| Objective:To investigate the effect of LncRNA SNHG1 on inhibiting the anticancer effect of baicalein on cervical cancer in vivo and in vitro,to explore the molecular mechanism of LncRNA SNHG1 to regulate miR-3127-5p/FZD4/Wnt/β-catenin signaling pathway,and to block the Wnt/β-catenin signaling pathway and prevent The role of LncRNA SNHG1 in the anticancer process of baicalein was specifically developed in this study.Methods:First,the cervical cancer cell lines HeLa and SiHa were treated with baicalein at different concentrations(0,25,50,100 μM)for 24 hours,and then the effect of baicalein on the viability of cervical cancer cells was tested by Glo cell viability experiment.By stably transfecting the overexpressed and knockdowned IncRNA SNHG1 plasmid,HeLa and SiHa cell lines with stable overexpression and knockdown of lncRNA SNHG1 were constructed.EDU incorporation test,Transwell test,Glo activity test,TUNEL test,TOPFLASH/FOPFLASH luciferase,PCR and WB were used for detection.A subcutaneous tumor-bearing experiment was performed in nude mice.3.0× 106 stable overexpression of LncRNA SNHG1 and control HeLa cells were seeded subcutaneously in nude mice.After subcutaneous tumor-bearing,tumor-bearing mice were injected intraperitoneally with 10μg baicalein or control(0.25%DMSO)per g of nude mouse body weight daily.The volume of the subcutaneous tumor was measured every 7 days,and the volume of the subcutaneous tumor was calculated according to "0.5× length × width × width".Secondly,in order to explore the possible molecular mechanism of LncRNA SNHG1 exerting its anticancer effect on baicalein,first of all,the bioinformatics analysis(http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/)was used to determine its subcellular localization.Using TargetScan prediction algorithm and ENCORI(http://starbase.sysu.edu.cn/),it was found that there is a miR-3127-5p binding site on LncRNA SNHG1.To verify whether miR-3127-5p can recognize LncRNA SNHG1 by recognizing this site,a dual luciferase reporting experiment was performed.To further verify the binding of LncRNA SNHG1 and miR-3127-5p,biotin-labeled LncRNA SNHG1 antisense probes were used to bind and enrich endogenous LncRNA SNHG1 and miRNAs bound to LncRNA SNHG1.In order to further prove the binding of LncRNA SNHG1 and miR-3127-5p,biotin-labeled wild-type LncRNA SNHG1 and miR-3127-5p-mutated LncRNA SNHG1 were transcribed in vitro,and then biotin-labeled wild-type,mutant LncRNA SNHG1 and HeLa Cell lysates were incubated and avidin magnetic beads were used to specifically enrich biotin-labeled LncRNA SNHG1 and miRNAs combined with LncRNA SNHG1.To verify whether LncRNA SNHG1 can regulate the binding and targeting of miR-3127-5p to FZD4,a double luciferase reporting experiment was carried out again.At the same time,the effect of LncRNA SNHG1 on FZD4 mRNA and protein expression was further examined.The TOPFlash/FOPFlash luciferase activity and the expression of target genes in the Wnt/β-catenin signaling pathway were measured to evaluate the activation of the Wnt/β-catenin signaling pathway.In order to further study the effect of baicalein on Wnt/β-catenin signaling pathway.LncRNA SNHG1 was stably overexpressed and treated with 50 μM baicalein for 48 hours,then TOPFlash/FOPFlash luciferase activity and Wnt/β-catenin target gene expression were detected.Third,LncRNA SNHG1 was stably overexpressed and control HeLa cells were treated with 100 μM baicalein and 25 μM ICG-001 for 24 hours,and then the cell proliferation was detected by Glo cell viability experiment,and the positive rate of cells was detected by EdU incorporation experiment The TUNEL experiment was performed to detect the apoptosis of the corresponding cells.50 μM baicalein and 12.5 μM ICG-001 were used to treat LncRNA SNHG1 stably overexpressed and control HeLa cells for 48 hours,while Transwell migration experiments were performed on the treated LncRNA SNHG1 stably overexpressed and control HeLa cells.Results:(1)Baicalin inhibited the progress of cervical cancer in-vivo and in-vitro:The SNHG1 over-expressed HeLa or SiHa cells were treated with different concentrations of baicalin.The cell viability decreased greatly in Vector group and SNHG1 group(P<0.05).Compared with Vector+control group,the ratio of EdU-positive cells decreased significantly in Vector+baicalein group(P<0.05)and the ratio of TUNEL-positive cells increased significantly in SNHG1+baicalein group(P<0.05),in which the migration rate was inhibited greatly(P<0.05).The expression of SNHG1 in SNHG1 group was significantly higher than that in Vector+control group(P<0.05).The SNHG1 knockdown HeLa or SiHa cells were treated with different concentrations of baicalin.Compared with shVec group,the cell viability decreased greatly in shSNHG1-1 or shSNHG1-2 group(P<0.05).The ratio of EdU-positive cells in shVec+baicalein group was lower and the ratio of TUNEL-positive cells was higher than that in shVec+Control group(P<0.05).Compared with shVec+Control group,the ratio of EdU-positive cells decreased and the ratio of TUNEL-positive cells increased greatly in shvec+baicalein,shSNHG1-1 or shSNHG1-2 group(P<0.05).And the cell migration was also inhibited greatly.In in-vivo experiment,compared with Vector+control group,the tumor volume decreased,the weight of the tumor lightened,the ratio of the Ki67-possitive cells decreased and the ratio of TUNEL-positive cells increased in Vector+baicalein group(P<0.05).However,no difference on the tumor volume,tumor weight,ratio of the Ki67-possitive cells and ratio of TUNEL-positive cells in SNHG1+baicalein group was observed,compared with Vector+control group.(2)LncRNA SNHG1 regulates miR-3127-5p/FZD4/Wnt/β-catenin signal pathway:The expression level of SNHG1 and miR-3127-5p was greatly higher in SNHG1 probe group than that in control group(P<0.05).However,no significant difference was observed between LacZ probe group and control group.The affinity between SNHG1 and miR-3127-5p was greater than SNHG1-mut or beads(P<0.05).There were four binding sites for miR-3127-5p on the 3’ untranslated region of FZD4.The results of double luciferase assay showed that the fluorescence intensity in SNHG1 group was greater than that in SNHG1-mut or Vector group(P<0.05).The double luciferase plasmid containing the 3’ untranslated region of FZD4 and the shRNAs against SNHG1 were transfected into the HeLa cells together.The fluorescence intensity in shSNHG1-1 or shSNHG1-2 group was lower than that in shvec+Control group(P<0.05).The expression level of FZD4,cyclin D1,c-Myc and the activity of TOPFlash/FOPFlash luciferase in SNHG1 group were higher than that in Vector or SNHG1-mut group(P<0.05).The expression level of FZD4,cyclin D1,c-Myc and the activity of TOPFlash/FOPFlash luciferase in shSNHG1-1 or shSNHG1-2 group were lower than that in shvec group(P<0.05).50 μM baicalin was used to incubated with the SNHG1 over-expressed HeLa cells for 48 hours,the expression level of FZD4,cyclin D1,c-Myc and the activity of TOPFlash/FOPFlash luciferase in Vector+baicalin group were lower than that in Vector+control or SNHG1+baicalin group(P<0.05).(3)Wnt/β-catenin signaling pathway inhibitor ICG-001 abolished the role of LncRNA SNHG1 in the anticancer process of baicalein:The results of the Glo cell viability experiment showed that compared with Vector+baicalein group,the ratio of Glo positive cells increased greatly in SNHG1+baicalin group(P<0.05).However,no significant difference was observed between SNHG1+baicalin+ICG-001 group and Vector+baicalein group.The results of EdU incorporation experiments showed that compared with Vector+baicalein group,the ratio of EdU positive cells increased greatly in SNHG1+baicalin group(P<0.05).However,no significant difference was observed between SNHG1+baicalin+ICG-001 group and Vector+baicalein group.The results of TUNEL experiments showed that compared with Vector+baicalein group,the ratio of TUNEL positive cells decreased greatly in SNHG1+baicalin group(P<0.05).However,no significant difference was observed between SNHG1+baicalin+ICG-001 group and Vector+baicalein group.The results of Transwell migration experiments showed that the number of migrating cells increased significantly in SNHG1+baicalin group(P<0.05).However,no significant difference was observed between SNHG1+baicalin+ICG-001 group and Vector+baicalein group.Conclusion:(1)Baicalein exerted anti-tumor effects against cervical cancer in-vitro and in-vivo,which was reversed by the introduction of LncRNA SNHG1.(2)LncRNA SNHG1 activated the Wnt/β-catenin signal pathway by up-regulating the expression of FZD4 through targeting miR-3127-5p;(3)Baicalein exerted anti-tumor effects by regulating the Wnt/β-catenin signal pathway.And LncRNA SNHG1 reversed the anti-tumor effects of baicalein through activating Wnt/β-catenin signal pathway. |
PDF Full Text Request