Functional Study Of Tumor-related MicroRNAs Using Antisense Techniques

Objective:We designed the specific antisense oligonucleotides against four of the knownhuman tumor-related microRNAs, to investigate the effects of these fourLipofectamine^TM2000 mediated antisense-microRNA-oligonucleotides (AMOs) onthe growth and apoptosis of human lung cancer cell line A549 and leukemia cell lineK562. In addition, we wanted to select the most effective anti-lung-cancer andanti-leukemia AMOs to further explore their anticancer mechanisms.Methods:1. Selection of the effective concentrations of AMO on human lung cancer cellline A549 and leukemia cell line K562 by Trypan blue stain assay(1) Preparation of the mixtures Lipofectamine^TM 2000-AMOs: Lipofectamine^TM 2000and commercially synthesized AMOs were mixed at a mass ratio of 2.5:1 before use.(2) At final concentrations of 0.05μmol/L, 0.1μmol/L, 0.2μmol/L, 0.3μmol/L and0.6μmol/L, separately transfect the Lipofectamine^TM 2000-AMO into human lungcancer cell line A549 and then determine their inhibitory effects on proliferation of thecells.(3) At final concentrations of 0.1μmol/L, 0.2μmol/L, 0.3μmol/L and 0.6μmol/L,separately transfect the Lipofectamine^TM 2000-AMO into leukemia cell line K562 andthen determine their inhibitory effects on proliferation of the cells. 2. Time-course determination of the inhibitory effects of AMO on growth ofhuman lung cancer cell line A549 and leukemia cell line K562(1) In a time-course with 24h, 48h and 72h time points and at a final concentration of0.3μmol/L, determine the inhibitory effects of AMOs on the growth of human lungcancer cell line A549.(2) In a time-course with 24h, 48h and 72h time points and at a final concentration of0.6μmol/L, determine the inhibitory effects of AMOs on the growth of leukemia cellline K562.3. Flowcytometric analysis of cell cycle and subdiploid changes in A549 and K562cell lines induced by AMOAfter separately incubated with AMOs for different times, the infected humanlung cancer cell line A549 and leukemia cell line K562 were flowcytometricallydetermined for the formation of subdiploids and changes in the cell cycles after PIdyed.4. Real-time PCR determination of microRNA levels in human lung cancer cellline A549 and leukemia cell line K562 after incubated with AMOAfter the two cell lines were separately incubated with AMOs for differentdurations, the cytosolic levels of miR-21 and miR-181a were determined by real-timePCR.Results:1. The optimal effective concentrations of AMO on human lung cancer cell lineA549 and leukemia cell line K562(1) AMOs began to exhibit their inhibitory effects on proliferation of human lungcancer cell line A549 at 0.1μmol/L, while the best effects were found to be at 0.3μmol/L.(2) The effective concentrations of both AMO-miR-21 and AMO-miR-181a werefound to start from 0.2μmol/L to show the inhibition of proliferation of leukemia cellline K562. At 0.3μmol/L and 0.6μmol/L their inhibitory effects were more evident, while their best effects were observed at 0.6μmol/L.2. Inhibitory effects of AMO on growth of human lung cancer cell line A549 andleukemia cell line K562(1) When compared with the control of randomly synthesized sequence, at a finalconcentration of 0.6μmol/L and after incubation for 72h, both AMO-miR-21 andAMO-miR-181a exhibited statistically significant inhibitory effects on proliferation ofleukemia cell line K562. These inhibitory effects of the two drugs were observedstronger with incubation time.(2) After incubated with AMOs for different durations at a final concentration of 0.3μmol/L, both AMO-miR-16 and AMO-miR-181a were found to exhibit inhibitoryeffects on growth of human lung cancer cell line A549. The inhibitory effectsappeared after 48h of incubation, became stronger with incubation time, and reachedthe maximum at 72h. Similar inhibitory effects were also observed for AMO-miR-21.(3) After incubation for different durations at a fmal concentration of 0.6μmol/L, bothAMO-miR-21 and AMO-miR-181a were found to exhibit inhibitory effects on growthof leukemia cell line K562. These inhibitory effects appeared after 48h of incubation,increased with incubation time, became more evident at 72h and even stronger at 96h.3. AMO-miR-21 and AMO-miR-181a increased the subdiploids in both humanlung cancer cell line A549 and leukemia cell line K562, and promoted apoptosisof the two cell lines(1) When compared with the control of randomly synthesized sequence, at a finalconcentration of 0.3μmol/L and after incubation for 24h, 48h and 72h, AMO-miR-16,AMO-miR-21 and AMO-miR-181a groups exhibited significant apoptotic peaks inhuman lung cancer cell line A549, indicating that the three AMOs promoted apoptosisof the cancer cells. Statistical analysis by group cooperation showed thatAMO-miR-21 and AMO-miR-181a had significantly better apoptosis-promotingeffects than others (P＜0.05).(2) When compared with the control of randomly synthesized sequence, at a finalconcentration of 0.6μmol/L and after incubation for 24h, 48h and 72h, bothAMO-miR-21 and AMO-miR-181a groups showed significantly increased subdiploid peaks in leukemia cell line K562, indicating that the two AMOs promoted apoptosisof the tumor cells. (P＜0.05).4. In human lung cancer cell line A549 and in leukemia cell line K562, the levelsof miR-21 and miR-181 were decreased respectively by AMO-miR-21 andAMO-miR-181aThe human lung cancer cell line A549 was incubated separately with AMOs ata final concentration of 0.3μmol/L for 48h, and the leukemia cell line K562 wasincubated separately with AMOs at a final concentration of 0.6μmol/L for 48h and72h. The total RNA was extracted for real-time PCR determination on the miR-21 andmiR-181 levels in the different group cells. It was found that the standard curve formiR-21 showed a well fitted strait line of the concentration versus C_T (8.30, 13.00,17.07, 0.98, 25.36,28.57), with a correlation of 0.998. Therefore, this standard curvewas used to calculate the microRNA levels in cells. Statistically compared with thecontrol of randomly synthesized sequence, after 48h of incubation AMO-miR-21 didnot show significant effects on the level of miR-21 in human lung cancer cell lineA549 (P＞0.05), while after incubation with AMO-miR-21 for 48h and 72h, leukemiacell line K562 was found to exhibit significantly decreased levels of miR-21(P＜0.05).Conlusions:1. The AMOs against microRNA can efficiently inhibit growth of human lung cancercell line A549 and leukemia cell line K562. Of those AMOs tested, AMO-miR-21 andAMO-miR-181a exhibited strong inhibitory effects on leukemia cell line K562, whileAMO-miR-16, AMO-miR-21 and AMO-miR-181a exhibited good inhibitory effectson human lung cancer cell line A549. These growth inhibitions of cancer cells by theAMOs showed well time course and dosage dependence.2. Apoptosis of tumor cells can be efficiently promoted by AMOs against microRNA.This may be one of the mechanisms by which AMOs inhibit growth of tumor cells.AMO-miR-21 and AMO-miR-181a promoted apoptosis of leukemia cell line K562, while AMO-miR-16, AMO-miR-21 and AMO-miR-181a promoted apoptosis ofhuman lung cancer cell line A549.3. Real-time PCR demonstrated that AMOs could decrease the levels of microRNA intumor cells. AMO-miR-21 could lower the levels of miR-21 in leukemia cell lineK562.4. The control of randomly synthesized sequence and the control of liposome hadnon-specific inhibitory effects on cell proliferation.