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Expression of Prolyl Isomerase Pin1 in Osteosarcoma and the Effect of Regulation on Cell Cycle

Author: ZhouLu
Tutor: YangShuGuang
School: Taishan Medical College
Course: Internal Medicine
Keywords: Pin1 Osteosarcoma Proliferation recombinant adenovirus expression vector cell cycle cyclinD1
CLC: R738.1
Type: Master's thesis
Year: 2011
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Abstract


ObjectiveOneogenesis is a multistep and multifactorial process both at genetic and epi-genentic levels, that results in uncontrolled cell proliferation, transformation, and cell death. Phosphorylation of proteins on serine/threonine residues preceding proline (pSer/Thr-Pro) is a key regulative mechanism for the control of cell proliferation and transformation. Pin1, a recently identified peptidyl-prolyl cis/trans isomerase (PPIase), has two domains: a PPIase domain responsible for isomerization and a WW domain, which functions as a binding element specific for pSer/Thr-Pro motifs. Through these two domains, Pin1 binds to and isomerizes specific pSer/Thr-Pro motifs and catalytically induces conformational changes after phosphorylation. Such conformational changes can have profound effects on the function of many Pin1 substrates, such as p53, cyclin D1, C-Jun, nuclear factor-κB, C-myc, E2F, andβ-catenin, thereby playing an important role in many cellular events, such as cell cycle progression, transcriptional regulation, RNA processing, and cell proliferation and differentiation. Now there have been little reports about the expression of Pinl in osteosarcoma tissue and the relationship with cyclinD1. So in current studies, we investigated whether the expression level of Pin1 and cyclinD1 may increase in osteosarcoma compared with in normal tissue. Thus we hypothesized that Pin1 and cyclinD1 may have important role in osteosarcoma generation and development.Methods(1) The expression of Pin1 in 25 cases of human osteosarcoma was detected with immunohistochemistry and Western-Blot; (2) Total RNA was extracted from cells, then the hPIN1 cDNA was amplified by RT-PCR; The full-length Pin1 cDNA was subcloned into pENTR vector; Using the pAd/CMV/V5-DEST kit, Pin1 adenovirus was generated according to the manufacturer’s protocol, and was further verified by automated sequencing; (3) Pin1 (103 MOI) was transfected into osteosarcoma Saos-2 and U2-OS cells. These cells were treated with Pin 1 inhibitor juglone and were collected before and after transfection. The curve of cell growth was made through MTT method. The cell growth cycle was detected by FACS. The expression of Pin1 and cyclinD1 were detected by Western Blot.Results(1) The expression of Pin1 was 72% in osteosarcoma; but no expression of Pin1 was detected in normal bone. The Pin1 substrate cyclinD1 andβ-catennin were also increased in osteosarcoma. (2) Pin1 gene was obtained by PCR method.The adenoviral entry and expression clones were identified correctly by automated sequencing.Those correct recombinant adenoviral expression plasmids transfected HEK293A cells to obtain the recombinant adenovirus, which can increase Pin1 in the cells. (3) The transfection by Pin1 virus significantly stimulated the proliferation of Saos-2 and U2-OS cells and led to cell cycle progression. (4) Pin1 can promote the substrate cyclinD1, cyclin E, CDK4 and CDK6, the inhibitor juglone reduced Pin1 level compared with normal cells.Conclusion:The levels of Pin1 in tumor tissue were significantly higher than in normal tissues. The Pin1 has relationships with proliferation and cell cycle progression and increase cyclinD1 expression. Pin1 may play an important role in tumor genesis and tumor progression, and may provide new target for gene therapy.

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