The Role And Mechanism Of RNAi-mediated Silencing Of WFDC2in The Progression Of Human Serous Ovarian Epithelial Cancer

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 (â… +â…¡) 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 (â… +â…¡) stage, suggesting HE4has potential for the discrimination between LGSC and HGSC, but its role in early diagnosis of OEC remains to be critically evaluated.