CAJ | 학술논문

目的构建人微小RNA145(miR-145)前体的真核表达载体,探讨其对胃癌细胞生物学行为的影响.方法根据人pre-miR-145序列,合成两条互补的寡核苷酸链,退火后连接入pPG-miR-EGFP载体,转化扩增后进行测序鉴定;重组质粒在阳离子脂质体Lipofectamine 2000的介导下,瞬时转染人胃癌细胞株SGC-7901,实时定量聚合酶链反应(PCR)检测细胞中miR-145表达水平,黏附实验和Transwell小室实验检测癌细胞游走、侵袭能力.结果　所构建的pPG-miR145-EGFP载体中插入基因片段与设计序列完全匹配;重组质粒转染SGC-7901细胞后,miR-145表达分别上调20.45倍(P＜0.01),细胞黏附能力分别下降51.5%(P＜0.01),细胞侵袭能力分别下降30.4%(P＜0.01).结论　成功构建人pre-miR-145真核表达载体,转染胃癌细胞过表达后,能抑制癌细胞的游走和侵袭能力.
목적 구건인미소RNA145(miR-145)전체적진핵표체재체,탐토기대위암세포생물학행위적영향.방법 근거인pre-miR-145서렬,합성량조호보적과핵감산련,퇴화후련접입pPG-miR-EGFP재체,전화확증후진행측서감정;중조질립재양리자지질체Lipofectamine 2000적개도하,순시전염인위암세포주SGC-7901,실시정량취합매련반응(PCR)검측세포중miR-145표체수평,점부실험화Transwell소실실험검측암세포유주、침습능력.결과　소구건적pPG-miR145-EGFP재체중삽입기인편단여설계서렬완전필배;중조질립전염SGC-7901세포후,miR-145표체분별상조20.45배(P＜0.01),세포점부능력분별하강51.5%(P＜0.01),세포침습능력분별하강30.4%(P＜0.01).결론　성공구건인pre-miR-145진핵표체재체,전염위암세포과표체후,능억제암세포적유주화침습능력.
Objective To construct the eukaryotic expression vector of miR-145 and explore its effects on the biological behavior of gastric cancer. Methods According to the pre-miR-145 sequence,two complementary oligonucleotide strands were synthesized and inserted into pPG-miR-EGFP vector,which was validated by nucleic acid sequencing. Under the induction of Lipofectamine 2000, the recombinant was transfected into SGC-7901 cells. The expression level of miR-145 was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). The cell adhesion and transwell assays were applied for the detection of in vitro migration and invasion of cancer cells. Results Sequencing analysis confirmed the correctness of the construction. Compared with negative control, the expression of miR-145 was upregulated by 20. 45 times (P ＜0. 01 ). Meanwhile,the cellular adhesion was downregulated by 51.5% (P ＜0. 01 ) ,and the invasive activity was downregulated by 30. 4% (P ＜0. 01 ). Conclusion The expression vector of premiR-145 was successfully constructed,and induced the miR-145 overexpression in gastric cancer cells, resulting in decrease of migration and invasion of cancer cells.