The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells

Area of Science:

• Oncology
• Molecular Biology
• Virology

Background:

• Cervical cancer is frequently caused by persistent infection with high-risk human papillomavirus (HPV) types, particularly HPV16.
• HPV oncoproteins E6 and E7 play crucial roles in viral replication and cellular transformation, driving cancer progression by inactivating tumor suppressor proteins like p53 and retinoblastoma (RB).
• MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are often dysregulated in cancer, with miR-182 implicated in various malignancies.

Purpose of the Study:

• To investigate the efficacy of simultaneously targeting HPV16 E6 and E7 oncoproteins using small interfering RNA (siRNA) and inhibiting miR-182 with an anti-miR-182 oligonucleotide in HPV16-positive cervical cancer cells.
• To evaluate the impact of these combined therapeutic strategies on cancer cell apoptosis, proliferation, and chemosensitivity.
• To assess the expression levels of key cellular targets, including p53, p21, and FOXO1, following the therapeutic interventions.

Main Methods:

• The HPV16-positive CaSki cervical cancer cell line was utilized for in vitro experiments.
• Cells were transfected with specific siRNA sequences targeting E6 and E7 oncoproteins, and/or anti-miR-182.
• Gene and protein expression levels were analyzed using real-time PCR, Western blot, and immunocytochemistry.
• Apoptosis rates were determined using Annexin-V-FITC staining, and cell viability was assessed via MTT assays.
• The effect of combined treatments with siRNAs, anti-miR-182, and cisplatin was evaluated.

Main Results:

• Individual transfection with E6-targeting siRNA (E6-1) and E7-targeting siRNA (E7-2) significantly increased apoptosis rates to 12.4% and 16%, respectively, after 48 hours.
• Transfection with anti-miR-182 alone also elevated the apoptosis rate to 12.7% in cervical cancer cells.
• Combination therapy using E6-1 siRNA with anti-miR-182 resulted in a 19.3% apoptosis rate, while E7-2 siRNA with anti-miR-182 led to a 26% apoptosis rate, demonstrating synergistic effects.
• Combined treatment with E6 and E7 siRNAs and cisplatin enhanced cancer cell sensitivity to cisplatin and reduced cell viability compared to cisplatin alone.
• The combination of cisplatin and anti-miR-182 showed no significant additional effect on cell viability or apoptosis compared to cisplatin monotherapy.

Conclusions:

• Targeting HPV16 E6 and E7 oncoproteins with siRNA, particularly in combination with anti-miR-182, represents a promising strategy for inducing apoptosis and reducing proliferation in cervical cancer.
• The synergistic effect observed with the combined siRNA and anti-miR-182 treatment highlights its potential as an enhanced therapeutic approach.
• Combined E6/E7 siRNA and cisplatin treatment improves chemosensitivity, suggesting a role for these agents in overcoming treatment resistance in cervical cancer.