CAJ | 학술논문

目的筛选并鉴定调控生育酚结合蛋白(TAP)的miRNAs,并观察其对前列腺癌细胞增殖的影响.方法生物信息学分析和双荧光报告筛选调控TAP的miRNAs；以前列腺组织总RNA1μg反转录并扩增TAP基因(SEC14L2,Gene ID:23541)及其3'端非翻译区(3’-UTR),克隆入p3 ×FLAG-CMVTM-7.1载体；miR-96类似物与TAP表达载体pCMV7.1-TAP、pCMV7.1-TAP-3’-UTR转染前列腺癌细胞DU-145,Western blot检测TAP的表达,噻唑蓝(MTT)法观察DU-145的增殖.结果 miR-96可作用于TAP的3’-UTR；pCMV7.1-TAP-3’-UTR分别经Not Ⅰ/BglⅡ、Sal Ⅰ/Xba酶切后电泳,在1200、1400 bp附近有目的条带,测序结果与Gene Bank报道一致.Western blot检测结果表明miR-96可使TAP表达下降65.1％ (P ＜0.05).MTT分析显示对照组以及空载体转染组的细胞吸光度(A)值分别为0.972 ±0.023、0.967±0.042;pCMV7.1-TAP转染组、pCMV7.1-TAP-3’-UTR转染组的A值分别为0.625±0.037、0.731±0.043,差异均有统计学意义(P＜0.05)；pCMV7.1-TAP-3’-UTR和miR-96共转染组的A值为0.914±0.034,与对照组比较,差异无统计学意义(P＞0.05).结论 miR-96可通过3’-UTR调节TAP的表达,并减弱TAP对前列腺癌细胞增殖的抑制作用.
목적 사선병감정조공생육분결합단백(TAP)적miRNAs,병관찰기대전렬선암세포증식적영향.방법 생물신식학분석화쌍형광보고사선조공TAP적miRNAs；이전렬선조직총RNA1μg반전록병확증TAP기인(SEC14L2,Gene ID:23541)급기3'단비번역구(3’-UTR),극륭입p3 ×FLAG-CMVTM-7.1재체；miR-96유사물여TAP표체재체pCMV7.1-TAP、pCMV7.1-TAP-3’-UTR전염전렬선암세포DU-145,Western blot검측TAP적표체,새서람(MTT)법관찰DU-145적증식.결과 miR-96가작용우TAP적3’-UTR；pCMV7.1-TAP-3’-UTR분별경Not Ⅰ/BglⅡ、Sal Ⅰ/Xba매절후전영,재1200、1400 bp부근유목적조대,측서결과여Gene Bank보도일치.Western blot검측결과표명miR-96가사TAP표체하강65.1％ (P ＜0.05).MTT분석현시대조조이급공재체전염조적세포흡광도(A)치분별위0.972 ±0.023、0.967±0.042;pCMV7.1-TAP전염조、pCMV7.1-TAP-3’-UTR전염조적A치분별위0.625±0.037、0.731±0.043,차이균유통계학의의(P＜0.05)；pCMV7.1-TAP-3’-UTR화miR-96공전염조적A치위0.914±0.034,여대조조비교,차이무통계학의의(P＞0.05).결론 miR-96가통과3’-UTR조절TAP적표체,병감약TAP대전렬선암세포증식적억제작용.
Objective To screen α-tocopherol associated protein (TAP) -regulating miRNAs and investigate its effect on the proliferation of prostate cancer cells.Methods Bioinformatics analysis and DualLuciferase Reporter Assay were perform to identify TAP-regulating miRNAs.TAP gene (SEC14L2) and its 3' -untranslated region ( 3' -UTR ) sequence were cloned into p3 × FLAG-CMVTM-7.1 expression vector.MiR-96 and TAP expression vector pCMV7.1-TAP,pCMV7.1-TAP-3'-UTR were transfected into prostate cancer cell line DU-145.The expression level of TAP was detected by western blotting and the cell proliferation was valued by methyl thiazol tetrazolium (MTT) assay.Results MiR-96 targets 3' -UTR of TAP.After double restriction enzyme digestion and agarose gel elecrophoresis of pCMV7.1-TAP-3'-UTR,the 1200 bp and 1400 bp purpose bands were sequenced,results was exactly the same with the sequence GeneBank had reported,indicating that the recombinant plasmids pCMV7.1-TAP-3 ' -UTR were successfully constructed.TAP protein expression level mediated by pCMV7.1-TAP-3' -UTR plasmid was down-regulated by miR-96 by 65.1％ (P ＜0.05).MTT assay showed that the cellular absorbance value (A value) of the control group and the empty vector transfection group was 0.972 ±0.023 and 0.967 ± 0.042 each; A value of pCMV7.1-TAP transfection group and pCMV7.1-TAP-3 ' -UTR transfection group was 0.625 ± 0.037 (P ＜ 0.05 ) and 0.731 ±0.043 (P ＜0.05) respectively；A value of pCMV7.1-TAP-3'-UTR and miR-96 co-transfection group was 0.914±0.034 ( P ＞ 0.05 ).Conclusion miR-96 can regulate the expression of TAP by targeting 3' -UTR and attenuates the effect of TAP on the proliferation of prostate cancer cells.