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.