Expression And Clinical Significance Of GINS Complex In Colorectal Cancer

[Background]Colorectal cancer (CRC) is one of the most common cancers in the world. Although the prognosis of colorectal cancer patients has improved because of multidisciplinary treatment program including surgery, cytotoxic drug and biological targeted therapy, the prognosis of colorectal cancer patients still remains very poor. The research data show that the improvement of colorectal cancer is not significant in the past three decades;5-year survival rates of colon and rectal cancer after surgery are still around70%and50%.The traditional diagnosis and treatment of which depends on clinical TNM stage and classic pathological.In clinical pathology, completely different fates and prognoses were observed in colorectal cancer patients with the same type, at the same stage, and even with the same treatment. Hence, there is an urgent need to understand the biological mechanisms that contribute to colorectal cancer development and progression so as to develop effective therapies, and to identify more effective, specific molecular markers of colorectal cancer so as to predict the outcome of colorectal cancer patients, to further standardize molecular classification model, and to guide individual treatment of colorectal cancer.The GINS complex, which is composed of four paralogous subunits Sld5, PSF1, Psf2, and Psf3, has been suggested to be involved in DNA replication because of its binding to DNA replication protein. Recent evidence has shown that GINS complex is frequently upregulated in several types of human cancer, indicating its diagnostic or prognostic significance for these cancers. And these studies showed that GINS complex was closely associated with tumor development, recurrence, and progression. It might be a useful prognostic marker for disease recurrence and progression in several types of human cancers. These findings may provide better strategy for the diagnosis and management of cancer.The expression and function of GINS complex in colorectal cancer and the clinical significance remain largely unknown. In the present study we employed quantitative reverse-transcription PCR analysis to detect the expression of GINS complex in a series of surgical specimens of colorectal cancer and analyzed the association between GINS complex expression and the clinicopathologic characteristics of colorectal cancer. In addition, we silenced PSF1expression by RNA interference (RNAi) in human colon cancer cell lines to investigate the functional role of PSF1in colon cancer development.[Objective]1, To investigate the expression and clinical significance of GINS complex (Go, Ichi, Nii, and San; five, one, two, and three, respectively, in Japanese) in colorectal cancer.2, To detect the expression of PSF1(partner of S1d five1) in colon cancer specimens, and to explore the effect of RNA interference targeting PSF1on the proliferation of colon cancer cells and its mechanism.[Methods]1, The expression of GINS complex which was composed of PSF1(partner of S1d five1), PSF2(partner of S1d five2), PSF3(partner of S1d five3) and SLD5(partner of S1d five5) in colorectal cancer specimens(n=76) were detected by real-time fluorescent quantitative polymerase chain reaction.2, Its correlation to the clinicopathological parameters and prognosis of colorectal cancer patients were also analyzed.3, The expression level of PSF1protein in colon cancer specimens was detected by western blot in40patients with colon cancer. 4, The shRNA(short hairpin RNA) plasmid targeting PSF1was transfected into LoVo, HT-29and HCT116with liposome. After that, the expression level of PSF1protein was investigated by western blot.5, The effect of PSF1shRNA plasmid transfection on cell proliferation and PSF2, PSF3and SLD5mRNA expression was measured by MTT assay, soft agar colony-formation assay and quantitative reverse transcription polymerase chain reaction.6, The effect of PSF1shRNA plasmid transfection on cell cycle and PSF2, PSF3and SLD5mRNA expression was measured by flow cytometry and quantitative reverse transcription polymerase chain reaction.[Results]1, The mRNA levels of PSF1, PSF2, PSF3and SLD5were significantly higher in colorectal cancer tissues than those in normal colorectal mucosa (P<0.01).2, PSF1mRNA expression was correlated with tumor size (P<0.01),PSF2mRNA expression was significantly associated with age (P<0.05) and lymphatic metastasis (P<0.05), no relationship was observed between PSF3mRNA expression and clinicopathological parameters, SLD5mRNA expression was correlated to lymphatic metastasis (P<0.01). Patients in PSF1, PSF2and SLD5high-expression group showed a poorer5-year overall survival rate than those in the low-expression group (P<0.05). Multivariate regression analyses showed that PSF1mRNA expression (P<0.05) was one of the independent factors for prognosis of colorectal cancer.3,The relative expression level of PSF1protein in colon cancer tissues was0.485±0.113, which was statistically higher than that in adjacent normal mucosa tissues(0.056±0.014,P<0.01).4, Western blot showed that the expression level of PSF1protein was significantly decreased in colon cancer cells transfected with PSF1shRNA plasmid (P<0.05).5, After PSF1shRNA plasmid transfection, cell proliferation was significantly suppressed, the soft agar colony-forming rates of LoVo, HT-29and HCT116cells were significantly lower than those in control groups(P<0.05).6, FACS analysis showed that the depletion of PSF1in LoVo, HT-29and HCT116 cells led to an increase in the fraction of cells in the S phase. After PSF1shRNA plasmid transfection, the expression level of PSF2, PSF3and SLD5mRNA was significantly decreased (P<0.05).[Conclusion]1, Overexpression of GINS complex in colorectal cancer is associated with the development and progression of colorectal cancer,2, The expression levels of PSF1mRNA may be a favorable independent prognostic biomarker for colorectal cancer patients.3, PSF1is significantly up-regulated in colon cancer tissues compared with adjacent normal mucosa tissues. ShRNA plasmid targeting PSF1can inhibit the expression of PSF1gene; suppress the proliferation of colon cancer cells, suggesting that it may be a new therapeutic target for colon cancer.