微小RNA-221/222海绵体载体的构建及验证

Construction and validation of micro RNA-221/222 sponge vector

目的构建微小RNA(mi R)-221/222海绵体载体并初步探讨其在口腔鳞状细胞癌(OSCC)细胞中的作用。方法利用mi RNA海绵体技术设计合成含3个has-mi R-221和3个hasmi R-222反义序列的DNA片段,经酶切连入p DC316-m CMV-EGFP质粒载体,合成mi R-221/222海绵体载体。将mi R-221/222海绵体载体转染入OSCC细胞系UM1,检测转染效率,实时荧光定量聚合酶链反应(PCR)检测mi R-221和mi R-222表达水平,Western blot检测转染后PTEN蛋白表达水平,Transwell实验检测细胞的侵袭能力。结果成功构建mi R-221/222海绵体载体,测序验证与设计序列完全一致;UM1各组细胞转染效率均在70%以上;实时荧光定量PCR显示,mi R-221/222海绵体载体组mi R-221和mi R-222表达水平较p DC316质粒组和UM1细胞组明显降低(t1=111.69,P1=0.001;t2=6.98,P2=0.002;t3=236.55,P3=0.001;t4=22.06,P4=0.001);Western blot显示,海绵体转染组较对照组PTEN蛋白水平表达明显升高;Transwell实验显示,海绵体转染组细胞侵袭能力较对照组减弱。结论实验成功构建了mi R-221/222海绵体载体,并初步验证其对OSCC细胞侵袭性的抑制作用,为后续mi R-221/222的功能研究奠定基础。

Objective To construct micro RNA（mi R）-221 / 222 sponge vector and investigate the effect of sponge transfection in oral squamous cell carcinoma cells. Methods Using the technology of mi RNA sponge, a DNA fragment contained three has-mi R-221 and three has-mi R-222 antisense sequences was designed and synthesized. The DNA fragment was cloned into the p DC316-m CMV-EGFP plasmid vector by enzyme digestion to construct the mi R-221 / 222 sponge vector. Then the mi R-221 /222 sponge vector was transfected into UM1 cells, and the transfection efficiency was detected. Real time quantitative PCR was used to test the expression level of the mi R-221 and mi R-222. The protein expression of phosphatase and tension homolog deleted on chromosome 10（PTEN） was analyzed by Western blot and Transwell assay to measure the invasion ability of cells. Results The mi R-221 / 222 sponge vector was constructed successfully, and it was validated to be entirely consistent with the designed sequence. The transfection efficiency of UM1 cells in each group was more than 70%. The results of real time quantitative PCR showed that the expression levels of the mi R-221 and mi R-222 in sponge transfected group were decreased compared to the positive control group and the negative control group（t1= 111.69,P1= 0.001;t2= 6.98,P2= 0.002;t3= 236.55,P3= 0.001;t4= 22.06,P4= 0.001）. Western blot showed that the expression of PTEN in the transfected sponge group was significantly increased compared with mock group and non-transfected group. Transwell assay indicated that cell invasion was inhibited by mi R-221 / 222 sponge transfection. Conclusions The mi R-221 / 222 sponge vector was successfully constructed and its inhibitory effect of the invasion of OSCC cells has been validated. This lays the foundation for further study on the function of mi R-221 / 222.