长非编码RNA HOXD⁃As2通路抑制直肠癌细胞增殖、转移作用机制研究

Study on mechanism of long noncoding RNA HOXD⁃As2 pathway inhibiting proliferation and metastasis of rectal cancer cells

目的探讨长非编码RNA(LncRNA)HOXD⁃As2通路抑制直肠癌细胞增殖、转移的潜在机制。方法在直肠癌HCT116与HT29细胞中,转染阴性设为对照组,转染LncRNA HOXD⁃As2过表达质粒设为HOXD⁃As2组;转染空载体Vector设为Vector组,转染HOXD8过表达质粒设为HOXD8组。采用荧光素酶报告实验检测LncRNA HOXD⁃As2与靶基因HOXD8的相互作用。检测并比较4组细胞活力、增殖、侵袭、细胞迁移水平、上皮⁃间质转化相关蛋白表达水平及磷酸化蛋白激酶B/蛋白激酶B(p⁃AKT/AKT)、磷酸化磷脂酰肌醇⁃3⁃激酶/磷脂酰肌醇⁃3⁃激酶(p⁃PI3K/PI3K)、磷酸化雷帕霉素机械靶蛋白/雷帕霉素机械靶蛋白(p⁃mTOR/mTOR)蛋白相对表达水平。结果与对照组相比,HOXD⁃As2组荧光素酶活性下降,而mRNA、蛋白相对表达情况增加,差异均有统计学意义(P<0.05)。与对照组相比,HOXD⁃As2组直肠癌细胞HCT116、HT29中HOXD8的mRNA与蛋白表达水平均显著上升,差异均有统计学意义(P<0.05)。与Vector组相比,HOXD8组直肠癌细胞HCT116、HT29细胞活力、细胞增殖、细胞侵袭、细胞迁移水平及神经性钙粘蛋白、基质金属蛋白酶9蛋白表达均显著下降,上皮性钙粘蛋白表达增加,差异均有统计学意义(P<0.05)。HOXD⁃As2组直肠癌细胞HCT116、HT29中p⁃AKT/AKT、p⁃PI3K/PI3K、p⁃mTOR/mTOR水平均低于对照组,差异均有统计学意义(P<0.05)。HOXD8组直肠癌细胞HCT116、HT29中p⁃AKT/AKT、p⁃PI3K/PI3K、p⁃mTOR/mTOR水平均低于Vector组,差异均有统计学意义(P<0.05)。结论LncRNA HOXD⁃As2通过与靶基因HOXD8相互作用,上调HOXD8的mRNA、蛋白表达水平,抑制PI3K/Akt通路,降低直肠癌细胞增殖与转移水平。

Objective To investigate the potential mechanism of long noncoding RNA(LncRNA)HOXD⁃As2 pathway inhibiting the proliferation and metastasis of rectal cancer cells.Methods In colorectal cancer HCT116 and HT29 cells,negative transfection was set as control group,transfected LncRNA HOXD⁃As2 overexpression plasmid was set as HOXD⁃As2 group.Transfection with empty Vector was set as vector group,transfection with HOXD8 overexpression plasmid was set as HOXD8 group.Luciferase reporting assay was used to detect the interaction between LncRNA HOXD⁃As2 and the target gene HOXD8.Cell viability,proliferation,invasion,cell migration,epithelial⁃mesenchymal transition related protein expression levels and phosphorylated protein kinase B/protein kinase B(p⁃AKT/AKT),phosphorylated phosphatidylinositol 3⁃kinase/phosphoinositide⁃3 kinase(p⁃PI3K/PI3K),phosphorylated mechanistic target of rapamycin/mechanistic target of rapamycin(p⁃mTOR/mTOR)protein relative expression levels of the 4 groups were detected and compared.Results Compared with the control group,the luciferase activity in HOXD⁃As2 group decreased,while the relative ex⁃pression of mRNA and protein increased,with statistical significance(P<0.05).Compared with the control group,the mRNA and pro⁃tein expression levels of HOXD8 in colorectal cancer cells HCT116 and HT29 in HOXD⁃As2 group were significantly increased,with statistical significance(P<0.05).Compared with Vector group,HCT116 and HT29 cell viability,cell proliferation,cell invasion,cell migration and the expression of N⁃Cadherin and matrix metalloproteinase 9 protein in HOXD8 group were significantly decreased,while E⁃Cadherin expression was increased,with statistical significance(P<0.05).The levels of P⁃Akt/AKT,P⁃PI3K/PI3K and P⁃mTOR/mTOR in colorectal cancer cells HCT116 and HT29 in HOXD⁃As2 group were lower than those in control group,with statistical signifi⁃cance(P<0.05).The levels of P⁃Akt/AKT,P⁃PI3K/PI3K and P⁃mTOR/mTOR in colorectal cancer cells HCT116 and HT29 in HOXD8 group were lower than those in Vector group,with statistical significance(P<0.05).Conclusion LncRNA HOXD⁃As2 interacts with target gene HOXD8 to up⁃regulate the mRNA and protein expression of HOXD8,inhibit PI3K/Akt pathway,and reduce the proliferation and metastasis of rectal cancer cells.