PMC

Cells were treated with a miRNA mimic or an miRNA inhibitor for each family member (miR-15a/15b/16/16-1*/424*) (10nM) and challenged with cisplatin (50 μM to 0 μM). miR-15a/15b/16/16-1*/424* all showed an increase in resistance when the miRNA was inhibited and an increase in sensitivity to cisplatin when the miRNA was increased.

A, Expression of miR-15 family members and miR-155 in KB-3-1 and KB-CP.5, with RT-PCR. Samples were normalized to U6. Using ANOVA statistical analysis, columns comparing expression of one miRNA between KB-3-1 to KB-CP.5 have a p-value ≤ 0.05. Columns comparing different miRNAs to each other in the same cell line have a p-value of 0.0001. Western blots showing expression of WEE1 and CHK1 in response to transfection with miR-15a/15b/16/16-1*/424* miRNA mimics and miRNA. KB-3-1 cells transfected with mimics (B) and inhibitors (C), and KB-CP.5 cells transfected with mimics (D) and inhibitors (E) were collected and protein extracted after 72 h.

A, Follow-up dose response analyses using additional siRNAs confirms sensitization mediated by the knockdown of ATR, CHK1, and WEE1 in KB-CP.5 cells compared to siNegative treated cells. B, Western blots showing normal expression of kinases. ATR, WEE1, and CHK1 have increased expression in resistant lines (KB-CP.5 and KB-CP20) compared to sensitive (KB-3-1). The increase in expression is dependent on the level of resistance. C, Small molecule kinase inhibitors PD 407824 (CHK1 and WEE1), MK 1775 (WEE1), and SB 218078 (CHK1) have concentration-dependent toxic effect on sensitive and resistant cell lines. D, Cells were treated with sub-toxic doses of kinase inhibitors (10nM) and challenged with cisplatin (50 μL to 0 μL). KB-3-1 sensitive cells had a 1.2 – 3.9 fold decrease in survival in cisplatin when the kinase function was inhibited with small molecule inhibitors. KB-CP.5 resistant cells showed less of an effect at 1.2 to 1.9 fold increase in sensitivity to cisplatin. CisPt = cisplatin.