The Preliminary Study Of Experession Of Ror2 In Osteosarcoma Tissues And Cells And The Effect Of Ror2 On Osteosarcoma Cell Biology Behavior

Osteosarcoma is the most common osteogenis malignant tumor.It is characterized by production of both osterid and bone by malignant spindle cells.The incidence of the disease is approximately from 1/100000 to 0.1/100000 in the world every year.Osteosarcoma most commonly develops at sites of rapid bone turnover,such as the distal femur,proximal tibia and proximal humerus.The incidence of osteosarcoma peaks in the children and adolescents periods.The onset of osteosarcoma is without obvious clinical symptoms and it is highly malignant and early possible lung metastasis,so the result of osteosarcoma is big harmfulness and the patients have high mortality rate.Thus,many orthopedist want to get early diagnosis and precise treatment for the disease.ROR2 is receptor tyrosine kinase-like orphan receptor.It belongs to the receptor tyrosine kinase family.In humans,mutations with in the ROR2 gene are responsible for brachydactyly type B,characterized by hypoplasia/aplasia of distant phalanges,and for Robinow syndrome characterize by short staturt,limb bone shortening segmental defects of the spine,and a dysmorphic facial appearance. Mice lacking Ror2 exhibit dwarfism, short limbs and tails, facial abnormalities, ventricular septal defects, and respiratory dysfunction resulting in neonatal lethality.Thus,ROR2 plays importment role in development morphogenesis,in particular in skeletal development.Recently researchs show ROR2 is closely related to some tumorigenesis and demonstrate inhibitors of specific tyrosine kinases regulate motility,colomy formation and invasiveness of osteosarcoma cells.All of which are critical components of tumorigenesis and/or metastasis.So we consider that ROR2 maybe has relationship with tumorigenesis and development of osteosarcoma.Part one:Expression of ROR2 in osteosarcoma tissues and cells and its clinical significanceObjective To investigate the expression and significance of ROR2 in human osteosarcomatous tissue and cells.Methods Western-bloting test was performed to detect the expression of ROR2 protein in fresh specimens of 4 osteosarcoma patients and 3 osteochondroma patient;Immunohistochemistry was performed to detect the expression of ROR2 protein in paraffin specimens of 55 osteosarcoma patients and 15 osteochondroma patients;RT-PCR test was performed to detect the expression of ROR2 mRNA in SaoS-2 osteosarcoma cells.Results①39 specimens showed positive staining of ROR2 in 55 osteosarcoma cases and the positive rate is 70.91%.3 specimens showed positive staining of ROR2 in 15 osteochondroma cases and the positive rate is 20.00%.The difference of positive rate was statistically significant.②The difference of ROR2 protein in Western-bloting test results was statistically significant.③The results of RT-PCR showed expression of ROR2 mRNA in SaoS-2 osteosarcoma cells.④The positive rate of ROR2 in the osteosarcoma patients with metastasis is 100.00%, however,the positive rate of ROR2 in the osteosarcoma patients without metastasis is 61.91%.The difference was significant.⑤The positive rate of ROR2 in the osteosarcoma patients on the Enneking I stage is 22.22%,that is 72.73% on the EnnekingⅡstage and 100.00% on the EnnekingⅢstage.The difference of the positive rate of ROR2 between EnnekingⅠstage and EnnekingⅡstage was significant,that between EnnekingⅠstage and EnnekingⅢstage was significant but between EnnekingⅡstage and EnnekingⅢstage was not significant.Conclusion①The expression of ROR2 protein and gene in osteosarcoma tissues and cells is high. It may effect the biology behavior of the osteosarcoma.Part two:The effects of small interfering RNA on ROR2 gene and protein expression in osteosarcoma cell sublineObjective To explore the effects of small interfering RNA on ROR2 gene and protein expression in osteosarcoma cell subline.Methods We designed and chemically synthesized sequence-specific siRNA targeting ROR2 gene and transfected siRNA into osteosarcomatous cell subline by using LipofectamineTM 2000, detected the change of ROR2 mRNA expression by RT-PCR and the change of ROR2 protein expression by Western-bloting.Results①ROR2 siRNA was synthesized successfully that confirmed by digestion and sequencing;②The RT-PCR test demonstrated the expression of ROR2 mRNA after transfection (0.19±0.02) was lower than the negative control group(0.55±0.04) and blank group(0.58±0.04), the differences were significant(P<0.05);③The Western-bloting test demonstrated the expression of ROR2 protein after transfection (0.18±0.03) was lower than the negative control group (0.66±0.04) and blank group (0.84±0.03), the differences were significant (P<0.05).Conclusions (1) The ROR2 siRNA was successfully synthesized and transfected into osteosarcomatous cell subline; (2)The ROR2 siRNA could successfully inhibit the expression of ROR2 mRNA and ROR2 protein.Part three:The effects of ROR2 on proliferation and invasion of osteosarcoma cell sublineObjective To investigate the effccts of ROR2on proliferation and invasion of osteosarcoma cell and demonstrate the function of ROR2 on osteosarcoma cell biology behaviorMethods We detected the change of proliferation and invasion of osteosarcoma cell by flow cytomety,Brdu-labeled test and Transwell test after taking effect of ROR2 RNAi on osteosarcoma cellResults①The flow cytomety demonstrated the cell proportion in S phase of transfection group (12.40±4.85) was lower than the negative control group(31.23±2.21)and blank group(26.50±2.07), the differences were significant (P<0.05),and the cell proportion in G1 phase of transfection group (81.47±6.65) was higher than the negative control group (57.87±4.65) and blank group (63.07±5.03), the differences were significant (P<0.05);②The Transwell test demonstrated the amount of exosomatic invasive osteosarcoma cell in transfection group(20.40±11.93) was lower than that of negative control group(37.80±6.83)and blank group(44.00±7.81), the difference was significant(p<0.05);③The Brdu-labeled test demonstrate the Brdu-labeled positive index in transfection group(0.14±0.03) was lower than that of negative control group(0.32±0.02)and blank group(0.28±0.03), the difference was significant(P＜0.05).Conclusion ROR2 is necessary for hman osteosarcoma cell proliferation and invasion,It is a significant role in the biology behavior of osteosarcoma and a potential target for mechanism of osteosarcoma pathogenesis and osteosarcoma specific treatment probably.