The Mechanistic Study Of Long Non-coding RNA CCAT2 And MiR-200b/VEGF In The Regulation Of Osteosarcoma

BackgroundOsteosarcoma(OSA)is a malignant tumor derived from the osteointerstitial cells and usually happened in adolescent.It is characteried by high rate of maligancy,easy to metastasis and poor prognosis.Long non-coding RNAs(lncRNAs)are a class of RNA transcripts with more than 200 nucleotides in length.Due to they have no opening reading frame(OFR),they can not code proteins.Recent years,lots of literatures reported that IncRNAs played oncogenic or tumor suppressive roles in multiple human cancers.Colon cancer-associated transcript 2(CCAT2)is a newly discovered lncRNA that exerts oncogenic function on a number of human cancers.ObjectiveThis study was aimed to investigate whehter CCAT2 participated in the initiation and development of osteosarcoma,as well as the intrinstic molecular mechanism related to miR-200b.Firstly,the expressions of CCAT2 and miR-200b in osteosarcoma tissues and corresponding paracancerous tissues,along with osteosarcoma cells and human normal osteoblasts were measured.Subsequently,the effects of silencing CCAT2 on proliferation,apoptosis,migration and invasion of osteosarcoma cells were analyzed.MethodsFirstly,osteosarcoma tissue samples and corresponding paracancerous tissue samples were collected,and human osteosarcoma MG63,U20S,US732,Saos-2 cells and human normal osteoblasts hFOB1.19 cells were cultured.qRT-PCR was carried out to test the expressions of CCAT2 and miR-200b.Then,MG63 and U20S cells with low CCAT2 expression were constructed by transfection with si-CCAT2.Cell counting kit-8(CCK-8)assay,5-bromodeoxyuridine(BrdU)incorporation assay,FITC-Annexin V/PI apoptosis assay,scratch assay and invasion chamber assay were utlized to detect the effects of CCAT2 silencing on viability,proliferation,apoptosis,migration and invasion of MG63 and U20S cells,respectively.Western blot was done for assessingthe protein levels of Cyclin Dl,cyclin-dependent kinase 6(CDK6),Bcl-2,Bax and Caspase 3 in MG63 and U20S cells after CCAT2 silencing.ResultsThe results of qRT-PCR showed that compared to paracancerous tissues,the expression of CCAT2 was significantly increased in osteosarcoma tissues(P<0.001),while the expression miR-200b was remarkably decresed in osteosarcoma tissues(P<0.01).Relative to human normal osteoblasts hFOB1.19 cells,the CCAT2 was highly expressed in osteosarcoma MG63,U20S,US732 and Saos-2 cells(P<0.05 or<0.01),while miR-200b was lowely expressed in osteosarcoma MG63,U20S,US732 and Saos-2 cells(P<0.05 or<0.01).Transfection of si-CCAT2 significantly silenced the expression of CCAT2 in MG63 and U20S cells(P<0.01).The results of CCK-8 assay showed that CCAT2 silencing remarkably reduced the viabilites of MG63 and U20S cells(P<0.05).The results of BrdU incorporation assay displayed that CCAT2 silencing notably inhibited the proliferation of MG63 and U20S cells(P<0.05 or<0.01),which were accompanied with the decreased protein levels of Cyclin D1 and CDK6 in MG63 and U20S cells(P<0.05 or<0.01).The results of FITC-Annexin V/PI apoptosis assay presented that CCAT2 silencing significantly induced MG63 and U20S cell apoptosis(P<0.01 or<0.001),which were accompanied with the decreased protein levels of Bcl-2(P<0.05 or<0.01)and increased protein levels of Bax and Cleaved-caspase 3 in MG63 and U20S cells(P<0.01 or<0.001).The results of scratch assay and invasion chamber assay pointed out that CCAT2 silencing dramatically reduced MG63 and U20S cell migration and invasion(P<0.01).Conclusions1.CCAT2 had high expression levels in osteosarcoma tissues and cells,while miR-200b had low expression levels in osteosarcoma tissues and cells.2.CCAT2 silencing inhibited the osteosarcoma MG63 and U20S cells viabilities,proliferation,migration and invasion,but promoted cell apoptosis.BackgroundMicroRNAs(miRNAs)are another type of RNA transcripts in cells with 20-25 nucleotides in length.They also can not code proteins.LncRNAs generally exhibit oncogenic or tumor suppressive roles in cancer cells via altering miRNAs expression.Previous literatures confirmed that overexpression of miRNA-200b(miR-200b)could inhibit osteosarcoma cell proliferation,migration and invasion.However,it is not clear that whether CCAT2 participates in the initiation and development of osteosarcoma and whether CCAT2 can regulate miR-200b expression in osteosarcoma cells.ObjectiveThe relationship between CCAT2 and miR-200b was used as an entry point to probe whether miR-200b took part in the effects of CCAT2 silencing on osteosarcoma cell proliferation,apoptosis,migration and invasion.Finally,to study the possible molecular targets of miR-200b.MethodsFirstly,qRT-PCR was performed to detece the expression of miR-200b in MG63 and U20S cells after CCAT2 silencing.Subsequently,the MG63 and U20S cells with low miR-200b expression were constructed by transfection with miR-200b inhibitor.BrdU incorporation assay,FITC-Annexin V/PI apoptosis assay,scratch assay and invasion chamber assay were utlized to detect the impacts of miR-200b knockdown on CCAT2 silencing-caused MG63 and U20S cell proliferation reduction,apoptosis enhancement,migration inhibition and invasion represssion,respectively.Western blot was done for measusing the protein levels of Cyclin D1,CDK6,Bcl-2,Bax and Caspase 3 in MG63 and U20S cells.Finally,the relationship between miR-200b and vascular endothelial growth factor(VEGF)in MG63 and U20S cells was analyzed by western blot and luciferase reporter gene assay.ResultsThe results of qRT-PCR showed that CCAT2 silencing significantly increased the miR-200b expression in MG63 and U20S cells(P<0.05 or<0.01).The results of BrdU incorporation assay displayed that by contrast with single CCAT2 silencing group,miR-200b inhibitor transfection remarkably promoted the proliferation of MG63 and U20S cells(P<0.05),which were accompanied with the increased protein levels of Cyclin D1 and CDK6 in MG63 and U20S cells(P<0.05).The results of FITC-Annexin V/PI apoptosis assay presented that miR-200b inhibitor transfection noticeably alleviated the CCAT2 silencing-caused MG63 and U20S cell apoptosis enhancement(P<0.05).Compared to single CCAT2 silencing group,miR-200b inhibitor transfection raised the Bcl-2 protein levels in MG63 and U20S cells(P<0.05),but declined the Bax and Cleaved-caspase 3 protien levels(P<0.05).The results of scratch assay and invasion chamber assay pointed out that miR-200b inhibitor transfection also weakened CCAT2 silencing-caused decreases of MG63 and U20S cell migratory and invasive abilities(P<0.05).Besides,western blot results showed that CCAT2 silencing remarkably reduced the protein levels of VEGF in MG63 and U20S cells(P<0.01),while miR-200b inhibitor transfection attenuated the decreases of VEGF protein levels aroused by CCAT2 silencing(P<0.05).The results of luciferase reporter gene assay displayed that co-transfection with miR-200b mimic and wild-type VEGF reporter plasmid significantly reduced the relative luciferase activity(P<0.01).Conclusions1.CCAT2 silencing up-regulated the miR-200b expressions in MG63 and U20S cells.2.Knockdown of miR-200b attenuated the influences of CCAT2 silencing on MG63 and U20S cell proliferation,apoptosis,migration and invasion.BackgroundExploring the functions of CCAT2 in the initiation and development of ostesarcoma,along with the intrinsic molecular mechanism involving in miR-200bObjectiveTo detect the activity of phophoinositide 3 kinase(PI3K)/protein kinase B(AKT)signaling pathway and adenine monophosphate activated protein kinase(AMPK)signaling pathway activities in osteosarcoma cells along with CCAT2 silencing and/or miR-200b knockdown.MethodsIn order to explore the influences of CCAT2 silencing and/or miR-200b knockdown on the activities of PI3K/AKT and AMPK signaling pathways in MG63 and U20S cells,western blot analysis was carried out to detect the protein levels of total PI3K,phosphorylated PI3K,total AKT,phosphorylated AKT,total AMPK and phosphorylated AMPK in MG63 and U20S cells after CCAT2 silencing and/or miR-200b inhibitor transfection.ResultsThe results of western blot showed that CCAT2 silencing notably repressed the PI3K/AKT and AMPK signaling pathways in MG63 and U20S cells through reducing the expression ratio of phosphorylated PI3K to total PI3K(p/t-PI3K,P<0.05),the expression ratio of phosphorylated AKT to total AKT(p/t-AKT,P<0.05 or<0.01)and the expression ratio of phosphorylated AMPKto total AMPK(p/t-AMPK,P<0.01).Moreover,miR-200b inhibitor transfection significantly alleviated the CCAT2 silencing-caused inactivation of PI3K/AKT and AMPK signaling pathways in MG63 and U20S cells through enhancing the p/t-PI3K(P<0.05 or<0.01),p/t-AKT(P<0.05)and p/t-AMPK(P<0.05)expressions.ConclusionsCCAT2 silencing inactivated PI3K/AKT and AMPK signaling pathways in MG63 and U20S cells by up-regulating miR-200b.