| [Objective] To study the effect and the mechanism of Artemether on hyperther mia-induced apoptosis in tongue squamous cell carcinoma Cal-27 cells.[Methods] (l)Cal-27 cells were cultured With DMEM containing 10% fetal bovine serum sugar medium, with 37 ℃ and 5% CO2 incubator.(2) Experimental groups: control group(Cal-27 cells), Artemether treated group(Cal-27 cells were treated with 0.1mg/ml Artemether), hyperthermia-induced group (Cal-27 cells were treated with 43 ℃ 80min), Artemether+hyperthermia-induced group(Cal-27 cells were treated with 0.1mg/ml Artemether+43℃ 80min). (3) The changes of cell morphology were observed by inverted microscope. (4) CCK8 assay was used to evaluate the inhibition rate of cellular proliferation. (5) Apoptosis rates of Cal-27 cells were analyzed with Annexin V-FITC/PI labeling through flow cytometry. (6) miRNA PCR assay chips were used to screen differentially expressed miRNAs, RT-PCR for validation. (7)The expressions of HSP90a were analyzed through Western blot. (8) The expressions of HSP90AA1 mRNA were tested by RT-PCR. (9) The regulating relationship between hsa -miR-134-5p (miR-134) and predicted target genes HSP90AA1 was analyzed through dual luciferase fluorescein enzymatic test. (10) Statistical analysis was carried out with SPSS 17.0.[Results] (1) After treated with hyperthermia or Artemether alone, Cal-27 cells showed obvious morphological changes of apoptosis. The cells became round and shrinkage. When treated with Artemether combined hyperthermia, the numb er of round and shrinkage cells increased significantly. (2) The inhibition rate of proliferation of Cal-27 cells in the Artemether group was (27.19±6.64)%,The inhibition rate of proliferation of Cal-27 cells in hyperthermia-induced group was (32.70±6.78)%, and Artemether+hyperthermia-induced group was (42.04 ±6:31)%. Artemether+hyperthermia-induced group was statistically significant differences compared with that of the group treated with hyperthermia or Arte mether alone (P<0.05), but there was no statistically significant differences bet ween hyperthermia and Artemether. (3) With Annexin V-HTC/PI labeling and an alysis by flow cytometry, the apoptosis rate in the control group was (2.65±0.6 0)%. The apoptosis rate in Artemether treated group was (13.86±1.47)%; The hyperthermia-induced group was (18.64±0.94)% and the Artemether+hyperther mia-induced group was (29.24±1.01)% at 24h after treatment. There was stati stically significant differences compared with that of the group treated with hy perthermia or Artemether alone (P<0.05). (4) MiR-134 in Artemether group ex pressing quantity was 10.68 times of normal group and miR-134 in the Arteme ther+hyperthermia-induced group was 6.68 times of normal group. Compared with the control group, hsa-miR-144-3p (miR-144), hsa-miR-153-5p (miR-153) and hsa-miR-32-5p(miR-32) expressions were decreased obviously in Artemeth er group and Artemether+hyperthermia-induced group. Expression amount of miR-144 in artemether group was 43% of the normal group, miR-153 was 4 5% of the normal group, miR-32 was 43% of the normal group. In Artemether +hyperthermia-induced group, miR-153 amounted to 22% of the normal grou p, the miR-32 amounted 40% of the normal group. The rest of miRNAs were no obvious change. The expressions of miR-134 were analyzed with RT-PCR. And in Artemether group, miR-134 relative expression was (2.17±0.18), Compa red with the normal control group, its expression increased (P< 0.05); In hype rthermia-induced group, miR-134 relative expression was (0.67 ±0.06), with no obvious difference on the normal group (P> 0.05); In Artemether+hyperther mia-induced group, the relative expression of miR-134 was (4.43±0.37), compar ed with normal group or hyperthermia-induced, its amounts of expressions we re significantly increased (P< 0.05). (5) The expressions of HSP90α were anal yzed through Western blot. In hyperthermia-induced group, the relative expressi on of HSP90α was (0.98±0.12). Compared with the normal control group(0.77 ± 0.05), its expression increased (P<0.05); And the relative expression of HSP9 0α was (0.48 ± 0.06) in Artemether treatment group and down-regulated compa red with the normal group (P<0.05); In Artemether+hyperthermia-induced gro up relative expression of HSP90α was (0.63±0.09). Compared with the hyperth ermia-induced group, its expression decreased (P< 0.05). (6) Compared with t he normal control group, the relative expression of HSP90AA1 mRNA was dec reased in artemether group that was (0.65±0.11) (P< 0.05); In hyperthermia-in duced group, relative expression of HSP90AA1 mRNA was (1.52±0.07), comp ared with normal group, its expression increased obviously (P< 0.05). In Arte mether+hyperthermia-induced group the expression was (1.03±0.11). Compa red with hyperthermia-induced group, the relative expression quantity significant ly decreased (P< 0.05). (7) It was found that miR-134 of HSP90AA1-3 ’UTR had significant inhibitory effect on the level of gene expression in Dual-Lucif erase Reporter Assay System (P< 0.05) while miR-134 of HSP90AA1-3 ’UTR mutant fragments had no obvious inhibitory effect (P> 0.05).[Conclusion] Artemether can inhibit HSP90 α expression through up-regulating hsa-miR-134-5p and then promotes hyperthermia-induced apoptosis in tongue squamous cell carcinoma Cal-27 cells. |
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