| Purpose:(1)To detect the relative expression of linc-ROR,miR-181a-5p and MEST in high-grade ovarian serous carcinoma,normal ovarian epithelial tissue and normal umbrella end of fallopian tube,and the relationship between them and clinicopathological features.(2)To explore the molecular mechanism of Linc-ROR targeting miR-181a-5p regulating the expression of MEST and promoting the malignant biological behavior of tumor from the perspective of cytology.Method:(1)From June 2019 to June 2021,36 cases of ovarian cancer tissues treated by surgery for high-grade serous ovarian cancer in the Affiliated Hospital of Qingdao University were collected as the research objects;26 cases of normal ovarian epithelial tissue and normal fimbria end of fallopian tube were collected as control.The relative expression levels of linc-ROR,miR-181a-5p and MEST in 36 cases of high-grade ovarian serous carcinoma,26 cases of normal ovarian epithelium and 26 cases of normal fimbria end of fallopian tube were detected by real-time fluorescence quantitative PCR(qRT-PCR),and the relationship between the expression of linc-ROR,miR-181a-5p and MEST and FIGO stage and lymph node metastasis of high-grade serous ovarian carcinoma was analyzed;Pearson correlation analysis was used to analyze the correlation between Linc-ROR and miR-181a-5p,and between miR-181a-5p and MEST.(2)The targeted binding sites between linc-ROR and miR-181a-5p and between miR-181a-5p and MEST were predicted by bioinformatics software;Double luciferase was used to verify the targeted combination of Linc-ROR and miR-181a-5p.(3)Linc-ROR siRNA and miR-181a-5p siRNA were synthesized by small interference RNA siRNA technology.Linc-ROR overexpression plasmids were synthesized and transfected into SKOV3 cells respectively.The experiment was divided into four groups: linc-ROR-p group(overexpression linc-ROR group),linc-ROR-NC group(Linc-ROR negative control group)Linc-ROR-si+miR-181a-5p-si group(knock down Linc-ROR and miR-181a-5p group at the same time),Linc-ROR-si group(knock down Linc-ROR group).The relative expressions of linc-ROR,miR-181a-5p and MEST in SKOV3 cells were detected by qRT-PCR;cell cloning test,cell scratch test and transwell test were used to detect the proliferation,migration and invasion of SKOV3 cells in four groups;Western blot was used to detect protein and Wnt pathway related protein E-cadherin,Vimentin and β-catenin relative expression levels.Result:(1)Compared with normal ovarian epithelial tissues(1.00 ± 0.21,1.03 ± 0.19)and normal fimbria end of fallopian tube(1.03 ± 0.21,1.01 ± 0.23),Linc-ROR and MEST were highly expressed in ovarian cancer tissues(3.07 ± 0.30,2.21 ± 0.41,F= 742.99,157.81,P < 0.01);Compared with normal ovarian epithelial tissue(1.01 ± 0.17)and normal fimbria end of fallopian tube(1.02 ± 0.22),miR-181a-5p was low expressed in ovarian cancer(0.48 ± 0.11,F= 742.99,P < 0.01).Compared with normal ovarian epithelial tissue(1.01±0.17)and normal fimbria end of fallopian tube(1.02±0.22),the expression of miR-181a-5p in ovarian cancer was low(0.48±0.11 F=97.24,P < 0.01).The relative expression levels of Linc-ROR,miR-181a-5p and MEST were related to FIGO stage and lymph node metastasis of ovarian cancer.The later the stage was,the higher the expression levels of Linc-ROR and MEST were,and the lower expression level of miR-181a-5p(t=7.93 ～ 10.08,P< 0.01).Pearson correlation analysis showed that Linc-ROR expression was negatively correlated with miR-181a-5p expression(r =-0.979,P < 0.001),but positively correlated with MEST expression(r = 0.989,P < 0.001).(2)The targeted binding sites between Linc-ROR and miR-181a-5p and between miR-181a-5p and MEST were analyzed by biological information.Double luciferase assay showed that miR-181a-5p inhibited the luciferase activity of Linc-ROR wild type,but had no effect on Linc-ROR mutant(P < 0.01).(3)qRT-PCR was used to detect the expression of miR-181a-5p and MEST in SKOV3 cells of the four groups.The results showed that when linc-ROR overexpression plasmid was successfully transfected into the cells,the expression levels of MEST in linc-ROR-p group increased significantly,which were 2.01±0.08 respectively,while the expression of miR-181a-5p decreased correspondingly,which was 0.23±0.05(t=28.08,30.54 all P < 0.01);When the expression level of Linc-ROR in Linc-ROR-si group was significantly inhibited,the expression levels of MEST decreased significantly,which were0.72±0.03 respectively,and the expression level of miR-181a-5p increased significantly to 1.98±0.07(t=10.58,28.53 all P < 0.01).When the expression of linc-ROR and miR-181a-5p in SKOV3 cells was inhibited at the same time,the expression levels of MEST in SKOV3 cells were not significantly changed compared with linc-ROR-NC group(t=0.19,1.54 P > 0.05).(4)The results of cell cloning experiment showed that the number of cloned cells in linc-ROR-p group was the highest(798.00 ± 12.27),the number of cloned cells in linc-ROR-si group was the lowest(200.80 ± 7.02)(t=41.80,51.35 all P < 0.01),while the number of cloned cells in linc-ROR-si + miR-181a-5p-si group was(497.20 ± 9.75),which had no significant change compared with linc-ROR-NC group(t=0.25 P > 0.05).The results of cell scratch test and transwell test showed that the migration rate and the number of transmembrane cells in linc-ROR-p group were the highest(81.01 ± 2.91)%,(223.40 ± 2.70),while the migration rate and the number of transmembrane cells in linc-ROR-si group were the lowest(31.81 ± 2.86)%,(55.24 ± 3.34),which were statistically significant compared with linc-ROR-NC group(t=16.15,43.54 t=10.79,36.33 all P < 0.01),The migration rate and the number of transmembrane cells in linc-ROR-si + miR-181a-5p-si group were(51.66 ± 3.19)%,(145.08 ± 2.80),respectively.There was no significant change compared with linc-ROR-NC group(t=0.14,0.17 P >0.05).(5)The results of immunoblotting showed that compared with linc-ROR-NC group,The expression of MEST and β-catenin and Vimentin increased by 0.91±0.02,0.72±0.01 and 0.63±0.02 respectively,and the expression of E-cadherin decreased by 0.18±0.02;in Linc-ROR-si group the expression of MEST,β-catenin and Vimentin decreased by0.29±0.01,0.17±0.01 and 0.31±0.02 respectively,and the expression of E-cadherin increased by 0.42±0.01 respectively(t=44.05,57.13,25.34,4.19;t=31.75,18.02,9.32,15.35 P < 0.01);After simultaneously knocking down the expression of Linc-ROR and miR-181a-5p in SKOV3 cells,The expression of MEST β-catenin,Vimentin and E-cadherin protein had no significant change compared with Linc-ROR-NC group(t=2.40,0.01,0.27,0.16 P > 0.05).Conclusion:(1)Linc-ROR is highly expressed in high-grade serous ovarian cancer,which is significantly negatively correlated with the low expression of miR-181a-5p.miR-181a-5p may be an important downstream target for Linc-ROR to regulate the invasion and metastasis of ovarian cancer and play its role in promoting cancer.(2)Linc-ROR may regulate the expression of MEST through competitive adsorption of miR-181a-5p and further activate Wnt/β-catenin pathway promotes the epithelial mesenchymal transformation of ovarian cancer,and then promotes the malignant biological behavior of ovarian cancer cell invasion and metastasis. |
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