CTHRC1增强结直肠癌细胞侵袭能力促进结直肠癌转移的机制研究

Mechanism of collagen triple helix repeat containing 1(CTHRC1) promoting metastasis by enhancing the invasiveness of colorectal cancer cells

目的:探讨胶原三股螺旋重叠蛋白1(collagen triple helix repeat containing 1,CTHRC1)对结直肠癌细胞生物学特性的影响及可能的调控机制。方法:采用实时定量PCR技术检测上调或抑制miR-520d-5P的表达,探讨其对基因CTHRC1的调控作用,双荧光素酶实验验证miR-520d-5P与基因CTHRC1的3'UTR区之间是否相互结合。采用Western blot法检测6株不同结直肠癌细胞系及临床结直肠癌配对标本中基因CTHRC1的蛋白表达水平。构建稳定转染基因CTHRC1的结直肠癌细胞系;CCK-8、Transwell等方法检测稳定转染目的基因后结直肠癌细胞的增殖、侵袭能力的变化。结果:在配对结直肠癌患者组织中发现,正常组织中miR-520d-5P的表达高于癌组织(P<0.001),基因CTHRC1的表达趋势相反(P<0.001),且两者的表达呈负相关。通过miR-520d-5P模拟类似物mimics及抑制物inhibitor的瞬时转染实验发现其对基因CTHRC1的蛋白表达有负向调节作用。双荧光素酶实验验证miR-520d-5P与CTHRC1的3'UTR之间存在相互结合。稳定转染基因CTHRC1后,细胞的增殖能力下降,但侵袭能力提高,差异具有统计学意义(P<0.05)。在配对结直肠癌标本中,原发癌组织中基因CTHRC1的蛋白表达水平高于正常组织(P=0.003)。结论:miR-520d-5P对基因CTHRC1蛋白表达具有负调控作用。基因CTHRC1可以增强结直肠癌细胞的侵袭能力,抑制结直肠癌细胞的增殖能力,且与miR-520d-5P具有临床相关性。

Objective:To analyze the effect of collagen triple helix repeat containing 1(CTHRC1)on the biological characteristics of the colorectal cancer cells and its possible regulatory mechanism.Methods:Real-time PCR was used to detect the mRNA levels of CTHRC1 by overexpression or inhibition of miR-520d-5P.Dual Luciferase assay was used to assess the binding of miR-520d-5P to the CTHRC1 3'-UTR.Western blot analysis was performed to detect the protein levels of CTHRC1 in 6 human colorectal cancer(CRC)cell lines and paired colorectal cancer tissues.Following the stable transfection of CTHRC1 in CRC cell lines,the proliferative and the invasive ability of CTHRC1 were analyzed by in vitro CCK-8 assay and invasion assay,respectively.Results:MiR-520d-5P expression in normal tissues was significantly higher than the paired cancer tissues(P<0.001),and the CTHRC1 expression in cancer tissues was significantly higher than the paired normal tissues(P<0.001),indicating a negative correlation between their expression.We also observed that the CTHRC1 protein levels were lower in the miR-520d-5P mimics group and higher in the miR-520d-5P inhibitor group.Dual Luciferase assay confirmed that miR-520d-5P targets the 3'-UTR of CTHRC1.In vitro CCK-8 assay demonstrated a decreased proliferation in CRC Cells(SW480,SW620)overexpressing CTHRC1.Results from the in vitro invasion assay showed that the cells transfected with CTHRC1 were significantly more invasive(P<0.05).The results showed that protein expression of gene CTHRC1 in primary cancer tissues was significantly higher than the paired normal tissues(P=0.003).Conclusions:CTHRC1 protein expression is negatively regulated by miR-520d-5P in colorectal cancer.Furthermore,CTHRC1 enhances the invasiveness and decreases the proliferation of CRC cell lines.