| Background:Osteosarcoma (OS) is the most common malignancy in children and adolescents. In 1879, Gross firstly reported a typical OS. Although there are many new chemotherapy drugs used to therapy OS, it is still a clinical challenge. In the past 20 years, the survival rate of patients with OS did almost not increase. Currently, molecular targeted therapy is the most promising therapy for cancer. In future, molecular targeted therapy can directly target against genes related to tumorigenesis, development and metastasis and so on. This method has high quality in selectivity, high affinity when combined with the target molecule, then efficiently and selectively kill tumor cells with less adverse effects. But so far, clinically, there is no any drug targeted gene in OS. Thus, future research on molecular mechanisms involving tumorigenesis and development and then to develop new drugs targeted genes in OS becomes urgent.OS genesis and development are closely related to the proto-oncogenes and tumor suppressor genes which are balancing in normal tissues. In OS, up to now, tumor suppressor genes, p53 and Rb are the most interesting. Two downstream genes of p53 gene, bax and puma determine, the induction of apoptosis pathway in cells. Apoptotic signaling makes them translocation from the cytoplasm to the mitochondria, inducing the release of cytochrome C, thereby resulting in cell apoptosis. A nuclear molecular, p21 is also a downstream molecular of p53. p21 plays a tumor suppressor role in the progress of cell cycle through inhibition of cell proliferation in OS. However, studies have shown that mutant p53 gene which loses a supervisory role in tumor cells results in a variety of human tumors, even plays a pro-oncogene role in vivo. Studies also showed p53 gene loss and mutation rate is as much as 75% in OS.Mutation or deletion of p53 gene easily loses its tumor suppressor function, and the role of Rb gene in OS is still controversial. Fortunately, p73 gene, a family member of p53 gene, was found in 1997. p73 gene encodes a product with similar structure to and function of p53 protein, which also have transcription activity similar to p53 protein. But different from p53, mutation and deletion of p 73 gene was rarely found.p73 can substitute p53 functions to induce the expression of endogenous p21, puma, bax genes. Thus,p73 is a very promising molecular target in OS. But p73 gene has many transcriptional variants, of which only TAp73 protein has an anticancer activity. But noted, our group and other scholars have found TAp73 transcriptional activity and antitumor functions are regulated by many phospho-kinases in different tumors. Thus, determining the kinases catalyzing TAp73 phospho-modification in OS is a facing challenge, and a key point to this event. As we know, these kinases will be the promising therapeutic molecular targets.Currently, as following the bioinformatics application in medical field, scientific researchers have opened up a broad prospects in tumor researches. In addition, the biochip of genes is another important application of bioinformatics in biomedical research. Biochips are carried out high-throughput testing interesting molecules according to the studies, and through scanning the image of hybridization chips, then processing image signals and finally obtain experimental data, which are reliable, extensive and accurate results. Up to now, many researchers successfully analyzed a lot of protein modification processes through the bioinformatic database and software, and ultimately confirmed by experimental verification. Thus, these bioinformatic databases and softwares are really effective research tools such as protein phosphorylation prediction software KinasePhos, Scansite, GPS and so on., Nobel laureate In 1991 W. Gilbert said, the traditional way to solve scientific problems is conducted only by experimental research which is time-consuming and laborious; but now, after the human genome deciphered, new way of biomedical research is about to change; it will make the scientists initiate from theoretical speculation, then go to lab experiments to verify or track these theoretical assumptions. Scientific study, therefore, is no longer blind without direction, no more than looking for a needle in a haystack.Therefore, the application of bioinformatics combining with experimental studies will result in brilliant achievements in the 21st century. And it will create an incalculable economic and social benefit. In this study, application of bioinformatic tools and database analysis combined with experimental research to screen phosphor-kinase and verify its catalysis of TAp73 protein phosphorylation in OS cells aims to explore the biological function of the interesting kinase and its molecular mechanism, as a preliminary observation molecular therapeutic effect for OS therapy.Purpose:Basing on the theoretical and experimental researches, it is to observe the expression of suppressor gene p73 and the phosphor-kinase profile in OS cells; to analyze the characteristics of modified TAp73 protein activity and screen the interesting phosphor-kinase catalyzing TAp73 protein phosphorylation Using bio-informatics and determining its functions in OS and biology effects for OS gene targeted therapy; to explore new targets and develop new methods for molecularly targeted OS gene therapy in clinical application.Method:1. Basing on bioinformatics analysis, it characterizes the known and unknown potential phosphor-sites in TAp73 protein, and their functional regulation.By searching bioinformatic database, the datasets of TAp73 protein phosphorylation were collect to analyze the characteristic of TAp73 protein modification and summarize the regulations of its function according to different environments and responding phosphor-kinases, providing the basis for later functional analysis. Furthermore, the newest version of the online bio-software, Scansite 3.0, was used to analyze all phosphor-kinases catalyzing potential phosphorylation sites in TAp73 protein and their applicable conditions.2. Determination of the phosphor-kinase expression profile and screening the interesting phosphor-kinase catalyzing TAp73 phosphorylation in OS cells by cDNA biochips.Total RNA was extracted from p53null Saos2 cells under normal culture. NanoDrop ND-1000 was used to quantify and quality total RNA. Denatured agarose gel electrophoresis was used to assess RNA integrity. Thereafter, the Invitrogen SuperScript ds-cDNA synthesis kit was used to reversely transcript 5 μg of total RNA into the first double-strand cDNA (ds-cDNA) by 100 pmol Oligo (dT) reverse primer. Then the ds-cDNA was quantified and qualified. The US NimbleGen Systems monochrome DNA labeling kit was used for ds-cDNA fluorescently labeling. And 4 μg labeled ds-cDNA was taken to incubate with NimbleGen Hybridization system, Roche NimbleGen 12 x 135K human genes microarray in which covers 45,033 referencing to the NCBI database, at 42 ℃ for 16 hours. After washed with the appropriate eluent, Axon GenePi x 4000B microarray scanner was used to scan the hybridization chips. The expression data from scanning was analyzed according to NimbleGen Gene Expression Analysis protocol, and subsequently screened the most promising phosphor-kinases. Finally, SYBR GREEN fluorescence Quantitative Real-time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) was used to verify the data from microarray experiments in OS cells. Polo-like kinase 2 (PLK2) was screened out as the most potential target gene in OS cell.3. The phosphor-sites predicting bioinformatic software, GPS 1.0, was used to analyse PLK2 catalyzing phosphor-sites in TAp73 protein, and experiments determined these results.The most promising phosphor-kinase, PLK2, was theoretically analyzed its catalyzing sites in TAp73 using bioinformatic software, GPS 1.0, in which the result provides a theoretical guide for subsequent experiments. Accordingly, co-immunoprecipitation (Co-IP) experiment was performed to determine whether PLK2 can physically bind to TAp73. Phosphor-tag Western Blot (WB) detected the endogenous TAp73 phosphorylation. WB Experimental groups include:control group, blank siRNA group, siPLK2 group, PLK2 inhibitor group (5μg/ml, the same below), blank siRNA+DNA damage drug (25μg/ml, the same below) group, PLK2 inhibitor +DNA damage drug group, siPLK2+DNA damage drug group, siPLK2+siTAp73 +DNA damage drug group. On the other hand, in vitro kinase assay was used to detect ectopic TAp73 or point-mutation TAp73 phosphorylation after incubated with PLK2. According to the analyzing result from bioinformatics, point-mutation TAp73 protein, TAp73(T27A) and TAp73(S48A) were produced, and their phosphorylation were also detected by phosphor-tag WB assay to determine the catalyzed site by PLK2.4. Mechanism of interaction between PLK2 and TAp73 at post translation level.RT-qPCR and WB were performed to detect whether TAp73 regulates PLK2 mRNA expression and protein translation in OS cells. On the other hand, whether PLK2 regulates TAp73 and its downstream gene expression were also detected by RT-qPCR and WB. Experimental groups include:control group, blank siRNA group, siPLK2 group, PLK2 inhibitor group, blank siRNA+DNA damage drug group, PLK2 inhibitor+DNA damage drug group, siPLK2+siTAp73+DNA damage drug group. At the same time, Protein half-life experiment and confocal immunofluorescence assay were conducted to observe the mechanism of interaction between TAp73 and PLK2; the experimental groups including the control and treatment groups.5. Effects of PLK2 interacting with TAp73 on OS at cell or animal levels.PLK2 plays a role in OS cell physiology through TAp73 was observed. CCK-8 cell proliferation assay detected the effects of PLK2 on OS cell proliferation via TAp73; Flow cytometer (FCM) was used to observe PLK2 affecting OS cell cycle progression; Terminal Deoxynucletidyl Transferase dUTP Nick End Labeling (TUNEL) experiment detected PLK2 regulating cell apoptosis through TAp73; Cell wound-healing assay was applied to observe PLK2 effects on cell invasiveness. These experiments’groups include:control group, blank siRNA group, siPLK2 group, PLK2 inhibitors group, siRNA+DNA damage drug treatment group, PLK2 inhibitor +DNA damage drug group, siPLK2+siTAp73+DNA damage drug group.Construction of an OS model on nude mouse was done. PLK2 inhibitor effecting on OS growth was observed. HE staining and immunohistochemical staining applied to OS tissue samples in order to preliminarily learn the cell and molecular pathology signatures of OS. Self-control of paired match experiments included 3 separate groups:Saos2 OS group treated with DNA damaging drug CDDP (25μg CDDP per g mouse weight, the same below); Saos2 OS group treated with CDDP+PLK2 inhibitors (5μg per g mouse weight, the same below); Saos2-TAp73KO OS group treated with PLK2 inhibitors. Each group has 6 nude mice. After successfully constructed an experimental OS model, pre-treated tumors’ size was recorded by measuring the maximum (a, mm) and minimum diameters (b, mm). Then totally 7 times drugs were administered in four weeks. Thereafter, these tumors’ sizes were measured again. Tumor volume was calculated using the formula V (mm3)=1/2ab2. Tumor volume changing from pro-treatment to post-treatment was used to assess therapeutic result. Preliminary observation of H&E staining and immunohistochemistry staining on OS tissue samples were performed. Cell and molecular pathology of osteosarcoma after treatment targeted to PLK2 were discussed, expecting to provide a guide for clinical applications in OS therapy.Result:1. The potential regulation of TAp73 phosphor-modified effects on its function was built up through summarizing all known phosphorylation sites in TAp73 protein. All unknown potentially catalyzed phosphor-sites in TAp73 protein were analyzed by bioinformatic software, Scansite 3.0.After retrieving three common biomedical databases in English such as Pubmed, Embase, Ovid database, and three common databases in Chinese, combining the result of online protein phosphorylation database PhophositePlus.com, total 14 phosphor-sites in TAp73 and their corresponding 11 phosphor-kinase were determined. Of that,5 locate at transactivation domain including TA1 and TA2, and 9 locate non-transactivation domain. Moreover, phosphorylation sites locate at TAp73 transactivation domains will be reduced its transcriptional activity; on the other hand, phosphorylation sites locate at TAp73 non-transactivation domains would positively regulate TAp73 functions.For unknown potential catalyzed phosphor-sites in TAp73 protein by all known phosphor-kinases in human cells, the latest version of protein phosphorylation prediction software Scansite 3.0 was run. Analyzing result under high threshold match shows that a total of 18 potential phosphor-sites,3 kinase-binding sites. Of 18 potentially modified sites, there are 2 catalyzed by acidic phosphor-kinase,7 by alkaline phosphor-kinase,2 by proline phosphor-kinase,7 by other kinase. In addition, the acid kinase catalytic sites located at TAp73 transactivation domain; and the rest had no obvious signature. According to the regulation mentioned above, the 2 potentially modified sites catalyzed by acid phosphor-kinase will negatively regulate TAp73 functional activity.2. Expression profile of phosphor-kinase in OS cell was successfully built up by scaning cDNA biochips. Phosphor-kinases with high expression level in OS were indicated. PLK2 would be the most potentiated phosphor-kinase targeted at TAp73 protein.Roche NimbleGen 12 x 135K human microarray covering 45,033 human genes from the NCBI database. The expression data on microarrays were analyzed using NimbleGen Gene Expression Analysis protocol and in accordance with US company NimbleGen Systems genetic data quality analysis. A qualified data was obtained. It is a total 269 phosphor-kinases from 434 reported phosphor-kinases in human cells was significantly expressed in OS cells according to the standard of expressing signal more than 200. Of that,77 phosphor-kinases were reported as cytoplasmic localization,48 nuclear,38 membrane,30 nuclear-cytoplasmic,24 membrane-cytoplasmic,6 cytoskeleton,3 mitochondrial,2 Golgi apparatus,2 ribosome,2 chromosome,1 centriole. Ten phosphor-kinases with the highest expression in OS cells were CK1, CAML1, CK2, CAML2, CDK8, PLK2, CLK3, MARK, CDK4, PLK1. Four acidic phosphor-kinases including CK1, CK2, PLK1, PLK2 are arranged at high level expression. Furthermore, in consistent with the microarray data, PLK2 was implied as a most potential phosphor-kinase targeting at TAp73 protein in OS cells independent of p53 gene using RT-qPCR validation.3. Through running the phosphorylation site-prediction software GPS 1.0, PLK2 catalyzing phosphor-site in TAp73 was successfully determined. In vitro and in vivo experiments confirmed PLK2 catalyzing TAp73 protein phosphorylation and its corresponding sites.Previous researches have demonstrated, in different context, acidic phosphor-kinase PLK1 and CK2 can catalyze phosphorylation of TAp73. The polo-like specific-site prediction software GPS 1.0 was run for PLK2 to analyze TAp73 protein sequence. Under high threshold match, results show a total of 4 potential phosphorylation sites and 2 kinase-binding sites. Combined the results of foregoing analysis, site T27 or S48 located TAp73 transactivation domain is the most potential site catalyzed by PLK2. Based on the above bioinformatics analysis, it is a hypothesis that PLK2 catalyzes TAp73 phosphorylation at site T27 or S48 located at transactivation domain, and thereby negatively regulating TAp73 function in OS. Thereafter, in consistence, Co-IP experiments determined PLK2 and TAp73 physical binding to each other. And TAp73 protein phosphorylation was detected by phos-tag WB which depended on TAp73 abundance in OS cells. Moreover, after TAp73 point-mutate (alanine residue substituting mutation-residue) or flag-TAp73 purified and incubated with flag-PLK2 conducting to in vitro kinase assay, the results indicated that flag-PLK2 can catalyze TAp73 and TAp73(T27A) phosphorylation but TAp73(S48A). Thus, PLK2 catalyzed TAp73 phosphorylation was successfully determined, and its catalyzing site is S48 locating at TA1 domain in TAp73, which is a new find.4. Both PLK2 and TAp73 interacted with each other at post-translation level.As mentioned above, bioinformatic analysis and molecular, assays determined PLK2 catalyzing phosphorylation of TAp73 under high abundance. Experiments in this part were supported in osteosarcoma cells PLK2 can inhibit transcriptional activity of TAp73, and both interacted with each other at post-translation level. Firstly, RT-qPCR results showed that comparing with in control group, in Saos2 cells under normal culture condition with low level of TAp73, mRNA relative expression level of TAp73 downstream genes, p21 and puma had no significant difference among in PLK2 inhibitor or siPLK2 treatment group (P> 0.05), as well as PLK2-vector transfected group (P> 0.05); but in DNA damage drug treated group, p21, puma mRNA levels were significantly increased (P< 0.01). In addition, compared with in single DNA damage drug-treated group, in siPLK2+DNA damage drug-treated group, PLK2 inhibitor+DNA damage drug treated group, they were significantly increased (P< 0.01); however, siPLK2+siTAp73+DNA Damage drug-treated group had no significant difference (P> 0.05). Differently, in MG63 cells under normal culture condition with high level of TAp73, single PLK2 inhibition would result in significantly increasing p21 and puma expression. Secondly, WB experimental results are consistent with the RT-qPCR results. Thirdly, Confocal immunofluorescence assay results in Saos2 cells showed that in the control group TAp73 and PLK2 fluorescence signals uniformly distributed in the cytoplasm and nucleus, whereas in siRNA silencing PLK2 gene, TAp73 fluorescence signals were mainly distributed in cell nucleus. Moreover, half-life assay of nascent PLK2 in Saos2 cells demonstrated that in maternal Saos2 cells PLK2 half-life was about 20 minutes, which were shorten to about 15 minutes in stable or transient siTAp73 transfected Saos2 cells.5. PLK2 regulates OS cell physiology through rich abundance of TAp73 protein, supporting PLK2 could be a new potential molecular target for OS therapy. A preliminary observation of nude mouse mode also indicated a promising insight for clinically therapy tumors with high expression of TAp73 protein.PLK2 regulates OS cell physiology through TAp73. Cell cycle progress tested in Saos2 cells by Flow Cytometer showed that compared with in control group, in the experimental groups treated with PLK2 inhibitors or siPLK2, G1 phase cell proportion was reduced (P< 0.01); while groups treated with DNA damage drugs, G1 phase cell proportion was significantly increased (P< 0.01). On the other hand, Compared with in single DNA damage drug-treated group, G1 phase cell proportion was significantly increased in siPLK2+DNA damage drugs treated groups, as well as PLK2 inhibitor+DNA damage drugs treated groups (P< 0.01). TUNEL assay showed that there are no significant difference among the groups of control, single PLK2 inhibition in the maternal Saos2 and U2OS cells(P> 0.05); percentage of apoptotic cells in DNA damage drug-treated group significantly increased (P< 0.01). Also, Comparing with in single DNA damage drug-treated group, PLK2 inhibition+ DNA damage drug-treated groups were significantly higher proportion of apoptotic cells (P< 0.01), but this situation disappeared in siPLK2+siTAp73+DNA damage drug-treated group.CCK-8 is a well kind of cell proliferation experiments, in which results showed that OS cell groups with low level of TAp73 treated with PLK2 inhibitor or siPLK2 was significantly increased in optical density (OD) (P< 0.05); single DNA damage drug-treatment would significantly reduce the cells OD (P< 0.01). Compared with single DNA damage drug-treated group, siPLK2+DNA damage drug-treated group was significantly decreased in OD value (P< 0.01), especially in Saos2 cells. In addition, in Saos2 cell wound-healing assay, single PLK2 inhibition healed the scars faster than maternal Saos2 cells. They would heal within three days. Cells treated with DNA damage drugs would be difficult to completely heal in 3 days. Cells treated with siPLK2+DNA damage drug could not heal scars at last, and accompanying with numerous apoptotic cells. But these cells pre-treated with siTAp73 can completely heal in 3 days.Experiments above at molecular and cell level showed PLK2 can affect OS cell survival and proliferation and migration under high abundance of TAp73. Nude mouse mode was used to preliminarily observe the therapeutic effect on OS when targeted at PLK2 in vivo and characterize OS in cell and molecular pathology. After treatment, tumor volume of TAP73wt OS in CDDP (25 μg per g mouse weight, the same below) treatment group, CDDP+PLK2 inhibitor (5μg per g mouse weight, the same below) treatment group were significantly reduced when respectively comparing their own pre-treatment volume (P<0.01). They shrank from pre-treatment volume 578.200 ± 74.780 mm3,537.976 ± 118.333 mm3 to post-treatment volume 87.835 ± 31.603 mm3,237.957 ± 122.884 mm3, respectively. So, tumor volume reduction in CDDP+PLK2 inhibitor treatment group is more evident. In contrast, tumor volume of TAp73null OS with single PLK2 inhibitor treatment grew up from pre-treatment 628.068 ± 201.036 mm3 to post-treatment 2280.518 ± 1050.480 mm3. This result indicated that therapy targeting at PLK2 can enhance CDDP therapeutic effects on TAp73wt OS, but no effect was observed in OS with absence of TAp73 expression. Besides, HE staining examination in cytopathology showed that TAp73wt OS cells after PLK2 inhibitor treatment were typically large in morphology and more proportion in cytoplasm and small nucleus, with more connective tissue, when comparing with TAp73null OS after PLK2 inhibitor treatment. Immunohistochemistry staining in molecular pathology results indicated that TAp73wt OS treated with CDDP combining PLK2 inhibitor have obvious positive nuclei TAp73 signals. Thus, these results provide an experimental basis and promising insight to future clinical patients with OS and other tumor. Conclusion:1. In this study, totally known 14 phospholylation sites in TAp73 and their corresponding kinase was determined after literature reviewing and bioinformatic analysis. The distribution signature of all known phosphor-sites in TAp73 protein is of which 5 locate at transactivation domain including TA1 and TA2, and 9 locate non-transactivation domain. The potential principle of TAp73 protein activity regulated by phosphorylating modification was found:phosphorylation sites in TAp73 at transactivation domains will be reduced its transcriptional activity; on the contrary, phosphorylation sites in TAp73 at non-transactivation domains would enhance TAp73 functions. This would not only provide a basis for function prediction of TAp73 protein chemically modified, but also to give an insight to future use of gene therapy targeted at TAp73.2. Applying the high throughput cDNA biochips to screen gene targets for OS therapy, this study builds up a phosphor-kinase expression profile and screens out 10 phosphor-kinases with high expression level in OS. This result provides a platform for future study on molecular mechanisms of OS genesis and development, and on signaling pathways in OS cells. Eventually, experiments also imply that CK1, CK2, PLK1 and PLK2 are the potential key phosphor-kinases in OS cells. Besides, in this research, PLK2 was first reported as a key regulator to TAp73 function by phosphorylation.3. This research based on a hypothesis derived from analysis of bioinformatics resource, which successfully guided the actual experiments. This provides a fresh direction to research mode. Furthermore, for the first time, it was found that PLK2 phosphorylates TAp73 with high abundance, and also determined a new phosphorylation site S48 in TAp73 located at TA domain which is in accordance with the principle mentioned above. Still, this research explore the mechanism of PLK2 interacting with TAp73 in OS cells:PLK2 physically binding to TAp73 prolong itself half-life, and prohibiting TAp73 nucleus translocation and function as a tumor suppressor to activate cell fate related genes, p21 and puma, expression through phosphorylation; so that OS cells rapidly go through G1 phase, inhibit apoptosis and accelerate cell proliferation and migration. Thus, PLK2 would be a potential therapeutic target in OS.4. It was found that, both in vitro and in vivo, PLK2 affects OS cell survival and proliferation through interacting with abundance of TAp73 upregulated by DNA damaging drugs, while PLK2 inhibition can rescue TAp73 anti-OS activity, and enhance DNA damaging drugs CDDP, ADM effects on anti-OS. However, in the absence of TAp73 or at low abundance of TAp73, it was not observed the therapeutic effect of PLK2 inhibition on OS cell physiology and OS tumor growth. Besides, preliminary observation of typical OS cell and molecular pathology was concluded. These results would provide the experimental and theoretic evidence to guide to clinically therapy OS and other malignant tumors with characteristic of high abundance of TAp73. |
PDF Full Text Request