The Expression Of TAp73 And Beclin1 In Human Osteosarcoma And Their Clinical Significance

Background and objection:Osteosarcoma is the most common primary bone malignant tumor, which commonly affects children, adolescents and young adults, the rate of disability and mortality is very high. At present, the main clinical treatment is surgery combined with neoadjuvant chemotherapy. But according to a worldwide study, in the past 30 years the overall efficacy of osteosarcoma did not significantly improve, the overall survival rate is about 60%, there is still high rate of postoperative tumor recurrence and metastasis, many patients died result in postoperative tumor metastasis, most of which are lung metastasis. Today, for the embarrassing situation, and the main reason is that osteosarcoma has become resistance to the current conventional chemotherapy drugs, which occurs bad outcome of chemotherapy after surgery, and patients’ prognosis is not ideal. Therefore, so many studies are applied in order to explore new treatment approaches, so as to improve the curative effect of osteosarcoma, that has become a hot research. In recent years, the study found that: the relationship between the regulation of autophagy and tumor occurrence and is much closely, for example, the differentiation and proliferation of tumor cells, cells apoptosis, drug resistance, tolerance to radiotherapy and tumor stem. Moreover, some scholars have found that autophagy also regulates the growth and resistance of osteosarcoma. However, the specific mechanism is how autophagy to adjust the growth and development of osteosarcoma? How to participate in the resistance of chemotherapy? Up to now, there was a few authoritative literatures on these regulatory mechanism, which was still a puzzle. So our research group believes that through the study of osteosarcoma cell autophagy adjustment mechanism, clarify autophagy in malignant tumor growth and the mechanisms of drug resistance. And then, through regulating autophagy inhibits osteosarcoma growth and promote chemotherapy sensitivity, which may be a way to increase the sensitivity of osteosarcoma chemotherapy.Autophagy is refers to the formation of autophagosomes from rough endoplasmic reticulum ribosome free attached bilayer packages of the abscission zone cell matter and cell domestic demand degradation of organelles, protein and other ingredients, and fused with lysosomes to form the autophagy lysosome. The contents of the degradation of the package, in order to realize the update of the cell’s metabolic needs and some organelles. Autophagy is generally activated in the body under stress conditions such as starvation, hypoxia, inflammation cells, and it is considered to be an important mechanism for cell survival, maintaining cell homeostasis. Although it is a protective mechanism, maintaining cell homeostasis, and excessive activation of autophagy can lead to cell death, called type Ⅱ programmed cell death. Autophagy can be seen in physiological and pathological processes in the organism, there is a close relationship between autophagy and tumor, the role of autophagy in tumor is two-way, both to promote tumor growth, can also inhibit tumor growth, the specific regulatory mechanism has not been fully elucidated. On one hand, some scholars think that autophagy can induce drug resistance and anti-radiation effect, promote tumor growth; on the other hand, there are some autophagy modulators used in tumor clinical trials, and achieved good results. What’s more, further research found that:Although autophagy can promote some tumors for their occurrence, growth, drug resistance, tolerance to radiation. As in other tumors, autophagy not only did not promote tumor, even inhibit tumor growth and progression. In recent years, the study of osteosarcoma also found that autophagy had a moderating effect on osteosarcoma, but the mechanism about how to adjust has not been illustrated. To explore the role of autophagy in osteosarcoma, looking for a precise pathway for the treatment of osteosarcoma by regulating the autophagy is the purpose of the following research.According to the studies:p53 participates in the regulation of autophagy in tumor cells. Rosenfeldt’s papers published in nature and New England J Med infer autophagy in tumor cells with wild type p53 can promote the effect of tumor growth, and in tumor cells with mutant p53, the regulation of autophagy is a kind of tumor suppression mechanism. Rao, Mansilla and Chakradeo also found that p53 is a key regulator of autophagy, in p53 deficient tumor cells, autophagy can increase the efficacy of anthracycline anticancer drugs. P53 is a kind of important antitumor gene, but p53 gene in malignant tumors generally found mutation or deletion. The deletion or mutation of p53 gene is an important genetic marker for osteosarcoma. In tumor cells and animal experiments, people found tumors with p53 gene deletion and transfected with recombinant wild type p53 gene can inhibit tumor growth and release drug resistance, but the human gene therapy in transfection is still efficiency, stability, specificity, carrier security still exist many problems, and the gene therapy strategy is still very difficult to be used in clinical.P73 is a new member of the p53 family, and most scholars believe that it is the ideal replacement for p53 to solve the above problems. The p73 gene is located on human 1st chromosome 1p36, and is found in many human cancer research, the majority of tumor gene deletion is common, but there are a number of tumor suppressor gene located in human 1p36 region. Because of the particularity of the location of p73 gene and its high homology with the p53 gene structure and function, so a majority of researchers believe the p73 gene is a new tumor suppressor gene. P73 protein widely exists in eukaryotic cells, it is involved in the regulation of cell cycle, cell apoptosis, cell growth, differentiation and aging, neural development process. In many tumors p73 involved in tumor drug resistance, anti-radiation, it can also regulate the sensitivity of tumor cells to chemotherapeutic drugs, improve the drug effect of chemotherapy. In addition, p73 is closely related with the DRAM, can also regulate autophagy through inducing the activation of DRAM. Even some literatures reported that p73 can directly regulate autophagy related factors, including Atg5 and Atg7.TAp73 is a full-length p73 subtype, with the similar structure and antitumor function to the wild type p53. TAp73 can promote the differentiation of osteosarcoma and malignant tumor suppression, inducing the activation of TAp73 by specific ways which can improve the sensitivity of tumors to chemotherapy and radiotherapy. But the existing studies reveal that regulation of autophagy involved TAp73 gene in tumor, but its mechanism has not yet been elucidated completely. The most important thing is that, compared with p53, the expression of TAp73 is very stable, very few mutations. A study found that the specific activation of TAp73 in p53 gene deletion tumor cells, TAp73 can activate some downstream signaling pathway of p53 gene, thereby inhibiting the tumor growth and development, and promote the apoptosis of tumor. Autophagy regulates tumors by inhibiting signaling pathways downstream of p53 gene family, p53 gene family and TAp73 are also involved in the regulation of anti-tumor signaling pathway. Therefore, whether there is some relationship between TAp73 and autophagy of tumor? To explore the expression of TAp73 and autophagy related gene in human osteosarcoma can do this to solve the mystery relationships of TAp73 and autophagy, which is helpful to further elucidate the specific mechanisms about TAp73 regulates autophagy, provide basis for further study.Autophagy related factor Beclinl homolog of yeast Atg6/Vps30 gene, also known as BECN1, plays a very important role in tumor cell autophagy. In 1998, Liang et al in the sinbis virus rats found a protein with a molecular weight of 60kDa, and interacting with the expression of bcl-2 gene product Bcl-2 and inhibiting sinbis virus to copy. They get this protein gene name Beclinl. Beclinl gene is located in human chromosome 17q21 and contains 12 exons and Beclinl protein contains three domains, BH3, CCD and ECD are included, these domains are the sites that Beclinl interact with other proteins. Beclinl is an essential molecule for autophagosome formation, it can mediate other autophagy protein located in the phagosome, and regulates formation and mature of autophagy in mammalian. In the process of autophagy Beclinl expression level is often a rising state. The overexpression of Beclinl induces the phosphatidylinositol 3 kinase family III up-regulated, and PI3KC1 (P110 alpha) and the expression of its downstream of p-PKB is down regulated and induced the activity of tumor cell autophagy, inhibits the growth and proliferation of tumor cells, produce anti-tumor effect. Beclin1 is autophagy regulation in the main one of the molecules, it can apply different regulation mechanisms for the regulation of autophagy activity. That is to say the expression of Beclin1 in the tumor is a kind of embodiment of the autophagic activity, can describe the autophagy activity of tumor by detecting the expression of Beclinl. The expression of Beclinl in clinical in many tumors there has been a lot of literature, but in human osteosarcoma and the expression of specific regulation has not been reported in detail.The advantages of our study design:TAp73 belongs to the p53 anti-tumor gene family. P53 mutation or deletion is one of the causes of OS, at the same time, it also induces drug resistance in osteosarcoma, and p53 is also a target for the treatment of osteosarcoma. This study was to explore the relationship between TAp73 and tumor autophagy regulation, trying to find an accurate way for the treatment of osteosarcoma by regulating autophagy. The study infers TAp73 expression is stable, less prone to mutation, and with a high similarity to the wt-p53 structure and antitumor function. With TAp73 as the breakthrough point, by exploring its relationship with autophagy, not only can regulate TAp73 stability and protein content, activate its specific function, but can also modulate autophagic activity, and ultimately achieve the inhibition of tumor growth. The results of this thesis provides the basis for the next step to explore the mechanism of the regulation of TAp73 on autophagy.This study intends to solve the following problems:(1) The expression of TAp73 and Beclinl protein impacts on overall survival in osteosarcoma patients? (2) In human osteosarcoma, whether the expression of TAp73 protein will influence the expression of Beclinl protein? (3) How the correlation between the expression of TAp73 and Beclinl in human osteosarcoma?To solve these problems, the experiment using immunohistochemistry detection the expression of TAp73 and Beclinl in human osteosarcoma, combined with the clinical data of patients with osteosarcoma, using statistical methods to analysis, the relationship between TAp73 and autophagy, and the effect of autophagy in osteosarcoma, so as to provide experimental basis for clinical application.Methods:48 osteosarcoma patients were collected for our study. Patients were followed up by regularly, through the ways of phone calls or appointment to the hospital, and wrote detailed records of all patients with osteosarcoma about their progress of the disease. Use immunohistochemistry way to detect the expression of TAp73 and Beclin1. Combined with clinical data, analysis the difference of the expression of TAp73 and Beclinl in gender, metastasis and 5 year survival rate, and use statistical methods to analyze their relationship in human osteosarcoma.Results:1. The expression of TAp73 and Beclinl is helpful to the patients’ prognosisIn human osteosarcoma, positive expression of TAp73 and Beclinl was positively correlated to patients’ survival time(r=0.673, P=0.000; r=0.388, P=0.003), TAp73 and Beclinl expression in patients with better overall survival than patients without the expression of TAp73 and Beclinl(80.8%vs50.0%, P=0.000; 80.0%vs44.4%, P=0.001), what’s more, the increasingly high degree of expression of TAp73、 Beclinl, patients with better prognosis. Results showed that the positive expression of TAp73 can inhibit malignant transformation and enhance sensitivity to chemotherapy drugs in osteosarcoma and help in the prognosis of the patients; the upregulation of autophagy activity helps to inhibit the development of osteosarcoma, induced autophagy cell death in osteosarcoma and improve the overall survival of patients.2. TAp73 induce the expression of Beclinl proteinIn human osteosarcoma TAp73 protein positive expression and negative expression, which impacts the expression level of Beclinl protein(28.35>19.95, Z=-2.146 P=0.032). And both proteins’ degree of expression were positively correlated(r=0.470, P=0.001). Results showed that TAp73 can induce the expression of Beclin1 protein and up-regulate the activity of autophagy.All experimental data were handled using statistical software SPSS20.0 statistical analysis. Rates between groups were compared by analysis of χ2. Correlation analysis using Spearman rank correlation analysis, Kaplan Meier method calculating the study in all patients overall survival rate, survival rate compared using the log-rank test. P<0.05 as statistical significance.Conclusion:1 Osteosarcoma patients with the expression of TAp73, Beclinl protein, which can inhibit tumor development, and help to improve patients’ overall survival;2 The expression of TAp73 and Beclinl and survival time of patients were correlated in osteosarcoma, and the higher degree of expression, and the better prognosis of patients.3 In clinical work, to detect the expression of TAp73 and Beclinl protein in osteosarcoma patients, help to assess the prognosis of the patients, and the adjustment of postoperative chemotherapy;4 The expression of TAp73 in osteosarcoma, can induce the expression of Becllinl protein, and up-regulates the autophagic activity of osteosarcoma cells;5 Whether TAp73 can be a new tumor therapeutic target by regulating autophagic activity in the treatment of osteosarcoma should be further explored, and next step may pay attention to the specific mechanism of how TAp73 regulates autophagic activity.