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骆锴冉.卵泡抑素样蛋白1在维持结肠癌细胞生长中的作用*[J].浙江中西医结合杂志,2020,30(10):
卵泡抑素样蛋白1在维持结肠癌细胞生长中的作用*
FSTL1 sustains colon cancer cell growth
投稿时间:2020-05-16  修订日期:2020-08-21
DOI:
中文关键词:  结肠癌发生  FSTL1  慢病毒  ERK1/2  AKT
英文关键词:colon tumorigenesis  FSTL1  lentivirus  ERK1/2  AKT
基金项目:
作者单位E-mail
骆锴冉 绍兴文理学院附属医院普外科 hosrml@qq.com 
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中文摘要:
      目的:研究卵泡抑素样蛋白1(FSTL1)在结肠癌中的表达及其功能。方法:利用western blot、PCR和免疫组化检测FSTL1在51例结肠癌标本和正常结肠上皮组织中的含量;通过蛋白质印迹法测定FSTL1在体外结肠癌细胞株(HT29、HCT116、DLD1)及人正常结肠上皮细胞株HCEC-1CT中的表达;将靶向FSTL1基因的小干扰RNA(small interfering 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,将两组细胞系通过裸鼠皮下注射法建立结肠癌裸鼠异种移植瘤模型,同时设置未感染细胞作为空白对照组,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在异种移植瘤组织中的表达;Western blot检测细胞通路蛋白(p-ERK1/2、p-JNK、p-AKT和IκB)的表达。结果:与正常结肠上皮组织相比较,FSTL1在结肠癌组织中的转录和翻译水平的表达均显著上调,差异有统计学显著性(P < 0.05);高表达FSTL1与结肠癌侵润深度和淋巴结转移显著相关(P < 0.05);FSTL1蛋白在3种结肠癌细胞株中的表达较HCEC-1CT细胞明显上调,差异均有统计学显著性(P < 0.05);与siRNA-NC组和对照组相比较,FSTL1蛋白在siRNA-FSTL1组细胞中的表达显著下调,干扰效率为88.4%,差异有统计学显著性(P < 0.05);siRNA-FSTL1组裸鼠异种移植瘤重量显著低于siRNA-NC组和对照组,抑瘤率为36.9%;siRNA-FSTL1组异种移植瘤中FSTL1、Ki-67、p-ERK1/2和p-AKT的相对表达量均低于siRNA-NC组和对照组,细胞凋亡率明显高于siRNA-NC组和对照组,差异均有统计学显著性(P < 0.05);与siRNA-NC组或对照组相比较,p-JNK和IκB在siRNA-FSTL1组异种移植瘤中的表达无显著改变,差异均无统计学显著性(P > 0.05)。结论:FSTL1在结肠癌细胞中的表达显著上调,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。
英文摘要:
      AIM: To investigated the expression and functional role of Follistatin-like protein 1 (FSTL1) in colon cancer. METHODS: Western blot, PCR and immunohistochemistry were employed to analyze the level of FSTL1 in colon cancer specimens and surrounding non-tumor tissues. Western blot was used to evaluate the expression of FSTL1 in colon cancer cell lines DLD1, HCT116 and HT29 as well as normal epithelial colon cell line HCEC-1CT. The expression of FSTL1 was dyregulated in DLD1 cells by infection of small interfering RNA (siRNA) lentivirus targeting FSTL1 (LV-siRNA-FSTL1) and negative control lentivirus (LV-siRNA-NC). Non-transfected cells were used as blank control (BC). Three stable cell lines were implanted subcutaneously into right flank of nude mice to establish the subcutaneous tumor model of human colon cancer. The weight of xenografts on the 28th day were executed and compared among the three groups. Immunohistochemistry was employed to analyze the levels of Ki-67 and FSTL1 in the xenografts, and the apoptosis of colon cancer cells in vivo was measured by Tunel method. The levels of p-ERK1/2, p-JNK, p-AKT and IκB were determined by western blot. RESULTS: A significant increase of FSTL1 was seen in human colon cancer specimens as compared to the surrounding non-tumor tissues and this occurred at both RNA and protein level, the difference was statistically significant (P < 0.05). It is also closely engaged to tumorSinfiltrationSdepth and lymphnode metastasis of colon cancer patients (P < 0.05). The relative level of FSTL1 was significantly up-regulated in colon cancer cell lines in comparison with normal epithelial colon cell line, the difference was statistically significant (P < 0.05). The relative expression of FSTL1 protein in siRNA-FSTL1 group was significantly lower than that in siRNA-NC group or control group, the difference was statistically significant (P < 0.05), the interference rate was 88.4%. The weight of subcutaneously implanted tumors was notably decreased in siRNA-FSTL1 group compared with siRNA-NC group or control group, the difference was statistically significant (P < 0.05), the tumor inhibition rate was 36.9%. The relative expression of FSTL1, Ki-67, p-ERK1/2 and p-AKT in siRNA-FSTL1 group were notably decreased compared to siRNA-NC group and control group, while the apoptosis rate was higher instead, with statistically significant difference observed (P < 0.05). Moreover, Knockdown of FSTL1 in colon cancer cells exerted no significant effect on the expression of p-JNK and IκB (P > 0.05). CONCLUSIONS: The expression of FSTL1 is notably increased in colon cancer and clearly associated with the growth of the subcutaneous colon xenografts in nude mice, which may be related to ERK1/2 and AKT signaling pathway, and FSTL1 can become a novel therapeutic target for colon cancer treatment in the future.
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