| Objective: Endometrial cancer(EC)is one of the most common gynecological cancers in developed countries,and its incidence is increasing year by year.Endometrioid adenocarcinoma is the most common pathological tissue type of EC,and other common pathological tissue types including mucinous adenocarcinoma,serous adenocarcinoma,clear cell carcinoma,mixed adenocarcinoma,squamous cell carcinoma,small cell carcinoma,and undifferentiated carcinoma.At present,the clinical treatment of EC is based on surgery,supplemented by radiotherapy,chemotherapy and other adjuvant treatments.With the continuous in-depth study of the molecular mechanism of EC,a few molecular-targeted drugs have entered the preliminary stage of clinical application,and obvious progress has been made.Therefore,the research on the mechanism of the occurrence and development of EC is of great significance for guiding moleculartargeted therapy of EC in clinical.The human PVT1 gene,also known as plasmacytoma variant translocation gene 1(plasmacytoma variant translocation gene 1),is located downstream of the c-myc gene in chromosome 8q24.It can produce a variety of non-codingRNAs and transcribe few miRNAs,including miR-1204,miR-1205,miR-1206,miR-1207-5p,miR-1207-3p and miR-1208,etc.In previous study,PVT1 was shown to be highly expressed in EC tissues and cell lines,and promote cell proliferation,invasion and migration.However,the mechanism of PVT1 involved in the occurrence and development of EC still needs further research.We predicted potential miR-612 binding sites in PVT1 by bioinformatics database These results imply that PVT1 may act as a sponge to capture miR-612.This study aims to identify the mechanism of PVT1 involved in the aggressive progression of EC,and to explore whether PVT1 affects the malignant biological behavior of EC cells through the PVT1/miR-612/CENP-H axis.Method: We collected raw Uterine Corpus Endometrial Carcinoma(USEC)datasets from TCGA database,and construct a PVT1-centered ceRNA network.Real time-PCR was used to detects the expression level of miR-612 in human endometrial cancer tissues and normal endometrial tissues.Then we analyzed the correlation between miR-612 expression and the clinicopathological parameters and prognosis of patients with endometrial cancer.Western blots were used to evaluated the expression level of CENPH protein in human EC tissues and normal endometrium.Meanwhile,we analyzed the correlation between CENP-H expression and clinicopathological parameters and prognosis of EC patients.After that,we estimated the relationship between miR-612 and CENP-H mRNA expression levels.Changes of miR-612 and CENP-H protein expression in hyperplastic endometrium and EC tissue were detected by in situ hybridization and immunohistochemical(IHC)respectively.We then conducted the diagnostic receiver operating curve(ROC)and prognostic operating curve to evaluate the diagnostic and prognostic value of CENP-H.After overexpression or knockdown of miR-612 in EC cells.Biological behaviors as cell proliferation,migration,invasion,apoptosis and cell cycle of EC cells were estimated by the CCK-8 assay,the edu assay,transwell invasion assay,the wound healing assay,Annexin V/PI apoptosis assay andRNAse/PI cell cycle assay,respectively.Similarly,we overexpressed or downexpressed the expression of CENP-H in EC cells,and evaluated the biological behaviors of EC cells,subsequently.We performed a series of assays to discribed malignancy of EC cells,after changing the expression of miR-612 and CENPH or PVT1 and miR-612 at the same time.To confirm the regulation of PVT1 on miR-612 and miR-612 on CENP-H,we upregulated or downregulated the expression of PVT1 and miR-612.The fluorescence in situ hybridization(FISH)assays was used to indicate the localization of PVT1 and miR-612 in EC cells.The dual luciferase assay was performed to verify the binding effects and binding sites of PVT1 on miR-612,and CENP-H on miR-612.Western blots showed the changes of CDK1 and downstream p-AKT/AKT,p-m TOR/m TOR.Next,we verified the functions of PVT1 and miR-612 in an EC xenograft mouse model.The results suggest that knockdown of PVT1 combined with overexpression of miR-612 significantly suppressed EC tumour growth in vivo.Results: The PVT1/miR-612/CENP-H/CDK1 axis played a vital role in the malignant progression of advanced EC.Mi R-612 was downregulated in EC tissues and acted as a tumour suppressor to inhibit cell proliferation,migration,invasion,and promote cell apoptosis.CENP-H was found overexpressed in EC tissues,and the expression level was correlated to diagnosis and prognosis of EC.Hyperactivated CENP-H promoted cell proliferation,migration,invasion,and inhibited cell apoptosis.Overexpressed CENP-H prevented the anti-tumour effects observed with upregulated miR-612;knockdown of miR-612 also suppressed the anti-tumour effects of downregulated PVT1.Knockdown of PVT1 together with upregulated miR-612 exerted the strongest anti-tumour effects in nude mice.These effects were mediated by CDK1 through modulation of the Akt/m TOR signaling pathway.In this study,we constructed a PVT1-centered ceRNA network,which showed that the PVT1/miR-612/CENP-H/CDK1 axis may play a critical role in the progression of endometrial cancer.Real time-PCR and western blots to evaluated the miR-612 expression and the mRNA and protein expression levels of CENP-H in endometrial cancer tissues and normal endometrium.Then,the correlation between the genes and the clinicopathological parameters and prognosis of EC patients was evaluated.The results showed that miR-612 was lower expressed in endometrial cancer tissues compared with normal endometrial tissues.As the disease stage increased,the expression level of miR-612 decreased significantly,and the 5-year survival rate of patients in the high miR-612 group was significantly higher than that of the low miR-612 group(91.3% vs 48.9%,P<0.05).However,the CENP-H mRNA and protein expression levels in endometrial cancer tissues were significantly higher than those in the control group.Moreover,the CENP-H expression levels in stage III-IV endometrial cancer tissues were higher than those in stage I-II EC tissues significantly.The 5-year survival rate of patients in the low CENP-H group was significantly better than that of the high CENP-H group(90.9% vs50.1%,P<0.01).The results of in situ hybridization assays showed that the expression level of miR-612 in hyperplastic endometrium and EC tissues of patients in stage I,II,III,and IV declined,while IHC assays showed that the expression of CENP-H protein in hyperplastic endometrium and EC tissues of patients in stage I,II,III,and IV rising.The expression levels in tissues and in stage I,II,III,and IV endometrial cancer tissues are increasing.Next,we conducted a diagnostic receiver operating curve(ROC)and a prognostic operating curve,by using the mRNA expression of CENP-H and the information of patient’s survival.The area under the curves(AUC)obtained was 0.796(95% CI=0.655-0.936,P<0.001)and 0.783(95%CI=0.657-0.908,P=0.002),respectively.It reveals that CENP-H has good diagnostic and prognostic value.After overexpression/knockdown of miR-612 in endometrial cancer cells(Ishikawa,HEC-1A),the malignant biological behavior of EC cells was evaluated.The results showed that overexpression of miR-612 EC cells Inhibited cell proliferation,migration,invasion and G2-M phase retention,and increased cellular apoptosis.Subsequently,the proliferation,migration,invasion and G2-M phase retention of endometrial cancer cells were inhibited,and apoptosis increased in the knockdown CENP-H group.Then we found that up-regulating the expression of CENP-H can partially restore the inhibition of cell proliferation,migration,invasion and G2-M phase retention caused by overexpression of miR-612,as well as increased cell apoptosis.It reveals that miR-612 inhibits the malignant biological behavior of endometrial cancer cells through CENP-H.Next,we found that down-regulating the expression of miR-612 in endometrial cancer cells can partially restore the suppression of malignant biological behavior caused by knocking down PVT1,which suggested that PVT1 promotes the malignant biological behavior of endometrial cancer cells through miR-612.To explore the mechanism of PVT1 and miR-612 affecting endometrial cancer cells,we up-expressed and down-regulated the expression of the two genes in EC cells respectively.Then we detected the expression of miR-612 and CENP-H.The results showed that PVT1 can negatively regulate miR-612,while positively regulate CENP-H,while miR-612 can negatively regulate CENP-H.FISH results showed that PVT1 and miR-612 were co-localized in EC cells,while the results of dual luciferase reporter genes showed that PVT1 and CENP-H can target miR-612 through the predicted binding sites.Western blots showed that when PVT1 or CENP-H was overexpressed,CDK1 expression and downstream p-AKT/AKT,p-m TOR/m TOR,while overexpression of miR-612 lead to reduction of CDK1 expression and p-AKT/AKT,p-m TOR/m TOR.Upregulated CDK1 could partially restore the low p-AKT/AKT and low p-m TOR/m TOR caused by knockdown of CENP-H.It is suggested that the PVT1/miR-612/CENP-H axis may activate the AKT/m TOR signal pathway by regulating CDK1.Next,we constructed an EC xenograft mouse model.Then we evaluated the volume changes of tumors and CENP-H expression.The results suggested that knockdown of PVT1 or overexpression of miR-612 inhibited tumor growth in vivo.The combination of Low PVT1 and High miR-612 group inhibited tumor growth to the greatest extent.Conclusion: The expression of miR-612 was down-regulated in endometrial cancer tissues,while the CENP-H expression was up-regulated.The expression levels were correlated to the clinical pathological parameters and prognosis of patients.PVT1 negatively regulated miR-612 to promote the malignant biological behavior of endometrial cancer cells.Mi R-612 negatively regulated CENP-H to inhibit the malignant biological behavior of endometrial cancer cells.The PVT1/miR-612/CENP-H/CDK1 regulatory axis plays a critical role in the progression of EC. |
PDF Full Text Request