RU 08. The growth inhibitory effect of 2'-fluoridedsp21 on human bladder cancer cell
Introduction and Objective: dsP21 is a synthesized 21-nucleotide double strand RNA (dsRNA) targeting the P21 promoter at position -322 relative to the transcription start site. Previous study has shown that dsP21 has antitumor effect in human bladder cancer cells by inducing P21 protein expression. To enhance its antitumor effect and bioavailability, we synthesized 2′-fluoride-dsP21 (M-dsP21), which uracil residues were modified. This study aimed to investigate the effect of M-dsP21 on the cancer cell proliferation and apoptosis.
Methods: Bladder cancer cells (T24, J82) were cultured in vitro. Transfection was performed by using dsRNA (50 nmol/L) and RNAi-max (Invitrogen). The bioavailability of M-dsP21 was investigated by labeling M-dsP21 with FAM. Cells were collected at time points 8 hour(h), 16, 24 and 32 h, subsequently observed and assessed under con-focal microscope. Cell morphology changes were directly observed after 3 days transfection. The cell growth assay was performed by MTT assay. The P21 mRNA and protein expression were investigated by Real-time PCR and Western-blot. The cell cycle and apoptosis were assessed by flow cytometry. Results: M-dsP21 showed higher penetrating capability and RNA enzymes resistance, and could be retained in cell longer than dsP21. Both M-dsP21 and dsP21 can induce the cell morphology change, ultimately lead to cell death after 3 days transfection. The cell growth was arrested by M-dsP21 tranfection, parallel with increased P21 protein expression, but not parallel with the mRNA level. Flow cytometry analysis revealed a significant increase in the G0-G1 population, G2-M-phase populations and the corresponding decrease S-phase populations (P<0.01), the same as the effect of dsP21 on the bladder cancer cells. M-dsP21 transfection showed more apoptosis cell population than mock, ds-control and dsP21 treatment.
Conclusions: M-dsP21 enhances the bioavailability and RNA enzymes resistance, has an inhibitory effect on human bladder cancer cell. The exact mechanism for this effect need profound investigation.
