ROR2在骨肉瘤组织及细胞中的表达及其对骨肉瘤细胞生物学行为影响的初步研究
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摘要
骨肉瘤(Oseteosarcoma)是除浆细胞瘤外最常见的恶性骨原发性肿瘤,其组织学特点是在多数情况下肿瘤细胞产生骨样或不成熟骨。骨肉瘤发病率各国统计数字差异较大,约1/10万—0.1/10万。骨肉瘤易于侵及生长迅速的干骺端,其典型的位置是在长管状骨,股骨远端及胫骨、肱骨的近端是最常见的发病部位,全部病人的50%—70%病变发生在膝关节周围。虽然骨肉瘤在各年龄组均有报道而且其发病率低于其他常见恶性肿瘤,但由于该病好发于青少年长骨时期,有研究证实75%的骨肉瘤于10岁—30岁人群发病。起病时无明显的临床症状,极易与外伤混淆,且恶性程度高早期即可能发生肺转移,故其危害性大死亡率很高。临床骨科工作者一直致力于对其防治的研究,希望寻找到骨肉瘤精确有效治疗的靶点。
ROR2 (Receptor Tyrosine Kinase-like Orphan Receptor)全称为受体酪氨酸激酶样孤核受体2,其属于受体酪氨酸激酶家族(RTKs),此激酶家族成员在动物发育的形态发生及组织分化过程中具有重要意义。它们参与调节细胞增殖、分化、粘附、迁移以及凋亡等多种细胞功能。ROR受体属于受体酪氨酸激酶家族中的孤核受体一类,在进化上非常保守。在哺乳动物其包含两种结构上相关的蛋白ROR1和ROR2。已有的研究证实ROR2在面部、四肢、心脏、大脑及肺等重要器官和组织中均有表达,对于神经系统和骨骼的发育具有重要作用。其在中枢神经系统可调节轴突的生长。在人类, ROR2基因的纯合子型突变可导致Robinow综合症,此种病症表现出多种骨骼发育不良：身材矮小、全身肢体短小、脊柱部分节段缺失及面部骨骼畸形。ROR2基因的杂合子型突变与以手指及足趾远节缺失为特征的brachydactyly type B综合症有关。在小鼠,ROR2基因的缺乏可导致侏儒症、肢体及尾巴短小、面部畸形、心室间隔缺损以及引起新生儿死亡的呼吸功能障碍。已有的研究显示ROR2可促进成骨细胞的增殖和分化,Real-time RT-PCR检测显示ROR2在人类骨髓间质干细胞中无表达,随其的成骨分化逐渐增高,而当其分化为定型的前成骨细胞时ROR2的表达达到峰值,而后又迅速下降并在正常骨细胞中表达消失。ROR2受体通过二聚化而激活可促进人类间质干细胞的成骨转化并在组织培养期间增加新的骨形成。ROR2在小鼠体内试验时也表现出很强的促进骨形成的能力。以上结果均提示ROR2在骨骼的发育过程中具有重要作用。而且研究证实ROR2对于骨骼发育的影响主要是通过与WNT家族蛋白结合,调节经典或非经典WNT信号通路来实现。
有研究显示ROR2与某些肿瘤的发生存在一定的关系。例如其在黑色素瘤组织中有较强的表达,还有研究证实ROR2是Hela宫颈癌细胞的促存活激酶。最新的研究显示在口腔鳞状细胞癌中发现ROR2的增强表达而且ROR2的高表达与其恶性程度相关以及ROR2可促进肾细胞恶性肿瘤的生长。ROR2与肿瘤之间的关系均与其可与Wnt蛋白结合,激活或调整某一WNT信号通路有关。而最近的研究显示WNT通路在放射诱导的的骨肉瘤中具有转录活性。同时WNT/β连环蛋白通路的拮抗剂姜黄素和PKF118-310在人类骨肉瘤细胞实验中显示了抗肿瘤活性。而且2008年有学者研究证实特异性酪氨酸激酶抑制剂(包括表皮生长因子受体抑制剂、胰岛素样生长因子-1受体抑制剂及met抑制剂等)可在体外抑制骨肉瘤细胞的活动能力、集落形成和侵袭能力。此结果显示酪氨酸激酶可能调节骨肉瘤的形成及转移,其可能成为骨肉瘤治疗的新靶点。
ROR2作为酪氨酸激酶家族成员的身份、其在部分肿瘤中的表达及其生物学行为、骨肉瘤的成骨特性和ROR2对于骨骼发育的影响及其通过WNT信号通路调节成骨细胞增殖及分化的生物学特点使得我们怀疑ROR2可能在骨肉瘤的发生发展过程中具有一定的作用,其可能促进了骨肉瘤的发生发展及恶性生物学行为。因此,ROR2可能成为骨肉瘤新的治疗靶点。针对ROR2和骨肉瘤关系的研究将为骨肉瘤的发病机制研究和治疗靶点选择提供一条全新的思路。
第一部分：ROR2在骨肉瘤组织及细胞中的表达及意义
目的：研究ROR2在骨肉瘤组织及细胞中的表达情况,探讨其表达的临床意义。
方法：使用Western-bloting实验方法检测4例骨肉瘤和3例骨软骨瘤患者新鲜原发灶标本中ROR2表达情况；使用免疫组化方法检测55例骨肉瘤患者和15例骨软骨瘤患者原发灶标本中ROR2表达情况并结合患者临床资料了解其临床意义；使用RT-PCR实验方法检验SaoS-2骨肉瘤细胞中ROR2mRNA表达情况。
结果：①通过免疫组织化学的方法检测55例骨肉瘤患者标本中有39例患者标本有ROR2阳性表达,其阳性表达率为70.91%,检测的15例骨软骨瘤患者标本有3例ROR2阳性表达,阳性表达率为20.00%,两者阳性表达率有显著性差异(x2=12.73 P<0.05)。②通过Western-bloting实验检测新鲜骨软骨瘤和新鲜骨肉瘤组织中ROR2蛋白表达的结果显示两者标本中ROR2蛋白表达有显著性差异(P<0.05)。③通过RT-PCR实验检测了SaoS-2骨肉瘤细胞株中ROR2基因表达情况,结果显示其内有ROR2基因表达。④检测了有或无转移的骨肉瘤患者肿瘤标本中ROR2表达的结果显示在有转移的患者标本中ROR2的阳性表达率为100.00%,而在无肿瘤转移患者标本中ROR2阳性表达率为61.91%,两者表达阳性率有显著性差异(X2=5.26P<0.05)。⑤检测Ennecking分期分别为I期、Ⅱ期、Ⅲ期的骨肉瘤患者肿瘤标本中ROR2表达的结果显示ROR2在骨肉瘤Enneking分期Ⅰ期表达阳性率为22.22%(2/9),Ⅱ期表达阳性率为72.73%(24/33),Ⅲ期表达阳性率为100.00%(13/13)。Ⅰ期与Ⅱ期间有显著性差异(X2=5.66 P<0.05),Ⅰ期与Ⅲ期间有显著性差异(x2=11.46P<0.05),而Ⅱ期与Ⅲ期间无显著性差异(X2=2.85 P>0.05)。
结论：①ROR2基因和蛋白在骨肉瘤细胞及组织中有较强表达,而且其可能影响骨肉瘤的生物学行为。
第二部分：小干扰RNA对骨肉瘤细胞株ROR2基因及蛋白表达水平的影响
目的：研究小干扰RNA对骨肉瘤细胞株ROR2基因及蛋白表达水平的影响
方法：使用化学合成法构建针对ROR2的小干扰RNA,通过脂质体法将其转染入骨肉瘤细胞株。使用RT-PCR和Western-bloting实验方法检验其对ROR2基因及蛋白表达水平的影响。
结果：①通过测序和酶切证实成功构建了针对ROR2的小干扰RNA；②通过RT-PCR实验检测了干扰前后SaoS-2骨肉瘤细胞株中ROR2基因表达情况,转染后干扰组ROR2 mRNA的表达水平(0.19±0.02)明显低于阴性对照组(0.55±0.04)和空白组(0.58±0.04),干扰组和阴性对照组比较有显著性差异(t=13.71,P<0.05),干扰组和空白组比较有显著性差异(t=14.86,P<0.05),而空白组和阴性对照组之间无显著性差异(t=1.24,P>0.05)。③通过Western-bloting实验检测了干扰前后SaoS-2骨肉瘤细胞株中ROR2蛋白表达情况,转染后干扰组ROR2蛋白的表达水平(0.18±0.03)明显低于阴性对照组(0.66±0.04)和空白组(0.84±0.03),干扰组和阴性对照组比较有显著性差异(t=7.61,<0.05),干扰组和空白组比较有显著性差异(t=6.37,P<0.05P),而空白组和阴性对照组之间无显著性差异(t=0.31,P>0.05)。。
结论：①成功地合成了ROR2 siRNA并将其转染入SaoS-2骨肉瘤细胞；②在国内首次发现ROR2 siRNA能下调SaoS-2骨肉瘤细胞ROR2基因和蛋白的表达水平。
第三部分：ROR2对骨肉瘤细胞增殖及侵袭力的作用
目的：研究ROR2对骨肉瘤细胞增殖及侵袭力的影响,证实其在骨肉瘤中的生物学作用
方法：通过细胞流式检验、Brdu标记实验和transwell侵袭实验了解ROR2 RNA干扰后骨肉瘤细胞增殖和侵袭力的变化。
结果：①细胞流式检测示RNAi后干扰组细胞中S期细胞所占百分比(12.40±4.85)明显低于阴性对照组(31.23±2.21)和空白组(26.50±2.07),干扰组和阴性对照组比较有显著性差异(t=6.12,P<0.05),干扰组和空白组比较有显著性差异(t=4.63,P<0.05),而空白组和阴性对照组之间无显著性差异(t=2.71,P>0.05)。RNAi后干扰组细胞中G1期细胞所占百分比(81.47±6.56)明显高于阴性对照组(57.87±4.65)和空白组(63.07±4.92),干扰组和阴性对照组比较有显著性差异(t=5.09,P<0.05),干扰组和空白组比较有显著性差异(t=3.86,P<0.05),而空白组和阴性对照组之间无显著性差异(t=1.32,P>0.05)。②Transwell侵袭实验示RNAi后干扰组侵袭细胞数(20.40±11.93)明显低于阴性对照组(37.80±6.83)和空白组(44.00±7.81),干扰组和阴性对照组比较有显著性差异(t=3.70,P<0.05),干扰组和空白组比较有显著性差异(t=2.83,P<0.05),而空白组和阴性对照组之间无显著性差异(t=1.34,P>0.05)。③Brdu标记实验结果示RNAi后干扰组标记指数(0.14±0.03)明显低于阴性对照组(0.32±0.02)和空白组(0.28±0.03),干扰组和阴性对照组比较有显著性差异(t=5.18,P<0.05),干扰组和空白组比较有显著性差异(t=6.70,P<0.05),而空白组和阴性对照组之间无显著性差异(t=2.34,P>0.05)。
结论：在国内首次证实ROR2 RNAi下调ROR2表达可明显抑制SaoS-2骨肉瘤细胞的增值和侵袭能力,揭示ROR2对骨肉瘤的增值和侵袭等生物学行为有重要影响,其可能为骨肉瘤发病机制研究和特异性治疗提供新的思路和靶点。
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 significance
Objective 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 subline
Objective 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 subline
Objective To investigate the effccts of ROR2on proliferation and invasion of osteosarcoma cell and demonstrate the function of ROR2 on osteosarcoma cell biology behavior
Methods 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 cell
Results①The 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.
ROR2 (Receptor Tyrosine Kinase-like Orphan Receptor)全称为受体酪氨酸激酶样孤核受体2,其属于受体酪氨酸激酶家族(RTKs),此激酶家族成员在动物发育的形态发生及组织分化过程中具有重要意义。它们参与调节细胞增殖、分化、粘附、迁移以及凋亡等多种细胞功能。ROR受体属于受体酪氨酸激酶家族中的孤核受体一类,在进化上非常保守。在哺乳动物其包含两种结构上相关的蛋白ROR1和ROR2。已有的研究证实ROR2在面部、四肢、心脏、大脑及肺等重要器官和组织中均有表达,对于神经系统和骨骼的发育具有重要作用。其在中枢神经系统可调节轴突的生长。在人类, ROR2基因的纯合子型突变可导致Robinow综合症,此种病症表现出多种骨骼发育不良：身材矮小、全身肢体短小、脊柱部分节段缺失及面部骨骼畸形。ROR2基因的杂合子型突变与以手指及足趾远节缺失为特征的brachydactyly type B综合症有关。在小鼠,ROR2基因的缺乏可导致侏儒症、肢体及尾巴短小、面部畸形、心室间隔缺损以及引起新生儿死亡的呼吸功能障碍。已有的研究显示ROR2可促进成骨细胞的增殖和分化,Real-time RT-PCR检测显示ROR2在人类骨髓间质干细胞中无表达,随其的成骨分化逐渐增高,而当其分化为定型的前成骨细胞时ROR2的表达达到峰值,而后又迅速下降并在正常骨细胞中表达消失。ROR2受体通过二聚化而激活可促进人类间质干细胞的成骨转化并在组织培养期间增加新的骨形成。ROR2在小鼠体内试验时也表现出很强的促进骨形成的能力。以上结果均提示ROR2在骨骼的发育过程中具有重要作用。而且研究证实ROR2对于骨骼发育的影响主要是通过与WNT家族蛋白结合,调节经典或非经典WNT信号通路来实现。
有研究显示ROR2与某些肿瘤的发生存在一定的关系。例如其在黑色素瘤组织中有较强的表达,还有研究证实ROR2是Hela宫颈癌细胞的促存活激酶。最新的研究显示在口腔鳞状细胞癌中发现ROR2的增强表达而且ROR2的高表达与其恶性程度相关以及ROR2可促进肾细胞恶性肿瘤的生长。ROR2与肿瘤之间的关系均与其可与Wnt蛋白结合,激活或调整某一WNT信号通路有关。而最近的研究显示WNT通路在放射诱导的的骨肉瘤中具有转录活性。同时WNT/β连环蛋白通路的拮抗剂姜黄素和PKF118-310在人类骨肉瘤细胞实验中显示了抗肿瘤活性。而且2008年有学者研究证实特异性酪氨酸激酶抑制剂(包括表皮生长因子受体抑制剂、胰岛素样生长因子-1受体抑制剂及met抑制剂等)可在体外抑制骨肉瘤细胞的活动能力、集落形成和侵袭能力。此结果显示酪氨酸激酶可能调节骨肉瘤的形成及转移,其可能成为骨肉瘤治疗的新靶点。
ROR2作为酪氨酸激酶家族成员的身份、其在部分肿瘤中的表达及其生物学行为、骨肉瘤的成骨特性和ROR2对于骨骼发育的影响及其通过WNT信号通路调节成骨细胞增殖及分化的生物学特点使得我们怀疑ROR2可能在骨肉瘤的发生发展过程中具有一定的作用,其可能促进了骨肉瘤的发生发展及恶性生物学行为。因此,ROR2可能成为骨肉瘤新的治疗靶点。针对ROR2和骨肉瘤关系的研究将为骨肉瘤的发病机制研究和治疗靶点选择提供一条全新的思路。
第一部分：ROR2在骨肉瘤组织及细胞中的表达及意义
目的：研究ROR2在骨肉瘤组织及细胞中的表达情况,探讨其表达的临床意义。
方法：使用Western-bloting实验方法检测4例骨肉瘤和3例骨软骨瘤患者新鲜原发灶标本中ROR2表达情况；使用免疫组化方法检测55例骨肉瘤患者和15例骨软骨瘤患者原发灶标本中ROR2表达情况并结合患者临床资料了解其临床意义；使用RT-PCR实验方法检验SaoS-2骨肉瘤细胞中ROR2mRNA表达情况。
结果：①通过免疫组织化学的方法检测55例骨肉瘤患者标本中有39例患者标本有ROR2阳性表达,其阳性表达率为70.91%,检测的15例骨软骨瘤患者标本有3例ROR2阳性表达,阳性表达率为20.00%,两者阳性表达率有显著性差异(x2=12.73 P<0.05)。②通过Western-bloting实验检测新鲜骨软骨瘤和新鲜骨肉瘤组织中ROR2蛋白表达的结果显示两者标本中ROR2蛋白表达有显著性差异(P<0.05)。③通过RT-PCR实验检测了SaoS-2骨肉瘤细胞株中ROR2基因表达情况,结果显示其内有ROR2基因表达。④检测了有或无转移的骨肉瘤患者肿瘤标本中ROR2表达的结果显示在有转移的患者标本中ROR2的阳性表达率为100.00%,而在无肿瘤转移患者标本中ROR2阳性表达率为61.91%,两者表达阳性率有显著性差异(X2=5.26P<0.05)。⑤检测Ennecking分期分别为I期、Ⅱ期、Ⅲ期的骨肉瘤患者肿瘤标本中ROR2表达的结果显示ROR2在骨肉瘤Enneking分期Ⅰ期表达阳性率为22.22%(2/9),Ⅱ期表达阳性率为72.73%(24/33),Ⅲ期表达阳性率为100.00%(13/13)。Ⅰ期与Ⅱ期间有显著性差异(X2=5.66 P<0.05),Ⅰ期与Ⅲ期间有显著性差异(x2=11.46P<0.05),而Ⅱ期与Ⅲ期间无显著性差异(X2=2.85 P>0.05)。
结论：①ROR2基因和蛋白在骨肉瘤细胞及组织中有较强表达,而且其可能影响骨肉瘤的生物学行为。
第二部分：小干扰RNA对骨肉瘤细胞株ROR2基因及蛋白表达水平的影响
目的：研究小干扰RNA对骨肉瘤细胞株ROR2基因及蛋白表达水平的影响
方法：使用化学合成法构建针对ROR2的小干扰RNA,通过脂质体法将其转染入骨肉瘤细胞株。使用RT-PCR和Western-bloting实验方法检验其对ROR2基因及蛋白表达水平的影响。
结果：①通过测序和酶切证实成功构建了针对ROR2的小干扰RNA；②通过RT-PCR实验检测了干扰前后SaoS-2骨肉瘤细胞株中ROR2基因表达情况,转染后干扰组ROR2 mRNA的表达水平(0.19±0.02)明显低于阴性对照组(0.55±0.04)和空白组(0.58±0.04),干扰组和阴性对照组比较有显著性差异(t=13.71,P<0.05),干扰组和空白组比较有显著性差异(t=14.86,P<0.05),而空白组和阴性对照组之间无显著性差异(t=1.24,P>0.05)。③通过Western-bloting实验检测了干扰前后SaoS-2骨肉瘤细胞株中ROR2蛋白表达情况,转染后干扰组ROR2蛋白的表达水平(0.18±0.03)明显低于阴性对照组(0.66±0.04)和空白组(0.84±0.03),干扰组和阴性对照组比较有显著性差异(t=7.61,<0.05),干扰组和空白组比较有显著性差异(t=6.37,P<0.05P),而空白组和阴性对照组之间无显著性差异(t=0.31,P>0.05)。。
结论：①成功地合成了ROR2 siRNA并将其转染入SaoS-2骨肉瘤细胞；②在国内首次发现ROR2 siRNA能下调SaoS-2骨肉瘤细胞ROR2基因和蛋白的表达水平。
第三部分：ROR2对骨肉瘤细胞增殖及侵袭力的作用
目的：研究ROR2对骨肉瘤细胞增殖及侵袭力的影响,证实其在骨肉瘤中的生物学作用
方法：通过细胞流式检验、Brdu标记实验和transwell侵袭实验了解ROR2 RNA干扰后骨肉瘤细胞增殖和侵袭力的变化。
结果：①细胞流式检测示RNAi后干扰组细胞中S期细胞所占百分比(12.40±4.85)明显低于阴性对照组(31.23±2.21)和空白组(26.50±2.07),干扰组和阴性对照组比较有显著性差异(t=6.12,P<0.05),干扰组和空白组比较有显著性差异(t=4.63,P<0.05),而空白组和阴性对照组之间无显著性差异(t=2.71,P>0.05)。RNAi后干扰组细胞中G1期细胞所占百分比(81.47±6.56)明显高于阴性对照组(57.87±4.65)和空白组(63.07±4.92),干扰组和阴性对照组比较有显著性差异(t=5.09,P<0.05),干扰组和空白组比较有显著性差异(t=3.86,P<0.05),而空白组和阴性对照组之间无显著性差异(t=1.32,P>0.05)。②Transwell侵袭实验示RNAi后干扰组侵袭细胞数(20.40±11.93)明显低于阴性对照组(37.80±6.83)和空白组(44.00±7.81),干扰组和阴性对照组比较有显著性差异(t=3.70,P<0.05),干扰组和空白组比较有显著性差异(t=2.83,P<0.05),而空白组和阴性对照组之间无显著性差异(t=1.34,P>0.05)。③Brdu标记实验结果示RNAi后干扰组标记指数(0.14±0.03)明显低于阴性对照组(0.32±0.02)和空白组(0.28±0.03),干扰组和阴性对照组比较有显著性差异(t=5.18,P<0.05),干扰组和空白组比较有显著性差异(t=6.70,P<0.05),而空白组和阴性对照组之间无显著性差异(t=2.34,P>0.05)。
结论：在国内首次证实ROR2 RNAi下调ROR2表达可明显抑制SaoS-2骨肉瘤细胞的增值和侵袭能力,揭示ROR2对骨肉瘤的增值和侵袭等生物学行为有重要影响,其可能为骨肉瘤发病机制研究和特异性治疗提供新的思路和靶点。
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 significance
Objective 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 subline
Objective 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 subline
Objective To investigate the effccts of ROR2on proliferation and invasion of osteosarcoma cell and demonstrate the function of ROR2 on osteosarcoma cell biology behavior
Methods 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 cell
Results①The 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.
引文
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