protein, which synergistically activated pRb protein and blocked G1/S transition. These results suggest that the cooperation of miR-26a/b and their host genes may provide a“double guarantee system”for the activation of pRb protein, which guards the cell from runaway proliferation.Taken together, our findings provide new insight into the regulatory network of cell cycle control and also provide new molecular targets for developing preventive and therapeutic strategies against cancer.