miR-183靶向CX3CL1基因调控肝癌细胞MICA/B表达和增加NK细胞对肝癌细胞杀伤作用

Molecular mechanism of miR-183 targeting CX3CL1 gene to regulate MICA/B expression in hepatocellular carcinoma cells and increase NK cell killing effect

目的探讨miR-183对肝癌细胞MHC-Ⅰ类相关蛋白A/B(MICA/B)表达和自然杀伤(NK)细胞杀伤作用影响及其分子机制。方法实验设置miR-con组、miR-183组、anti-miR-con组、anti-miR-183组、pcDNA组、pcDNA-CX3CL1组、antimiR-183+si-con组、anti-miR-183+CX3CL1组、对照(NC)组。实时荧光定量PCR(RT-qPCR)检测miR-183和CX3CL1 m RNA表达水平;蛋白质印迹(Western blot)法检测CX3CL1、MICA和MICB蛋白表达;流式细胞术检测肝癌细胞表面MICA/B的表达;NK细胞与肝癌细胞共培养后,细胞计数试剂盒8(CCK-8)法检测NK细胞对肝癌细胞的杀伤作用;酶联免疫吸附法(ELISA)法检测肝癌细胞与NK细胞共培养后培养液中肿瘤坏死因子-α(TNF-α)和干扰素γ(IFN-γ)水平;荧光素酶报告实验检测miR-183和CX3CL1的靶向关系。结果肝癌组织和肝癌细胞中miR-183高表达,CX3CL1、MICA和MICB低表达。干扰miR-183表达和过表达CX3CL1,肝癌细胞中MICA、MICB阳性表达率显著升高,与NK细胞共培养后TNF-α和IFN-γ水平显著升高,NK细胞对肝癌细胞的杀伤率显著升高(P<0.05)。miR-183靶向调控CX3CL1,沉默CX3CL1逆转了干扰miR-183对肝癌细胞MICA/B表达和NK细胞杀伤作用的影响。结论抑制表达miR-183可增加肝癌细胞MICA/B表达,增加NK细胞对肝癌细胞的杀伤作用,其机制可能与CX3CL1有关。

The purpose of this experiment was to investigate the effect miR-183 on the expression of MHC-Ⅰ related protein A/B(MICA/B) and the killing effect of natural killer(NK) cells against liver cancer cells. Hun3 B liver cancer cells were divided into miR-con group, miR-183 group, anti-miR-con group, anti-miR-183 group,pcDNA group, pcDNA-CX3 CL1 group, anti-miR-183+si-con group, anti-miR-183+CX3 CL1 group and normal control group. Real-time fluorescence quantitative PCR(RT-qPCR) was used to detect the mRNA expressions of miR-183 and CX3 CL1 in liver cancer cells;Western blot was used to detect the expression of CX3 CL1 protein, and flow cytometry was used to detect the expression of MICA/B on the surface of liver cancer cells. NK cells were isolated from peripheral blood, and co-culture with liver cancer cells of the above groups. Then cell counting kit 8(CCK-8) method was used to detect the killing effect of NK cells on liver cancer cells;enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ) in the culture medium of liver cancer cells and NK cells after co-culture. miR-183 was co-transfected with CX3 CL1 wild-type and mutant luciferase vectors, and the targeting relationship between miR-183 and CX3 CL1 was detected by luciferase report experiment. Data showed that miR-183 was highly expressed in liver cancer cells, while CX3 CL1, MICA and MICB were lowly expressed. miR-183 expression interference or CX3 CL1 overexpression could significantly promote the positive expression rates of MICA and MICB in liver cancer cells, un-regulate the levels of TNF-α and IFN-γ after co-culture with NK cells, and increase the killing rate of NK cells on liver cancer cells. miR-183 targeted and regulated CX3 CL1. And after interfering with the expression of miR-183 and CX3 CL1, the positive expression rates of MICA and MICB in liver cancer cells were significantly reduced, the levels of TNF-α and IFN-γ were significantly reduced, and the killing rate of NK cells on liver cancer cells was significantly reduced. The above data indicate that inhibiting the expression of miR-183 may increase the expression of MICA/B in liver cancer cells by increasing the expression of CX3 CL1, thus increase the killing effect of NK cells on liver cancer cells.