RNAi介导的WFDC2基因沉默在人卵巢浆液性上皮癌发展中的作用及其机制的初步研究
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摘要
目的：
1.构建WFDC2基因稳定低表达的人浆液性卵巢癌细胞株SKOV3,体外检测WFDC2基因沉默对肿瘤细胞增殖、凋亡、侵袭和迁移能力的影响。
2.构建裸鼠皮下移植瘤模型,观察肿瘤生长、转移特点的变化。
3.从表皮生长因子受体(epidermal growch factor receptor,EGFR)等信号通路研究低表达WFDC2显著抑制SKOV3生长的机制。
4.立足乳清酸蛋白(WAP)家族具有抑制环的结构上的共性和调节蛋白酶活性的功能上的共性,探讨1)WFDC2基因编码的人附睾蛋白4(HE4)是否能和MMP-9(基质金属蛋白酶-9)和MMP-2结合；2)HE4蛋白对已经激活的的MMPs酶活性是否存在调节作用,在蛋白水平探讨HE4蛋白与基质金属蛋白酶相互作用对卵巢癌侵袭能力影响的机制。
5.立足卵巢癌疾病的“异质性”,应用卵巢浆液性癌“两级分级系统”(two-tier system),对比HE4蛋白在低级别浆液性癌(low-grade serous carcinoma,LGSC)和高级别浆液性癌(high-grade serous carcinoma,HGSC)组织和血清中的差异,为进一步研究WFDC2基因和HE4蛋白的功能提供新思路。
方法：
1.以慢病毒pLKO.1为载体,构建人WFDC2基因特异的小干扰序列shRNA,筛选有效的序列,通过酶切、连接、转化和筛选,获得WFDC2基因沉默的慢病毒载体后转染SKOV3,经RT-PCR和Western-blot鉴定。嘌呤霉素压力筛选获取WFDC2基因稳定低表达的细胞株后,分为4组进行体外实验,即两株WFDC2基因稳定低表达的细胞株(OEC-si5和OEC-si8)、空载体细胞株(OEC-mock)和未处理的细胞株(OEC),MTT检测细胞增殖、Annexin/PI法流式细胞仪检测细胞凋亡、Transwell法检测细胞侵袭、迁移,Westernblot法检测EGFR)通路中相关蛋白的变化和MMP-9、MMP-2的改变。
2.选择5号稳定株进行体内实验,分3组：未处理组(OEC)、转入空载体组(OEC-mock)和转染shRNA组(OEC-si5),组织块移植法建立二代卵巢癌荷瘤裸鼠模型。观察各组肿瘤生长、转移特点的变化,免疫组化检测HE4蛋白,细胞核增殖抗原(PCNA)、微淋巴管密度的改变。
3.Westernblot检测体外实验细胞中PI-3K-Akt-STAT3通路中Akt、p-Akt、 STAT3、p-STAT3和ERK-JNK-Jun-Fos通路中相关蛋白ERK、pERK、JNK、 c-Jun、c-Fos表达量的变化,探讨影响增殖和侵袭的机制。
4.通过免疫共沉淀研究HE4蛋白和基质金属蛋白酶MMP-9、MMP-2是否有结合；激活SKOV3培养上清中的MMP-2和MMP-9后,再和MMPs的特异性底物共同孵育,明胶酶谱法和底物裂解法研究HE4蛋白对MMP9、MMP2酶活性的影响。
5.以两级分级系统将60例浆液性卵巢癌分为低级别LGSC和高级别HGSC亚型,ELISA检测血清中HE4蛋白和同位素法检测血清CA125的水平,免疫组化检测HE4蛋白在卵巢癌组织中的表达水平,对比HGSC和LGSC血清、组织中HE4蛋白表达的差异。
结果：
1.体外实验。1)基因测序表明寡核苷酸成功插入到预计位点。慢病毒感染SKOV3细胞后与对照组比较,HE4的mRNA和蛋白表达量明显降低,提示成功构建了的特异性抑制人WFDC2基因的真核表达载体,并经抗生素压力筛选形成稳定株;2)增殖：敲低WFDC2基因后,SKOV3细胞株增殖显著减慢、PCNA表达量显著降低；细胞周期出现G0/G1期阻滞、cyclinD1蛋白表达量显著降低；3)凋亡：转染5号shRNA的细胞株(OEC-si5)的早期细胞凋亡率升高(P<0.05),晚期凋亡和坏死率无差异。凋亡基因Bax、Bcl-2、Casp3、TP53、Fas/FasL和凋亡蛋白bax、bcl-2和Caspase-3无显著变化。4)侵袭和迁移：Transwell侵袭和迁移细胞数显著下降(P<0.05)；5)信号通路：ERK-JNK-Jun-Fos通路中相关蛋白pERK、JNK、c-Jun、c-Fos的表达量均显著下降,基质金属蛋白酶MMP-9表达量下降。
2.动物实验。1)生长曲线。转染shRNA组(OEC-si组)自移植3天后,与空白对照组(未处理组OEC、转入慢病毒空载体组OEC-mock)开始出现差异(P<0.01),而OEC和OEC-mock之间肿瘤体积一直无差异(P>0.05)；2)OEC-si组皮下移植瘤的生长速度显著慢于空白对照组(P<0.05)；3)OEC-si组首次出现可见肿瘤的时间晚于空白对照组(P<0.05);4)实验终点时肿瘤重量：OEC-si组肿瘤重量低于空白对照组。5)OEC-si组织的HE4、PCNA表达量、微淋巴管密度均低于空白对照组。6)实验组和对照组均未发现淋巴结转移。
3.体外细胞实验WFDC2基因沉默抑制细胞生长的机制。WFDC2基因后,Akt、p-Akt、STAT3和p-STAT3表达量无变化；总ERK无变化,磷酸化ERK表达量降低伴随着JNK2、p-c-Jun和c-Fos表达量的降低。
4.HE4蛋白与基质金属蛋白酶。免疫共沉淀显示HE4和MMP-9、MMP-2有结合；HE4显著抑制SKOV3细胞培养液上清中MMP-9和MMP-2的活性,以MMP-9最明显；抑制率与HE4的浓度呈正相关。
5.临床研究。高级别浆液性癌(HGSC)组织中HE4蛋白阳性率显著高于低级别浆液性癌(LGSC)(P<0.01); HGSC的血清中HE4蛋白的水平高于LGSC(P<0.01)；早期FIGO(Ⅰ+Ⅱ期)病例中,HGSC和LGSC组织中HE4蛋白表达量有差异(P<0.05),但血清中HE4蛋白的水平无差异(P>0.05)。
结论：
1. WFDC2基因参与促进SKOV3细胞增殖、迁移、侵袭等行为。ERK-JNK-Jun-Fos信号通路参与了该过程；不能确定WFDC2基因与凋亡的关系。WFDC2基因可能在人卵巢浆液性上皮癌进展中起重要作用,
2. WFDC2基因沉默显著抑制人卵巢癌SKOV3细胞株在裸鼠体内的生长。
3.HE4蛋白可以和MMP-9、MMP-2结合,对激活的的MMP-9的酶活性存在明显的抑制作用,该作用和HE4的浓度呈正相关；
4.HE4在鉴别低、高级别浆液性癌有潜在意义,但不能肯定血清HE4对高级别浆液性癌的早期诊断的价值,需要慎重对待目前文献过于肯定HE4早期诊断价值的结论。
OBJECTIVES:
1. To investigate effect and mechanisms of silencing human WFDC2(HE4) gene on biological behavior changes such as cell proliferation,apoptosis, migration and invasion of human serous ovarian cancer cell line SKOV3.
2. To establish subcutaneous transplanted model of human ovarian carcinoma in nude mice with cells stablely transfected with lentiviral WFDC2gene sequence of small interfering shRNA,and to explore its influence on their biological properties.
3. To illustrate the mechanisms of significantly inhibited growth of tumor cells by silencing WFDC2gene from different perspectives of signaling as ERGF pathways.
4. To explore possible mechanisms of decreased invasiveness by silencing WFDC2gene from the perspective of cobinding and interaction between HE4protein and matrix metalloproteinases basing on similarities of molecular structure of WAP family and their protease inhibitory function.
5. To investigate the difference of HE4protein in paraffin section of patients with low-grade serous (LGSC) and high-grade serous carcinoma (HGSC) serous ovarian cancer by two-tier grading system basing on the heterogeneity of OEC.
MATERIALS AND METHODS:
1. Lentiviral WFDC2gene sequence of small interfering siRNA was stablely transfected into SKOV3identified by RT-PCR and western-blot. Proliferation, apoptosis, migration and invasion assays of ovarian cancer cell line were measured by MTT, Annexin/PI doubdle staining by flow cytometry and transwell assay respectively. Protein alteration as EGFR signaling pathway were measured by western-blot analysis.
2. An subcutaneous xenotransplanted tumor model of human ovarian carcinoma in nude mouse was establised and tumor volume were detected by continuous measurement to draw the tumor growth curve.Tumor weight were measured at the end point of the in vivo experiment.It aimed to observe the effect of silencing WFDC2on the tumor progression characteristics as growth(tumor growth curve,tumor weight), metastasis and matrix metalloproteinases (MMP)-9expression. HE4protein,proliferating cell nuclear antigen(PCNA), lymphatic microvessel density,(LMVD) were detected by immunohistochemistry (IHC) and in situ apoptosis of tumor cells were measured by TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) analysis.
3. Signaling pathway associated protein expression such as PI-3K-Akt-STAT3and ERK-JNK-Jun-Fos in OEC cell lines was analyzed by means of Western-blot analysis.
4. Co-Immunoprecipitation(Co-IP) was performed to prove whether there was protein cobinding between HE4and matrix metalloproteinases (MMP-9, MMP-2). MMP-9and MMP-2were activated by APMA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid),a MMPs activator and their activity was assessed by SDS-PAGE enzymograph and enzyme substrates pyrolysis cultured with HE4.
5. Low-grade serous (LGSC) and high-grade serous carcinoma (HGSC) serous ovarian cancer were defined by the two-tier grading system, serum levels of HE4and carbohydrate antigen125(CA125) were measured by ELISA and radioisotope method respectively in60serous ovarian cancer patients to compare the difference of HE4protein in paraffin section of patients with LGSC and HGSC.
RESULTS:
1. Lentiviral WFDC2gene sequence of small interfering siRNA was stablely transfected into SKOV3and identified by RT-PCR and Western-blot. Gene sequencing showed that the oligonucleotides were successfully inserted into the expected site. Proliferation of ovarian cell line was significantly inhibited(P <0.05),G0/G1phase was arrested by the cell cycle(P<0.01) and capacity of the migration and invasion decreased significantly (P<0.01). Proteins and phosphorylated protein involed in ERK-JNK-Jun-Fos protein pathway such as p-ERK,c-Jun,c-Fos decreased and protease protein involved in tissue remoding as matrix metalloproteinases MMP-9,MMP-2decreased after WFDC2silencing.
2. Human ovarian carcinomas transplanted subcutaneously in nude mice were established successfully. It was earlier to see the visible tumors in control group than WFDC silencing group (P<0.05).Tumor weight was significantly smaller in WFDC2silencing group than that of control groups,which coincide with results in vitro. Although no evidence of metastasis was found, Lymphatic microvessel density (LMVD) was significantly smaller in WFDC2silencing group than that of control groups It has been confirmed that WFDC2gene silencing significantly inhibit the growth of ovarian cancer growth and play a potential role in lymph node metastasis.
3. There was no significant change on Akt,p-Akt, STAT3or p-STAT3, proteins expression after WFDC2silencing.It suggested that inhibiting tumor cell growth was not by the classical PI-3K-Akt-STAT3signaling pathway. Decreased phosphorylated ERK, JNK2,c-Jun and c-Fos were detected,which implied the ERK-JNK-Jun-Fos signaling pathway would work.
4. Binding between HE4and matrix metalloproteinases (MMP-9, MMP-2) were confirmed by co-immunoprecipitation analysis. Culturing with HE4protein,the enzyme activity of MMP-2and MMP-9were inhibited and active matrix metalloproteinases were significantly suppressed. Effect of matrix metalloproteinases inhibition is associated with concentration of HE4protein.
5.There was significant difference in HE4protein between LGSC and HGSC, and there was not significant difference in FIGO (Ⅰ+Ⅱ) stage by serum analysis It suggested that HE4could probably be a biomarker for the discrimination between LGSC and HGSC. But its role in early diagnosis remained to be reevaluated.
CONCLUSIONS:
1. WFDC2gene plays an important role in ovarian cancer development involved in regulating the proliferation which were significantly suppressed after WFDC2silencing, but the exact effect on apoptosis can not be determined yet.The ERK-JNK Jun-Fos pathways are involved in the inhibition of proliferation and invasion of ovarian tumor cells.
2. WFDC2gene silencing plays a potential role in regulating capability of invasion and motility of ovarian cancer cells,which is essential to lymph node metastasis.
3. There is evidence of protein binding between HE4and matrix metalloproteinases MMP-9, MMP-2, which can inhibit protease hydrolysis function of MMP-9, MMP-2.Effects of inhibition are correlated with concentration of HE4. It remains to be clarified whether HE4-MMPs interaction plays a protrusive or protective role in ovarian cancer development.
4. Although there is significant difference in serum HE4levels between LGSC and HGSC, the result is similar within FIGO (Ⅰ+Ⅱ) stage, suggesting HE4has potential for the discrimination between LGSC and HGSC, but its role in early diagnosis of OEC remains to be critically evaluated.
1.构建WFDC2基因稳定低表达的人浆液性卵巢癌细胞株SKOV3,体外检测WFDC2基因沉默对肿瘤细胞增殖、凋亡、侵袭和迁移能力的影响。
2.构建裸鼠皮下移植瘤模型,观察肿瘤生长、转移特点的变化。
3.从表皮生长因子受体(epidermal growch factor receptor,EGFR)等信号通路研究低表达WFDC2显著抑制SKOV3生长的机制。
4.立足乳清酸蛋白(WAP)家族具有抑制环的结构上的共性和调节蛋白酶活性的功能上的共性,探讨1)WFDC2基因编码的人附睾蛋白4(HE4)是否能和MMP-9(基质金属蛋白酶-9)和MMP-2结合；2)HE4蛋白对已经激活的的MMPs酶活性是否存在调节作用,在蛋白水平探讨HE4蛋白与基质金属蛋白酶相互作用对卵巢癌侵袭能力影响的机制。
5.立足卵巢癌疾病的“异质性”,应用卵巢浆液性癌“两级分级系统”(two-tier system),对比HE4蛋白在低级别浆液性癌(low-grade serous carcinoma,LGSC)和高级别浆液性癌(high-grade serous carcinoma,HGSC)组织和血清中的差异,为进一步研究WFDC2基因和HE4蛋白的功能提供新思路。
方法：
1.以慢病毒pLKO.1为载体,构建人WFDC2基因特异的小干扰序列shRNA,筛选有效的序列,通过酶切、连接、转化和筛选,获得WFDC2基因沉默的慢病毒载体后转染SKOV3,经RT-PCR和Western-blot鉴定。嘌呤霉素压力筛选获取WFDC2基因稳定低表达的细胞株后,分为4组进行体外实验,即两株WFDC2基因稳定低表达的细胞株(OEC-si5和OEC-si8)、空载体细胞株(OEC-mock)和未处理的细胞株(OEC),MTT检测细胞增殖、Annexin/PI法流式细胞仪检测细胞凋亡、Transwell法检测细胞侵袭、迁移,Westernblot法检测EGFR)通路中相关蛋白的变化和MMP-9、MMP-2的改变。
2.选择5号稳定株进行体内实验,分3组：未处理组(OEC)、转入空载体组(OEC-mock)和转染shRNA组(OEC-si5),组织块移植法建立二代卵巢癌荷瘤裸鼠模型。观察各组肿瘤生长、转移特点的变化,免疫组化检测HE4蛋白,细胞核增殖抗原(PCNA)、微淋巴管密度的改变。
3.Westernblot检测体外实验细胞中PI-3K-Akt-STAT3通路中Akt、p-Akt、 STAT3、p-STAT3和ERK-JNK-Jun-Fos通路中相关蛋白ERK、pERK、JNK、 c-Jun、c-Fos表达量的变化,探讨影响增殖和侵袭的机制。
4.通过免疫共沉淀研究HE4蛋白和基质金属蛋白酶MMP-9、MMP-2是否有结合；激活SKOV3培养上清中的MMP-2和MMP-9后,再和MMPs的特异性底物共同孵育,明胶酶谱法和底物裂解法研究HE4蛋白对MMP9、MMP2酶活性的影响。
5.以两级分级系统将60例浆液性卵巢癌分为低级别LGSC和高级别HGSC亚型,ELISA检测血清中HE4蛋白和同位素法检测血清CA125的水平,免疫组化检测HE4蛋白在卵巢癌组织中的表达水平,对比HGSC和LGSC血清、组织中HE4蛋白表达的差异。
结果：
1.体外实验。1)基因测序表明寡核苷酸成功插入到预计位点。慢病毒感染SKOV3细胞后与对照组比较,HE4的mRNA和蛋白表达量明显降低,提示成功构建了的特异性抑制人WFDC2基因的真核表达载体,并经抗生素压力筛选形成稳定株;2)增殖：敲低WFDC2基因后,SKOV3细胞株增殖显著减慢、PCNA表达量显著降低；细胞周期出现G0/G1期阻滞、cyclinD1蛋白表达量显著降低；3)凋亡：转染5号shRNA的细胞株(OEC-si5)的早期细胞凋亡率升高(P<0.05),晚期凋亡和坏死率无差异。凋亡基因Bax、Bcl-2、Casp3、TP53、Fas/FasL和凋亡蛋白bax、bcl-2和Caspase-3无显著变化。4)侵袭和迁移：Transwell侵袭和迁移细胞数显著下降(P<0.05)；5)信号通路：ERK-JNK-Jun-Fos通路中相关蛋白pERK、JNK、c-Jun、c-Fos的表达量均显著下降,基质金属蛋白酶MMP-9表达量下降。
2.动物实验。1)生长曲线。转染shRNA组(OEC-si组)自移植3天后,与空白对照组(未处理组OEC、转入慢病毒空载体组OEC-mock)开始出现差异(P<0.01),而OEC和OEC-mock之间肿瘤体积一直无差异(P>0.05)；2)OEC-si组皮下移植瘤的生长速度显著慢于空白对照组(P<0.05)；3)OEC-si组首次出现可见肿瘤的时间晚于空白对照组(P<0.05);4)实验终点时肿瘤重量：OEC-si组肿瘤重量低于空白对照组。5)OEC-si组织的HE4、PCNA表达量、微淋巴管密度均低于空白对照组。6)实验组和对照组均未发现淋巴结转移。
3.体外细胞实验WFDC2基因沉默抑制细胞生长的机制。WFDC2基因后,Akt、p-Akt、STAT3和p-STAT3表达量无变化；总ERK无变化,磷酸化ERK表达量降低伴随着JNK2、p-c-Jun和c-Fos表达量的降低。
4.HE4蛋白与基质金属蛋白酶。免疫共沉淀显示HE4和MMP-9、MMP-2有结合；HE4显著抑制SKOV3细胞培养液上清中MMP-9和MMP-2的活性,以MMP-9最明显；抑制率与HE4的浓度呈正相关。
5.临床研究。高级别浆液性癌(HGSC)组织中HE4蛋白阳性率显著高于低级别浆液性癌(LGSC)(P<0.01); HGSC的血清中HE4蛋白的水平高于LGSC(P<0.01)；早期FIGO(Ⅰ+Ⅱ期)病例中,HGSC和LGSC组织中HE4蛋白表达量有差异(P<0.05),但血清中HE4蛋白的水平无差异(P>0.05)。
结论：
1. WFDC2基因参与促进SKOV3细胞增殖、迁移、侵袭等行为。ERK-JNK-Jun-Fos信号通路参与了该过程；不能确定WFDC2基因与凋亡的关系。WFDC2基因可能在人卵巢浆液性上皮癌进展中起重要作用,
2. WFDC2基因沉默显著抑制人卵巢癌SKOV3细胞株在裸鼠体内的生长。
3.HE4蛋白可以和MMP-9、MMP-2结合,对激活的的MMP-9的酶活性存在明显的抑制作用,该作用和HE4的浓度呈正相关；
4.HE4在鉴别低、高级别浆液性癌有潜在意义,但不能肯定血清HE4对高级别浆液性癌的早期诊断的价值,需要慎重对待目前文献过于肯定HE4早期诊断价值的结论。
OBJECTIVES:
1. To investigate effect and mechanisms of silencing human WFDC2(HE4) gene on biological behavior changes such as cell proliferation,apoptosis, migration and invasion of human serous ovarian cancer cell line SKOV3.
2. To establish subcutaneous transplanted model of human ovarian carcinoma in nude mice with cells stablely transfected with lentiviral WFDC2gene sequence of small interfering shRNA,and to explore its influence on their biological properties.
3. To illustrate the mechanisms of significantly inhibited growth of tumor cells by silencing WFDC2gene from different perspectives of signaling as ERGF pathways.
4. To explore possible mechanisms of decreased invasiveness by silencing WFDC2gene from the perspective of cobinding and interaction between HE4protein and matrix metalloproteinases basing on similarities of molecular structure of WAP family and their protease inhibitory function.
5. To investigate the difference of HE4protein in paraffin section of patients with low-grade serous (LGSC) and high-grade serous carcinoma (HGSC) serous ovarian cancer by two-tier grading system basing on the heterogeneity of OEC.
MATERIALS AND METHODS:
1. Lentiviral WFDC2gene sequence of small interfering siRNA was stablely transfected into SKOV3identified by RT-PCR and western-blot. Proliferation, apoptosis, migration and invasion assays of ovarian cancer cell line were measured by MTT, Annexin/PI doubdle staining by flow cytometry and transwell assay respectively. Protein alteration as EGFR signaling pathway were measured by western-blot analysis.
2. An subcutaneous xenotransplanted tumor model of human ovarian carcinoma in nude mouse was establised and tumor volume were detected by continuous measurement to draw the tumor growth curve.Tumor weight were measured at the end point of the in vivo experiment.It aimed to observe the effect of silencing WFDC2on the tumor progression characteristics as growth(tumor growth curve,tumor weight), metastasis and matrix metalloproteinases (MMP)-9expression. HE4protein,proliferating cell nuclear antigen(PCNA), lymphatic microvessel density,(LMVD) were detected by immunohistochemistry (IHC) and in situ apoptosis of tumor cells were measured by TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) analysis.
3. Signaling pathway associated protein expression such as PI-3K-Akt-STAT3and ERK-JNK-Jun-Fos in OEC cell lines was analyzed by means of Western-blot analysis.
4. Co-Immunoprecipitation(Co-IP) was performed to prove whether there was protein cobinding between HE4and matrix metalloproteinases (MMP-9, MMP-2). MMP-9and MMP-2were activated by APMA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid),a MMPs activator and their activity was assessed by SDS-PAGE enzymograph and enzyme substrates pyrolysis cultured with HE4.
5. Low-grade serous (LGSC) and high-grade serous carcinoma (HGSC) serous ovarian cancer were defined by the two-tier grading system, serum levels of HE4and carbohydrate antigen125(CA125) were measured by ELISA and radioisotope method respectively in60serous ovarian cancer patients to compare the difference of HE4protein in paraffin section of patients with LGSC and HGSC.
RESULTS:
1. Lentiviral WFDC2gene sequence of small interfering siRNA was stablely transfected into SKOV3and identified by RT-PCR and Western-blot. Gene sequencing showed that the oligonucleotides were successfully inserted into the expected site. Proliferation of ovarian cell line was significantly inhibited(P <0.05),G0/G1phase was arrested by the cell cycle(P<0.01) and capacity of the migration and invasion decreased significantly (P<0.01). Proteins and phosphorylated protein involed in ERK-JNK-Jun-Fos protein pathway such as p-ERK,c-Jun,c-Fos decreased and protease protein involved in tissue remoding as matrix metalloproteinases MMP-9,MMP-2decreased after WFDC2silencing.
2. Human ovarian carcinomas transplanted subcutaneously in nude mice were established successfully. It was earlier to see the visible tumors in control group than WFDC silencing group (P<0.05).Tumor weight was significantly smaller in WFDC2silencing group than that of control groups,which coincide with results in vitro. Although no evidence of metastasis was found, Lymphatic microvessel density (LMVD) was significantly smaller in WFDC2silencing group than that of control groups It has been confirmed that WFDC2gene silencing significantly inhibit the growth of ovarian cancer growth and play a potential role in lymph node metastasis.
3. There was no significant change on Akt,p-Akt, STAT3or p-STAT3, proteins expression after WFDC2silencing.It suggested that inhibiting tumor cell growth was not by the classical PI-3K-Akt-STAT3signaling pathway. Decreased phosphorylated ERK, JNK2,c-Jun and c-Fos were detected,which implied the ERK-JNK-Jun-Fos signaling pathway would work.
4. Binding between HE4and matrix metalloproteinases (MMP-9, MMP-2) were confirmed by co-immunoprecipitation analysis. Culturing with HE4protein,the enzyme activity of MMP-2and MMP-9were inhibited and active matrix metalloproteinases were significantly suppressed. Effect of matrix metalloproteinases inhibition is associated with concentration of HE4protein.
5.There was significant difference in HE4protein between LGSC and HGSC, and there was not significant difference in FIGO (Ⅰ+Ⅱ) stage by serum analysis It suggested that HE4could probably be a biomarker for the discrimination between LGSC and HGSC. But its role in early diagnosis remained to be reevaluated.
CONCLUSIONS:
1. WFDC2gene plays an important role in ovarian cancer development involved in regulating the proliferation which were significantly suppressed after WFDC2silencing, but the exact effect on apoptosis can not be determined yet.The ERK-JNK Jun-Fos pathways are involved in the inhibition of proliferation and invasion of ovarian tumor cells.
2. WFDC2gene silencing plays a potential role in regulating capability of invasion and motility of ovarian cancer cells,which is essential to lymph node metastasis.
3. There is evidence of protein binding between HE4and matrix metalloproteinases MMP-9, MMP-2, which can inhibit protease hydrolysis function of MMP-9, MMP-2.Effects of inhibition are correlated with concentration of HE4. It remains to be clarified whether HE4-MMPs interaction plays a protrusive or protective role in ovarian cancer development.
4. Although there is significant difference in serum HE4levels between LGSC and HGSC, the result is similar within FIGO (Ⅰ+Ⅱ) stage, suggesting HE4has potential for the discrimination between LGSC and HGSC, but its role in early diagnosis of OEC remains to be critically evaluated.
引文
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