MicroRNA-142-3p Regulating TET2 And Its Effect On Proliferation Of Human Ovarian Cancer Cell Line SKOV-3

Objective: To construct the lentiviral vectors,which can efficiently expresses miR-142-3p,to study its role on ten-eleven translocation2(TET2)gene;and to investigate its effect about the proliferation of ovarian cancer cell line SKOV-3,describes the molecular mechanism of miR-142-3p in the pathogenesis of ovarian cancer,and provide a theoretical basis and new ideas for the research on clinical treatment of ovarian cancer.Methods:1 According to the sequence of miR-142-3p from human,at the same time with a stem loop sequence with small forward reverse RNA sequence base vector synthesis double strand,inserted into the virus packaging required on the application of double enzyme digestion and nucleotide sequence of sequence two experimental method to verify the construction results,lentivirus using liposome transfection packaging,constructed by the second generation lentivirus three plasmid vector transfection,and co transfected into HEK-293 cell lines,the virus titer was determined after the success of the packaging,we synthesized miR-142-3p inhibitor reagent.2 Using micro RNA.miRanda and Target Scan target prediction database software to protential target genes of miR-142-3p,and there may be targeting the relationship between TET2 as a target gene,according to the 3 'untranslated region(3'UTR)of TET2 by the specific primers and amplified the sequence,then construct into the p MIR-Report expression vector and by enzyme digestion and sequencing method after successful verification,using Lipofectamine.p Sico R-miR-142-3p and p MIR-Report-TET2 were transfected into 293 cells,while the design of the blank control group and inhibition group,using dual luciferase reporter assay system to verify the miR-142-3p targeting TET2 relationship.3 The LV-miR-142-3p lentivirus and miR-142-3p inhibitor were successfully infected with SKOV-3 cell line respectively.The infection results were observed after 24 h,and the cells were collected.The cells were m RNA and totalprotein expression level and the expression of TET2 were assayed by real-time PCR application miR-142-3p m RNA,detection of the expression levels of TET2 protein using Western blot,then detection of gray value,The proliferation effects of miR-142-3p in SKOV-3 cell lines were analyzed by MTT assay.Using the statistical analysis SPSS20.0 software.Result:1 The recombinant plasmids of p Sico R-miR-142-3p were constructed successfully,and Lentivirus particles with over expressed miR-142-3p were successfully packaged and the virus titer was 2.66×105 pfu/m L.2 The network database showed the presence of miR-142-3p and TET2 binding sites,and the dual luciferase reporter system officially miR-142-3p targeting TET2 3'UTR,and inhibit the expression of the gene,p Sico R-miR-142-3p over expression vector group,TET2 relative luciferase activity was about 4.09 times lower than the wild type mutant(P<0.05);miR-142-3p inhibitor group,TET2 luciferase activity increased compared to wild type and mutant.Compared with the wild type alone,the p Sico R-miR-142-3p overexpression group decreased about 4.52 times(P<0.05)compared with the p Sico R/NC group,and increased about times(P<0.05)compared with the over expression group(p Sico R-miR-142-3p).3 The Lentivirus Expression group of miR-142-3p expression level compared with normal control group increased by about 17.5 times(P<0.05),and TET2 m RNA expression levels compared with normal control group by7.8 times(P<0.05);miR-142-3p transfected with the miR-142-3p expression level of inhibitor group and normal control group compared by about 18 times(P<0.05),and TET2 the expression level of m RNA was increased by about3.9 times(P<0.05).4 Western blot showed that TET2 protein expression was significantly decreased compared with normal group,transfection of miR-142-3p inhibitorgroup the expression of TET2 protein increased,the gray value of detection is more intuitive to show the expression level of miR-142-3p virus TET2 protein after stimulation.5 MTT test confirmed that miR-142-3p could significantly inhibit the proliferation of SKOV-3.Conclusions: We successfully constructed lentiviral vector expressing miR-142-3p,and further confirmed that over expression of miR-142-3p can effectively inhibit the expression of TET2 gene and protein in SKOV-3 cells and inhibit the proliferation of SKOV-3 cells in m RNA cells.To provide new ideas and research targets for gene therapy of ovarian cancer in the future.