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宫颈癌是妇女中最常见的恶性肿瘤之一,在发展中国家尤为常见,位居各种恶性肿瘤之首[1-2]。我国每年死于宫颈癌的患者有五万余人[3]。宫颈癌是一种感染性的疾病,大量分子生物学研究和流行病学结果证实,高危型人类乳头状瘤病毒感染是宫颈癌发生的必要条件之一[4-6]。辅助外科手术的放疗是治疗宫颈癌的常用手段。然而,中晚期宫颈癌患者会对放疗产生耐辐射性,对放疗逐渐变得不敏感,需加大治疗照射剂量,然而剂量的增加会给肿瘤患者带来比较严重的不良反应,降低了患者的生活质量[7-8]。因此,研究宫颈癌辐射耐受产生的机制,改变放射敏感性调节相关基因的表达水平,靶向地增加宫颈癌的放疗敏感性,一直是宫颈癌放疗临床和基础研究的热点。
乙肝病毒X 蛋白结合蛋白(hepatitis B X-inter-acting protein,HBXIP)是一种细胞组成型表达的蛋白,首次于肝癌细胞株HepG2 中被发现,通过酵母双杂交技术筛选作为乙肝病毒编码的蛋白HBx 的作用因子。HBXIP 基因定位于人染色体1p13.3,其开放阅读框可编码173 个氨基酸,相对分子质量约为19 000[9]。研究结果显示,HBXIP蛋白在多种恶性肿瘤疾病中都显著高表达,包括乳腺癌、肺癌、宫颈癌、结肠癌以及前列腺癌[10-11]。同时,HBXIP 作为一个多功能的调节蛋白,可通过不同的分子机制促进肿瘤细胞的增殖和迁移,抑制肿瘤细胞的凋亡,进而促进肿瘤的发生与发展[12-15]。我们选用宫颈癌ME-180细胞系为研究对象,研究人为降低HBXIP基因表达对宫颈癌ME-180细胞放射敏感性的影响,为探索宫颈癌放疗增敏的新途径提供实验依据和理论基础。
HBXIP蛋白表达对宫颈癌细胞的增殖能力及放射敏感性的影响
Effects of HBXIP protein expression on the proliferation and radiosensitivity of cervical cancer cells
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摘要:
目的探讨用siRNA敲降乙肝病毒X蛋白结合蛋白(HBXIP)的表达对宫颈癌ME-180细胞的增殖能力及放射敏感性的影响。 方法根据不同的处理方法,分别按两种分组方式进行分组。(1)把宫颈癌ME-180细胞分为4组:空白对照组、4 Gy γ射线照射组、HBXIP-siRNA转染组以及HBXIP-siRNA+γ射线照射联合组。采用MTT和克隆形成实验法来检测细胞增殖;采用qRTPCR检测凋亡相关蛋白Bcl-2及Bid的表达;Western blot检测蛋白激酶AKT的磷酸化水平。(2)把宫颈癌ME-180细胞分为3组:空白对照组、HBXIP-siRNA单独处理组、HBXIP-siRNA和AKT共转染组。对3组细胞进行不同剂量的γ射线照射,并采用克隆形成实验方法检测细胞生长。采用Student t-test对数据进行统计学分析,P < 0.05表示差异有统计学意义。 结果 MTT实验和克隆形成实验结果显示,与γ射线照射组相比,HBXIP-siRNA转染+γ射线照射联合组的宫颈癌ME-180细胞增殖率明显降低(t=11.63、12.17,均P < 0.01),并伴随抑凋亡蛋白Bcl-2表达的降低(t=10.88,P < 0.01)和促凋亡蛋白Bid表达的增高(t=9.31,P < 0.01)。γ射线照射明显上调了HBXIP蛋白的表达水平和AKT蛋白的磷酸化水平,而转染HBXIP-siRNA则抑制了γ射线照射导致的AKT磷酸化水平的升高。另外,与HBXIP-siRNA单独处理组相比,HBXIP-siRNA和AKT共转染组中HBXIP-siRNA对宫颈癌ME-180细胞增殖的影响显著降低(t=8.96,P < 0.01)。 结论降低HBXIP蛋白表达可以抑制辐照诱导的AKT磷酸化水平,进而降低宫颈癌ME-180细胞的增殖能力,同时增强其放射敏感性。 -
关键词:
- HBXIP /
- RNA, 小分子干扰 /
- 宫颈肿瘤 /
- 细胞增殖 /
- 辐射耐受性
Abstract:Objective To explore the effects of (hepatitis B X-interacting protein) HBXIP downregulation on the proliferation and radiosensitivity of cervical cancer ME-180 cells. Method According to the different treatment methods, cervical cancer ME -180 cells were divided into different groups:(1) The cervical cancer ME -180 cells were divided into 4 groups:control group, 4 Gy γ ray irradiation group, HBXIP-siRNA transfection group and HBXIP-siRNA transfection+γ ray irradiation group. cervical cancer ME-180 cell proliferation was detected by MTT and clonogenic assays. The expression of Bcl-2 and Bid mRNA was detected by quantitative real-time polymerase chain reaction, and the phosphorylation of AKT protein was measured by Western blot analysis. (2) The cervical cancer ME-180 cells were divided into 3 groups:control group, HBXIP-siRNA transfection group and HBXIP-siRNA+AKT transfection group. Then the three groups of cells were irradiated with different doses of γ ray, and the cervical cancer ME-180 cell proliferation was detected by clonogenic assay. Statistical significance of the results was determined by SPSS statistical software and analyzed by Student t-test. P < 0.05 were considered statistically significant. Results MTT and clonogenic assays showed that, compared with the cells irradiated alone, the cervical cancer ME-180 cells irradiated in the presence of HBXIP-siRNA had significantly decreased proliferation (t=11.63, 12.17, P < 0.01). The decreased proliferation was accompanied by a decreased expression of Bcl-2 protein (t=10.88, P < 0.01) and an increased expression of Bid protein (t=9.31, P < 0.01). The transfection with HBXIP-siRNA inhibited the increased HBXIP protein expression and AKT phosphorylation, which were caused by radiation. The enhanced AKT expression significantly reduced the HBXIP -siRNA inhibition of cervical cancer ME-180 cell proliferation after irradiation as compared with that of the HBXIP-siRNA alone (t=8.96, P < 0.01). Conclusion HBXIP down -regulation reduced the proliferation and increased the radiosensitivity of cervical cancer ME-180 cells by mediating AKT activation. -
Key words:
- HBXIP /
- RNA, small interfering /
- Uterine cervical neoplasms /
- Cell proliferation /
- Radiation tolerance
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图 1 MTT 实验(A)和克隆形成实验(B)检测 γ 射线(4 Gy)和(或)HBXIP-siRNA 对宫颈癌 ME-180 细胞生长的影响 图中,A: 与 4 Gyγ 射线照射组相比, HBXIP-siRNA+γ 射线照射联合组可以明显抑制细胞生长, 且生长程度的差异有统计学意义(24 h、 48 h、 72 h), ** : t=9.83、 10.28、 11.63, 均 P<0.01;B: 与 4 Gyγ 射线照射组相比, HBXIP-siRNA+γ 射线照射联合组可以明显抑制细胞生长, 且生长程度的差异有统计学意义(24 h、 48 h), * : t=9.83 和 10.28, 均 P<0.05; **: t= 12.17, P<0.01。 HBXIP: 乙肝病毒 X 蛋白结合蛋白。
Figure 1. Effects of 4 Gy γ ray and (or) HBXIP-siRNA on growth of cervical cancer ME-180 cells was detected by MTT (A) and clonogenic (B) assays.
图 2 HBXIP-siRNA 和 γ 射线照射对宫颈癌 ME-180 细胞中 Bcl-2 和 BidmRNA 表达水平(A)以及蛋白表达水平(B)的影响 图中, 与空白对照组比较, HBXIP-siRNA 转染组的细胞出现了一定程度的 Bcl-2 mRNA 表达降低和 Bid mRNA 表达增加, *: t=4.97、 5.62, 均 P<0.05; 与空白对照组比较, HBXIP-siRNA+γ 射线照射联合组中, Bcl-2 mRNA 的表达水平出现了明显降低, 而 Bid mRNA 的表达水平出现了明显增加, 同时两者之间表达量的差异有统计学意义, **: t=10.88、 9.31, 均 P<0.01。 HBXIP:乙肝病毒 X 蛋白结合蛋白。
Figure 2. Effects of HBXIP-siRNA and radiation on expression of Bcl-2 and Bid mRNA (A) and protein (B) in cervical cancer ME-180 cells.
图 4 HBXIP siRNA 和 pCMV-AKT 共转染对 ME-180 细胞放射敏感性的影响 图中, 与单独照射组细胞相比, HBXIPsiRNA 转染明显提高了细胞的放射敏感性, 而共转染了HBXIP-siRNA 和 pCMV-AKT 的细胞则出现明显放射敏感性下降, **: t=11.24、 8.96, 均 P<0.01。 HBXIP: 乙肝病毒 X 蛋白结合蛋白。
Figure 4. Effects of HBXIP siRNA 和 pCMV-AKT co-transfection on radiosenstivity of cervical cancer ME-180 cells.
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