Research Of Cyclin G2-mediated Regulation On Wnt Pathway And Cell Proliferation

In eukaryotic cells, cell cycle is governed by cyclin-dependent protein kinases (CDKs) whose activities are regulated by cyclins and CDK inhibitors (CKIs). Cyclin, CDK and CKI form a regulating network to coordinate cell cycle transition. The 8 species of cyclins reported in mammal cells, cyclins A to H, share a conserved amino acid sequence of about 100 residues called cyclin box. The altered expression of variable cyclins is associated with the deregulation of the cell cycle and the uncontrolled cell proliferation in cancer cells.Cyclin G2 is encoded by cyclin G2 gene (CCNG2) located at 4q21.1, with a typical cyclin box sequence which usually mediates the combination with CDK in N-terminal. The nucleotide sequence of cyclin G1 and cyclin G2 are 60%identical. Unlike cyclin G1, cyclin G2 contains a C-terminal PEST motif, which links to the degradation of the cyclins, suggesting that cyclin G2 expression is tightly regulated through cell cycle. The mRNA expression of cyclin G2 fluctuates with the cell cycle, peaking in the late S phase, falling in G2/M transition. Cyclin G2 displays higher expression in the cerebellum, thymus, spleen, prostate and kidney. Recent studies have shown that cyclin G2 is a nucleocytoplasmic shuttling protein which combines with PP2A. Using chips and small-molecule inhibitors, researchers have found that the expression of cyclin G2 would have impacted through c-Jun amino-terminal kinase (JNK) pathways and mammalian target of rapamycin (mTOR) pathways, but the mechanism by which cyclin G2 mediates inhibition on cell cycle remains unknown.To further investigate the function of cyclin G2, we screened the interacting proteins of cyclin G2 using Yeast Two-hybrid System and found 35 non-repeated positive clonies. Functional prediction based on sequence alignments and bioinformatics of these clonies suggested that cyclin G2 might play a negative role in Wnt pathway. The canonical Wnt signaling pathway is very conservative in the process of evolution. Wnt binds to Frizzled in membrane and leads to the recruitment of Dishevelled (Dvl), resulting in the disruption of the destruction complex and consequently the accumulation of P-catenin. The accumulated P-catenin translocates into the nucleus and activates the transcription of target genes through interacting with T-cell factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors.In this paper we provided evidences that the overexpression of cyclin G2 inhibited cell proliferation. Besides, we found that overexpressed cyclin G2 downregulatedβ-catenin, and inhibited transcriptional activity and target gene expression of Wnt pathway. Finally we demonstrated that exogenous cyclin G2 promoted P-catenin phosphorylation and degradation through the ubiquitin degradation pathway, leading to the negative regulation of Wnt pathway by cyclin G2.Materials and methods1. Comparison of CCNG2 mRNA levels between gastric mucoca and gastric cancer cell lines with RT-PCR.Total RNA was extracted from gastric mucoca cell line GES-1 and gastric cancer cell line SGC-7901 and MGC-803. Then, semi-quantitative reverse transcription-PCR was performed to detect mRNA expression levels of CCNG2 gene.2. Investigation of cell proliferation regulated by exogenous cyclin G2 using trypanblau assay.Cyclin G2 was overexpressed in GES-1 and SGC-7901 by transitient transfection, and trypanblau assay was carried out to detect the effects of cyclin G2 on cell proliferation, respectively.3. Detection of P-catenin expression in cyclin G2 overexpressing cells using Western blots.Cyclin G2 was overexpressed in HT-29, HeLa and COS-7 cells using retro viral system, followed by Western blots to detect P-catenin expression levels.4. Alteration of transcription factor activity downstream of the Wnt pathway in cyclin G2 overexpressing cells with dual-luciferase assay.Cyclin G2 was overexpressed in COS-7 cells, and TCF/LEF transcriptional relative activity impacted by exogenous cyclin G2 was detected using dual-luciferase assay.5. Investigation of c-Myc expression in cyclin G2 overexpressing cells using Western blots.Overexpression of cyclin G2 was performed using retroviral system in HT-29 cells, followed by Western blots to detect c-Myc expression, one of the downstream targets of Wnt pathway.6. Exploration of the mechanisims by which cyclin G2 promoted P-catenin degradation.Cyclin G2 was overexpressed in HT-29 cells followed by Western blots to investigateβ-catenin phosphorylation. Moreover, the proteasome inhibitor MG-132 was applied to inhibitβ-catenin ubiquitination degradation with equal-volume DMSO as control, and detectedβ-catenin using Western blots and indirect immunofluorescence.Results1. CCNG2 gene expresses was down regulated in gastric cancer cell lines compared to gastric mucoca cell line.Semi-quantitative reverse transcription-PCR was performed to detect mRNA expression of CCNG2 gene in GES-1, SGC-7901 and MGC-803 cells. The results indicated that cylin G2 expression is down-regulated in gastric cancer cell lines compared to gastric mucoca cell line (P<0.05).2. Ectopic expression of cyclin G2 inhibited cell proliferation.Trypanblau assay was performed to detect the effects of exgenous cyclin G2 on cell proliferation in GES-1 and SGC-7901 cells. The results showed that cyclin G2 inhibits cell proliferation in vitro.3. Overexpression of cyclin G2 reduced P-catenin expression in a variety of cell lines.Cyclin G2 was overexpressed in HT-29, HeLa and COS-7 cells, followed by Western blots to detectβ-catenin expression regulated by cyclin G2. The results showed thatβ-catenin, the key factor of Wnt pathway, was significantly reduced in HT-29, HeLa, COS-7 cells overexpressing cyclin G2, which indicates cyclin G2 inhibited the expression ofβ-catenin.4. Overexpression of cyclin G2 inhibited the transcriptional activity of Wnt pathway.Cyclin G2 was overexpressed in COS-7 cells, and TCF/LEF transcriptional activity infulunced by exogenous cyclin G2 was investigated using dual-luciferase analysis system. The results showed that TCF/LEF transcriptional activity significantly decreased compared to control, suggested that cyclin G2 inhibited the transcriptional activity of Wnt pathway. 5. Overexpression of cyclin G2 inhibited the expression of c-Myc, downstream targets of Wnt pathway.Cyclin G2 was overexpressed in HT-29 cells using retroviral system, followed by Western blots to detect the expression of c-Myc, one of the downstreams targets of Wnt pathway. The results demonstrated that transient expression of cyclin G2 inhibited c-Myc expression.6. Cyclin G2 promotedβ-catenin degradation through the ubiquitin pathway.Cyclin G2 was overexpressed in HT-29 cells, P-catenin phosphorylation was detected by Western blots and found that cyclin G2 significantly up-regulatedβ-catenin phosphorylation. Ubiquitin degradation was inhibited using proteasome inhibitor MG-132, and detectedβ-catenin expression by Western blots and indirect immunofluorescence. The results demonstrated that cyclin G2 promoted P-catenin degradation through the ubiquitin pathway.Conclusion1. Exogenous cyclin G2 inhibited cell proliferation.2. Overexpression of cyclin G2 downregulated expression ofβ-catenin, the key factor of Wnt pathway, and inhibited transcriptional activity and target gene expression of Wnt pathway.3. Cyclin G2 overexpression promotedβ-catenin phosphorylation and degradation through the ubiquitin degradation pathway, leaded to the negative regulation of Wnt pathway by cyclin G2.