The Role And Mechanism Of CARLo-5 In Endometrial Carcinoma

Endometrial carcinoma(EC)is one of the most common malignancies of the female reproductive system.It is estimated that 63400 new cases were diagnosed in 2015 in China with about 21.8% deaths.The incidence of EC is rising and the incidence rate in young women is increasing.In the United States,EC became the second gynecological cancer following breast cancer with about 60050 new cases in 2016.EC seriously affects women's health and life.Although the current diagnosis and treatment techniques have been very developed,the molecular mechanisms of EC remains unkown.Long non-coding RNA(lncRNA)is a kind of transcript that is more than 200 nt in length but can not encode proteins.A large number of evidences suggest that lncRNAs play essential functions in the regulation of biological processes and development of various diseases.CARLo-5 is a recently found lncRNA with 2628 nucleotides in length which locats near the enhancer of the proto-oncogene myc gene.Recent studies reported that CARLo-5 is up-regulated in colon cancer,gastric cancer and other cancerous tissues and palys important role in cancer development and progression with potential value in the diagnosis,prognosis and treatment of multiple cancers.However the expression,functions and its mechanism of CARLo-5 in EC have never been reported.We detected the expression level of CARLo-5 gene by Real-time PCR,and detected the expression level of cell cycle relative proteins CDK2 and CDKN1 A by western blot in this study.Then we analyzed the relationship between CARLo-5 expression level and clinicopathological features and overall survival time of EC patients,and CDK2,CDKN1 A proteins expression.We knocked down the CARLo-5 expression in EC cells,then investigated the effects on biological behaviours of EC cells in vitro by MTS,Transwell migration,Matrigel invasion assay,clone formation assay and flow cytometry.For exploring the mechanism of CARLo-5 in the carcinogenesis and progression of EC,we detected the effect of CARLo-5 low-expression on the gene and protein level of CDK2 and CDKN1 A,then we detected the effect of CARLo-5 low-expression on the proteins level of matrix metalloproteinase MMP2 and MMP9.The EC cell line with stable low expression of CARLo-5 was constructed by RNAi lentivirus technique.Then we detected the effect of CARLo-5 low expression on the proliferation of EC cell in vivo by nude mice xenograft model.Our study firstly investigates the effect and mechanism of CARLo-5 in the occurrence and development of endometrial carcinoma.Our results are beneficial to elucidate the molecular mechanism of endometrial cancer,so as to provide experimental basis for gene diagnosis and treatment of endometrial cancer.Part one: The expression of CARLo-5 and its clinical significance in endometrial carcinomaObjective: To investgate the relationship between CARLo-5 expression and clinicopathological features and overall survival time in endometrial carcinoma patients through detecting the expression level of CARLo-5 in normal endometrial tissues(NET)and endometrial carcinoma tissues.To investgate the relationship between CARLo-5 expression and cell cycle correlated proteins CDK2 and CDKN1 A through detecting the expression level of CDK2 and CDKN1 A proteins in EC and NET.Methods:1 Real-time PCR was adopted to detect the expression of CARLo-5 in 108 endometrioid carcinoma tissues and 66 normal endometrial tissues.2 Western blot was adopted to detect the expression of cell cycle correlated proteins CDK2 and CDKN1 A.3 The correlation between CARLo-5 expression level and clinicopathological features and overall survival time in endometrial carcinoma patients was analyzed.4 The correlation between CARLo-5 expression level and cell cycle correlated proteins CDK2 and CDKN1 A expression was also analyzed.Results:1 The expression of CARLo-5 in EC and NET.Real-time PCR demonstrated that the expression level of CARLo-5 in EC were higher than those in NET(P<0.001).2 The relationship between CARLo-5 expression and clinicopathological features of EC patients.The expression level of CARLo-5 was associated with FIGO stage and lymph node metastasis(P < 0.05),but not related to age,histology,histological grade,myometrial invasion,and lymphovascular space invasion(P>0.05).3 The relationship between CARLo-5 expression and overall survival time of EC patients.The patients with higher CARLo-5 expression had shorter overall survival time(43.0±14.1months)than patients with lower CARLo-5 expression(52.5±9.1 months)(P<0.05).4 Expression of cell cycle correlated proteins CDK2 and CDKN1 A in EC.Western-blot showed that the expression levels of CDK2 protein in EC(9.32±0.69)was higher than NET(1.00±0.54)(P<0.001),but the expression levels of CDKN1 A protein in EC(0.71±0.14)was lower than NET(1.00±0.11)(P<0.001).5 The relationship between CARLo-5 expression and CDK2,CDKN1 A protein expression.The patients with higher CARLo-5 expression had higher CDK2 protein expression(r=0.30,P=0.043)and lower CDKN1 A protein expression(r=-0.28,P=0.046)than patients with lower CARLo-5 expression(P<0.05).Conclusions:1 Expression level of CARLo-5 in endometrial carcinoma tissues is significantly increased.These date suggests that CARLo-5 may play a essensial role in EC.2 High expression of CARLo-5 was related to the poor prognosis of endometrial cancer patients.These results suggest that CARLo-5 may be an important molecular marker for the prognosis of endometrial carcinoma.3 The expression level of CDK2 in EC is significantly increased,but the expression level of CDKN1 A in EC is significantly reduced.The expression of CARLo-5 was significantly correlated with the expression of CDKN1 A and CDK2.These results suggest that CARLo-5 may play an important role in the regulation of endometrial carcinoma by regulating the expression of CDK2 and CDKN1 A proteins.Part two: Effects of CARLo-5 on the biological behavior of endometrial cancer cells and the regulation mechanismsObjective: To investigate the functions of CARLo-5 in endometrial carcinoma cells and its possible mechanisms.Methods:1 The proliferation ability of EC cells with CARLo-5 low-expression were detected by MTS.2 The migration and invasion ability of EC cells with CARLo-5 low-expression were detected by transwell migration and matrigel invasion chamber assays,respectively.3 Effects of knockdown of CARLo-5 on the clone forming ability of EC cells was detected by colony forming assay.4 Effects of CARLo-5 konckdown on cell cycle distribution in endometrial cells were detected by flow cytometry.5 Expression levels of genes and proteins of CDK2 and CDKN1 A in EC cells with CARLo-5 low-expression were detected by Real-time PCR and Western-blot.6 Expression levels of MMP2 and MMP9 proteins in EC cells with CARLo-5 low-expression were detected by Western blot.Results:1 Effects of CARLo-5 konckdown on proliferation ability of EC cells.The MTS assay demonstrated that the proliferation ability of HEC-1-B and KLE with low-expression of CARLo-5 was significantly low(P<0.05).2 Effects of CARLo-5 konckdown on proliferation and invasion ability of EC cells.Transwell migration and wound healing experiments showed that the migration ability of EC cells(HEC-1-B and KLE)with CARLo-5 low-expression was significantly decreased(P < 0.05).Matrigel invasion assay showed that the invasion ability of EC cells(HEC-1-B and KLE)with CARLo-5 low-expression was significantly decreased(P<0.05).3 Effects of CARLo-5 konckdown on the clone forming ability of EC cells.Colony formation assay demonstrated that low-expression of CARLo-5 could supress the colony formation potential of HEC-1-B and KLE(P<0.05).4 Effects of CARLo-5 konckdown on EC cell cycles.Flow cytometry demonstrated that HEC-1-B and KLE cells number of G0/G1 phase were significantly increased,however the cells number of S phase were significantly decreased(P<0.05).5 Effects of CARLo-5 low-expression on the expression of CDKN1 A and CDK2.Real-time PCR and Western blot showed that the expression level of CDK2 mRNA and protein in the EC cells with low-expression of CARLo-5 was significantly decreased(P<0.05),while the expression level of CDKN1 A protein and mRNA was significantly increased(P<0.05).6 Effects of CARLo-5 low-expression on the expression level of MMP2 and MMP9 proteins.Western blot showed that the expression level of CDK2 and CDKN1 A proteins was significantly decreased in the EC cells with low-expression of CARLo-5(P<0.05).Conclusion:1 CARLo-5 can promote the proliferation and invasion of EC cells,CARLo-5 is expected to become the new target to the treatment of EC.2 Knockdown of CARLo-5 promotes the GO/GI arrest,inhibits CDK2 expression and promotes the expression of CDKN1 A,suggesting that CARLo-5 may inhibit G0/G1 arrest,by regulating the expression levels of CDK2 and CDKN1 A,then promote the proliferation of EC cells.3 Knockdown of CARLo-5 can inhibit the expression of MMP9 and MMP2 proteins in EC cells,suggesting that the promotion of EC metastasis by CARLo-5 may be related to the up regulation of MMP2 and MMP9 expression.Part three: Effects of CARLo-5 low-expression on the viability of endome-trial carcinoma cells in nude mice and its possible mechanismObjective: To investigate the effect of CARLo-5 knockdown on the proliferation ability of EC cell HEC-1-B in nude mice,and to explore the mechanism of CARLo-5 in the development of EC in vivo.Methods:1 To construct an EC cell line with stable CARLo-5 low-expression by transfecting HEC-1-B cell with a lentivirus construct containing desired vector.2 EC cells with CARLo-5 knockdown were injected into nude mice models to investigate the effect of CARLo-5 knockdown on the tumor growth.3 Expression levels of cell cycle related proteins CDK2 and CDKN1 A in nude mice transplanted tumors were detected by immunohistochemistry.Results:1 Effect of CARLo-5 knockdown on the proliferation ability of EC cells in vivo.Nude mice transplanted tumors showed,compared with NC group(499.74±98.61mm3),that tumor growth was significantly decreased in the shRNA-1 group(157.66±37.93mm3)and shRNA-2 group(102.81±58.33mm3)(P<0.05).2 Expression levels of CDK2 and CDKN1 A in nude mice transplanted tumors.Expression levels of CDK2 proteins was lower in CARLo-5 knockdown groups,but expression levels of CDKN1 A proteins was higher in CARLo-5 knockdown groups(P<0.05).Conclusions:Knockdown of CARLo-5 can significantly inhibit the proliferation of EC cells in vivo.The mechanism may be that CARLo-5 inhibit the expression of CDKN1 A protein and promote CDK2 protein expression,and then promote the proliferation of EC cells in vivo.