ABSTRACT
@# 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 mRNAexpression 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 mRNAexpressions 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 mRNAexpression 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.