In-vitro growth assays PC3. Methods: PC3 and DU145 Human Prostate cancer cell-lines (Charles River / NCI Developmental Therapeutics Program) were seeded in clear-6, clear-12 or black-96 well plates at 5000, 2000, or 1000 cells/well, respectively, in RPMI 1640 + 2mM L-glutamine ( 11875-093, Life Technologies, Grand Island, NY) + 10% FBS + 50 units/ml penicillin and 50ug/ml streptomycin (30-001-CI, Mediatech, Manassas, VA) and incubated at 370 C and 5% CO2 overnight. HPS or control oligonucleotides (Sigma-Aldrich, Woodland, TX, / Eurofins MWG Operon, Huntsville, AL) and Calcitriol were applied without DMSO to the well-plates which then were incubated at time-courses. The media was slowly aspirated and ATPlite one-step (6016731, Perkin-Elmer, Akron OH) solution was added to the wells. A Perkin-Elmer Victor III plate reader set for 120 seconds of 3.0 mm of orbital shaking lysed the cells and then measured the luminescent of individual wells. Growth Rates did not change in DU-145.
Conclusion: S4 and s5 point mutations removed the miR-125 extended seed sequence and restored normal growth; therefore, the growth decline in PC3 is related to miR-125 activity on HuR or on other miR-125 related activities. S6 and s7 restored the miR-125 extended seed in the oglionucleotide and restored the growth declins in PC3. Thus, blocking miR-125 activity is responsible for PTEN deficient PC3 in-vitro growth rate declines. Mir-9 and mir-133 are not related to PC3 growth rates declines. S7 removed both miR-9 and miR-133 seed sequences from the HPS. S8 has the extended seed for miR-125 and reduced PC3 growth rates like the full 22 NT HPS. Thus, the HPS is bio-active because the in-vitro growth rate of PC3 Human Prostate cancer cells is decreased. The specific microRNA activity, HuR expression related to mutant HPS, and other factors still need to be explored.