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子宫内膜癌是发生于子宫内膜的一种上皮性恶性肿瘤,是最常见的女性生殖系统肿瘤之一[1]。脆性组氨酸三联体(fragile histidinetriad, Fhit)基因是活跃的第一位脆性位点的抑癌基因,它对于肿瘤的发生及进展起着重要的负性调控作用,在多种肿瘤中都有不同程度的表达缺失且具有相关性[2]。RNA激活(RNA activation,RNAa)系将靶向目的基因启动子区域的双链RNA分子(dsRNA)导入肿瘤细胞,RNAa是小双链RNA引导的Argonaute蛋白参与的转录基因激活机制,这种小双链RNA被称为小激活RNA(saRNA)[3]。本研究检测Fhit基因在子宫内膜癌中的表达及其与临床病理因素的相关性,拟运用RNA激活上调人子宫内膜癌细胞株(Ishikawa, ISK)中Fhit基因的表达,探讨其对子宫内膜癌细胞增殖和侵袭、迁移能力的影响。现作报道。
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结果显示,正常子宫内膜组织中的细胞质内有阳性染色表达,子宫内膜癌组织中的细胞质内阳性染色表达减弱甚至缺失,2组差异有统计学意义(P < 0.01)(见图 1和表 1)。
组织学类型 n 阴性 阳性 χ2 P 正常子宫内膜组织
子宫内膜癌组织40
1357
10633
2950.22 < 0.01 表 1 Fhit在不同组织中的表达
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子宫内膜癌中Fhit表达阳性率FIGO分期Ⅲ+Ⅳ高于FIGO分期Ⅰ+Ⅱ、肌层浸润>1/2高于肌层浸润≤1/2、有淋巴结转移高于无淋巴结转移,差异均有统计学意义(P < 0.05~P < 0.01),阳性率与年龄无明显相关性(P>0.05)(见表 2)。
因素 n 阴性 阳性 χ2 P 年龄/岁 < 50
≥5026
10916
6510
440.03 >0.05 组织学分级 G1 44 26 18 3.03 >0.05 G2 58 42 16 G3 33 25 8 FIGO分期 Ⅰ+Ⅱ
Ⅲ+Ⅳ70
6540
5030
155.93 < 0.05 肌层浸润 ≤1/2
>1/266
6939
5327
164.88 < 0.05 淋巴结转移 无
有92
4355
3637
77.64 < 0.01 表 2 Fhit表达与子宫内膜癌临床病理特征的联系
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与子宫内膜癌相比,正常子宫内膜中Fhit蛋白表达显著增高(见图 2)。2组间差异有统计学意义(P < 0.01)(见表 3)。细胞转染48 h后与阴性对照组和空白对照组相比,实验组Fhit蛋白表达显著增高(见图 3),各组间差异有统计学意义(P < 0.01)(见表 4)。
分组 Fhit相对表达量 t P 子宫内膜癌组织
正常子宫内膜组织0.44±0.02
0.80±0.0211.06 < 0.01 表 3 子宫内膜癌组织与正常子宫内膜组织中Fhit的表达比较(x±s)
分组 Fhit相对表达量 F P 空白对照组 0.21±0.02 阴性对照组 0.23±0.01 14.78 < 0.01 实验组 1.04±0.11 表 4 RNAa转染后ISK细胞中Fhit的表达比较(x±s)
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细胞转染48 h后实验组mRNA表达水平为0.86±0.15,显著高于阴性对照组的0.34±0.01和空白对照组的0.33±0.01(P < 0.01)。
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CCK-8检测结果显示,细胞转染24、48、72、96、120 h抑制率分别为5.3%、15.6%、37.2%、43.3%和54.8%,与阴性对照组相比,实验组细胞增殖减慢,生长明显受抑制,差异有统计学意义(P < 0.05)(见图 4)。
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Transwell小室实验显示,与空白对照组和阴性对照组相比,实验组细胞的侵袭和迁移能力有明显下降(P < 0.01)(见表 5、图 5)。
分组 穿膜细胞数量(侵袭) 穿膜细胞数量(迁移) 空白对照组 179.0±6.9 111.3±7.1 阴性对照组 162.7±8.9 108.3±7.8 实验组 41.2±8.1 38±7.2 F 265.20 94.95 P < 0.01 < 0.01 表 5 Fhit对ISK细胞侵袭和迁移能力的影响(x±s)
Fhit基因在子宫内膜癌增殖、侵袭中的作用和临床意义
Role and clinical significance of Fhit gene in the proliferation and invasion of endometrial carcinoma
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摘要:
目的探讨Fhit基因在子宫内膜癌中的表达情况,并通过RNA激活上调Fhit表达后观察其对子宫内膜癌细胞增殖和侵袭、迁移的影响。 方法收集135例子宫内膜癌组织和40例正常子宫内膜组织,使用免疫组织化学和Western blotting检测Fhit基因的表达情况并分析Fhit表达与临床病理特征的联系。构建Fhit-saRNA表达载体,转染至子宫内膜癌ISK细胞株,建立上调Fhit基因表达的子宫内膜癌细胞株。Western blotting和RT-PCR验证转染效果。通过CCK-8和Transwell试验分析转染前后增殖、迁移、侵袭能力的差异。 结果免疫组织化学和Western blotting结果均显示在子宫内膜癌组织中Fhit基因表达与正常子宫内膜组织相比明显降低甚至缺失(P < 0.01);CCK-8结果显示Fhit基因表达上调后子宫内膜癌细胞生长明显减慢,与未转染组比较差异有统计学意义(P < 0.01);Transwell试验结果显示Fhit基因表达上调后子宫内膜癌细胞迁移和侵袭能力显著减弱,空白对照组的穿膜细胞数显著高于转染组(P < 0.01)。 结论相比于正常子宫内膜组织,Fhit基因在子宫内膜癌组织中表达较低且与临床病理特征相关。在子宫内膜癌细胞株中通过saRNA上调Fhit后,其增殖、迁移、侵袭能力受到明显抑制。 Abstract:ObjectiveTo explore the expression level of Fhit gene in endometrial carcinoma, and observe its effects on the proliferation, invasion and migration of endometrial carcinoma cells through the RNA activation up-regulating the expresson of Fhit. MethodsThe expression levels of Fhit protein in 135 endometrial carcinoma tissues and 40 normal endometrial tissues were detected using immunohistochemistry and Western blotting.The correlation between Fhit expression and clinicopathological features was analyzed.The Fhit-saRNA expression vector was constructed, and transfected into endometrial carcinoma ISK cell line, and the endometrial carcinoma cell line with Fhit up-regulating expression was established.The transfection effects were verified using Western blotting and RT-PCR.The differences of the proliferation, migration and invasion abilities between before and after transfection were analyzed using CCK-8 and Transwell assays. ResultsThe results of immunohistochemistry and Western blotting showed that the expression levels of Fhit in endometrial carcinoma tissue significantly reduced or even missed compared with normal endometrial tissue(P < 0.01).The results of CCK-8 showed that the growth of endometrial carcinoma significantly slowed down after the up-regulating of Fhit expression, and the difference of which between transfection group and non-transfection group was statistically significant(P < 0.01).The results of Transwell assay showed that the migration and invasion abilities of endometrial carcinoma cells after the level of Fhit up-regulating significantly weakened, and the number of transmembrane cells in blank control group were significantly higher than that in transfection group(P < 0.01). ConclusionsCompared with normal endometrial tissues, the expression level of Fhit in endometrial carcinoma tissues is lower, and related to the clinicopathological features.After up-regulating the Fhit expression by RNA activation in endometrial carcinoma cell line, the abilities of proliferation, migration and invasion of cells are significantly inhibited. -
Key words:
- endometrial carcinoma /
- fragile histidinetriad /
- RNA activation
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表 1 Fhit在不同组织中的表达
组织学类型 n 阴性 阳性 χ2 P 正常子宫内膜组织
子宫内膜癌组织40
1357
10633
2950.22 < 0.01 表 2 Fhit表达与子宫内膜癌临床病理特征的联系
因素 n 阴性 阳性 χ2 P 年龄/岁 < 50
≥5026
10916
6510
440.03 >0.05 组织学分级 G1 44 26 18 3.03 >0.05 G2 58 42 16 G3 33 25 8 FIGO分期 Ⅰ+Ⅱ
Ⅲ+Ⅳ70
6540
5030
155.93 < 0.05 肌层浸润 ≤1/2
>1/266
6939
5327
164.88 < 0.05 淋巴结转移 无
有92
4355
3637
77.64 < 0.01 表 3 子宫内膜癌组织与正常子宫内膜组织中Fhit的表达比较(x±s)
分组 Fhit相对表达量 t P 子宫内膜癌组织
正常子宫内膜组织0.44±0.02
0.80±0.0211.06 < 0.01 表 4 RNAa转染后ISK细胞中Fhit的表达比较(x±s)
分组 Fhit相对表达量 F P 空白对照组 0.21±0.02 阴性对照组 0.23±0.01 14.78 < 0.01 实验组 1.04±0.11 表 5 Fhit对ISK细胞侵袭和迁移能力的影响(x±s)
分组 穿膜细胞数量(侵袭) 穿膜细胞数量(迁移) 空白对照组 179.0±6.9 111.3±7.1 阴性对照组 162.7±8.9 108.3±7.8 实验组 41.2±8.1 38±7.2 F 265.20 94.95 P < 0.01 < 0.01 -
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