| BackgroundOsteosarcoma is the most common bone malignant tumor with high grade malignancy and strong invasion,mostly occurred in adolescent,easily happening early pulmonary metastasis,its prognosis is poorer than others.A single treatment curative effect was poor previously,the 5-year survival rate was less than 20%.Accompanied with the further study on the neoadjuvant chemotherapy and limb-salvage surgery,a growing number of patients with osteosarcoma preserved body and prolong life,but so far there is still a lack of targeted in diagnosis markers,while poor differentiation osteosarcoma rely on morphology is difficult to make definite pathological diagnosis.The malignant biological behavior of tumor such as recurrence and metastasis is still the main factors that largely affects the prognosis of patients,causes the low survival rate of osteosarcoma patients,and causes the failure of treatment in patients with osteosarcoma.Currently,there is still no significant breakthrough in the related basic research about the mechanism of metastasis and etiology of osteosarcoma,and there is no new drugs and new treatment mode in clinic,so looking for a new breakthrough point,and the study on the mechanisms of invasion and metastasis in osteosarcoma is very important.It is the common feature of tumor cells to inhabit cell apoptosis and promote the viability of tumor cells,so cell apoptosis is the main mechanism of chemotherapy and radiotherapy mediates killing tumor cells.The mechanism of normal cells’ apoptosis is restrained,leading to the occurrence of tumors,and the mechanism disorder of tumor cells’ apoptosis causes proliferation of tumor.Both of them are core issues of the restrictions for the further development of the clinical treatment and the basic study on tumor.At its most basic,the key to the issue is that apoptosis suppressor genes including IAPs family are involved in and coordinate with each other.Now,IAP family is just one of research hotspots.Inhibitor of apoptosis proteins(IAPs)codes a group of proteins of dependency structure.Members in this family,the unique endogenous protein that regulate and start Caspase and its effector mechanism,restrain cell apoptosis mainly through bonding or restraining special aspartic acid proteolytic enzyme that contains cysteine,namely Caspase.Till now,eight members,NAIP,XIAP(ILP-1/MIHA),cIAP-1(HIAP-2/MIHB),cIAP-2(HIAP-1/MI-HC),Survivin(TIAP),Apollon(Bruce),ILP-2(Ts-IAP)and Livin(ML-IAP/KIAP),of IAPs family have been found in human body,among which Survivin is discovered in recent years.Because of its unobvious expression in normal human tissue and specific and high expression in malignant tumor tissue,Survivin has been taken as an important factor in connecting cell cycle with cell apoptosis,gradually becoming a research hotspot in osteogenic sarcoma field.Survivin gene is screened out from human gene library by Ambrosini utilizing the cDNA of effector cell protease receptorl,ECPR1,with total length of 15kb,locating at chromosome 17q25,containing 4 exons and 3 introns,coding to generate a Survivin protein of 142 amino-acids,which is an AIP protein with the smallest molecular weight that has ever discovered.Not only has the function on inhabitation of cell apoptosis,Survivin gene also participates in mitosis,cytoplasmic division,regulation and control of cell cycle and formation of blood vessel for tumor metastasis:(1)The function on inhabitation of cell apoptosis:① Through amino acid residue Trp67,Pro33 and Cys84 related to apoptosis inhabitation in BIR functional area,Survivin directly acts on Caspase(cysteine-aspanic-acid-protease)-3,Caspase-7,inhabits their activities,and block cells’ apoptosis process.② Survivin forms Survivin-CDK4 complex by combining with cell cycle regulatory factors CDK4,releasing P21 from CDK4 complex to combine with Caspase-3,accordingly to inhabit Caspase indirectly and prevent cell apoptosis.③Recently,it is discovered that Survivin is the substrate in mitotic phase of cyclin-dependent kinases p34cdc-cyclinB1,after phosphorylation,Survivin Thr34 combines with caspase-9 and inhabits the latter’s activity,and prevents the cell apoptosis signal transduction of Caspase-9 dependence.(2)The function on regulation and control of cell cycle:after combining with CDK4,Survivin inhabits the formation of CDK4/p16(INK4a)complex competitively,further leads to the excitation of CDK2/cyclin E and Rb’s phosphorylation,and thus speeds up the conversion from G1 period to S period.For the specific expression in G2/M period,Survivin lacks of RING Zinc finger at its carboxyl terminal,which is replaced by an a helical structure consisting of 40 amino-acids in length of 6.5nm,at the early phase of mitosis,by means of the structure aforesaid,Survivin occurs specific saturable reaction with the microtubules of spindle,therefore preventing the cell apoptosis induced byDNAdue to its damage or mutation,promoting the abnormal proliferation of transformed cell through mitosis.(3)The function on participating in blood vessel formation:Survivin is the protective gene in VEGF induced blood vessel formation,playing an important role in the formation of vascular endothelial cell and new vessels.O’onnor et al.verified VEGF and bFGF could induce the expression of vascular endothelial cell Survivin(the expression quantity raises 16 times),while inflammatory factors,such as TNF and IL-1,do not have this function.Through researches,Tran et al.discovered that Survivin could effectively maintain the structure of micro vascular network,promote the protection function for VEGF’s endothelial cell,resist the apoptosis function of chemotherapy drugs to vascular endothelial cell.By means of researches,Coma et al.discovered that the Survivin’s ASODN could eliminate VEGF factors against the tumor necrosis or resistance function of apoptosis induced by ceramide,leading to apoptosis of the endothelial cells and rapid degradation of blood capillary,thus showing the anti-apoptosis function of VEGF to the endothelial cell was mainly realized through inducing the expression of Survivin.Meanwhile,there was a positive correlation between Survivin’s expression and VEGF’s,presenting the two played a synergistic role in the process of blood vessel generation of osteosarcoma.Thus,it can be seen that occurrence,development and transfer of osteosarcoma is a very complicated process,and the pathogenesis of osteosarcoma has not been clarified yet.Since the disorder of cell’s growth and apoptosis pathway is beneficial to tumor’s growth and invasion,namely growth and development of tumor rely on the balance between the proliferation and apoptosis of tumor cells,the mechanism of cell’s apoptosis may play a great role in the process of osteosarcoma’s occurrence and development.However,the effective target has not been discovered so far.As a highly conserved mechanism of cell death,cell apoptosis is also called programmed cell death(PCD),possessing characteristics in the aspects of histopathology,biochemistry and morphology.On one hand,under the physiological status,environmental stability in tissues and developmental process of embryonic cells rely on apoptosis program playing roles,while on the other hand,inhabitation of apoptosis can cause the abnormal lengthening of cell’s life time and the gather of mutant proinsulin gene,which induce tumor’s formation,Promote tumor’s transfer,and enhance tumor’s chemotherapy resistance.Current study found that the abnormal expression of Survivin gene in osteosarcoma tissue may directly promote tumor cells to escape from growth monitoring,and inhabit the apoptosis of tumor cells.Some scholars found that the staining magnitude of Survivin protein in bone sarcoma tissue is clearly stronger than that in osteochondroma and normal bone tissues.The histopathology discovered that in Eunecking of osteosarcoma,Survivin protein’s expressions in Ⅱ A and Ⅱ B period were lower than that in Ⅲ period,prompting that Survivin protein’s expression was closely related to the grade malignancy of bone tumor,periodization of osteosarcoma and tumor’s differentiation.Furthermore,Survivin protein’s over-expression is related to the infiltration and transfer of osteosarcoma patients.What’s more,some studies adopts Cox multivariate regression model to eonduct evaluations for the prognosis of chemotherapy sensitivity prior to adjuvant chemotherapy of Surviving expression level of osteosarcoma patients,proving the up-regulation of Survivin protein’s expression is closely related to the occurrence and development of osteosarcoma tissue,besides,Survivin may become a sole marker of osteosarcoma diagnosis and judgment prognosis.Due to selective high expression of apoptosis inhibiting protein Survivin in tumor tissues,we hypothesized that inhibition of Survivin may induce spontaneous apoptosis of osteosarcoma cells,our previous experiments confirmed that the expression of Survivin increased significantly in osteosarcoma cell line U20S,SAOS and MG63.Compared with the normal tissues,the expression of Survivin in osteosarcoma tissues have been confirmed in our subsequent experiments.We also found that the Sirvivin antisense oligonucleotide technology can reduce Survivin expression in osteosarcoma cells,inhibit osteosarcoma cell proliferation and presents the concentration dependence,thereby inducing apoptosis of osteosarcoma.Based on the results of previous research,we further put forward the hypothesis that whether there is a kind of downstream signaling pathways component,that Survivin can effect on osteosarcoma cells in vitro through it,and whether inhibition of Survivin can affect the growth of osteosarcoma in vivo.Tumor’s transfer includes tumor cells migrate in stroma and touch vessels,tumor cells breakthrough vascular endothelium and the basilar membrane under the endothelium,and enter into blood stream where survive,and tumor cells go out of blood vessel,survive and proliferate in tissue,and form metastasis.Thus,the tumor metastasis was involved in the adhesion between cells and cells,cells and extracellular matrix.And this kind of mutual recognition and interaction depend on CAMs-mediating.Int is one of the important members of the CAMs family,it is the dimers molecules that both alpha and beta subunits form by non covalent bond connection.Current study found that receptor family,including at least 25 alpha subunit and 11 beta subunit,to form more than dozens of different Int.Both alpha and beta subunits form by three parts:long extracellular segment(amino end),transmembrane segment,and short segments of the intracellular(carboxyl end).The extracellular section of alpha subunit recognized the ECM RGD sequence,mediated adhesion between cells and ECM,and the intracellular section of beta subunit connected to the cytoskeleton.Int play its role by binding to the ligands,so Int can be divided into three categories according to different ligands recognition.Int alpha 5 discussed in this paper recognized non-RGD sequence,its ligand is the basement membrane proteins such as layer adhesion proteins(LN),collagen protein(CO),fiber connection(FN),etc.Collagen protein is the main composition of extracellular matrix of bone tissue.Recently,people found the expression level of Int regulated osteogenesis and osteolysis in the bone tissue,suggest that Int is related to a lot of bone metabolic diseases even the pathological mechanism of osteogenesis and osteolysis.In addition to mediating the adhesion between cell and basement membrane,cell and cell,Int can transmit signals into the cells by binding to its ligand,then affect gene expression,and finally effect the biological behaviour of cells such as metabolism proliferation,apoptosis,and gene transduction.With the literature,Caspase-3 play a mediating role in the process of Int alpha 5 and Survivin effect on osteosarcoma cells proliferation;Int alpha 5 and Survivin may block the apoptosis mediated by various stimulation process with directly inhibiting Caspase-3.All of the three talked above possibly participate in malignant biological behavior of osteosarcoma cells.So,Int is likely to impact on the growth,development,invasion,and metastasis of osteosarcoma which involved coordination and interaction with Survivin.Compared with the technology of ASODN,RNA interference technology has advantages of high transfection efficiency,strong specificity,weak potential toxicity,sufficient action time,simple and convenient experimental preparation and operation.The technology is first found and proved to be a kind of process that dsRNA-mediated homologous RNA degradation during the study on antisense RNA in caenorhabditis elegans(c.elegans)by Fire in 1998.It has gradually be accepted as authoritative experimental techniques and treatments.Its principle is that silencing specific target genes induced by dsRNA in sequence of the biological cells were used to block the activity of the target gene quickly.SiRNA is a Intermediate product of RNAi process,and it is essential for RNAi exerts its effect.At present there are two kinds of siRNA:synthesis of RNA interference fragment in vitro andDNAinterference carrier.Due to the former transfection efficiency and the defects of instantaneous effect in RNAi,we often use the RNAi technology byDNAinterference carrier.Tong-tao Yang et al.synthesized the specific RNA interference segments of Survivin gene,through the experiment of growth in osteosarcoma cells in vitro and tumor transplantation in vivo,they confirmed that specific siRNA for Survivin can obviously promote the apoptosis of osteosarcoma cells.It suggest that in the experimental treatment of osteosarcoma,RNAi can not only inhibit the expression of oncogenes,knockdown the oncogenes activated by point mutations and suppress gene amplification,but also inhibit the expression of fusion gene and other tumor related gene.Zou et al.found that using RNA interference technology in combination with chemotherapy drugs such as etoposide,cisplatin and doxorubicin can significantly inhibit the growth of MG63 cells and increase the sensitivity of chemotherapy.Their experiment showed that the RNA interference technology for Survivin can enhance curative effect of chemotherapy,which can reduce the dose of chemotherapy drugs and the drug adverse reactions,finally realize the effective inhibition in the growth of osteosarcoma cells,and show the superiority of inhibition of Survivin gene in combination with chemotherapy in the treatment of osteosarcoma.In conclusion,we study on the relationship among Survivin,Int alpha 5 and malignant behavior characteristics in osteosarcoma,evaluate the expression of Int alpha 5 in osteosarcoma cells after the treatment of Survivin RNA interference technology,analyze the regulating relationship between Survivin and Int alpha 5,observe the effect of the above treatment on the growth of osteosarcoma tissue in vivo,clear whether Survivin can effect on osteosarcoma cells in vitro and in vivo by Int alpha 5,and make both Survivin-SiRNA and Int alpha 5 specific antibody become a new way of targeted therapy for osteosarcoma.Part Ⅰ:Preparation of Survivin-SiRNA transfected cell aiming at osteosarcoma MG-63 cell line and the related research in vitro.objective:To prepare the Survivin-SiRNA transfected cell aiming at osteosarcoma MG63 cell line,and compare and analyze the expressions of Survivin in osteosarcoma cell after transfection,to verify the effect of transfection.Method:Design the SIRNA specifically targeting to Survivin,and transfect osteosarcoma MG63 cells,at the same time set negative control group(scramble RNA transfection)and blank control group(empty lipoplast transfection)in same concentration for comparison.Then we use western blot technique to detect the expressions of Survivin protein after transfection of each MG63 cell.Result:The detection result of western blot shows that the specific Survivin protein bands existed in MG63 cell lysis solution.Survivin-SiRNA could decrease the expressions of Survivin protein in MG63 cells.After transfection,the expression of Survivin protein were obviously lower than negative control group.Conclusion:Survivin-SiRNA targetling to Survivin significantly reduced the expression of Survivin protein in osteosarcoma MG63 cell,farther confirmed that Survivin-SiRNA could inhibit the expression of Survivin.The preparation of this kind of transfected cell lines accumulated relevant experience for further experiments and laid a theoretical foundation.Part Ⅱ:The effect of Survivin-SiRNA on invasion and migration of osteosarcoma MG63 cellsobjective:To explore the effect of Survivin-SiRNA on invasion and migration of osteosarcoma MG63 cells.Method:Design the SiRNA specifically targeting to Survivin,and transfect osteosarcoma MG63 cells,at the same time set negative control group(scramble RNA transfection)in same concentration for comparison.Then we use transwell assay and wound healing assay to detect the invasion and migration after transfection of each MG63 cell.Result:Transwell assay showed that eompared with negative eontrol group,the invading quantity of osteosarcoma MG63 cells after Survivin-SiRNA transfection decreased significantly(P<0.01),and wound healing assay showed that compared with negative control group,the migrating quantity of osteosarcoma MG63 cells to the wounding area within 48h after Survivin-SiRNA transfection decreased significantly(P<0.01).Conclusion:Survivin-SiRNA significantly inhibited the invasion and migration of osteosarcoma MG63 cell,further confirmed that the inhibition of the expression of Survivin can affect the development of osteosarcoma cells.It suggest that Survivin is not only closely related to orthotopic growth of osteosarcoma,but also may be involved in the process of distant metastasis of osteosarcoma.Part Ⅲ:The effect of Survivin-SiRNA on the expression of Int α5 in osteosarcoma MG63 cellsobjective:To explore the effect of Survivin-SIRNA on the expression of Int a5 in osteosarcoma MG63 cells,to test whether Int alpha 5 is the downstream signaling pathways component of Survivin.Method:Design the SIRNA speciflcally targeting to Survivin,and transfect osteosarcoma MG63 cells,at the same time set negative control group(scramble RNA transfection)in same concentration for comparison.Then we use flow cytometric analysis and immunofluorescence to detect the expression of Int alpha 5 after transfection of each MG63 cell.Result:Flow cytometric analysis and immunofluorescence showed that compared with negative control group,the expression of Int alpha 5 in osteosarcoma MG63 cell after Survivin-SiRNA transfection was significantly suppressed(P<0.01).Conclusion:Survivin-SiRNA significantly inhibited the expression of Int alpha 5 in osteosarcoma MG63 cell,further confirmed that the targeted-inhibition of the expression of Survivin can reduce the expression of Int alpha 5 in osteosarcoma MG63 cell.It suggest that Survivin may be an upstream regulator for Int alpha 5,and Survivin can effect on osteosarcoma cells by Int alpha 5 in vitro.Part Ⅳ:The effect of targeting therapy for Int a5 on invasion and migration of osteosarcoma MG63 cellsobjective:To prepare the Int alpha 5-SiRNA transfected cell aiming at osteosarcoma MG63 cell line.After verifying the effect of transfection,we explore the effect of Int alpha 5-SiRNA and Int alpha 5 antibody on invasion and migration of osteosarcoma MG63 cells.Method:Design the SiRNA specifically targeting to Int alpha 5,and transfect osteosarcoma MG63 cells,at the same time set negative control group(scramble RNA transfection)in same concentration for comparison.Then we use flow cytometric analysis and immunofluorescence to verify the effect of transfection.Finally,we use transwell assay and wound healing assay to detect the invasion and migration after Int alpha 5-SiRNA transfection and antibody blocking of each MG63 cell.Result:Transwell assay showed that compared with negative control group,the invading quantity of osteosarcoma MG63 cells after Int alpha 5-SiRNA transfection and antibody blocking decreased significantly(P<0.01),and wound healing assay showed that compared with negative control group,the migrating quantity of osteosarcoma MG63 cells to the wounding area within 48h after antibody blocking Int alpha 5 decreased significantly(P<0.01).Additionally,the migration of cells in the wound healing assay was over 2 fold lower in a5 integrin knockdown cells than scramble RNA transfected cells after Int alpha 5-SiRNA transfection.Conclusion:Both Int alpha 5-SiRNA and Int alpha 5 antibody significantly inhibited the invasion and migration of osteosarcoma MG63 cell,On the one hand,it proved that the expression of Int alpha 5 correlated with invasion and metastasis of osteosarcoma cells.On the other hand,it also suggest targeting the expression of Int alpha 5 can directly reach curative effect,to provide theory basis for updating the clinical treatment of osteosarcoma.Part Ⅴ:The effect of Survivin-SiRNA on the experiments of osteosarcoma MG63 cells in vivo.objective:To prepare the Survivin-SiRNA transfected cell aiming at osteosarcoma MG-63 cell line and transplant the transfected cells into nude mice in vivo.After the establishment of the animal models of osteosarcoma,we observe its growth in vivo.Method:Design the SiRNA specifically targeting to Survivin,and transfect osteosarcoma MG63 cells,at the same time set negative control group(scramble RNA transfection)in same concentration for comparison.Then MG63 cells transfected with either the scramble RNA or siRNA targeting survivin were inoculated into the left and right flanks of nude mice,respectively.Three weeks after inoculation,the mice were sacrificed and the tumors were weighed.To observe the tumor volume,calculate the mean tumor weight for comparison.Result:After three weeks,the mean tumor weight and volume was quantified.Survivin knockdown tumors were significantly smaller and lighter than those produced by negative control group(P<0.01).Conclusion:Survivin-SiRNA significantly inhibited the growth of osteosarcoma tissue,further confirmed that the inhibition of the expression of Survivin can reduce the development of osteosarcoma tissue.It suggest that Survivin play an important role in the process of proliferation and reproduction in osteosarcoma. |
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