MicroRNA-501-5p Promotes Cervical Cancer Cell Proliferation,Migration And Invasion By Targeting CYLD

Background Cervical cancer is one of the common gynecological deadly malignancies worldwide.Despite significant declines in the incidence and mortality rate of cervical cancer,it remains the 4th most common cancer.microRNAs(miRNAs)is a class of non-coding,single-stranded small RNA molecules having a length of 18-24 nucleotides that bind to the 3’UTR of the target m RNAs by complementary base pairing,resulting in the target m RNA degradation or translation inhibition.Over the past decade,microRNAs(miRNAs)as an important regulator have attracted more attention in the oncology area including cervical cancer.microRNA-501-5p(miR-501)is a non-conserved miRNA emerging as a potential key player in tumorigenesis.Current evidences indicate that miR-501 is overexpressed in some malignancies including liver cancer,gastric cancer and lung cancer acting as an oncomir by promoting cell proliferation.In addition,high miR-501 expression could be a poor overall survival predictor in gastric cancer patients.However,the impacts and underlying mechanisms of miR-501 on the cervical cancer have not been explored.Cylindromatosis(CYLD)gene is map on chromosome 16q12.1 and encodes a deubiquitinating enzyme.CYLD gene mutations are associated with familial cylindromatosis(FC),multiple familial trichoepithelioma(MFT)and multiple myeloma.The reduced copy number of CYLD is reported in kidney cancer,HCC,and glassy cell carcinoma cell lines of the uterine cervix.Meanwhile,suppressed CYLD gene expression is found in the colorectal cancer,breast cancer and lung cancer.Thus,CYLD is considered as a tumor suppressor.Moreover,CYLD has been reported to bind to IKK gamma,a component of the Ikappa B kinase(IKK)complex,and negatively regulate the nuclear factor-kappa B(NF-κB)activity.A previous study has confirmed that CYLD is a direct target of miR-501 in hepatocellular carcinoma(HCC)cells.miR-501 enhances the proliferation of HCC cells through decreased CYLD and increased cyclin D1 and c-myc expressions.Objectives(1)To evaluate the effects of miR-501 on the cell proliferation,migration and invasion in the cervical cancer.(2)To confirm the regulatory correlation between miR-501 and CYLD and determine the underlying mechanism of miR-501 function in cervical cancer.Methods(1)Cervical cancer He La cells were transfected with miR-501 mimic(miR-501)and nonspecific miRNA(NC),miR-501 inhibitor(501i)and non-specific miRNA inhibitor(NCi),or small interfering RNA(si RNA)against CYLD(si CYLD)using lipofectamine 2000.(2)miR-501 expression was assessed by Taqman real-time quantitative RT-PCR(q RT-PCR)method.The expression of CYLD m RNA was analyzed by SYBR Green q RT-PCR method.CYLD,Bcl-2,Bax,NF-kB p65 and phosphorylated p65(p-p65)proteins were examined by Western blot analysis.(3)CCK-8 and colony formation assays were used to determine the effects of miR-501 on the proliferation of He La cells.(4)The scratch wound healing assay and Transwell migration and invasion assays were performed to evaluate the migratory and invasive potentials of He La cells.(5)The apoptosis rate was detected by flow cytometry assay.Results(1)The Western blot results showed that up-regulation of miR-501(miR-501)miR-501 induced significant decreased CYLD protein compared with non-transfected He La cells(Control)group and NC group(P<0.01),while similar results were observed in the si CYLD group(P<0.01).The real-time q RT-PCR analysis also demonstrated that CYLD m RNA level in miR-501 group and si CYLD group were significantly lower than in Control group and NC group(P<0.01).Knockdown of miR-501 resulted in significant up-regulation of CYLD both at m RNA and protein levels compared with the negative controls(P<0.01).(2)CCK-8 assays showed a significant increase in cell proliferation in the miR-501 and si CYLD groups compared to the Control and NC groups(P<0.01).The average colony count in the miR-501 group(689.3 ± 32.3)and si CYLD group(646.0 ± 29.6)was significantly higher than in the Control group(379.0 ± 13.0)and NC group(363.0 ± 13.9)(P<0.01).On the contrary,the significant decrease in cell proliferation was observed in 501 i group compared with the negative controls(P<0.05).Similar results were obtained in the average colony count in 501 i group(207.7 ± 10.7)compared with Control(651.7 ± 15.4),NCi(644.2 ± 17.6),and 501i+si CYLD(659.0 ± 21.5)(P<0.01).(3)Scratch wound healing assay showed that there was a significant decrease in wound area of miR-501 and si CYLD groups compared to Control and NC groups(P<0.01).Conversely,the wound area was larger in 501 i group compared to Control,NCi and 501i+si CYLD groups(P<0.01).The numbers of migrated He La cells penetrating through Transwell chamber without Matrigel in the miR-501 and si CYLD groups(99.0 ± 5.6;93.0 ± 3.5)were dramatically higher than in the Control group(30.7 ± 3.3)and NC group(33.3 ± 3.2)(P < 0.01).Similar results were observed in the Transwell invasive assay,and the invading cells in the miR-501(86.4 ± 3.7)and si CYLD groups(81.7 ± 5.0)were dramatically higher than in the Control group(26.7 ± 2.0)and NC group(28.3 ± 1.8)(P<0.01).On the contrary,knockdown of miR-501 inhibited the number of He La cells penetrating through the membrane with or without Matrigel(P<0.01).(4)The results of Annexin V/Propidium Iodide apoptosis assay showed a significant decreased cell apoptotic percentage in miR-501 and si CYLD groups(1.55% ± 0.10%;1.45% ± 0.05%)as compared with Control group(2.49% ± 0.12%)and NC group(2.41% ± 0.07%)(P<0.05).On the contrary,cells death number in 501 i group(10.65% ± 0.47%)was remarkably higher than in the Control group(2.47% ± 0.02%),NC group(2.39% ± 0.11%)and 501i+si CYLD group(2.19% ± 0.07%)(P<0.01).The proteins studies showed that the expressions of Bcl-2,NF-kB p65 and p-p65 were increased by miR-501 up-regulation and si CYLD knockdown comparing with Control and NC,whereas the expression of BAX was decreased.Conclusions(1)CYLD is downregulated by miR-501 at both m RNA and protein levels in the cervical cancer He La cells.(2)miR-501 promotes cervical cancer cell proliferation,migration and invasion,while inhibited apoptosis,at least partially via downregulating CYLD and subsequently activating NF-kB p65.(3)miR-501 might be a potential therapeutic target for cervical cancer.