BRAF激活的长链非编码RNA在结直肠癌中的表达及功能

Expression and function of BRAFactivated long noncoding RNA in colorectal cancer

    摘要
  • 摘要: 目的：探讨BRAF激活的长链非编码RNA(BANCR)在结直肠癌中的表达，以及对结直肠癌HCT116细胞生物学功能的影响。
    方法：收集2012年3月至2013年6月南京医科大学第一附属医院56例结直肠癌手术切除标本(包括癌组织和癌旁组织)，采用实时荧光定量PCR(qRTPCR)检测56例标本中BANCR的表达情况(BANCR高表达组28例、BANCR低表达组28例)，并分析其表达水平与临床病理因素的关系；用慢病毒介导shRNA 1和shRNA 2分别干扰HCT116细胞BANCR表达后，将HCT116细胞分为4组：干扰组1(转染慢病毒载体携带LV shRNA 1)、干扰组2(转染慢病毒载体携带LV shRNA 2)、阴性对照组(转染无意义序列的重组慢病毒载体)和空白组(仅以RPMI 1640培养基培养)，分别采用CCK-8法、流式细胞法和Transwell法检测各组细胞增殖、凋亡和迁移的能力。计量资料以x±s表示，两组间比较采用u检验，多组间均数比较采用单因素方差分析和重复测量的方差分析，两两比较采用LSD-t检验，多因素分析采用二元Logistic回归模型，分类资料采用χ2检验。结果：qRT-PCR检测结果显示：56例结直肠癌组织中BANCR相对表达量为1.6±0.4，高于癌旁组织的0.9±0.7，两者比较，差异有统计学意义(u=1 020.000，P<0.05)。单因素分析结果显示：BANCR的高表达与淋巴结转移、肿瘤分期相关(χ2=4.595，7.487，P<0.05)。多因素分析结果显示：淋巴结转移和肿瘤分期Ⅲ～Ⅳ期是影响BANCR高表达的独立危险因素(OR=4.000，5.914，95%可信区间：1.230～12.900，1.685～20.760，P<0.05)。qRT-PCR检测结果显示：干扰组1、干扰组2、阴性对照组、空白组细胞中BANCR相对表达量分别为0.25±0.04、0.20±0.06、0.96±0.04、0.98±0.03，4组比较，差异有统计学意义(F=271.610，P<0.05)。CCK8法检测结果显示：各组细胞培养至第6天，干扰组1、干扰组2、阴性对照组的细胞增殖率分别为80.6% ±0.9%，4组比较，差异无统计学意义( F=2.881，P >0.05)。Transwell法检测结果显示：干扰组1、干扰组2、阴性对照组、空白组的穿膜细胞数为(135±29)个、(107±18)个、(240±24)个、(245±22)个，4组比较，差异有统计学意义( F=45.194，P <0.05)。结论：BANCR在结直肠癌组织中高表达，其高表达与淋巴结转移和肿瘤分期相关。BANCR能促进HCT116细胞迁移，可能成为结直肠癌诊断和判断预后的重要分子标志物。

     

    Abstract: Objective:To investigate the expression of BRAF activated long non coding RNA (BANCR) in colorectal cancer, and the influence of BANCR on the biological function of HCT116 cells.
    Methods:Fifty six samples of colorectal cancer specimen (including the cancer tissues and precancerous tissues) were obtained at the First Affiliated Hospital of Nanjing Medical University from March 2012 to June 2013. The expressions of BANCR in all the specimens were detected by qRT PCR (28 cases in the BANCR high expression group and 28 cases in the BANCR low expression group). The relationship between the expressions of BANCR and the clinicopathological factors of colorectal cancer was analyzed. The HCT116 cells were divided into 4 groups after interfering BANCR with lentiviral mediated shRNA-1 and  shRNA-2：interference group 1 (HCT116 cells transfected with LV-shRNA-1), interference group 2 (HCT116 cells transfected with LV-shRNA-2), negative control group (HCT116 cells transfected with lentivirus vector with nonsense sequence) and blank control group (HCT116 cells cultured in RPMI 1640 medium). The proliferation, apoptosis and migration of HCT116 cells in the 4 groups were detected by CCK8 assay, flow cytometry and Transwell assay, respectively. The comparison between the 2 groups was analyzed by u test, and multiple groups were compared by one way analysis of variance, repeated measurement analysis of variance and LSD-t test. Multivariate analysis was done by Logistic regression model. The difference between categorical data was compared by chi-square test. 
    Results:The relative expression of BANCR in the cancer tissues was 1.6±0.4, which was significantly higher than 0.9±0.7 of the precancerous tissues (u=1 020.000, P<0.05). The result of univariate analysis showed that the high expression of BANCR was correlated with the lymph node metastasis and tumor stage (χ2=4.595, 7.487, P<0.05). The result of multivariate analysis showed that lymph node metastasis and tumor stage (stage Ⅲ-Ⅳ) were the independent risk factors influencing the high expression of BANCR(OR=4.000, 5.914, 95%CI: 1.230-12.900, 1.685-20.760，P <0.05). The relative expressions of BANCR of the interference group 1, interference group 2, negative control group and the blank control group were 0.25±0.04, 0.20±0.06, 0.96±0.04, 0.98±0.03, with significant difference among the 4 groups ( F=271.610, P <0.05) . The cell proliferation rates at day 6 of the interference group 1, interference group 2 and the negative control group were 80.6% ± 0.9%, with no significant difference among the 4 groups ( F=2.881, P >0.05). The numbers of transmembrane cells of the interference group 1, interference group 2, negative control group and the blank control group were 135±29, 107±18, 240±24 and 245±22, with significant difference among the 4 groups ( F=45.194, P <0.05). 
    Conclusions:BANCR was overexpressed in the HCT116 cells, and the BANCR overexpression was correlated with lymph node metastasis and tumor stage. BANCR can promote the migration of HCT116 cells. BANCR could be an important biomarker for the diagnosis and prognosis of colorectal cancer.

     

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