| BackgroundOsteosarcoma is the most common primary malignant bone tumor,which occurs mostly in adolescents.It has high inclination of invasion and metastasis,leading to a poor prognosis.Since 1970s,the five year survival rate of osteosarcoma has gradually increased to more than 65%due to the application of neoadjuvant chemotherapy.However,since 90s,the therapeutic outcome of osteosarcoma has entered so-called"plateau",and the survival rate has not been significantly improved.Metastasis of osteosarcoma is the main cause of final death.It is important to understand the mechanism of metastasis to improve the therapeutic outcome.When cells are detached from extracellular matrix,a special form of programmed cell death,called anoikis,can be induced.Under physiological conditions,anoikis can prevent the detached cells from adhering to the incorrect location and play an important defensive role.The tumor cells come to existance in the form of primary foci.After they are detached from the extracellular matrix,a few of them have the ability to resist anoikis,and can survive in the condition of suspension and then transfer to other organs through blood and lymph.It is widely accepted that anoikis is an important basis for metastasis and invasion in the process of tumor development.Tumor cells have the ability to resist anoikis through epithelial mesenchymal transition and activation of anti-apoptotic signaling pathways,and microRNAs are greatly involved in these processes.MicroRNA is a class of non-coding small RNA molecules.MiRNA gene is transcribed by RNA polymerase ?,DROSHA,DICER and other enzymes,and eventually transformed into mature miRNA.Mature miRNA usually binds to the 3'untranslated region of target mRNA,and regulates gene expression post-transcriptionally.A large number of studies have shown that miRNA not only participates in the physiological processes,for instance,embryonic development,cell differentiation,proliferation and apoptosis,but also can act as oncogene or anti-oncogene,and plays a key role in the process of tumor development.The miR-451 gene is located on chromosome seventeenth,which is dysregulated in many malignancies and affects tumor progression through different mechanisms.However,the effect and possible mechanism of miR-451 in the anoikis resistance of osteosarcoma cells has not been elucidated.Chapter 1miR-451 mediates anoikis resistance in osteosarcoma cellsObjectives:To explore the effect of miR-451 in the anoikis resistance of osteosarcoma cells,so as to find new targets for diagnosis and treatment.Methods:Osteosarcoma U20S and Saos-2 cell lines were used.Adherent culture was used to simulate the growth state of primary tumor cells of osteosarcoma,which was called adherent group.The anoikis model was established to simulate the growth state of metastatic tumor cells of osteosarcoma,called anoikis model group.The expression levels of miR-451 in both groups were detected by real-time PCR.Pre-miR-451 plasmids were constructed and transfected into U20S cells.U20S cells were divided into blank control group,reagent transfected group,miR-451 NC group and miR-451 plasmid transfected group.The expression level of miR-451 in each group was detected by fluorescence quantitative PCR,the rate of cell apoptosis was detected by flow cytometry,the invasion ability was tested by transwell assay,and the migration ability was tested by wound healing assay.Results:1.The cells in adherent group were adherent growth in single layer,and pseudopodia were observed.The cells in the anoikis model group grew in suspension,tightly clustered,and no cell adherence was observed.The model was built successfully.2.The expression level of miR-451 in anoikis model group was lower than that in adherent group,and the difference was statistically significant(p<0.05).3.The plasmids were successfully constructed.The results of fluorescence quantitative PCR showed that the expression level of miR-451 in the miR-451 plasmid transfected group was higher than that in the blank control group,the reagent transfected group and the miR-451 NC group significantly(p<0.05),and there was no significant statistical difference between the blank control group,the reagent transfected group and the miR-451 NC group in the miR-451 expression level(p>0.05).4.The anoikis model was established in U20S cells of the blank control group,reagent transfected group,miR-451 NC group and miR-451 plasmid transfected group.Flow cytometry showed that the apoptosis rate of the miR-451 plasmid transfected group was higher than that in the blank control group,the reagent transfected group and the miR-451 NC group significantly(p<0.05).Transwell assay showed that the number of cells passing through membrane in miR-451 plasmid transfected group was lower than that in the blank control group,the reagent transfected group and the miR-451 NC group significantly(p<0.05).Wound healing assay showed that the migration distance of U20S cells in the miR-451 plasmid transfected group decreased significantly at 24 hours and 48 hours,compared with the blank control group,the reagent transfected group and the miR-451 NC group(p<0.05).Conclusions:1.In osteosarcoma U20S and Saos-2 cell lines,the expression level of miR-451 decreased in anoikis model group.2.Upregulating the expression level of miR-451 in U20S cells,the apoptosis rate increased,the invasion ability decreased,and the migration ability decreased.Chapter 2miR-451 regulates Rab14 and PI3K/Akt in osteosarcoma cellsObjectives:To explore the mechanism of miR-451 mediated anoikis resistance in osteosarcoma cells,so as to identify potential tumor biomarkers for early diagnosis,and to provide new targets for treatment.Methods:Bioinformatics were used to predict the possible target genes of miR-451;dual luciferase reporter assay was used to verify the interaction between miR-451 and the target gene;real time quantitative PCR and Western blot were used to detect the expression of mRNA and protein of target gene;the expression level of PI3K/Akt signaling pathway was detected by Western blot.Results:1.After retrieving the databases,we predicted that Rabl4 may be one of the target genes of miR-451.The results of dual luciferase reporter assay in U20S and Saos-2 cells showed that miR-451 mimics could significantly reduce the luciferase activity of wild type Rab14 3'UTR,while the mutant did not decrease,indicating that miR-451 could bind to 3'UTR of Rab14.2.Real-time PCR detected the change of Rab14 mRNA level after transfection of miR-451 mimics,and the results showed that the expression level of Rab14 mRNA decreased significantly in U20S and Saos-2 cell lines(P<0.01).The effect of upregulation of miR-451 expression on target gene protein was verified by Western blot,and the results showed that miR-451 could significantly reduce the protein expression level of Rab14 in U20S and Saos-2 cell lines.3.The expression levels of p-PI3K,p-ERK and p-AKT were detected by Western Blot.The results showed that the expression levels of p-PI3K,p-ERK and p-AKT in miR-451 mimics group were significantly decreased in U20S and Saos-2 cell lines,and the difference was statistically significant(P<0.05).Conclusions:1.Rab14 is one of the target genes of miR-451.2.miR-451 can inhibit the expression of Rab14 by binding 3'UTR and inhibit the anoikis resistance of osteosarcoma cells by regulating PI3K/Akt pathway. |
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