| Part Ⅰ:Semaphorin4D expression in ovarian cancer (tissues and SKOV-3) and its relationship with clinicopathologicalObjectiveTo detect Sema 4D expression in ovarian cancer tissues andcell lines SKOV-3, and exploration relationshipwith clinicopathological features and function in ovarian cancer progression effect.MethodsTo detect Sema 4D of tissue of normal ovarian, benignovarian tissue and ovarian malignancies by immunohistochemistry. By RT-PCR and Western blot detection Sema 4D of SKOV-3 and IOSE80 differencesexpression levels. SPSS 19.0 statistical softwarepackage for data processing and analysis, measurement datausing mean ± standard deviation, immunohistochemistryusing a chi-square test (Chi-square Test), t test was used tocompare to two groups, P<0.05 was considered statistically significant analysis.Results1.Sema4D protein in normal ovarian tissue, benign ovarian tissueand ovariancancer tissue expression rates were40%,53.3% and 90%, the difference between groups was significant (x2=32.845, P=0.000, P<0.05).2. Sema4D proteins in the early (Ⅰ-Ⅱ) and late(Ⅲ-Ⅳ) ovarian carcinoma positive rate was 67% and 100%respectively, the difference was significant(X2=7.938,P=0.047 P<0.05). 3.SKOV-3 and IOSE80 2-△△Ct value ofSema4D mRNA were:1.828±0.260 and0.00317±0.00124, there was a significantdifference (P=0.0022, P<0.05). Western bolt results show Sema4Dprotein/β-actin:1.20±0.09and0.0038±0.0009,there was a significant difference(P=0.0003, P<0.05).ConclusionSema4D highly expressed in ovarian cancer tissues andcells. And positively correlated with the degree of malignancy.Part Ⅱ:miR-214 expression and function in SKOV-3ObjectiveTo detect the expression levels of miR-214 in SKOV-3, and toinvestigate the effect on function (proliferation, apoptosis andinvasion) of SKOV-3.MethodsTo detect and compare miR-214of two cell lines by RT-PCR. Bylentiviral infection method, get continued overexpression of miR-214 in SKOV-3, using CCK-8 was observed changes inovarian cancer cell proliferation, changes in flow cytometryapoptosis in ovarian cancer cell lines, Transwell chamberexperiments to observe fine changes in cell invasion ability.Results1.Between SKOV-3 and IOSE80 of miR-214 expression levelswere: 0.172±0.023 and 0.569±0.041, the difference was significant(P=0.0011, P<0.05).2. The relative expression oflentiviral infection group was higher than control group,there was significantdifference (P=0.0029, P<0.05).3. Therelative expression of miR-214 infected group was higher thanthe control group, there was a significant difference (P=0.0016, P<0.05). Blank control comparison with body controlgroup, the difference was not significant (P=0.1133, P>0.05).4. Among the three groups of AI were:the experimentalgroup (12.802±0.284) higher than the control group(7.638±0.338)%, the difference was significant (P<0.01). Theexperimental group than body control group (7.866±0.177)%, the difference was significant (P<0.001). Two control groups were nosignificant (P>0.05).5. Invasion assay resultsshowed the experimental group penetrate Matrigel Gray was 21.253 ± 5.023 lower than the blank control group59.309 ±5.132, the difference was significant (P<0.01). Theexperimental group than body control group 55.031 ±5.238, thedifference was significant (P<0.01).Blank control group andbody control group, the difference was not significant (P>0.05).ConclusionThe expression of miR-214 in SKOV-3 wasdecreased levels. The overexpression level of miR-214 inhibit the proliferation and invasion of cells and promote cellapoptosis.Part III:miR-214 negative regulation the expression of Sema4D in SKOV-3ObjectiveTo predict and verify Sema4D is target genes of miR-214, miR-214 through targeted Sema4D to regulate function of SKOV-3.MethodsAccording to the miRNA target gene database to predicte and screen of miR-214 target genes is Sema4D,and to detecte up-regulated and down-regulated of miR-214 of SKOV-3 of Sema4D mRNA and protein expression changes by RT-PCR and Western blot, and to verify by the dual luciferase reporter gene assay.Results1. To predicte Sema 4D is a potentialtarget genes of miR-214by bioinformatic databases. Establish high expression of miR-214SKOV-3 mimics and low expression of miR-214 SKOV-3inhibitor.2. SKOV-3 mimics and SKOV-3mimics control group Sema4DmRNA were: 0.067± 0.011 and0.390±0.095, mimics group than controlgroup significantly decreased, the difference was significant (P= 0.028, P<0.05).3. SKOV-3 inhibitor and SKOV-3 inhibitor control groupSema4DmRNA were: 0.955 ± 0.039 and 0.532 ±0.131, inhibitor group than controlgroup significantly higher, the difference wassignificant(P= 0.037, P<0.05).4. Sema 4D of SKOV-3 mimics groupthan SKOV-3 mimics control group lower, the difference was significant (P<0.01).5. Sema 4D of SKOV-3 inhibitor groupthan SKOV-3 inhibitor control group higher, the difference was significant (P<0.01).6. Dualluciferase reporter gene results display:in SKOV-3 CPC miR-214mimics transfected with wild-type 3’-UTR time zone recombinant vector, luciferase activity of cellstransfected with wild-type than the separate group wassignificantly inhibited,Gluc/seAP ratios were 0.411 ±0.013,3.408 ± 0.211, the difference was significant (P= 0.0001, P<0.05).7. When co-transfectedwith miR-214 inhibitor group and 3’-UTR region of wild-typerecombinant vector, cell fluorescence Light luciferase activitycompared with the wild-type transfected alone significantly higher, Gluc/seAP ratios were 8.777± 0.421,3.408 ± 0.211, the difference was significant(P= 0.0003, P<0.05).8. The co-transfected with miR-214 mimicsand mutant recombinant vector 3’-UTR region of the cell, theluciferase activity of cells transfected with the morealone. Therewere no significant changes in the mutantGluc/seAP ratios were7.107 ± 0.317,7.260 ± 0.086, the difference was no significant (P= 0.667, P>0.05).9. Co-transfectedwith miR-214 inhibitor and mutant 3’-UTR region cells, theluciferase activity compared with the recombinant vector alonetransfected mutant showed no significant change, Gluc/seAPratios were 7.654 ± 0.196,7.260 ± 0.086, the difference was no significant(P= 0.139, P>0.05).ConclusionSema4D is the target gene of miR-214, miR-214 can down-regulatetheexpression levels of Sema4D mRNA and protein, which play arole in the negative regulation of the target gene transcription andtranslation levels. |
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