微小RNA-218通过生存素对胆管癌细胞的作用

Study on the correlation of the effect of microRNA -218 through Survivin on cholangiocarcinoma

目的检测微小RNA（miR）-218对胆管癌细胞的侵袭的影响及miR-218通过凋亡抑制基因生存素（Survivin）对胆管癌细胞的作用。方法培养两种胆管癌细胞株（CCLPl）和（QBC939），将miR-218mimics（miR-218激动剂）及阴性对照转染CCLPl和QBC939细胞，设立miR-218组、阴性对照组及空白对照组，采用BDMartigel基底膜基质胶侵袭室测定其侵袭性，每组随机抽取3个结果。将CCLPl和QBC939细胞分别共转染miR-218mimics和pMIR-Survivin、miR-218mimics和pMIR-Survivin-mut。分别设立miR-218组、阴性对照组及空白对照组，每组随机抽取3个结果，酶标仪检测荧光素酶活性，探讨miR-218与Survivin的相关性。结果CCLPl细胞株中miR-218组细胞数为（28．33±4．16）个，阴性对照组细胞数为（72．00±8．72）个，NC组细胞数为（74．33±4．04）个；QBC939细胞株中miR-218组细胞数为（7．33±2．08）个，阴性对照组细胞数为（22．00±4．36）个，NC组细胞数为（22．00±4．00）个；与阴性对照组和NC组比较，转染miR-218mimics处理能显著抑制胆管癌细胞的侵袭能力，差异有统计学意义（CCLPl细胞株和QBC939细胞株其P值分别为0．000、0．004）。荧光素酶活性检测结果显示，QBC939野生型miR-218组荧光素酶活性为1．489±0．079，阴性对照组为2．825±0．068，NC组为2．747±0．081；QBC939突变型miR-218组荧光素酶活性为3．078±0．138，阴性对照组为2．944±0．144，NC组为2．807±0．090；CCLPl野生型miR-218组荧光素酶活性为1．322±0．039，阴性对照组为2．362±0．129，NC组为2．368±0．154；CCLPl突变型miR一218组荧光素酶活性为2．202±0．073，阴性对照组为2．274±0．250，NC组为2．202±0．073；野生型的3’端非编码区（3’UTR）的报告基因载体的荧光素酶活性与其阴性对照组及空白对照组比较显著下降，差异有统计学意义（CCLPl细胞株和QBC939细胞株其P值分别为0．000、0．000），突变型无显著变化。结论miR-218抑制胆管癌细胞的侵袭，在胆管癌的发生发展中起负性调节作用。miR-218可直接与Survivin3’UTR相互作用，miR-218对于胆管癌的负性调节作用可能是直接通过Survivin发挥作用。

Objective To study the effect of microRNA -218 （miR -218 ） on the invasion of cholangiocarcinoma cells by Survivin. Methods Two eholangiocarcinoma cell lines （CCLPI） and （QBC939） were cuhured, and transfected with miR -218 mimicss. Negative control group and blank con- trol group were set up. The invasiveness was determined via BD Martigel basement membrane matrix gel in- vasion Chamber. 3 results were randomly selected from each group. The CCLP1 and QBC939 cells were co transfected with miR- 218 mimic and pMIR- Survivin, miR- 218 mimic and pMIR- Survivin- mut, re- spectively, 3 results were randomly selected from each group, and the luciferase activity was detected by enzyme labelling instrument, and the correlation between miR- 218 and Survivin was discussed. Results The numbers of CCLP1 cells transfected with miR- 218, cells in negative control group, and cells in NC group were （28.33 ±4. 16）, （72. 00 ± 8.72） and （74. 33±4. 04） cells, respectively; The numbers of QBC939 cells transfected with miR -218, cells in negative control group, and cells in NC group were （7.33 ±2. 08）, （22. 00±4. 36） and （22. 00 ±4.00） cells, respectively. Results showed that transfec- tion of miR- 218 mimics can inhibit the invasion ability of cholangiocarcinoma （The P values of CCLP1 cell line and QBC939 cell line were 0. 000, 0. 004）. Luciferase activity detection results showed that lucif- erase activity in QBC939 wild type miRNA -218 group was 1. 489± 0. 079, lnciferase activity in negative control group was 2. 825± 0. 068, luciferase activity in NC group was 2. 747± 0. 081, luciferase activity in QBC939 mutant miRNA-218 group was 3. 078 ± 0. 138, luciferase activity in negative control group was 2. 944 ± 0. 144, the luciferase activity of the NC group was 2. 807 ± 0. 090, the luciferase activity of the CCLPI wild type miRNA - 218 group was 1. 322 ± 0. 039, the Iuciferase activity of the negative control group was 2. 362 ± 0. 129, the luciferase activity of the NC group was 2. 368±0. 154, the luciferase activi- ty of the CCLPI mutant miRNA -218 group was 2. 202±0. 073, and the negative control group luciferase activity was 2. 274± 0. 250, the activity of luciferase in group NC was 2. 202± 0. 073. The luciferase activ- ity of the wild type 3＇ untranslated regions （3＇ UTR） of the reporter gene vector was significantly lower than that of the negative control group and the blank control group （ The P values of CCLP1 cell line and QBC939 cell line were 0. 000, 0. 000 ）. While there was no significant change in the mutation type. Conclusion MiR- 218 inhibits the invasion of cholangiocarcinoma cells and plays a negative regulatory role in the development of cholangiocarcinoma, miR -218 can interact with Survivin 3＇ UTR directly. Thus miR -218 can have a negative regulatory effect on cholangiocarcinoma through the Survivin directly.