微小RNAlet-7c通过靶定细胞周期蛋白依赖激酶6抑制肝癌细胞增殖

MicroRNA let - 7c inhibits proliferation of hepatocellular carcinoma cells by targeting cydin - dependent kinase 6

目的观察微小RNA（miRNA，miR）let-7c对肝癌细胞增殖的影响并验证其靶基因。方法实时定量聚合酶链反应（Real-timePCR）检测人肝癌细胞MHCC-97L、HepG2、HCCLM3、SMMC-7721及人肝细胞L02中let-7c的表达。使用Lipofectamine^TM 2000脂质体将miRNA转染人HCCLM3细胞，设转染let-7c的细胞为let-7c组，转染阴性对照miRNA的细胞为对照组，空白组未进行转染，细胞计数试剂盒（CCK-8）法检测let-7c对细胞增殖的影响，流式细胞术检验各组细胞周期的差异，Westernblot检测转染后细胞周期蛋白依赖激酶6（CDK6）的蛋白表达情况。将CDK6的3’端非编码区（3’-UTR）连接人pMIR-REPORT荧光素酶载体，建成荧光素酶报告基因。将miRNA、荧光素酶报告基因、海肾荧光素酶质粒共转染人293T细胞，检测转染后荧光素酶的活性，验证let-7c对3’-UTR的结合作用。结果肝癌细胞MHCC07L、HepG2、HCCLM3、SMMC-7721中的let-7c表达量均低于L02，表达量分别为（3．22±0．08）×10^-4、（2．34±0．11）×10^-4、（1．854-0．03）×1010^-4、（3．03±0．11）×10^-4及（4．37±0．09）×10^-4（P〈0．05）。let-7c组细胞在转染后48、72、96h的吸光度值均低于对照组及空白组（P〈0．05），而对照组与空白组比较差异无统计学意义（P〉0．05）。转染后72h，let-7c组细胞处于G1期的比例为（56．95±2．40）％，均高于另外两组（P〈0．05），而另外两组比较差异无统计学意义（P〉0．05）。转染后48h，let-7c组CDK6蛋白的表达量均低于另外两组（P〈0．05），而另外两组比较差异无统计学意义（P〉0．05）。双荧光素酶报告基因检测发现let-7c与对照miRNA比较，能抑制荧光素酶的活性（P〈0．05）。结论let-7c在肝癌细胞中的表达低于正常肝细胞，let-7c能抑制肝癌细胞的增殖，并使细胞阻滞于G1期，CDK6是其调控的靶基因。

Objective To explore let -7c＇ s influence on the proliferation of human hepatocellular carcinoma cells and to clarify its target gene. Methods The let -7c expression in human hepatoeellular carcinoma cells （MHCC -97L, HepG2, HCCLM3, SMMC -7721 ） and human hepatic cell L02 were de- tected by real- time quantitative polymerase chain reaction （Real -time PCR）. MicroRNAs （miRNAs） were transfected into HCCLM3 ceils via LipofectamineTM2000. The cells were divided into three groups： let-7c group transfeeted with let -7e, negative control group transfected with negative control miRNA, blank control group transfected with no miRNAs. The proliferation of ceils was assessed via cell counting kit - 8 （ CCK - 8）, cell cycle of each group was detected by flow cytometry, Western blotting was used to evaluate the protein expression of cyclin dependent kinase 6 （CDK6） after transfection. The 3 ＇ untranslat- ed region （ 3＇ - UTR） of CDK6 containing the putative binding sites of let - 7c was inserted into a firefly luciferase reporter vector named pMIR - REPORT. MiRNAs, pMIR - REPORT and renilla luciferase con- trol plasmid were transfected into 293T cells together, the Dual - Luciferase Reporter Assay System was used to assay the lueiferase activity. Results The expression level of let -7c in MHCC -97L, HepG2, HCCLM3, SMMC-7721were （3.22±0.08） ×10^-4, （2.34±0.11） ×10^-4, （1.85±0.03） ×10^-4 and （3.03 ± 0. 11 ） × 10^-4 respectively, lower than that in L02 [ （4. 37 ± 0. 09） × 10^-4, p 〈 0.05 ]. The absorbances of let - 7c group were lower than those of the other two groups at 48, 72, 96 h after transfec-tion respectively （ P 〈 0. 05 ）, there was no statistical difference between the other two groups （ P 〉 0. 05 ）. The percentage of G1 stage in let -7c group was （56. 95 ±2.40）% at 72 h after transfection, higher than that of the other two groups （ P 〈 0. 05 ） , and there was no statistical difference between the other two groups （P 〉 0. 05 ）. The protein level of CDK6 in let -7c group was lower than that of the other two groups at 48 h after transfection （P 〈 0. 05 ） and there was no statistical difference between the other two groups （P 〉 0. 05 ）. The Dual- Luciferase Reporter Assay System revealed that let- 7c could suppress the lucif- erase＇ s activity compared with negative control miRNA （ P 〈 0. 05 ）. Conclusion The expression level of let- 7c is lower in the hepatocellular carcinoma cells compared with normal hepatic cell. let -7c can inhibit the proliferation of hepatocellular carcinoma cells and cause a delay in G1 -S transition, CDK6 is its target gene.