卵泡抑素样蛋白1在维持结肠癌细胞生长中的作用

Follistatin-like Protein 1 Sustains the Growth of Colon Cancer Cells

目的研究卵泡抑素样蛋白1(FSTL1)在结肠癌细胞表达及其功能。方法体外通过蛋白质印迹法测定FSTL1在结肠癌细胞株(HT29、HCT116、DLD1)及人正常结肠上皮细胞株HCEC-1CT表达量;将靶向FSTL1基因的小干扰RNA(siRNA)慢病毒(LV-siRNA-FSTL1)和阴性对照慢病毒(LV-siRNA-NC)分别感染DLD1细胞,将两组细胞系通过皮下注射法建立结肠癌异种移植瘤裸鼠模型,同时设置未感染细胞作为空白对照(BC)组,观察接种4周后三组异种移植瘤重量,Tunel法测定体内结肠癌细胞凋亡;免疫组织化学法检测FSTL1和Ki-67在异种移植瘤组织表达;Western blot检测细胞外调节蛋白激酶(ERK1/2)、氨基末端激酶(JNK)、蛋白激酶B(Akt)和核因子κB抑制蛋白(IκB)表达。结果FSTL1蛋白在三种结肠癌细胞株表达较HCEC-1CT细胞明显上调(P均<0.01);siRNA-FSTL1组、siRNA-NC组和BC组FSTL1蛋白相对表达量分别为(0.12±0.03)、(0.97±0.19)和(1.05±0.18),差异有统计学意义(P均<0.01),干扰效率88.42%;siRNA-FSTL1组裸鼠异种移植瘤重量(0.53±0.07)g显著低于siRNA-NC组(0.84±0.15)g和BC组(1.15±0.26)g,差异有统计学意义(P均<0.01),抑瘤率53.91%;siRNA-FSTL1组细胞凋亡率(16.82±2.33)%明显高于siRNA-NC组(2.93±0.44)%和BC组(3.51±0.32)%,差异均有统计学意义(P均<0.01);与siRNA-NC组比较,siRNA-FSTL1组异种移植瘤FSTL1、Ki-67、p-ERK1/2和p-Akt相对表达量分别降低(58.42±4.53)%、(61.61±6.82)%、(43.23±3.92)%和(47.52±5.14)%,差异有统计学意义(P均<0.05);与siRNA-NC组比较,p-JNK和IκB在siRNA-FSTL1组异种移植瘤表达无显著改变(P>0.05)。结论FSTL1在结肠癌细胞表达显著上调,敲除FSTL1基因可显著抑制结肠癌裸鼠异种移植瘤生长,其作用机制可能与ERK1/2和Akt通路相关,FSTL1有望成为结肠癌基因治疗新的作用靶点。

Objective To investigate the expression and functional role of Follistatin-like protein 1(FSTL1)in colon cancer.Methods The expression of FSTL1 in colon cancer cell lines(HT29,HCT116,DLD1)and human normal colon epithelial cell line HCEC-1CT was determined by Western blot in vitro;small interfering RNA(siRNA)lentivirus targeting FSTL1 gene(LV-siRNA-FSTL1)and negative control lentivirus(LV-siRNA-NC)were infected with DLD1 cells,respectively.Nude mice models of xenograft tumor of colon cancer were established by subcutaneous injection of two groups of cell lines,and uninfected cells were set as blank control(BC)group.After 4 weeks of inoculation,the weight of xenograft tumor in three groups was observed,and apoptosis of colon cancer cells in vivo was measured by TUNEL;the expression of FSTL1 and Ki-67 in xenograft tumor tissue was detected by immunohistochemistry;Western blot was used to examine the expression of the extracellular regulatory protein kinase(ERK1/2),N-terminal kinase(JNK),protein kinase B(Akt)and nuclear factor B inhibitor protein(IκB).Results The expression of FSTL1 protein in the three colon cancer cell lines was significantly higher than that of HCEC-1CT cells(P<0.01,respectively);the relative expression of FSTL1 protein in the siRNA-FSTL1 group,siRNA-NC group and BC group were(0.12±0.03),(0.97±0.19)and(1.05±0.18),the difference was statistically significant(P<0.01,respectively),the interference efficiency was 88.42%.The weight of nude mice xenograft tumor in the siRNA-FSTL1 group[(0.53±0.07)g]was significantly lower than those of siRNA-NC[(0.84±0.15)g]and BC group[(1.15±0.26)g],the difference was statistically significant(P<0.01,respectively),and the tumor inhibition rate was 53.91%.The apoptosis rate of siRNA-FSTL1 group[(16.82±2.33)%]was obviously higher than those of siRNA-NC group[(2.93±0.44)%]and BC group[(3.51±0.32)%],the difference was statistically significant(P<0.01,respectively).Compared to siRNA-NC group,The relative expression of FSTL1[(58.42±4.53)%],Ki-67[(61.61±6.82)%],p-ERK1/2[(43.23±3.92)%]and p-Akt[(47.52±5.14)%]of xenograft tumors in siRNA-FSTL1 group decreased significantly(P<0.05,respectively),and the expression of p-JNK and IκB had non-significantly changed.Conclusion The expression of FSTL1 was significantly up-regulated in colon cancer cells.Knockout of FSTL1 gene significantly inhibited the growth of xenografts of colon cancer in nude mice.The mechanism may be related to ERK1/2 and Akt pathway.FSTL1 is expected to be a new target of gene therapy for colon cancer.