The Function Of P55PIK On Cell Proliferation And Its Post-transcription Regulatory Mechanism In Colorectal Cancer Cells

Object:Previously, we have proved that p55PIK could bind to Rb protein through its N terminal24amino acids (short for N24), promote DNA synthesis and cell cycle progression. However, we also found that p55PIK could regulate DNA synthesis and cell cycle progression in Rb-deficient cells, but the mechanism is unkown. In this study, we try to determine the function of p55PIK on regulating DNA synthesis and cell cycle progression in Rb-deficient cells, and its mechanism of cell cycle regulating via Rb-independent pathway.Methods:First, p55PIK was over-expressed (or was down-regulated) by Adenovirus of p55PIK (or by siRNA of p55PIK) in thyroid cancer FTC236cells which were Rb-deficient naturally, DNA synthesis and cell cycle distribution were detected by BrdU/PI incorporation, cell proliferation were detected by cell counting. Then, we constructed the nude mouse xenograft model, and observed the influence of p55PIK on colorectal cancer HT29Rb-/-cell tumorigenicity in vivo. Furthermore, we detected the expression of p55PIK in colorectal cancer tissue and nomal tissue by Immunohistochemistry. Moreover, we explored the relationship between p55PIK and proliferating cell nuclear antigen (PCNA) through Immunofluorescence (IFC)、Co-immunoprecipitation (IP) and pull-down assay. Finally, we over-expressed p55PIK by Adenovirus (or inhibit the function of p55PIK by N24), and detected the effect of p55PIK on the binding ability of PCNA and DNA polymerase8by Quantitative co-immunoprecipitation (QIP).Results:Over-expressing p55PIK could promote DNA synthesis and accelerate cell cycle progression at S phase and increase cell proliferation, while down-regulating p55PIK restrain DNA synthesis and cell cycle progression and inhibit cell proliferation in FTC236cells. P55PIK was overexpression in colorectal cancer tissue compared to nomal tissue, and over-expressing p55PIK could promote HT29Rb-/-tumorgenicity in nude mouse xenograft model in vivo. P55PIK could directly bind to PCNA via its N terminal24amino acids, overexpressing p55PIK increased the combination of PCNA and DNA polymerase δ while inhibiting the function of p55PIK by N24decreased the combination of PCNA and DNA polymerase δ.Conclusion:p55PIK is overexpression in colorectal cancer tissue compared to normal tissue. P55PIK directly binds to PCNA. Over-expressing p55PIK promotes DNA synthesis and accelerates cell cycle progression at S phase through increasing the combination of PCNA and NDA polymerase δ while inhibiting the function of p55PIK by N24restrains DNA synthesis and blocks cell cycle progression through decreasing the combination of PCNA and DNA polymerase δ. Object:Previously, we have found that p55PIK is overexpression in colocrectal cancer tissue compared to normal tissue. Moreover, we have proved that overexpressing p55PIK promotes cell proliferation through accelerating cell cycle progression and DNA synthesis, and cleared the mechanism of p55PIK on promoting cell proliferation. In this, we try to find the mechanism of p55PIK expression regulating through its post-transcripition pathway.Methods:Colorectal cancer cells were transfected with miR-148b mimics or inhibitor, the expression of p55PIK was detected by western blot and real-time PCR, DNA synthesis and cell cycle progression was observed by BrdU/PI incorporation, cell proliferation was determined by cell counting. LoVo cells were transfected with miR-148b overexpressing or down-regulating plasmid, and were filtered by Puromycin to be stable cell line. Then, we constructed the nude mouse xenograft model by the stable cells, and observed the influence of miR-148b on colorectal cancer LoVo cell tumorigenicity and its effect on p55PIK expression in vivo. Furthermore, we constructed dual luciferase reporter plasmid of p55PIK3’Untranslated region, detected the effect of miR-148b mimics or inhibitor on the activity of p55PIK3’-UTR dual luciferase reporter plasmid. Mutating the binding sites of p55PIK3’-UTR and miR-148b, detected the effect of miR-148b on its activity. Finally, we try to determine whether the function of miR-148b on DNA synthesis and cell cycle progression is dependent on p55PIK by the "rescue" experiments.Results:Overexpressing miR-148b decreased p55PIK protein level, but not the mRNA level, inhibited DNA synthesis and blocked cell cycle at G0/G1phase. While low-expressing miR-148b increased p55PIK protein, promoted DNA synthesis and accelerated cell cycle progression to S phase. MiR-148b regulated p55PIK protein level by targeting p55PIK3’-UTR directly. In vivo study, we also found that overpressing miR-148b suppressed LoVo cell tumorigenicity while down-regulating miR-148b promoted LoVo cell tumorigenicity in nude mouse xenografts model. Moreover, miR-148b also regulated p55PIK protein level in vivo. Overpressing p55PIK could remove the function of miR-148b on inhibiting DNA synthesis and blocking cell cycle progression.Conclusion:miR-148b could inhibit colorectal cancer cell DNA synthesis and block cell cycle progression via suppressing p55PIK protein level by targeting p55PIK3’-UTR directly. MiR-148b inhibits colorectal cancer cells proliferation and tumorigenicity by inhibiting DNA synthesis and blocking cell cycle progression. The function of miR-148b on DNA synthesis and cell cycle progression is dependent on p55PIK. Object:Previously, we have found the function of p55PIK on cell proliferation and its mechanism, moreover, we have investigated that miR-148b could regulate p55PIK expression at protein level by targeting p55PIK3’-UTR directly in colorectal cancer cells. But the mechanism of miR-148b expression regulatory is not cleared. In this study, we try to find the mechanism of miR-148b expression regulating in colorectal cancer cells.Methods:First, we found that p53maybe a potential transcription factor of miR-148b through bioinformatics analysis. Then we constructed p53overexpressing plasmid and overexpressing p53or low expressing p53(p53siRNA) in colorectal cancer cells detected miR-148b expression through real-time PCR. Furthermore, we constructed dual luciferase reporter plasmid of miR-148b promoter active region, detected the effect of p53on the activity of miR-148b promoter active region dual luciferase reporter plasmid. Then mutating the binding sites of miR-148b promoter active region and p53, detected the effect of p53on its activity. Finally, we detected the relationship between p53and miR-148b promoter active region through chromatin immunoprecipitation.Results:Overexpressing p53significantly promoted miR-148b expression, while low expressing p53obviously inhibited miR-148b expression. Overexpressing p53significantly increased the activity of miR-148b promoter active region luciferase reporter gene, while down-regulateing p53decreased the activity of miR-148b promoter active region luciferase reporter gene. Neither overexpressing nor low expressing p53had effect on the activity of miR-148b promoter active region luciferase reporter gene. P53bind to miR-148b promoter active region directly.Conclusion:p53increases miR-148b expression via promotes its promoter activity through binding to miR-148b promoter directly. Object:Previously, we have cleared the function and mechanism of miR-148b on p55PIK expression regulatory in colorectal cancer cells. And we have also determined the function and mechanism of p53on miR-148b expression regulatory. In this study, we will try to find whether p53could regulate p55PIK expression through miR-148b.Methods:First, colorectal cancer cells were transfected p53plasmid ro p53siRNA to overexpress or low express p53, p55PIK protern level or mRNA level was detected. Then, we detected the expression of miR-148b and p55PIK in HCT116wild type (HCT116WT)and p53null cells (HCT116p53-/-) and investigated the influence of p53on miR-148b and p55PIK expression in HCT116p53-/-cells. Furthermore, p53was overexpressed or low expressed and miR-148b expression was intervened simultaneously in colorectal cancer cells, p55PIK protein level was detected by western blot. Moreover, we detected the expression of p53, miR-148b and p55PIK in8cell lines and analyzed the relationship amount p53, miR-148b and p55PIK expression. Finally, we detected the expression of p53, miR-148b and p55PIK in clinical colorectal cancer tissue and normal tissue and analyzed the relationship of p53, miR-148b and p55PIK expression.Results:Overexpressing p53suppressed while downexpressing p53promoted p55PIK protein level, but had no effects on p55PIK mRNA level. Overexpressing miR-148b could destroy the effect of p53on increasing p55PIK expression while low expressing miR-148b could restore p55PIK expression which was decreased by p53. In8colorectal cancer cell lines, the expression of miR-148b was down-regulated in cells which p55PIK were overexpressed, while the expression of p53was low expression. Whereas, the expression of miR-148b was up-regulated in cells which p55PIK was low expression, while p53 expression was overexpression. In clinical samples, p55PIK was up-regulated in colorectal cancer tissues, while miR-148b and p53expression were decreased compared to normal tissues. In9fresh tissues, p55PIK was overexpression in tumor tissues while miR-148b and p53were low expression in tumore tissues compared to normal tissues.Conclusion:p53suppresses p55PIK expression through promoting miR-148b expression in colorectal cancer cells. The expression of p55PIK and miR-148b is negative related, and the expression of p55PIK and p53is negative related, while the expression of miR-148b and p53is positive related in colorectal cancer tissues.