目的:探究长链非编码RNA（long non-coding RNA，lncRNA）HIT与骨肉瘤细胞顺铂（cisplatin，DDP）抵抗的关系及其上皮间充质转化（epithelial-mesenchymal transition，EMT）的相关机制。方法：选择天津医院骨科2017 年6 月至2018 年6 月42 例骨肉瘤组织及相应癌旁组织（距癌变边缘>5 cm）标本，采用qRT-qPCR 检测骨肉瘤组织及相应癌旁组织中HIT 及EMT标志物Snail、E-钙黏蛋白（E-cadherin）mRNA的表达水平。构建DDP抵抗的人骨肉瘤U2OS细胞株及人肾上腺293T细胞株作为抵抗组及对照组，采用慢病毒转染两组表达靶向HIT的siRNA载体于抵抗组细胞中作为干扰A组及B组，同时转染HIT过表达载体及空白载体构建HIT 过表达的U2OS细胞株及空白U2OS细胞株分别作为过表达组及空白组。MTT实验检测各组细胞DDP半抑制浓度（IC50），qRT-PCR实验检测各组细胞中HIT、Snail 及E-cadherin mRNA的表达水平，Western blotting 检测各组细胞Snail 及Ecadherin的表达水平，RNA结合蛋白免疫沉淀（RNA-IP）实验检测U2OS 及293T 细胞中HIT 与Snail 蛋白分子的结合情况。结果：骨肉瘤组织中HIT及Snail mRNA表达显著高于癌旁组织、E-cadherin mRNA表达显著低于癌旁组织，且骨肉瘤组织中HIT与E-cadherin mRNA表达呈显著负相关(均P<0.01)；抵抗组细胞DDP IC50显著高于对照组、干扰A组及B组细胞，过表达组DDP IC50显著高于空白组(均P<0.01)；抵抗组细胞HIT 表达显著高于对照组，干扰A组及B组细胞HIT 表达显著低于抵抗组及对照组，过表达组HIT表达显著高于空白组（均P<0.05 或P<0.01）；抵抗组细胞Snail 、E-cadherin mRNA表达水平显著高于或低于对照组、干扰A组及干扰B组，过表达组E-cadherinmRNA显著低于空白组；抵抗组细胞Snail 蛋白表达水平显著高于对照组、干扰A组及B组细胞，E-cadherin 蛋白表达水平则显著低于对照组、干扰A 组及B 组细胞，过表达组Snail 蛋白表达水平显著高于空白组(P<0.05或P<0.01）；在U2OS及293T细胞中，免疫磁珠介导Anti-snail 抗体下拉HIT的表达水平显著高于对照IgG抗体下拉HIT的表达水平（P<0.01）。结论：HIT通过正调控Snail蛋白水平，抑制E-cadherin的转录活性，从而促进骨肉瘤细胞EMT及DDP抵抗的发生。

Objective: To investigate the relationship between long non-coding RNA (lncRNA) HIT and cisplatin (DDP) resistance in osteosarcoma cells and the mechanism related to epithelial-mesenchymal transition (EMT). Methods: 42 pairs of osteosarcoma tissues and corresponding para-cancerous tissues (more than 5 cm away from the edge of cancer tissues) were collected at the Department of Orthopedics, Tianjin Hospital during June 2017 to June 2018. Quantitative Real-time PCR (qRT-PCR) was used to detect the mRNA expression of HIT and EMT related markers (Snail and E-cadherin) in the collected tissues. The DDP-resistant osteosarcoma U2OS cell line was constructed and human adrenal 293T cell line was used as control. Two sets of siRNA vectors targeting HIT loaded on lentivirus were transfected into cells with DDP-resistance as the interference group A and group B. Meanwhile, the U2OS cell line was transfected with HIT full-length vector and blank vector respectively, as over-expression group and blank group. The DDP 50% inhibitory concentration (IC50) was detected by MTT assay. qRT-PCR was used to detect the mRNA expressions of HIT, Snail and E-cadherin.Western blotting was used to detect the protein expressions of Snail and E-cadherin. RNA binding protein immunoprecipitation (RNAIP)assay was used to clarify the combination of HIT and Snail protein in the U2OS and 293T cells. Results: The mRNA expressions of HIT and Snail in osteosarcoma tissues were significantly higher than those in para-cancerous tissues, while the mRNA expression of Ecadherin was significantly lower than that in the paracancerous tissues. The mRNA expression of HIT and E-cadherin in osteosarcoma tissues was negatively correlated (all P<0.01). The DDP IC50 in the DDP-resistance group was significantly higher than that in the control group, interference group A and B, and the DDP IC50 in over-expression group was significantly higher than that in blank group (all P<0.01). The expression of HIT in resistance group was significantly higher than that in the control group, and the HIT expressions in interference group A and B were significantly lower than that in DDP-resistance and control group; moreover, the expression of HIT in over-expression group was significantly higher than that in blank group (all P<0.05 or P<0.01). The mRNA expression of Snail in DDPresistance group was significantly higher than that in the control group and interference group A and B, while the mRNA expression of E-cadherin in DDP-resistance group was significantly lower than that in the control group and interference group A and B; and the mRNA expression of E-cadherin in over-expression group was significantly lower than that in blank group. The protein expression of Snail in the DDP-resistance group was significantly higher than that in the control group and interference group A and B,while E-cadherin protein expression was significantly lower; and protein expression of Snail in over-expression group was significantly higher than that in blank group (all P<0.05 or P<0.01). The expression of HIT in the U20S and 293T cells treated by anti-Snail antibody induced by immunomagnetic beads was significantly higher than that in the cells treated by IgG antibody (P<0.01). Conclusion: HIT can promote EMT and cisplatin-resistance in osteosarcoma cells through up-regulation of Snail protein and inhibition of E-cadherin transcription activity.

天津市卫生局科技基金资助项目（No.2013KR17）;天津市天津医院科技基金资助项目（No.TJYY1508）