宫颈癌组织DcR3的表达与高危型人乳头状瘤病毒感染的相关性研究

韩晶; 苗卉; 冯向先

doi:10.16462/j.cnki.zhjbkz.2019.01.018

宫颈癌组织DcR3的表达与高危型人乳头状瘤病毒感染的相关性研究

doi: 10.16462/j.cnki.zhjbkz.2019.01.018

韩晶¹,
苗卉¹,
冯向先^2, ,

1.
046000 长治, 长治医学院附属和平医院生殖科
2.
046000 长治, 长治医学院预防医学系

基金项目:

卫生部"十一五"科技重大专项子项目 200902002-7

详细信息

通讯作者:
冯向先, E-mail: xfeng66@163.com

中图分类号: R51;R181
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出版历程
- 收稿日期: 2018-07-10
- 修回日期: 2018-09-30
- 刊出日期: 2019-01-10

Relationship between the expression of DcR3 and high-risk human papilloma virus infection in cervical carcinoma

1.
Department of Reproduction Heping Hospital Affiliated to Changzhi Medical College, Changzhi 046000, China
2.
Department of Preventive Medicine, Changzhi Medical College, Changzhi 046000, China

Funds:

Major Scientific and Technological Subprojects of the 11th Five-Year Plan of the Ministry of Health 200902002-7

More Information

Corresponding author: FENG Xiang-xian, E-mail: xfeng66@163.com

摘要

摘要: 目的观察宫颈癌组织诱捕受体3（decoy receptor 3，DcR3）表达与高危型人乳头状瘤病毒（high-risk human papilloma virus，HR-HPV）感染的相关性。方法分别采用免疫组织化学（streptavidin-perosidase，SP）法和第2代杂交捕获法检测35例正常宫颈组织（normal cervical tissues，NCE）、39例宫颈上皮内瘤变（cervical intraepithelial neoplasm，CIN）及44例宫颈癌组织（cervical squamous epithelial carcinoma，CSES）DcR3和HR-HPV的表达。设计并合成靶向HR-HPV16 E7基因的HR-HPV16 E7小干扰RNA（siRNA）和阴性对照HR-HPV16 E7 siRNA，脂质体法转染SiHa细胞，实时荧光定量聚合酶链式反应（real-time polymerase chain reaction，RT-PCR）法和蛋白质印迹（western blot，WB）法检测HR-HPV16 E7基因沉默对DcR3 mRNA和蛋白表达的影响，四甲基偶氮唑盐比色（methylthiazolyldiphenyl-tetrazolium bromide，MTT）法检测细胞增殖情况。结果 NCE、CIN及CSES组DcR3蛋白阳性表达率分别为8.6%（3/35）、48.7%（19/39）、77.3%（34/44），表达呈逐渐增高趋势，差异具有统计学意义（χ²=36.942，P<0.001）。NCE、CIN及CSES组HR-HPV阳性检出率分别为5.7%（2/35）、56.4%（22/39）、93.2%（41/44），差异具有统计学意义（χ²=60.322，P<0.001）。DcR3表达与HR-HPV感染呈正相关（r=0.893，P=0.004）。HR-HPV基因沉默后DcR3在mRNA和蛋白水平的表达均明显降低，且细胞增殖受到抑制。结论 DcR3在宫颈癌中异常高表达，其表达升高与HR-HPV感染密切相关，提示DcR3可能参与了宫颈癌的发生发展。siRNA技术成功诱导HR-HPV基因沉默可通过下调DcR3的表达抑制人宫颈癌SiHa细胞增殖。
- 宫颈癌 /
- DcR3 /
- 人乳头瘤状病毒
Abstract: Objective To explore the relationship between the expression of decoy receptor 3 (DcR3) and high-risk human papilloma virus(HR-HPV) infection in cervical carcinoma. Methods Immunohistochemistry and hybird capture Ⅱ assay were used to detect the expression of DcR3 and HR-HPV in 35 cases of normal cervical tissues(NCE), 39 cases of cervical intraepithelial neoplasm(CIN) and 44 cases of cervical squamous epithelial carcinoma(CSES). Specific HR-HPV16 E7 siRNA and nonspecific HR-HPV16 E7 siRNA were synthesized and transfected to SiHa cells by Lipofectamine. The expression of DcR3 at mRNA and protein levels was examined by real-time polymerase chain reaction and western blot. The growth inhibition was examined by MTT assay. Results In NCE, CIN and CSES, the positive expression rates of DcR3 were 8.6%(3/35), 48.7%(19/39) and 77.3%(34/44), respectively, and the expression intensity was increasing(χ²=36.942, P<0.001). In NCE, CIN and CSES, the infection rates of HR-HPV were 5.7%(2/35), 56.4%(22/39) and 93.2%(41/44), respectively(χ²=60.322, P<0.001). The protein expression of DcR3 was positively correlated with the infection of HR-HPV in CSES(r=0.893, P=0.004). Conclusions DcR3 was highly expressed in cervical cancer. Its expression was positively correlated with HR-HPV infection, which may contribute to the occurrence and development of cervical cancer. HR-HPV silencing inhibited cellular growth and proliferation by down-regulating the expression of DcR3.
- Cervical carcinoma /
- DcR3 /
- Human papilloma virus

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图 1 NCE、CIN及CSES中DcR3蛋白表达(SP×200)

Figure 1. Representative images showing DcR3 expression in NCE, CIN and CSES(SP×200)

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图 2 Western blot检测SiHa细胞中DcR3蛋白表达

Figure 2. Detection of DcR3 expression in SiHa cells using western blot

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表 1 HR-HPV16 E7、DcR3、β-actin引物序列及扩增产物长度

Table 1. Sequence of the primer and product length

基因	位置	引物序列(5′-3′)	PCR产物长度(bp)
HR-HPV16 E7	上游	ATGCATGGAGATACACCT	279
	下游	TTATGGTTTCTGAGAACA	279
DcR3	上游	GTCGCTGCTGTGCCGGTGT	287
	下游	AAGCCCGTGCCTCCTCCTCA	287
β-actin	上游	ACAATGTGGCCGAGGACTTT	141
	下游	ACAATGTGGCCGAGGACTTT	141

下载: 导出CSV

表 2 不同宫颈组织DcR3表达及HR-HPV16/18感染情况比较[n(%)]

Table 2. The DcR3 expression and the HR-HPV16/18 infection in NCE, CIN and CSES[n(%)]

组别	例数	阳性数
组别	例数	DcR3	HR-HPV16/18
NCE组	35	3(8.6)	2(5.7)
CIN组	39	19(48.7)^a	22(56.4)^a
CSES组	44	34(77.3)^{a, b}	41(93.2)^{a, b}
注：^aCSES组、CIN组与NCE组比较P<0.05；^bCSES组与CIN组比较P<0.05。

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表 3 DcR3、HR-HPVl6/18在宫颈癌中表达的相关性

Table 3. Relationship of DcR3 expression and the HR-HPV16/18 infection in CSES

HPVl6/18感染	DcR3表达		合计
HPVl6/18感染	阳性	阴性	合计
阳性	30	6	36
阴性	2	6	8
合计	32	12	44
注：r=0.893，P=0.004。

下载: 导出CSV

表 4 转染HR-HPV16 E7 siRNA后HR-HPV16 E7 mRNA的表达

Table 4. Effect of HR-HPV16 E7 siRNA transfection on mRNA expression of HR-HPV16 E7 in SiHa cells

组别	β-actin	HR-HPV18 E7	相对灰度值
实验组	60.32±1.41	10.72±1.13	0.18±0.12^ab
对照组	66.18±2.05	46.24±1.28	0.70±0.31
空白组	62.54±1.87	43.33±1.22	0.69±0.34
注：^a实验组与空白组比较P<0.05；^b实验组与对照组比较P<0.05。

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表 5 HR-HPV16 E7 siRNA对DcR3 mRNA表达的影响

Table 5. Effect of HR-HPV16 E7 siRNA transfection on mRNA expression of DcR3 in SiHa cells

组别	β-actin	DcR3	相对灰度值
实验组	68.56±2.21	31.21±1.76	0.46±0.08^ab
对照组	73.14±2.87	59.25±1.84	0.81±0.12
空白组	74.12±2.98	61.17±2.05	0.83±0.13
注：^a实验组与空白组比较P<0.05；^b实验组与对照组比较P<0.05。

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表 6 HR-HPV16 E7 siRNA对DcR3蛋白表达的影响

Table 6. Effect of HR-HPV16 E7 siRNA transfection on protein expression of DcR3 in SiHa cells

组别	β-actin	DcR3	相对灰度值
实验组	156.32±5.64	62.53±3.58	0.40±0.06^ab
对照组	163.33±5.94	137.19±4.77	0.84±0.14
空白组	161.31±5.26	138.73±4.89	0.86±0.19
注：^a实验组与空白组比较P<0.05；^b实验组与对照组比较P<0.05。

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