CAJ | 학술논문

目的：观察上调及下调microRNA-195（miR-195）对裸鼠皮下荷SHG-44人脑胶质瘤生长的影响并探讨其机制。方法使用脂质体瞬时转染法将miR-195 mimics和inhibitor分别转入人脑胶质瘤细胞系SHG-44，同时设立空白对照组和阴性对照组；实时荧光定量PCR（qRT-PCR）检测转染前后miR-195的含量变化；流式细胞法检测各组细胞周期分布；裸鼠成瘤实验观察miR-195对体内胶质瘤增殖的影响；肿瘤组织HE染色观察病理学改变；Western blot、免疫组织化学染色检测肿瘤组织周期相关蛋白P21、Cyclin D1的表达变化。结果 qRT-PCR测得转染mimics后miR-195的表达水平提高19倍左右，而转染inhibitor后miR-195的表达水平降低为空白对照组的42%；与空白对照和阴性对照组相比，miR-195mimics组细胞阻滞于G0/G1期（P＜0.05）；而miR-195 inhibitor组则相反（P＜0.05）；裸鼠成瘤实验显示，miR-195 mimics组与空白对照组和阴性对照组相比，肿瘤生长速度减慢，体积减小（P＜0.05），而miR-195 inhibitor组则结果相反（P＜0.05）；HE染色结果显示miR-195 mimics组肿瘤组织异型性下降，新生血管数减少，而miR-195 inhibitor组则相反；Western blot检测示与空白对照组和阴性对照组比较，miR-195 mimics组P21表达上调，而Cyclin D1表达下调，miR-195 inhibitor组结果相反（P均＜0.05）；免疫组化染色检测显示，与空白对照组和阴性对照组比较，miR-195 mimics组中P21表达阳性率较高，而Cyclin D1的表达阳性率较低（P均＜0.05），miR-195 inhibitor组情况则相反（P均＜0.05）。结论 MiR-195可使P21表达升高，下调Cyclin D1的表达，阻滞G0/G1期向S期转换，从而抑制人脑胶质瘤细胞SHG-44的增殖能力。
목적：관찰상조급하조microRNA-195（miR-195）대라서피하하SHG-44인뇌효질류생장적영향병탐토기궤제。방법사용지질체순시전염법장miR-195 mimics화inhibitor분별전입인뇌효질류세포계SHG-44，동시설립공백대조조화음성대조조；실시형광정량PCR（qRT-PCR）검측전염전후miR-195적함량변화；류식세포법검측각조세포주기분포；라서성류실험관찰miR-195대체내효질류증식적영향；종류조직HE염색관찰병이학개변；Western blot、면역조직화학염색검측종류조직주기상관단백P21、Cyclin D1적표체변화。결과 qRT-PCR측득전염mimics후miR-195적표체수평제고19배좌우，이전염inhibitor후miR-195적표체수평강저위공백대조조적42%；여공백대조화음성대조조상비，miR-195mimics조세포조체우G0/G1기（P＜0.05）；이miR-195 inhibitor조칙상반（P＜0.05）；라서성류실험현시，miR-195 mimics조여공백대조조화음성대조조상비，종류생장속도감만，체적감소（P＜0.05），이miR-195 inhibitor조칙결과상반（P＜0.05）；HE염색결과현시miR-195 mimics조종류조직이형성하강，신생혈관수감소，이miR-195 inhibitor조칙상반；Western blot검측시여공백대조조화음성대조조비교，miR-195 mimics조P21표체상조，이Cyclin D1표체하조，miR-195 inhibitor조결과상반（P균＜0.05）；면역조화염색검측현시，여공백대조조화음성대조조비교，miR-195 mimics조중P21표체양성솔교고，이Cyclin D1적표체양성솔교저（P균＜0.05），miR-195 inhibitor조정황칙상반（P균＜0.05）。결론 MiR-195가사P21표체승고，하조Cyclin D1적표체，조체G0/G1기향S기전환，종이억제인뇌효질류세포SHG-44적증식능력。
Objective To observe the effects of up-regulated expression and down-regulated expression of miR-195 on the SHG-44 human glioma xenograft growth, and explore the possible mechanism. Methods MiR-195 mimics(Group A) and inhibitor(Group B) were transfected into SHG-44 cell by Lipofectamine RNAiMAX. At the same time, the blank control group(Group C) and negative control group(Group D) were established. MiR-195 expression level was observed by Real-time PCR. Flow cytometry was used to monitor changes in cell cycle. Study the effects of miR-195 on glioma proliferation in vivo by xenograft experiment. Pathological changes of the glioma tissues were observed by HE staining. Western blot and immunohistochemistry were used to detect the expression changes of P21 and Cyclin D1 in removed tumor specimens. Results After transfection of miR-195 mimics, Real-time PCR showed that miR-195 expression level increased about 19 times. However, miR-195 expression level of inhibitor group were decreased to 42.3%of blank control group. Compared with Group C and Group D, cells of Group A were blocked in the G0/G1 phase (P<0.05). Group B, however, the result was opposite (P<0.05). Xenograft experiment showed that the volume in Group A was significantly smaller than of those in Group C and Group D (P<0.05) and the result of Group B was to the contrary (P<0.05). HE staining indicated that decreased heteromorphism and reduced number of new vessels in Group A were noted as compared with those in Group C and Group D and the result of Group B was opposite. Western blot showed that the expression levels of P21 in Group A was significantly up-regulated as compared with that in Group C and Group D, while the expression of Cyclin D1 was down-regulated. Group B, however, the situation was opposite. Immunohistochemistry revealed that expression of P21 in Group A was overexpression, while the expression levels of Cyclin D1 was down-regulated (P<0.05). However, the result of Group B was opposite (P<0.05). Conclusion Overexpression of miR-195 can efficiently block in G0/G1 phase to S phase transition and inhibit the growth of human glioma cell SHG-44 in vivo, probably through up-regulate P21 expression of and down-regulate Cyclin D1 expression.