转录因子FOXC2在宫颈癌发生的研究
摘要
子宫颈癌（cervical cancer）是女性生殖器最常见的恶性肿瘤，其发病率居高不下,每年新增宫颈癌病例1/3在中国，宫颈癌发病呈青年化趋势。特别严重困扰了中国中青年女性的生殖健康。2013年中国卫生和计划生育委员会发布的《2011中国卫生统计年鉴》统计，2010年中国城乡宫颈癌死亡率为2.45/10万~4.31/10万。因此早期发现、早期治疗宫颈癌能有效地延长病人生存期，降低病死率。
大量的研究表明，人乳头瘤病毒（HPV）感染是宫颈癌最常见的发病因素，其感染率高达99.7%。但是HPV感染后到最终发展成宫颈癌是多阶段、多种途径、多分子因素综合作用的结果。近年来肿瘤发生的胚胎源性越来越受到重视，有研究已发现胚胎发育相关性基因Oct-4、Twist、VEGF-C、Wnt等在宫颈癌发生、发展中起重要作用。FOXC2是胚胎发生早期发挥重要作用的转录调控因子，与胚胎淋巴管的发生、血管的发生、形态塑建、结构分化密切相关。2007年国外有报道，FOXC2基因在乳腺癌细胞中易位表达，FOXC2蛋白能够使乳腺癌细胞获得间质形态，使细胞的迁移和侵袭能力增加。2011年又有文献报道，FOXC2与食道鳞癌的侵袭转移有关，FOXC2靶向RNA干扰可以降低食道鳞癌细胞的增殖迁移能力。因此，研究FOXC2在宫颈癌增殖与侵袭力、淋巴管形成上的作用和作用模式，探讨宫颈癌组织淋巴管发生机制，对解明宫颈癌发生过程、预防宫颈癌淋巴转移及发现新的肿瘤诊治方法有重要的意义。
目的：
本文通过分析FOXC2基因在子宫颈癌病变组织、宫颈癌细胞中的表达水平、siRNA靶向干扰宫颈癌细胞FOXC2基因表达对其生物学行为的影响等研究，试图探讨FOXC2在宫颈癌增殖与侵袭力、癌组织血管与淋巴管形成以及宫颈癌远处转移等方面上的作用及调控机制，以期发现新的预防宫颈癌远处转移和诊治靶点。
方法：
1、用免疫组织化学方法研究FOXC2在人正常宫颈组织、CIN以及宫颈癌中的表达模式，分析FOXC2表达与宫颈癌临床病变程度的相关性；
2、在细胞水平用实时荧光PCR方法、Western Blot分析了人Hela、SiHa宫颈癌细胞及小鼠U14细胞中FOXC2表达模式；
3、合成了FOXC2基因序列的siRNA表达载体，利用脂质体法转染入人Hela、SiHa宫颈癌细胞、小鼠U14宫颈癌细胞；
4、用实时荧光PCR及Western Blot方法分析了FOXC2siRNA对宫颈癌细胞FOXC2基因表达的抑制作用；
5、CCK8法分析干扰前后宫颈癌细胞增殖活性，用划痕法检测干扰前后宫颈癌细胞体外迁移能力的改变。
结果：
1、25%的正常宫颈组织表达低强度的FOXC2蛋白，65%的CIN病例宫颈组织表达中等强度的FOXC2蛋白；而91.8%的宫颈癌组织表达高强度的FOXC2蛋白，其中宫颈腺癌组织全部高强度表达FOXC2蛋白。
2、宫颈鳞癌I期患者中FOXC2阳性表达率为88.5%，II期为100%，与正常宫颈及宫颈CIN患者比较差异显著，P<0.05。宫颈癌II期患者FOXC2表达强度高于I期患者FOXC2表达强度，两者均显著高于正常妇女，P<0.05。
3、宫颈鳞癌癌灶中存在新生血管，其周边组织存在丰富的新生微淋巴管，均有FOXC2表达阳性。
4、淋巴转移者癌组织FOXC2表达强度高于未发生淋巴转移者，但两组比较差异无显著性，P>0.05。
5、人宫颈癌HeLa、SiHa细胞和小鼠宫颈癌U14细胞均高水平的表达FOXC2mRNA和FOXC2蛋白表达，各种细胞间FOXC2表达无明显差异。
6、FOXC2-siRNA可抑制HeLa、SiHa和U14细胞FOXC2mRNA的表达，抑制率分别达到76.2%、75.7%和90.8%。
7、FOXC2-siRNA完全抑制HeLa、SiHa细胞FOXC2蛋白的表达。
8、FOXC2-siRNA作用48h后，Hela细胞增殖开始能力减弱；72h后Hela细胞和SiHa细胞增殖能力均明显减弱；U14细胞增殖能力也明显减弱。
9、 FOXC2-siRNA作用24h后，HeLa、SiHa细胞侵袭能力均显著低于对照组（P<0.05）。
结论：
1、宫颈癌病变组织存在FOXC2蛋白，FOXC2蛋白的表达水平及表达强度与宫颈癌的病变程度呈正相关，证明FOXC2蛋白参与了宫颈癌的发生发展过程。
2、FOXC2参与了宫颈鳞癌新生微血管和新生微淋巴管的形成，表明与宫颈鳞癌的远处转移有密切关系。
3、FOXC2shRNA可抑制宫颈癌细胞的增殖能力。
4、FOXC2shRNA可抑制宫颈癌细胞的体外迁移能力。5、FOXC2基因可能成为治疗宫颈癌的分子靶点。
Cervical cancer is the common malignant tumor of female reproductivesystem, each year, there are100000new cases of cervical cancer in China,1/5ofthe global cases. Tumor registration report newly increased cases of cervicalcancer are about135000cases in2013, whose incidence remains high.According to China's health and family planning commission issued2011Chinahealth statistics yearbook of statistics,in2010cervical cancer mortality rate is3.40/10thousand in big cities, and4.31/100thouand in small and medium-sizedcities,2.45/10thousand in rural area. cervical cancer mortality rate is relativelylow in young women, less than1.0/10thousand, but after entering the age of35,the mortality rate doubled and increased year by year,which threatenedmiddle-aged and old women seriously. Cervical cancer occurred secretively,progress rapidly,therefore,early detection of primary lesions and effectivetreatment is effective to prolong patient life time and reduce the fatality rate.
Tumor cells transfer from the primary tumor to distant organs just like thatcell migration in embryonic development. Embryonic transcription regulatoryfactor FOXC2has relation with occurrence morphological remodeling andstructure differentiation of lymphatic vessels. This factor expresses differently inthe tumor happening which play an important role in early stage of embryonicdevelopment. In2007someone found that ectopic expression of FOXC2inbreast cancer make breast cancer cells acquire interstitial morphology and moreability to invade an migrate.In2011and others found that FOXC2is associatedwith invasion and metastasis of esophageal squamous cell carcinomas, FOXC2targeted RNA interference can reduce esophageal squamous cancer cells abilityto proliferate and migrate. given the EMT in primary tumor cells migrated to thecirculatory system or lymphatic system, people can change process by blocking EMT to prevent the occurrence of infiltration and the formation of metastases，provide opportunities for the precious treatment of patients with cervical cancer.
Therefore to investigate the role of FOXC2in tumor formation and modeof action on the proliferation and invasion, lymphatic and lymph tumorlymphan-giogenesis and embryonic tissue tube. Whether the same mechanismProcess to have cervical cancer,The prevention of cervical cancer with lymphmetastasis and the new tumor diagnosis and treatment method.There is veryimportant significance.
Objective:
Forkhead box C2(FOXC2) is a family of winged helix transcription factorhaving a DNA-binding domain of a member.Report is closely related to tumorvasculature,tumor growth,invasion and metastasis. But FOXC2has no studied incervical cancer tumor growth and invasion.Therefore we studied the expressionof FOXC2in human cervical cancer，and its relation with clinical data，in orderto investigate the role of FOXC2in The occurrence and development of cervicalcancer.
Method:
1、The expression of FOXC2pattern by immunohistochemistry study onin human normal cervical tissue, CIN and cervical carcinoma, analysis of thecorrelation between FOXC2expression and cervical carcinoma lesion severity.
2、The expression of FOXC2pattern by RT-PCR, Western Blot analysis onHela, SiHa human cervical cancer cells and U14mouse cervical cancer cells.
3、The synthesis of siRNA fOXC2gene sequences expression vector, usinglipofectin transfected into Hela, SiHa cells and U14cells.
4、Inhibitory effect of FOXC2siRNA in cervical cancer cell was analyzedby RT-PCR and Western Blot method.
5、Analysis of the proliferation on cervical cancer cell activity interference before and after by CCK8method; Scratch detection interference before andafter on cervical cancer cells in vitro migration of change.
Result:
1、The low intensity expression of FOXC2in normal cervical tissue of25%,expressed moderate levels of FOXC2in65%cases of CIN, the high strengthexpression of FOXC2protein in91.8%cases of cervical cancer tissues;the highstrength expression of FOXC2protein in all cervical adenocarcinoma tissue.
2、The positive expression of FOXC2in cervical squamous cell carcinomain I stage was88.5%, II stage was100%, significantly compared with thenormal cervix and differences in CIN patients, P<0.05. Expression of FOXC2patients with cervical carcinoma stage II strength higher than I stage, both weresignificantly higher than that of normal cervical tissue, P <0.05.
3、The expression of FOXC2positive in squamous cell carcinoma tumorangiogenesis, the new micro lymphatic vessel and abundant in the surroundingtissue.
4、FOXC2expression in lymph node metastasis cancer tissue intensity washigher than that without lymph metastasis, but the two groups had no significantdifference, P>0.05.
5、High level expression of FOXC2mRNA and expression of FOXC2protein in HeLa, SiHa cells and U14cells, all cells showed no differencebetween the expression of FOXC2.
6、FOXC2-siRNA can inhibit the expression of FOXC2mRNA in HeLa,SiHa and U14cell, inhibition rate reached76.2%,75.7%and90.8%respectively.
7、The FOXC2-siRNA completely inhibited expression of FOXC2proteinin HeLa, SiHa cells.
8、FOXC2-siRNA48h after, Hela cell proliferation capacity decreased;72h began Hela and SiHa cell proliferation was decreased significantly,theproliferation of U14cells was significantly decreased.
9、FOXC2-siRNA24h after, HeLa and SiHa cells migration and invasionwere significantly lower than those in the control group (P<0.05).
Conclusion:
1、The expression of FOXC2protein in cervical carcinoma tissues, and theexpression of FOXC2protein expression in strength and cervical cancerpathological changes is positively related to the degree, we prove that FOXC2protein participate in the occurrence anddevelopment of cervical cancer.
2、 FOXC2participated in the formation of cervical squamous cellcarcinoma the new microvascular and the new micro lymphatic vessel,show andcervical squamous cell carcinoma of distant metastasis is closely related.
3、We found that FOXC2shRNA can inhibit the proliferation of cervicalcancer cells.
4、 We found the migration ability of FOXC2shRNA can inhibit thecervical cancer cells in vitro.
5、We found that the FOXC2gene could be a molecular target for thetreatment of cervical cancer.
大量的研究表明，人乳头瘤病毒（HPV）感染是宫颈癌最常见的发病因素，其感染率高达99.7%。但是HPV感染后到最终发展成宫颈癌是多阶段、多种途径、多分子因素综合作用的结果。近年来肿瘤发生的胚胎源性越来越受到重视，有研究已发现胚胎发育相关性基因Oct-4、Twist、VEGF-C、Wnt等在宫颈癌发生、发展中起重要作用。FOXC2是胚胎发生早期发挥重要作用的转录调控因子，与胚胎淋巴管的发生、血管的发生、形态塑建、结构分化密切相关。2007年国外有报道，FOXC2基因在乳腺癌细胞中易位表达，FOXC2蛋白能够使乳腺癌细胞获得间质形态，使细胞的迁移和侵袭能力增加。2011年又有文献报道，FOXC2与食道鳞癌的侵袭转移有关，FOXC2靶向RNA干扰可以降低食道鳞癌细胞的增殖迁移能力。因此，研究FOXC2在宫颈癌增殖与侵袭力、淋巴管形成上的作用和作用模式，探讨宫颈癌组织淋巴管发生机制，对解明宫颈癌发生过程、预防宫颈癌淋巴转移及发现新的肿瘤诊治方法有重要的意义。
目的：
本文通过分析FOXC2基因在子宫颈癌病变组织、宫颈癌细胞中的表达水平、siRNA靶向干扰宫颈癌细胞FOXC2基因表达对其生物学行为的影响等研究，试图探讨FOXC2在宫颈癌增殖与侵袭力、癌组织血管与淋巴管形成以及宫颈癌远处转移等方面上的作用及调控机制，以期发现新的预防宫颈癌远处转移和诊治靶点。
方法：
1、用免疫组织化学方法研究FOXC2在人正常宫颈组织、CIN以及宫颈癌中的表达模式，分析FOXC2表达与宫颈癌临床病变程度的相关性；
2、在细胞水平用实时荧光PCR方法、Western Blot分析了人Hela、SiHa宫颈癌细胞及小鼠U14细胞中FOXC2表达模式；
3、合成了FOXC2基因序列的siRNA表达载体，利用脂质体法转染入人Hela、SiHa宫颈癌细胞、小鼠U14宫颈癌细胞；
4、用实时荧光PCR及Western Blot方法分析了FOXC2siRNA对宫颈癌细胞FOXC2基因表达的抑制作用；
5、CCK8法分析干扰前后宫颈癌细胞增殖活性，用划痕法检测干扰前后宫颈癌细胞体外迁移能力的改变。
结果：
1、25%的正常宫颈组织表达低强度的FOXC2蛋白，65%的CIN病例宫颈组织表达中等强度的FOXC2蛋白；而91.8%的宫颈癌组织表达高强度的FOXC2蛋白，其中宫颈腺癌组织全部高强度表达FOXC2蛋白。
2、宫颈鳞癌I期患者中FOXC2阳性表达率为88.5%，II期为100%，与正常宫颈及宫颈CIN患者比较差异显著，P<0.05。宫颈癌II期患者FOXC2表达强度高于I期患者FOXC2表达强度，两者均显著高于正常妇女，P<0.05。
3、宫颈鳞癌癌灶中存在新生血管，其周边组织存在丰富的新生微淋巴管，均有FOXC2表达阳性。
4、淋巴转移者癌组织FOXC2表达强度高于未发生淋巴转移者，但两组比较差异无显著性，P>0.05。
5、人宫颈癌HeLa、SiHa细胞和小鼠宫颈癌U14细胞均高水平的表达FOXC2mRNA和FOXC2蛋白表达，各种细胞间FOXC2表达无明显差异。
6、FOXC2-siRNA可抑制HeLa、SiHa和U14细胞FOXC2mRNA的表达，抑制率分别达到76.2%、75.7%和90.8%。
7、FOXC2-siRNA完全抑制HeLa、SiHa细胞FOXC2蛋白的表达。
8、FOXC2-siRNA作用48h后，Hela细胞增殖开始能力减弱；72h后Hela细胞和SiHa细胞增殖能力均明显减弱；U14细胞增殖能力也明显减弱。
9、 FOXC2-siRNA作用24h后，HeLa、SiHa细胞侵袭能力均显著低于对照组（P<0.05）。
结论：
1、宫颈癌病变组织存在FOXC2蛋白，FOXC2蛋白的表达水平及表达强度与宫颈癌的病变程度呈正相关，证明FOXC2蛋白参与了宫颈癌的发生发展过程。
2、FOXC2参与了宫颈鳞癌新生微血管和新生微淋巴管的形成，表明与宫颈鳞癌的远处转移有密切关系。
3、FOXC2shRNA可抑制宫颈癌细胞的增殖能力。
4、FOXC2shRNA可抑制宫颈癌细胞的体外迁移能力。5、FOXC2基因可能成为治疗宫颈癌的分子靶点。
Cervical cancer is the common malignant tumor of female reproductivesystem, each year, there are100000new cases of cervical cancer in China,1/5ofthe global cases. Tumor registration report newly increased cases of cervicalcancer are about135000cases in2013, whose incidence remains high.According to China's health and family planning commission issued2011Chinahealth statistics yearbook of statistics,in2010cervical cancer mortality rate is3.40/10thousand in big cities, and4.31/100thouand in small and medium-sizedcities,2.45/10thousand in rural area. cervical cancer mortality rate is relativelylow in young women, less than1.0/10thousand, but after entering the age of35,the mortality rate doubled and increased year by year,which threatenedmiddle-aged and old women seriously. Cervical cancer occurred secretively,progress rapidly,therefore,early detection of primary lesions and effectivetreatment is effective to prolong patient life time and reduce the fatality rate.
Tumor cells transfer from the primary tumor to distant organs just like thatcell migration in embryonic development. Embryonic transcription regulatoryfactor FOXC2has relation with occurrence morphological remodeling andstructure differentiation of lymphatic vessels. This factor expresses differently inthe tumor happening which play an important role in early stage of embryonicdevelopment. In2007someone found that ectopic expression of FOXC2inbreast cancer make breast cancer cells acquire interstitial morphology and moreability to invade an migrate.In2011and others found that FOXC2is associatedwith invasion and metastasis of esophageal squamous cell carcinomas, FOXC2targeted RNA interference can reduce esophageal squamous cancer cells abilityto proliferate and migrate. given the EMT in primary tumor cells migrated to thecirculatory system or lymphatic system, people can change process by blocking EMT to prevent the occurrence of infiltration and the formation of metastases，provide opportunities for the precious treatment of patients with cervical cancer.
Therefore to investigate the role of FOXC2in tumor formation and modeof action on the proliferation and invasion, lymphatic and lymph tumorlymphan-giogenesis and embryonic tissue tube. Whether the same mechanismProcess to have cervical cancer,The prevention of cervical cancer with lymphmetastasis and the new tumor diagnosis and treatment method.There is veryimportant significance.
Objective:
Forkhead box C2(FOXC2) is a family of winged helix transcription factorhaving a DNA-binding domain of a member.Report is closely related to tumorvasculature,tumor growth,invasion and metastasis. But FOXC2has no studied incervical cancer tumor growth and invasion.Therefore we studied the expressionof FOXC2in human cervical cancer，and its relation with clinical data，in orderto investigate the role of FOXC2in The occurrence and development of cervicalcancer.
Method:
1、The expression of FOXC2pattern by immunohistochemistry study onin human normal cervical tissue, CIN and cervical carcinoma, analysis of thecorrelation between FOXC2expression and cervical carcinoma lesion severity.
2、The expression of FOXC2pattern by RT-PCR, Western Blot analysis onHela, SiHa human cervical cancer cells and U14mouse cervical cancer cells.
3、The synthesis of siRNA fOXC2gene sequences expression vector, usinglipofectin transfected into Hela, SiHa cells and U14cells.
4、Inhibitory effect of FOXC2siRNA in cervical cancer cell was analyzedby RT-PCR and Western Blot method.
5、Analysis of the proliferation on cervical cancer cell activity interference before and after by CCK8method; Scratch detection interference before andafter on cervical cancer cells in vitro migration of change.
Result:
1、The low intensity expression of FOXC2in normal cervical tissue of25%,expressed moderate levels of FOXC2in65%cases of CIN, the high strengthexpression of FOXC2protein in91.8%cases of cervical cancer tissues;the highstrength expression of FOXC2protein in all cervical adenocarcinoma tissue.
2、The positive expression of FOXC2in cervical squamous cell carcinomain I stage was88.5%, II stage was100%, significantly compared with thenormal cervix and differences in CIN patients, P<0.05. Expression of FOXC2patients with cervical carcinoma stage II strength higher than I stage, both weresignificantly higher than that of normal cervical tissue, P <0.05.
3、The expression of FOXC2positive in squamous cell carcinoma tumorangiogenesis, the new micro lymphatic vessel and abundant in the surroundingtissue.
4、FOXC2expression in lymph node metastasis cancer tissue intensity washigher than that without lymph metastasis, but the two groups had no significantdifference, P>0.05.
5、High level expression of FOXC2mRNA and expression of FOXC2protein in HeLa, SiHa cells and U14cells, all cells showed no differencebetween the expression of FOXC2.
6、FOXC2-siRNA can inhibit the expression of FOXC2mRNA in HeLa,SiHa and U14cell, inhibition rate reached76.2%,75.7%and90.8%respectively.
7、The FOXC2-siRNA completely inhibited expression of FOXC2proteinin HeLa, SiHa cells.
8、FOXC2-siRNA48h after, Hela cell proliferation capacity decreased;72h began Hela and SiHa cell proliferation was decreased significantly,theproliferation of U14cells was significantly decreased.
9、FOXC2-siRNA24h after, HeLa and SiHa cells migration and invasionwere significantly lower than those in the control group (P<0.05).
Conclusion:
1、The expression of FOXC2protein in cervical carcinoma tissues, and theexpression of FOXC2protein expression in strength and cervical cancerpathological changes is positively related to the degree, we prove that FOXC2protein participate in the occurrence anddevelopment of cervical cancer.
2、 FOXC2participated in the formation of cervical squamous cellcarcinoma the new microvascular and the new micro lymphatic vessel,show andcervical squamous cell carcinoma of distant metastasis is closely related.
3、We found that FOXC2shRNA can inhibit the proliferation of cervicalcancer cells.
4、 We found the migration ability of FOXC2shRNA can inhibit thecervical cancer cells in vitro.
5、We found that the FOXC2gene could be a molecular target for thetreatment of cervical cancer.
引文
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