Studies On Expression And Function Of MICAL-L2and ACTN4in Epithelial Ovarian Cancer

Objective: To explore the functions and mechanisms of MICAL-L2and its bindingprotein α-Actinin4coded by ACTN4gene in the development and metastasis of ovariancancer by epithelial ovarian cancer tissues, in vitro and in vivo experiments.Methods:(1) By detecting the protein expression of MICAL-L2in ovarian cancertissues and normal tissues using immunohistochemical methods in ovarian cancer tissuemicroarray to explore the correlationship between MICAL-L2and ovarian cancer,(2)Stable transfection with specific MICAL-L2shRNA was constructed using lentivirusexpression transfection technology. CCK8assay, wound healing assay, transwell migrationassay and transwell invasion assay were used to detect cell proliferation, migration andinvasion ability in ovarian cancer cells transfected with MICAL-L2shRNA.(3)Immunofluorescence technique was used to investigate the changes of cell morphologyand EMT markers in MICAL-L2knockdown cells.(4) Western blottingting assay was usedto detect the protein expression of EMT related markers and the downstream molecule ofsignal pathway in MICAL-L2knockdown groups and control groups.(5) Real-time PCRwas used to detect the mRNA expression of EMT related transcriptional factors.（6）DualLuciferase Reporter assay was used to detect TOP/FOP activity of Wnt pathway.(7) Thecorrelation of MICAL-L2and ACTN4was detected by RT-PCR and siRNA assays.(8)TCGA data was analyzed to explore the relationship between ACTN4and ovariancancer.(9) MTT and FCM (flow cytometry) assay were performed to detect the relationshipand mechanism of ACTN4on ovarian cancer cells.Results:(1) The results of immunohistochemistry in tissue microarray showed thatthe higher expression rate of MICAL-L2in ovarian cancer tissues（68.60%） wasobviously higher than in normal ovary tissues （16.67%） The difference was statistically significant (P <0.0001).In the tumor tissues, the expression intensity ofMICAL-L2is positively correlated with FIGO stage (P=0.011) and histological grade(P=0.009) more advanced FIGO stage and histological grade of ovarian cancer,higherexpression of MICAL-L2protein.(2) Compared with control groups, the abilities of proliferation, migration andinvasion were significantly reduced in MICAL-L2knockdown cells, the difference wasstatistically significant(P <0.01).（3）The results of immunofluorescence showed that thecells with MICAL-L2-shRNA appear from fibroblast-like mesenchymal morphologicalshape to planar epithelial cell morphological shape, the junctions between cell and cellbecame more closely,along with epithelial marker E-cadherin increased and mesenchymalmarker vimentin decreased.(4) western blottingting results showed that the protein level ofE-cadherin was increased,while vimentin was decreased. The expression of nuclearβ-catenin and Wnt/β-catenin downstream target gene Cyclin-D1were decreased, whilecytoplasm and p-β-catenin have no changes.(5) The mRNA levels of SNAIL, SLUG,TWIST, ZEB1and ZEB2, key transcription factors that promotes EMT,were determinedby RT-PCR. Results showed that TWIST, ZEB1and ZEB2were significantly decreased inMICAL-L2knockdown cells, while SNAIL and SLUG remained unchanged. thedifference was statistically significant(P <0.05)（6）TOP/FOP flash reporter plasmids wereused to measure Wnt/β-catenin transcriptional activity, results showed that knocking downMICAL-L2decreased TOPflash activity, but not FOPflash activity.(7) RT-PCR andsiRNA assay were used to explore the correlation of MICAL-L2and ACTN4,resultsshowed that ovarian cancer cell with higher expression of MICAL-L2also has higherexpression of ACTN4, after knock down of MICAL-L2using siRNA, the expression levelof ACTN4was also decreased.(8) The analysis of TCGA data showed that ACTN4wasassociated with the prognosis of ovarian cancer patients.The patients with altered ACTN4expression have poor prognosis. the difference was statistically significant（P<0.01）.(9)the sensitivity of cells transfected with ACTN4-siRNA to chemotherapeutic agents paclitaxel was significantly increased compared with the control groups (P <0.05) theapoptotic rates were also higher than that of the control groups.Conclusion:(1) MICAL-L2expression was increased in ovarian cancer tissues and correlated withFIGO stage and histological grade;(2) MICAL-L2promoted ovarian cancer cells proliferation, migration and invasionabilities.(3) MICAL-L2induced ovarian cancer epithelial-mesenchymal transition.(4)MICAL-L2inducing ovarian cancer epithelial-mesenchymal transition mayberegulated by activating signal pathway of Wnt/β-catenin and the activity of Rac1.(5) ACTN4closely related to poor prognosis of ovarian cancer,ACTN4mayparticipate in the chemosensitivity of ovarian cancer by inhibiting apoptosis of ovariancancer cells.(6) Silencing of MICAL-L2and ACTN4may provide new targets forclinical treatment.