Effects Of MiR-145 On Proliferation,Migration And Invasion Of Cervical Carcinoma Cells Through Targeting RCAN3

Objective: To detect the expression of miR-145 and RCAN3 in cervical cancer cells and explore their effects on the proliferation,migration and invasion of cervical cancer cells.Methods:1.RCAN3 expression was analyzed in 306 squamous carcinoma of the cervix tissues and 13 normal healthy tissues from TCGA and GTEX databases.Patients were divided into high and low expression groups according to the median expression level of RCAN3,and the survival rates of the two groups were analyzed by Kaplan-Meier survival analysis.Cox regression analysis were carried out to assess the potential function of RCAN3.2.MirBase and Target Scan were used to find the important regulatory genes of RCAN3.Dual luciferase reporter assay was carried out to verify that whether miR-145 was the upstream regulator of RCAN3.The expression in cervical squamous carcinoma and influence on survival of miR-145 were analyzed by bio-informatics.The expression of miR-145 in different cells was detected by q RT-PCR.In order to better perform functional experiments,CaSki with relatively high expression level was selected for knockout test,and C-33 A with relatively low expression level was selected for over-expression test.Western blot was used to detect the changes of up-regulated/down-regulated miR-145 on RCAN3 expression level.3.In order to further explore whether miR-145 can play its function on proliferation by regulating RCAN3,we carried out CCK-8 and colony formation experiments.Then we researched the influences of the miR-145/RCAN3 axis on the proliferation,invasion and migration of cervical cancer cells by Transwell assay.Results:1.RCAN3 was highly expressed in cervical squamous carcinoma and the survival time of low expression group was significantly longer than that of high expression group(P<0.05).Univariate analysis showed that the expression of RCAN3,clinical stage,pathologic-T,pathologic-M and pathologic-N could be used as prognostic factors of cervical squamous carcinoma.Multivariate analysis showed that only pathologic-T could be used as an independent prognostic factor of cervical squamous carcinoma.2.MiR-145 was a regulatory gene of RCAN3.The expression of miR-145 in cervical squamous carcinoma tissues was significantly decreased(3.52±0.51 vs 5.02±0.51,P<0.0001),and the 5-year overall survival was significantly reduced in those with low expression of miR-145(57.5% vs74.3%,P<0.05).The expression level of miR-145 was lower in cervical squamous carcinoma patients with high clinical stage,tumor diameter?4cm,lymph node metastasis and high-risk HPV infection(P<0.05,P<0.05,P<0.05).In cervical cancer cell lines(CaSki,He La and C-33A),the expression of miR-145 was significantly lower than that in the control group(P<0.01,P<0.01,P<0.01).Up-regulation of miR-145 significantly reduced the luciferase activity of WT-RCAN3 vector(0.16±0.03 vs 0.41±0.04,P<0.01)and the expression of RCAN3 in C-33 A cells(0.63±0.07 vs 2.19±0.14,P<0.01),while inhibition of miR-145 significantly enhanced the activity(0.64±0.07 vs0.41±0.04,P<0.01)and the expression of RCAN3 in CaSki cells(8.34±0.72 vs2.22±0.11,P<0.01).3.The number of cell proliferation,invasion and migration in C-33 A cells of the miR-145 mimic group significantly decreased(P<0.01,P<0.01,P<0.01),while the pc DNA3.1-RCAN3 group increased(P<0.01,P<0.01,P<0.01).However,miR-145 mimic + pc DNA3.1-RCAN3 group partially reversed promotion effect caused by the PCDNA3.1-RCAN3 group(P<0.01,P<0.01,P<0.01).The number of cell proliferation,invasion and migration in CaSki cells of miR-145 inhibitor group significantly increased(P<0.01,P<0.01,P<0.01),while the si-RCAN3 group decreased(P<0.01,P<0.01,P<0.01).However the inhibition effect caused by si-RCAN3 group could be partially reversed by miR-145 inhibitor + si-RCAN3 group(P<0.01,P<0.01,P<0.01).Conclusion:1.RCAN3 is highly expressed in cervical squamous carcinoma,and the prognosis of patients with high expression is poor.2.MiR-145 is low expressed in cervical squamous carcinoma,and the prognosis of patients with low expression is poor.3.MiR-145 can specifically bind to RCAN3 and negatively regulate the expression of RCAN3 protein.4.Up-regulation of miR-145 can partially reverse the promotion effect of up-regulation of RCAN3 expression on proliferation,invasion and migration of cervical cancer cells.5.Down-regulation of miR-145 expression can partially reverse the inhibitory effect of down-regulation of RCAN3 expression on proliferation,invasion and migration of cervical cancer cells.