miR-582-5p靶向调控AKT3对甲状腺乳头状癌细胞增殖和凋亡的影响

Effect of miR-582-5p targeting AKT3 on proliferation and apoptosis of papillary thyroid carcinoma cells

目的探讨miR-582-5p在人甲状腺乳头状癌(PTC)组织中的表达及其靶向调控AKT3对PTC细胞增殖和凋亡的影响。方法选取2017年1月至2017年10月海南省肿瘤医院手术切除的PTC组织及癌旁组织(距离肿瘤边缘1~2 cm)标本各10例,采用实时定量聚合酶链反应(PCR)检测PTC组织和癌旁组织中miR-582-5p表达;培养人PTC K1细胞,待细胞生长融合度达到50%~60%时分为miR-582-5p mimics组、miR-582-5p反义miRNA寡核苷酸(AMO)组、mimics对照组和AMO对照组,进行细胞转染;细胞转染后,采用实时荧光定量PCR检测4组K1细胞中miR-582-5p表达,四甲基偶氮唑盐法检测4组K1细胞活性,平板克隆形成实验检测4组K1细胞克隆能力,锥虫蓝染色检测4组K1细胞存活率,末端脱氧核苷酰基转移酶介导性d UTP切口末端标记法检测4组K1细胞凋亡情况,Western blot法检测4组K1细胞中miR-582-5p靶基因AKT3蛋白表达。结果 PTC组织和癌旁组织中miR-582-5p相对表达量分别为0. 372±0. 237、1. 010±0. 083,PTC组织中miR-582-5p相对表达量显著低于癌旁组织(P <0. 05)。miR-582-5p mimics组、mimics对照组、AMO对照组和miR-582-5p AMO组K1细胞中miR-582-5p相对表达量分别为14. 143±2. 318、1. 063±0. 214、1. 041±0. 372、0. 435±0. 145; miR-582-5p mimics组K1细胞中miR-582-5p相对表达量显著高于mimics对照组、miR-582-5p AMO组和AMO对照组(P <0. 05),miR-582-5p AMO组K1细胞中miR-582-5p相对表达量显著低于AMO对照组和mimics对照组(P <0. 05),mimics对照组与AMO对照组K1细胞中miR-582-5p相对表达量比较差异无统计学意义(P> 0. 05)。细胞培养0 h时4组K1细胞活性比较差异无统计学意义(P> 0. 05)。细胞培养12、24、36、48 h时,miR-582-5p mimics组K1细胞活性显著低于miR-582-5p AMO组、mimics对照组和AMO对照组(P <0. 05),miR-582-5p AMO组K1细胞活性显著高于mimics对照组和AMO对照组(P <0. 05),mimics对照组与AMO对照组K1细胞活性比较差异无统计学意义(P> 0. 05)。mimics对照组、miR-582-5p mimics组、AMO对照组、miR-582-5p AMO组K1细胞克隆数量分别为18. 47±5. 25、5. 10±4. 97、19. 53±6. 45、32. 63±7. 74; miR-582-5p mimics组K1细胞克隆数量显著少于mimics对照组、AMO对照组和miR-582-5p AMO组(P <0. 05),miR-582-5p AMO组K1细胞克隆数量显著多于AMO对照组和mimics对照组(P <0. 05),mimics对照组与AMO对照组K1细胞克隆数量比较差异无统计学意义(P> 0. 05)。mimics对照组、miR-582-5p mimics组、AMO对照组和miR-582-5p AMO组K1细胞存活率分别为(76. 46±13. 53)%、(42. 64±10. 85)%、(77. 82±10. 35)%、(83. 63±13. 53)%; miR-582-5p mimics组K1细胞存活率显著低于mimics对照组、AMO对照组和miR-582-5p AMO组(P <0. 05),miR-582-5p AMO组K1细胞存活率显著高于AMO对照组和mimics对照组(P <0. 05); mimics对照组与AMO对照组K1细胞存活率比较差异无统计学意义(P> 0. 05)。mimics对照组、miR-582-5p mimics组、AMO对照组和miR-582-5p AMO组K1细胞凋亡率分别为(23. 35±9. 64)%、(42. 63±13. 38)%、(22. 85±9. 74)%、(10. 84±3. 64)%; miR-582-5p mimics组K1细胞凋亡率显著高于mimics对照组、AMO对照组和miR-582-5p AMO组(P <0. 05),miR-582-5p AMO组细胞凋亡率显著低于AMO对照组和mimics对照组(P <0. 05),mimics对照组与AMO对照组K1细胞凋亡率比较差异无统计学意义(P> 0. 05)。mimics对照组、miR-582-5p mimics组、AMO对照组和miR-582-5p AMO组K1细胞中AKT3蛋白相对表达量分别为1. 000±0. 005、0. 531±0. 162、1. 010±0. 071、1. 432±0. 141; miR-582-5p mimics组K1细胞中AKT3蛋白相对表达量显著低于mimics对照组、AMO对照组和miR-582-5p AMO组(P <0. 05),miR-582-5p AMO组K1细胞AKT3蛋白相对表达量显著高于AMO对照组和mimics对照组(P <0. 05),mimics对照组与AMO对照组K1细胞AKT3蛋白相对表达量比较差异无统计学意义(P> 0. 05)。结论 miR-582-5p在PTC组织中表达下调; miR-582-5p可通过靶向调控AKT3的表达而抑制人PTC细胞的增殖,并促进其凋亡。

Objective To investigate the expression of miR-582-5p in human papillary thyroid carcinoma （PTC） and the effect of miR-582-5p targeting AKT3 on the proliferation and apoptosis of PTC cells.Methods Ten PTC tissue specimens and ten paracancerous tissue specimens （1-2 cm away from the edge of the tumor） were collected in Hainan Cancer Hospital from January 2017 to October 2017.The expression of miR-582-5p in PTC and paracancerous tissues was detected by real-time quantitative polymerase chain reaction （PCR）.Human PTC K1 cells were cultured and divided into miR-582-5p mimics group,miR-582-5p anti-miRNA oligonucleotide（AMO） group,mimics control group and AMO control group when the cell fusion rate reached 50%-60%.After cell transfection,the expression of miR-582-5p in K1 cells of the four groups was detected by real-time fluorescence quantitative PCR,the activity of K1 cells was detected by methyl thiazolyl tetrazolium method,The colony formation ability of K1 cells was detected by flat plate clone formation test.the survival rate of K1 cells was detected by trypanosoma blue staining,the apoptosis of K1 cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method,and the expression of miR-582-5p target gene AKT3 protein of K1 cells was detected by western blot method.Results The relative expression of miR-582-5p in PTC tissues and paracancerous tissues was 0.372±0.237 and 1.010±0.083,respectively.The relative expression of miR-582-5p in PTC tissues was significantly lower than that in the paracancerous tissues （ P 〈0.05）.The relative expression of miR-582-5p in K1 cells of miR-582-5p mimics group,mimics control group,AMO control group and miR-582-5p AMO group was 14.143±2.318,1.063± 0.214,1.041±0.372 and 0.435±0.145,respectively.The relative expression of miR-582-5p in K1 cells of miR-582-5p mimics group was significantly higher than that in the mimics control group,miR-582-5p AMO group and AMO control group （ P 〈0.05）.The relative expression of miR-582-5p in K1 cells of the miR-582-5p AMO group was significantly lower than that in the AMO control group and mimics control group （ P 〈0.05）.There was no significant difference in the relative expression of miR-582-5p between the mimics control group and AMO control group （ P 〉0.05）.There was no significant difference in the activity of K1 cells between the four groups when the K1 cells were cultured for 0 hour （ P 〉0.05）.When the K1 cells were cultured for 12,24,36,and 48 hours,the activity of K1 cells in the miR-582-5p mimics group was significantly lower than that in the miR-582-5p AMO group,mimics control group and AMO control group （ P 〈0.05）;the activity of K1 cells in the miR-582-5p AMO group was significantly higher than that in the mimics control group and AMO control group （ P 〈0.05）;there was no significant difference in the activity of K1 cells between the mimics control group and AMO control group （ P 〉0.05）.The number of K1 cell clones in the mimics control group,miR-582-5p mimics group,AMO control group and miR-582-5p AMO group was 18.47±5.25,5.10±4.97,19.53±6.45 and 32.63±7.74,respectively.The number of K1 cell clones in the miR-582-5p mimics group was significantly less than that in the mimics control group,AMO control group and miR-582-5p AMO group （ P 〈0.05）.The number of K1 cell clones in the miR-582-5p AMO group was significantly higher than that in the AMO control group and mimics control group （ P 〈0.05）.There was no significant difference in the number of K1 cell clones between the mimics control group and AMO control group （ P 〉0.05）.The survival rate of K1 cells in the mimics control group,miR-582-5p mimics group,AMO control group and miR-582-5p AMO group was （76.46±13.53）%,（42.64±10.85）%,（77.82±10.35）% and （83.63±13.53）%,respectively.The survival rate of K1 cells in the miR-582-5p mimics group was significantly lower than that in the mimics control group,AMO control group and miR-582-5p AMO group （ P 〈0.05）.The survival rate of K1 cells in the miR-582-5p AMO group was significantly higher than that in the AMO control group and mimics control group （ P 〈0.05）.There was no significant difference in the survival rate of K1 cells between the mimics control group and AMO control group （ P 〉0.05）.The apoptosis rate of K1 cells in the mimics control group,miR-582-5p mimics group,AMO control group and miR-582-5p AMO group was （23.35±9.64）%,（42.63±13.38）%,（22.85±9.74）% and （10.84±3.64）%,respectively.The apoptosis rate of K1 cells in the miR-582-5p mimics group was significantly higher than that in the mimics control group,AMO control group and miR-582-5p AMO group （ P 〈0.05）.The apoptosis rate of miR-582-5p AMO group was significantly lower than that in the AMO control group and mimics control group （ P 〈0.05）.There was no significant difference in the apoptosis rate of K1 cells between the mimics control group and AMO control group （ P 〉0.05）.The relative expression of AKT3 protein in K1 cells of the mimics control group,miR-582-5p mimics group,AMO control group and miR-582-5p AMO group was 1.000±0.005,0.531±0.162,1.010±0.071 and 1.432±0.141,respectively.The relative expression of AKT3 protein in K1 cells of the miR-582-5p mimics group was significantly lower than that in the mimics control group,AMO control group and miR-582-5p AMO group （ P 〈0.05）.The relative expression of AKT3 protein in K1 cells of the miR-582-5p AMO group was significantly higher than that in the AMO control group and the mimics control group （ P 〈0.05）.There was no significant difference in the relative expression of AKT3 protein between the mimics control group and AMO control group （ P 〉0.05）.Conclusion The expression of miR-582-5p ss down-regulated in PTC tissues.miR-582-5p can inhibit the proliferation of human PTC cells and promote its apoptosis through targeting AKT3 expression.