Expression And Role Of IRSp53 In The Malignant Behavior Of Epithelial Ovarian Tumor

Ovarian cancer is a common malignant tumor of female reproductive system,although its incidence is lower than cervical cancer and endometrial cancer,the mortality is the highest among the three tumors.Ovarian cancer mortality is so high mainly due to: 1)although cancer antigen 125(CA125)and human epididymis protein 4(HE4)detection have a great clinical value for diagnosis of ovarian cancer,the ovaries located in the pelvic cavity,a deep anatomical position,when the lesions occur,patients are often not self-conscious symptoms,more than 70% patients are diagnosed to advaced ovarian cancer when they visit the doctor;2)ovarian cancer is wide spread intra-abdominal and distant metastases.In recent years,the patients received comprehensive treatment of combined radiation and chemotherapy,targeted therapy,but the recurrence rate of ovarian cancer remains highiy;3)caused drug resistance.Studies have found that ovarian cancer of platinum,taxol and other chemotherapy drug resistance;4)ovarian cancer pathogenesis is not clear,targeted therapy research lag behind.The invasion and metastasis of ovarian cancer is a huge challenge to clinical doctors and the scientific researchers.Ovarian tissue composition is complicated,ovarian tumor pathological type many,Epithelial ovarian tumor accounted for about 60% of primary ovarian tumors,and Epithelial ovarian cancer(EOC)account for more than 85% of ovarian malignant tumors.Therefore,this paper discuss mechanisms of invasion and metastasis of EOC.The occurrence of malignant tumor development is a complex process of multi-factor regulation,including cell migration ability,tumor angiogenesis,tumor microenvironment,cell proliferation and apoptosis regulation and so on.In recent years,the correlation between the cells' ability to migrate and tumor invasion or metastasis has become a hotspot.Research has shown that tumor invasion and metastasis is positively related to the cells' ability to migrate.Cell migration depends on the formation of the pseudopodia,filopodia and lamellipodia are actin dynamics structure foundations,participating in the cytoskeleton rearrangement.The study found that insulin receptor tyrosine kinase substrate p53 protein(IRSp53)is a key protein to control cell migration ability and mediate pseudopodia formation.IRSp53 contains I-BAR(Inverse-Bin-Amphiphysin-Rvs),CRIB(Cdc42 / Rac-interactive binding motif),SH3(Src homology 3)and WH2(WASP-homology 2)domains.The I-BAR domain of IRSp53 induces membrane tabulation of vesicles and dynamic membrane protrusions.The IRSp53 SH3 domain interacts with proteins(e.g.Mena,N-WASP,m Dia1 and Eps8)that regulate actin filament formation.And,CRIB domain interact with small GTPase Rac or Cdc42.It suggest that IRSp53 plays an important role in regulating actin dynamics and cell membrane morphology.Early study found that there may exist Cdc42 / Rac1-IRSp53-WAVE signal pathway in ovarian cancer.This study explores IRSp53 expression in epithelial ovarian cancer,the role of IRSp53 on proliferation,invasion and metastasis in ovarian cancer,to detect the possibility that IRSp53 is a target for the targeted therapy of ovarian cancer.PART ONE THE EXPRESSION AND CLINICAL SIGNIFICATION OF IRSP53 IN EPITHELIAL OVARIAN TUMORObjectives:To investigate the expression of IRSp53 and the possible correlation between IRSp53 expression and clinicopathologic features.Methods:(1)Immunohistochemical staining was conducted in 22 normal ovaries,16 benign ovarian tumors,15 borderline ovarian tumors and 78 epithelial ovarian cancer paraffin specimens.What is more,the association of IRSp53 expression with clinicopathologic features was analysed in 78 epithelial ovarian cancer cases.(2)Western blot analysis was performed in 8 normal ovaries,11 benign ovarian tumors,11 borderline ovarian tumors and 23 epithelial ovarian cancer fresh tissues.(3)Survival analysis was conducted by Kaplan-Meier to study the correlation between IRSp53 expression and survival.Results:(1)According to the immunohistochemical staining,IRSp53 was high expression in EOC paraffin specimens,but in normal ovaries,benign ovarian tumor and borderline ovarian tumor,mainly for low or no expression,compared to EOC,the difference was statistically significant(p < 0.05).In EOC,the expression of IRSp53 relatated to advanced FIGO stage,grade,postoperative residual tumor size and serum CA125 level.IRSp53 expression in FIGO ?/? was significantly higher than FIGO ?/?(p<0.05),IRSp53 expression in low grade of EOC was higher than the high grade(p<0.05),IRSp53 expression in satisfied postoperative residual tumor diameter(< 0.5 cm)of EOC was higher than that dissatisfied(> 0.5 cm)(p<0.05),IRSp53 expression in high serum CA125 levels(> 35 u/ml)of EOC patients was higher than that serum CA125 level low(< 35 u/ml)(p<0.05).But the expression of IRSp53 was not relate to patients' age,ascites,tumor size and pathological type(p>0.05).(2)According to the Western blotting,IRSp53 expressed in normal ovaries,benign ovarian tumor,borderline ovarian tumor and EOC respectively 0.240±0.050,0.308±0.354,0.467±0.135 and 1.203±0.080,the difference was statistically significant(p<0.05).(3)According to the Kaplan-Meier analysis,patients with IRSp53 high expression had a lower overall survival compared with those with IRSp53 low or no expression(p<0.05).Conclusion:IRSp53 is high expression in EOC.IRSp53 high expression correlates with advanced tumor FIGO stage,low tumor grade,dissatisfied postoperative residual tumor size and high serum CA125 level.And,IRSp53 high expression is related to an unfavorable OS.IRSp53 may play an important role in EOC malignant behavior.PART TWO CONSTRUCTION OF SHRNA LENTIVIRAL VECTOR SILENCING IRSP53 AND STABLE TRANSFECTED SKOV3 CELL LINE SCREENINObjectives:To construct sh RNA lentiviral vector for silencing IRSp53 and screen a stable transfected SKOV3 cell line for further studies.Methods:(1)design and synthesis of 3 specific sh RNA sequences(IRSp53 sh RNA-1,2,3)targeted IRSp53 gene and a negative control sequence and DNA sequencing was used to identify recombinant plasmids.(2)the recombinant plasmid and PV112 were transfected into 293 T cells.(3)Real-time PCR was performed to test IRSp53 expression of stable transfection SKOV3 cells.Results:(1)DNA sequencing showed that the lentiviral vectors plasmids for silencing IRSp53 was built successfully.(2)stable transfeced SKOV3 cells was constructed and screened.(3)the Real-time PCR showed that IRSp53 m RNA expression significantly lower in transfeced SKOV3 cells,the difference was statistically significant(p < 0.05).Conclusions:Specific sh RNA sepuence for silencing IRSp53 has successfully designed and synthesized and slected the most effective sequence.Further,stable transfected SKOV3 cell line has generated and screened successfully.PART THREE RNA INTERFERENCE SILENCING IRSP53 GENE IMPACT ON THE MALIGNANT BEHAVIOR OF SKOV3Objectives:To study the impact of IRSp53 gene silencing with RNAi on the cell proliferation,invasion and migration of SKOV3.Methods:(1)MTT assay was performed to test the cell proliferation ability.(2)Transwell assay was used to detect the cell vertical migration ability.(3)scratch healing assay(wound healing assay)was used to detect the cell ateral migration ability.(4)Soft agar clone formation assay was performed to examine the cell anchorage-independent growth ability.Results:(1)MTT assay showed that,compared with SKOV-CON and SKOV-NC,cell proliferation of SKOV3-KD was restrained(p<0.05),RNAi silencing IRSp53 gene restrain cell proliferation ability.(2)Transwell assay showed,RNAi silencing IRSp53 gene suppress cell vertical migration ability,SKOV-CON or SKOV-NC vs SKOV3-KD(p<0.05).(3)Scratch healing assay showed,RNAi silencing IRSp53 gene suppress cell ateral migration ability,SKOV-CON or SKOV-NC vs SKOV3-KD(p<0.05).(4)Soft agar clone formation assay showed,the clone number of SKOV3-KD after 21 d was less(p<0.05),RNAi silencing IRSp53 gene reduce cell anchorage-independent growth ability.Conclusions:Gene silencing IRSp53 with RNA interference reduces the cell proliferation ability,the cell vertical migration ability,the cell ateral migration ability and the cell anchorage-independent growth ability of SKOV3.IRSp53 may regulate the malignant behaviors of EOC.