| Objects and significances:Phosphoprotein 32 (pp32) was first discovered by Malek in 1990. Pp32 is a member of leucine-rich acidic nuclear proteins (Leucine-rich acidic nuclear protein, LANPs) family including PHAPI, Mapmodulin, I1PP2A, APRIL, HPPCn, pp32r1, pp32r2 and so on. Pp32 consists of two structual domains, and the N-terminal structure is composed of LRRs (Leucine-rich repeats) structure including five leucines, which is a recognized conformation of interaction between proteins. The C-terminal structure is an acid tail with high levels of aspartic acid and glutamic acid. The stretching C-terminal tail and the N-terminal LRRs zone whose three-dimensional conformation is spherical, and make the entire protein is similar to a tadpole. A typical nuclear localization signal NLS (nuclear localization signal) KRKR is in the C-terminal acidic region and it is an important structural foundation of shuttling between nucleus and cytoplasm.Pp32, as a multifunctional protein, is involved in many physiological events which include cell proliferation, apoptosis, mRNA transport, and transcription. Pp32 has been isolated several times, and has therefore been assigned a variety of names: pp32 (phosphoprotein 32), Anp32a (acidic nuclear phosphoprotein32a), PHAPI (putative HLA-associated protein I), mapmodulin, Lanp (leucine-rich nuclear protein), and I1PP2A (inhibitor 1 protein phosphatase 2A).The study of pp32 functions at present is focused on the following aspects:(1) Pp32 can inhibit the malignant transformation of a variety of proto-oncogenes in rat fibroblasts, including ras, E1a, mutant p53, myc and so on. (2) Pp32 promotes the apoptosis of human cervical carcinoma cells Hela and human breast cancer cells HMECs. (3) Pp32 inhibits the activity of PP2A. (4) As a ligand of mRNA binding protein HuR,pp32 affects the transport of pp32 from cell nucleus to cytoplasm. (5) As a constituent of INHAT (inhibitors of acetyltransferase), pp32 is involved in histone acetylation and phosphorylation and regulating the structure of chromosomes. (6) Pp32 is involved in cytoskeleton dynamics. (7) Pp32 is involved in substance transportion in cells and signal transduction. (8) It is involved in tissue development. Some research showed that 72 h after the hypoxia, HepG2 and Huh7 cells, which typically look like epithelia with well-developed cell junctions, acquired spindle shapes and generally lost cell contacts. To further test whether the morphologic changes in hypoxic HepG2 and Huh7 cells represent EMT (epithelial-mesenchymal transition), so the morphologic changes were detected in HepG2 cells overexpressing pp32 compared to wide type HepG2 cells. We speculated that pp32 affect tumor cell motility and migration. Tumor metastasis is defined as the whole process that malignant neoplasm cells shed and leave original sites, reach other parts through the blood or lymphatic system, continue to grow, and then form new lesions. The new lesion is named after metastatic focus, and the original tumor is called primary focus. The major tumors have the transferability which is the main obstacle to cure cancer patients clinically.The complex process that a tumor cell obtains abilities of invasion and spread and survives at metastatic sites is regulated by genic and cellular levels. Studies of relations between pp32 and tumor metastasis probably provide useful clues for further gene therapy. This object majors in studying the following two aspects: 1,Pp32 inhibits hepatocellular carcinoma cell migration characteristics. 2,Pp32 infects Pc3M cell migration characteristics.Materials and methods:The protein level of pp32 was detected by western blots experiments in 15 cell lines including HepG2, SMMC7721, MHCC 97H, SK-hep1, MSC, Ad-293, and so on. In order to identify roles of pp32 in hepatoma carcinoma cells migration, we resuscitated HepG2- pcDNA3.1 cells and HepG2-pcDNA3.1-pp32 cells previously constructed, we sifted cells with 800μg/ml G418 for three weeks, and then we sustaining cultured cells with 400μg/ml G418. The ability of cell migration was detected by transwell assays, and the ability of cell adhesion was detected by adhesion experiments. The ability of cell proliferation was determined by clongenic assays, and cell apoptosis was determined by flow cytometries. The expression of vinculin in HepG2, HepG2-pcDNA3.1 and HepG2-pp32 were detected. Whereafter, the impact of pp32 on the metastasis of HepG2 cells of nude mice in vivo was primarily examined by experiments of injecting cells via tail vein. The impact of pp32 on the proliferation of HepG2 cells of nude mice in vivo was examined by tumor xenograft experiments.In addition, some studies have found that there are pp32 mutants in prostate such as pp32r1 and pp32r2, so we identified the impact of pp32 on the highly metastatic prostate cancer cell lines Pc3M. We shaked the bacilli of pcDNA3.1 and pcDNA3.1-pp32 previously saved, after the extraction of plasmids, plasmid pcDNA3.1 and plasmid pcDNA3.1-pp32 were transfected into Pc3M cells, after 48 hours we sifted cells with 700μg/ml G418 for three weeks in order to construct stable Pc3M-pcDNA3.1 and Pc3M-pcDNA3.1-pp32 cell lines. Pc3M-pcDNA3.1 and Pc3M-pcDNA3.1-pp32 cells were cultivated with 350μg/ml G418. We have identified the impact of pp32 on Pc3M cells traits in aspects of morphologic changes, migration, adhesion and proliferation in vitro.Results: Western blots showed that the expression of pp32 was detected in all of 15 cell lines. Pp32 over-expression reduced the migration and adhesion abilities of HepG2 cells. Pp32 inhibited the expression of vinculin in HepG2 cells. In vivo primary experimental results showed that pp32 might inhibit HepG2 cells metastasis in nude mice. Compared to HepG2-pcDNA3.1 cells, the tumor size formed by HepG2-pp32 cells in the nude mice armpit had no significant differences.Pc3M cell lines stable over-expressing pp32 (Pc3M-pp32) were obtained, pp32 could lead to shape changes of Pc3M cells, but did not affect their malignant phenotypes. Pp32 also led to promotion of the migration and adhesion of human prostate cancer cells Pc3M. The role of pp32 in cells migration of HepG2 and Pc3M cells is different, which was very interesting and gives us challenges.Conclusions and analysis:Pp32 is a multifunctional protein, which not only plays a role as a nuclear protein, but sometimes pp32 is located in the cytoplasm to take action. It is not only involved in tissue development, signal transduction, transcriptional regulation and other physiological processes, but also can promote cell apoptosis, inhibit tumor growth, inhibit the activity of PP2A and have other functions. Our studies showed that pp32 could inhibit cell migration characteristics of hepatoma carcinoma cells lines HepG2, but pp32 could stimulate Pc3M cell migration. Studies of pp32 regulating cell migration are not only conducive to the development of tumor biology, but also contribute to find new pathways to the treatment of human cancer. |
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