单酰甘油脂肪酶对人肝细胞癌裸鼠移植瘤的影响及其机制

Influence of monoacylglycerol lipase on growth of nude mice xenograft tumor of human hepatocellular carcinoma and related mechanism

目的探讨单酰甘油脂肪酶(MAGL)在人肝细胞癌(HCC)裸鼠移植瘤生长中的作用和机制。方法移植的SMMC-7721细胞株分为SMMC-7721WT组(未处理)、SMMC-7721MAGL-KD组(MAGL沉默)、SMMC-7721MAGL-OE组(MAGL过表达)和SMMC-7721Vector组(空载体转染) 4组。将27只雄性BALB/c裸鼠分为4组建立裸鼠皮下移植瘤模型,即A组(注射SMMC-7721WT组细胞株,n=12)、B组(注射SMMC-7721MAGL-KD组细胞株,n=5)、C组(注射SMMC-7721MAGL-OE组细胞株,n=5)及D组(注射SMMC-7721Vector组细胞株,n=5),其中A组又分为A1(正常饲喂,n=4)、A2[(高脂饲喂(HFD)+JZL184(MAGL的特异性抑制剂,n=4)和A3 (HFD饲喂,n=4) 3个亚组。观察并比较4组肿瘤体积变化,瘤体内增殖细胞核抗原(PCNA)、金属基质蛋白酶(MMP) 2、血浆溶血性磷脂酸(LPA)和前列腺素E2(PGE2)的表达水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果 MMC-7721WT组、SMMC-7721MAGL-KD组、SMMC-7721MAGL-OE组3组间MAGL蛋白相对表达水平比较差异有统计学意义(0. 377±0. 026 vs 0. 182±0. 055 vs 0. 689±0. 019,F=33. 382,P <0. 001),SMMC-7721MAGL-KD组MAGL蛋白表达水平显著低于SMMC-7721WT组(P <0. 05),SMMC-7721MAGL-OE组显著高于SMMC-7721WT组(P <0. 05);A、B、C、D 4组间裸鼠皮下移植瘤大小比较差异有统计学意义[(4236. 125±1284. 283) mm^3vs (1883. 375±552. 977) mm^3vs (10 146. 061±1842.264) mm^3vs (4307. 452±2070. 708) mm^3,F=6. 804,P=0. 023],C组的裸鼠皮下移植瘤的生长速度比A组更快(P <0. 05),而B组比A组慢(P <0. 05);A、B、C 3组间PCNA和MMP2水平比较差异均有统计学意义(PCNA:25 843. 821±4201. 310 vs 17 426. 95±5139. 202 vs 39 753. 103±5721. 444,F=21. 482,P <0. 001;MMP2:52 841. 621±4339. 253 vs 35 511. 451±8251. 423 vs 68 274. 731±6418. 594,F=11. 526,P <0. 001),B组PCNA和MMP2水平均明显低于A组(P值均<0. 05),而C组均高于A组(P值均<0.05);A1、A2、A3 3组间肿瘤体积比较差异有统计学意义[(23 476. 289±483. 872) mm^3vs (18 593. 851±1385. 805) mm^3vs (37 703.198±2925. 254) mm^3,F=47. 371,P=0. 004],与A1组相比,A3组的裸鼠皮下移植瘤体积增长速度更快(P <0. 05),A2组明显受到抑制(P <0. 05);A1、A2、A3 3组间PGE2水平比较差异有统计学意义[(0. 109±0. 023)μmol/L vs (0. 056±0. 010)μmol/L vs(0. 168±0. 024)μmol/L,F=16. 492,P <0. 001],A3组PGE2水平明显高于A1组(P <0. 05),A2组明显低于A1组(P <0. 05);B、C、D 3组间PGE2水平比较差异有统计学意义[(0. 069±0. 025)μmol/L vs (0. 175±0. 023)μmol/L vs (0. 096±0. 019)μmol/L,F=31. 550,P <0. 001],B组PGE2水平明显低于D组(P <0. 05),C组明显高于D组(P <0. 05)。结论 MAGL可能通过调控PGE2促进裸鼠HCC皮下移植瘤的生长,提示MAGL可能成为未来治疗HCC的潜在靶点。

Objective To investigate the role and mechanism of action of monoacylglycerol lipase( MAGL) on the growth of nude mice xenograft tumor of human hepatocellular carcinoma( HCC). Methods The transplanted SMMC-7721 cells were divided into SMMC-7721 WT group( without treatment),SMMC-7721 MAGL-KDgroup( with MAGL silencing),SMMC-7721 MAGL-OEgroup( with MAGL overexpression),and SMMC-7721 Vectorgroup( transfected with empty vector). A total of 27 male BALB/c nude mice were randomly divided into group A( 12 mice injected with the cells in the SMMC-7721 WTgroup),group B( 5 mice injected with the cells in the SMMC-7721 MAGL-KD group),group C( 5 mice injected with the cells in the SMMC-7721 MAGL-OEgroup),and group D( 5 mice injected with the cells in the SMMC-7721 Vectorgroup). The mice in group A were further divided into groups A1( control group),A2( treated with high-fat diet and JZL184,a specific inhibitor of MAGL),and A3( fed with high-fat diet),with 4 mice in each group. The four groups were compared in terms of the change in tumor volume and the expression of proliferating cell nuclear antigen( PCNA),metal matrix proteinase-2( MMP-2),lysophosphatidic acid( LPA),and prostaglandin E2( PGE2) in tumor. A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the SNK-q test was used for further comparison between two groups. Results There was a significant difference in the relative protein expression of MAGL between the SMMC-7721 WTgroup,the SMMC-7721 MAGL-KDgroup,and the SMMC-7721 MAGL-OEgroup( 0. 377 ± 0. 026 vs 0. 182 ± 0. 055 vs 0. 689 ± 0. 019,F = 33. 382,P < 0. 001);compared with the SMMC-7721 WTgroup,the SMMC-7721 MAGL-KDgroup had significantly lower protein expression of MAGL and the SMMC-7721 MAGL-OEgroup had significantly higher expression( P < 0. 05). There was a significant difference in the size of subcutaneous xenograft tumor between groups A,B,C,and D( 4236. 125 ± 1284. 283 mm3 vs 1883. 375 ± 552. 977 mm3 vs 10 146. 061 ± 1842. 264 mm3 vs 4307. 452 ± 2070. 708 mm3,F= 6. 804,P = 0. 023). Group C had a lower growth rate of subcutaneous xenograft tumor than group A( P < 0. 05),and group B had a higher growth rate than group A( P < 0. 05). There were significant differences between groups A,B,and C in the levels of PCNA( 25 843.821 ± 4201. 310 vs 17 426. 95 ± 5139. 202 vs 39 753. 103 ± 5721. 444,F = 21. 482,P < 0. 001) and MMP-2( 52 841. 621 ± 4339. 253 vs35 511. 451 ± 8251. 423 vs 68 274. 731 ± 6418. 594,F = 11. 526,P < 0. 001);group B had significantly lower levels of PCNA and MMP-2 than group A( P < 0. 05),and group C had significantly higher levels than group A( P < 0. 05). There was a significant difference in tumor volume between groups A1,A2,and A3( 23 476. 289 ± 483. 872 mm^3 vs 18 593. 851 ± 1385. 805 mm^3 vs 37 703. 198 ± 2925. 254 mm^3,F = 47. 371,P = 0. 004). Compared with group A1,group A3 had a significantly higher growth rate of subcutaneous xenograft tumor( P < 0. 05) and group A2 had a significantly lower growth rate( P < 0. 05). There was a significant difference in the level of PGE2 between groups A1,A2,and A3( 0. 109 ± 0. 023 μmol/L vs 0. 056 ± 0. 010 μmol/L vs 0. 168 ± 0. 024 μmol/L,F = 16. 492,P < 0. 001);group A3 had a significantly higher level of PGE2 than group A1( P < 0. 05),and group A2 had a significantly lower level than group A1( P < 0.05). There was a significant difference in the level of PGE2 between groups B,C,and D( 0. 069 ± 0. 025 μmol/L vs 0. 175 ± 0. 023 μmol/L vs 0. 096 ± 0. 019 μmol/L,F = 31. 550,P < 0. 001);group B had a significantly lower level of PGE2 than group D( P < 0. 05),and group C had a significantly higher level than group D( P < 0. 05). Conclusion MAGL can promote the growth of subcutaneous xenograft tumor of HCC by regulating PGE2,suggesting that MAGL might become a potential target for HCC treatment in future.