The Effect And Mechanism Of Knockdown Osteosclerotin On The Proliferation And Migration Of Choroidal Melanoma Cells

【Objective】Uveal melanoma is one of the most common primary intraocular malignant tumors.Choroid is the most common site,which is called choroid melanoma(CM).At present,the most commonly used method for the treatment of this disease is still enucleation,but whether it can effectively improve the relative survival rate of patients after surgery is still controversial.Therefore,the tumor is a serious disease affecting human health.As the pathogenesis of choroidal melanoma has not been fully elucidated,the clinical effect is not good.MicroRNAs(miRNAs) are small single stranded noncoding RNAs that exist in biology.Their main function is to regulate the expression of genes after transcription.Mature miRNA can trigger the degradation of mRNA or inhibit the translation of protein by binding to specific target mRNA.Studies have shown that most miRNAs are closely related to cell metabolism and participate in the process of human tumor development,while abnormal miRNAs usually appear as carcinogens or tumor suppressors,regulating the occurrence of various human tumors.Osteosclerotin is a negative regulatory factor in the process of bone formation.It is encoded under the guidance of the SOST gene,and it is an inhibitor of Wnt signaling pathway.It has been found that overexpression of SOST can promote Wnt signaling to drive bone metastasis and invasion of prostate cancer.Based on the above point of view,the purpose of this study is to explore the pathogenesis of choroidal melanoma,understand its potential signal transduction pathway,further explore the changes of proliferation and migration function of choroidal melanoma cells after sot gene knockdown,and its potential molecular regulatory mechanism.【Methods】Human choroidal melanoma cells(MUM-2C) were cultured by cell culture in vitro.The cells in logarithmic growth stage were randomly divided into two groups.One group was transfected with human SOST interference lentivirus to construct the SOST-knockdown stable cell line(SOST-knockdown group),the other group was not treated with SOST-knockdown as a negative control(SOSTNC group).CCK-8 kit was used to detect the proliferation activity of the two groups of cells;cell scratch technology was used to detect the migration movement of the two groups of cells;miRNAs were extracted from the two groups of cells of SOST-kockdown and SOSTNC by miRNAs extraction kit,gene sequencing was carried out,and the specific miRNAs were found according to the sequencing results,and then Wnt/β-catenin regulated by SOST gene was screened according to the enrichment analysis of KEGG The target miRNAs in these two groups of cells were detected by real-time fluorescence quantitative PCR.RT-qPCR and Western Blot were used to detect the content and expression of β-catenin in the two groups of cells.MUM-2C cells were randomly divided into two groups,which were transfected with micro RNA-744-5p-mimics and its negative control(mimics-NC).RT-qPCR and Western Blot were used to detect the content and expression of β-catenin.【Results】The results of CCK-8 method showed that the proliferation activities of the two groups increased with the increase of time.At the same time point,the proliferation activities of the cells in the group were significantly lower than those in the group of SOSTNC(P<0.05);the results of scratch test showed that compared with0 h,the migration amount of the cells in the group of SOST-knockdown at 24 h was significantly lower than that in the group of SOSTNC.According to the miRNAs gene sequencing results of the two groups of SOST-knockdown and SOSTNC cells,on the Wnt/β-catenin pathway regulated by SOST gene,micro RNA-744-5p with high mobility was selected as the target miRNA;RT-qPCR verification results showed that the micro RNA-744-5p content in the cells of the SOST-knockdown group was significantly lower than that of the SOSTNC group(P<0.05);The results of RT-qPCR and Western Blot showed that the content of β-catenin protein in SOST-knockdown group was significantly lower than that in SOSTNC group(P<0.05),and the expression level was also significantly lower than that in SOSTNC group;the results of RT-qPCR and Western Blot after micro RNAs transfection showed that the content of β-catenin protein mRNA in MUM-mimics group was significantly higher than that in MUM-NC group(P<0.05),and the expression level was also significantly higher than that in MUM-NC group.【Conclusion】When the SOST gene was knocked down by human SOST lentivirus transfection,the proliferation activity and migration ability of choroidal melanoma cells were significantly reduced;the molecular weight of micro RNA-744-5p was significantly reduced;the content and expression of β-catenin protein in Wnt/β-catenin signaling pathway regulated by Sost gene were significantly reduced.When micro RNA-744-5p was overexpressed,the content and expression of β-catenin protein in Wnt/β-catenin signaling pathway regulated by SOST gene in choroidal melanoma cells increased significantly.