Research On The Effects Of ARHI Gene On Biological Behavior Of Osteosarcoma Cells And Related Mechanism

Osteosarcoma is a common primary malignant bone tumor during childhood and adolescence. Early can occur broad lung metastases, high mortality, is one cause of death in children and adolescents. The overall incidence statistics are quite different in different countries, about 10/100 thousand to 1/100 thousand. Although the current new adjuvant chemotherapy and surgical techniques has been further developed and significantly improved the survival of patients with osteosarcoma, but some patients’ chemotherapy effect is still poor, prone to recurrence and metastasis. In recent years,with the development of molecular biology theory and experimental techniques,in-depth research into gene therapy of osteosarcoma has turned to experimental stage;research of expression of tumor associated genes from the molecular level has important significance to the occurrence, evolution, outcome and treatment of tumor.In recent years, several new progresses occurred in the research of tumor signal transduction pathways and biological mechanisms of gene targeting therapy that provides a new way to prevention and treatment of osteosarcoma.ARHI(aplasia ras homologue member I), is a maternal imprinted genes. It is also called NOEY2, was first discovered in 1999. Recent studies showed that the protein encoded by ARHI indicated different expression in multiple tissues of human such as pancreas, breast and ovary. However, it is manifested in the loss of expression or down-regulated in many tumors like pancreatic cancer, breast cancer, ovarian cancer,liver cancer, and bladder cancer, which suggesting that ARHI gene is relevant to the occurrence and development of tumor. ARHI gene as a tumor suppressor gene plays an important role to the regulation of growth and proliferation, invasion and migration of tumor cell, even the induction of apoptosis and interventions of cellular signal transduction pathways. ARHI as a new target gene provides a new thinking way tomolecular tumor gene therapy. Application of ARHI gene in tumors is still in the initial stage, there is no relevant research literature concerned with ARHI gene’s expression in osteosarcoma and effects of gene ARHI to osteosarcoma’s biological characteristics in the field of domestic and international.For these reasons, The goals of this experiment are to reseach the ARHI gene expression in osteosarcoma cells by constructing the osteosarcoma MG-63, U2 OS and SAOS-2 cells which stably expressed by target gene ARHI, and to furtherly discuss the effects of ARHI gene on biological characteristics of osteosarcoma tumor cells and its mechanism, meanwhile hope to provide more support and theoretical basis for gene diagnosis and treatment of osteosarcoma.The first part: the expression and significance of gene ARHI in bone sarcoma cells and normal osteoblast.Objective: To study the expression of ARHI in bone sarcoma cells and the normal osteoblast, explore the biological significance of ARHI which expressed in bone sarcomaMethods: Three kinds of bone sarcoma cell line(MG-63, U2 OS, SAOS-2) and human immortalized osteoblast h FOB1.19. Using RT-PCR and Western blot method to detect the m RNA and protein expression of ARHI gene.To reveal whether there is ARHI gene expression in the normal osteoblast and its expression changes in bone sarcoma cells through the statistical analysis of experimental results.Results:(1)Using RT-PCR and Western blot method respectively to detect the ARHI gene’s expressionin osteosarcoma cell line, result showed that there are different degrees of ARHI gene expression in these cell line.(2) The RT-PCR showed that the ARHI m RNA’s expression level was significantly lower than normal osteoblasts in osteosarcoma cell line, MG-63 cells droped observably, and the expression level had significant difference(P < 0.05).(3) Results detected by Western blot method: the ARHI protein’s expression levels of osteosarcoma cell line below normal osteoblast, MG-63 cells down in the most significant, and the expression level had markedly difference(P < 0.05).Conclusion: the gene ARHI showes generally lower expression in osteosarcoma cell line, which prompts that the expression may be closely related to the development of osteosarcoma and would affect the biological behavior of osteosarcoma.The second part: Establishment and identification of stable positive clone’s osteosarcoma cell lines transfected by ARHI gene.Objective: To establish and identificate a model of osteosarcoma cells which can stably express the target gene ARHI, In order to lay the foundation for studying the effects of ARHI gene on osteosarcoma tumor cells’ s biological behavior.Methods: To construct eukaryotic expression vector pc DNA3.1-ARHI(carrying the ARHI gene coding sequence) by using cloning technology, after making the ARHI gene recombinant plasmid pc DNA3.1-ARHI and Light physical grain pc DNA 3.1 be transformed into E. coli, and plasmid extraction,identification of been purified; To establish a stable ARHI gene transfected osteosarcoma cell line models by using liposome method transiently to transfect osteosarcoma cell line MG-63, U2 OS,SAOS-2 and by selecting G418. To detect target gene ARHI’s expression level changes in m RNA and protein before and after transfection of osteosarcoma MG-63,U2 OS, SAOS-2 cells through RT-PCR and Western blot method.Results(1) transfection of Osteosarcoma cell line by both Eukaryotic expression plasmid pc DNA3.1-ARHI and Empty vector pc DNA3.1 gained neomycin resistance;Through G418 selection, established a stable gene transfection ARHI osteosarcoma cell line.(2) through RT-PCR and Western blot method, we find that after transfection of osteosarcoma cell line by recombinant plasmid pc DNA3.1-ARHI, the purpose gene ARHI’s expression in m RNA and protein level were significantly higher(P < 0.05),compared with empty carrier group and transfection group relatively. While empty carrier group and not transfection group ARHI expression was not significantly different(P > 0.05).Conclusion: we had successfully builded the osteosarcoma cell line which could stably express the purpose gene ARHI by using gene cloning technology.The third part: ARHI gene’s regulation on osteosarcoma cell’s proliferation,apoptosis, invasion, migration in vitro study.Objective: To study the purpose gene ARHI’s effects on osteosarcoma cell’s proliferation, apoptosis and migration, to confirm its biological role in osteosarcoma.Methods: To take osteosarcoma cell lines which could stably express exogenous pc DNA3.1-ARHI, empty carrier pc DNA3.1 and non-transfected osteosarcoma cells as the research object.(1) To analyze gene ARHI’s effects on proliferation of osteosarcoma cell through the growth curve, and MTT method;(2) To detect gene ARHI’s impact on apoptosis of osteosarcoma through the Annexin V- FITC/PI double staining method.(3) To study gene ARHI’s impact on invasion and migration of osteosarcoma cells by Transwell Chambers attack experiment and Transwell Chambers migration.Results:(1) the data analysis from osteosarcoma MG-63,U2 OS,SAOS-2 cells’ growth curve shows that growth rate of ARHI cell is significantly slower than empty carrier group, gene ARHI inhibits the growth of osteosarcoma cells, compared with empty vector group, has obvious difference(P<0.05) especially in 72 ~ 96 hours.(2)To detect cell growth by MTT method,we find that ARHI group cell’s growth slowed significantly, it has obvious growth inhibition for MG-63, U2 OS and SAOS-2 cells,ARHI group compared with empty vector group, has obvious difference(P<0.05).(3)To detect osteosarcoma cell’s apoptosis by using the Annexin V-FITC/PI double staining methods and to quantitatively analyze osteosarcoma MG-63 cell apoptosis rate, we find that the percentage of apoptotic cells in ARHI group of MG-63 was29.7%, the empty carrier was 4.31%, compared with empty vector group, gene ARHI significantly increased apoptosis percentage(P > 0.05)(4) it was found that MG-63cells’ invasive ability significantly decreased in the group of ARHI compared with empty carrier(P < 0.05) through Transwell invasion experiments; It was found that MG-63 cells ’ migration ability decreased obviously in the group of ARHI compared with empty carrier(P < 0.05) via Transwell migration experiment.Conclusion:(1) gene ARHI can inhibit osteosarcoma cells’ proliferation;(2) gene ARHI can induce osteosarcoma cells’ apoptosis;(3) gene ARHI can inhibit the osteosarcoma cell’s invasion and migration;(4) gene ARHI is a tumor suppressor gene in its process of development.The fourth part: The effects of small interfering RNA on gene ARHI of osteosarcoma cells, the protein expression, cell proliferation, apoptosis and its effects on PI3K/Akt signal pathway.Objective: To study the effects of small interfering RNA on gene ARHI’s expression level in osteosarcoma cell MG-63, and on cell proliferation, apoptosis; to study the effects of gene ARHI on PI3K/Akt signaling pathway and Caspase apoptosis signaling pathways.Methods: Using chemical synthesis to build the small interfering RNA of ARHI,transfecting dharmafect 4 transfection reagents into the osteosarcoma MG-63 cells highly expressed by ARHI.(1) Using the RT-PCR, Western blot method, proliferation experiment determined by MTT and Flow cytometry Annexin V-FITC / PI double staining method to detect the effects of these methods on ARHI gene, protein expression level and cell proliferation, apoptosis(2) Using the Western blot method to detect the effects of gene ARHI on p-Akt and Akt protein expression in PI3K/Akt signal transduction pathway;(3) Using Caspase-Glo ? test 3/7 ARHI experiment to detect the effects of ARHI gene on Caspase 3 activity.Results:(1) successly constructed the small interfering RNA of gene ARHI(2)It was showed that after 48 h transfection of MG-63 cells by ARHI si RNA detected by RT-PCR, the ARHI m RNA expression level in ARHI si RNA transfection cells group fell by about 92%, with significant difference(P < 0.05), compared with the control si RNA transfection group;(3) It was detected by Western blot method that the ARHI protein expression level of MG-63 cells in ARHI si RNA transfection group reduced,with obvious difference(P < 0.05), compared with control si RNA transfection group;(4) it was detected by MTT method that the proliferation of ARHI si RNA transfection cells group is significantly increased, with significant difference(P < 0.05), compared with control si RNA transfection cells group;(5) it was showed that after 48 hours ARHI si RNA transfection the MG-63 cells apoptosis rate 4.8% was significantly lower than control si RNA transfection group 31.1%, with obvious difference(P <0.05);(6) 72 hours after MG- 63 cells transfected by gene ARHI, through observing the expression of p-Akt,It was showed that after transfection of osteosarcoma cells by gene ARHI the phosphorylation of p-Akt protein expression significantly lowered,compared with empty carrier; But the ARHI gene silence caused by RNAi significantly raised p-Akt protein expression, while p-Akt protein expression of the control si RNA transfection group has no obvious change;(7) The activity of Caspase-3 both in gene ARHI transfection cells group and ARHI si RNA transfection cells group had no significant change which was detected by Caspase-Glo? 3/7 test(P > 0.05).Conclusion:(1) ARHI si RNA was successfully synthesized and transfected into MG-63 cells of osteosarcoma;(2) ARHI si RNA could reduce the ARHI gene and protein expression level of osteosarcoma MG-63 cell, promote cell proliferation and inhibit apoptosis;(3) gene ARHI reduced p-Akt protein expression,inhibited PI3K/Akt signal transduction pathway associated with osteosarcoma;(4) The apoptosis of MG-63 cell caused by gene ARHI may have no relationship with Caspase pathway,the specific mechanisms need to be further studied.