ABSTRACT
Objective To analyze the expression of Kruppel-like factor 4 (KLF4 ) and CD44 in esophageal squamous cell carcinoma (ESCC) tissues and adjacent non-cancerous tissues ,and to investigate their effects on the prognosis .Methods From June 2012 to September 2016 ,in The Second People′s Hospital of Nanyang ,a total of 100 patients with ESCC who receiving operation were selected .The ESCC tissues and the adjacent non-cancerous tissues (control) of the patients were collected .The expression levels of KLF4 and CD44 protein were detected by immunohistochemistry .The expressions of KLF4 and CD44 at mRNA and protein level of 50 paired fresh tissues were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting ,respectively . T-test ,chi-square ,Kaplan-Meier method and Pearson correlation analysis were performed for statistical analysis .Results The positive expression rate of KLF4 protein in ESCC tissues was 55% (55/100) ,which was lower than that in adjacent non-cancerous tissues (77% ,77/100) ,and the difference was statistically significant (χ2 =10 .778 ,P=0 .001) .The positive expression rate of CD44 protein in ESCC tissues was 81% (81/100) ,which was higher than that in adjacent non-cancerous tissues (11% ,11/100) ,and the difference was statistically significant (χ2=112 .600 ,P<0 .01) .The expression level of KLF4 mRNA in 43 cases was lower than that in adjacent non-cancerous tissues ,the expression level of CD44 mRNA in 46 cases was much higher than that of adjacent non-cancerous tissues ,and the differences were statistically significant (χ2 =51 .837 and 70 .563 ,both P< 0 .01) .There were statistically significant differences in positive expression rates of KLF4 in cancer tissues between different gender , differentiation degree , invasion depth ,TNM stage and lymph node metastasis (all P<0 .05) .Similarly there were statistically significant differences in positive expression rates of CD 44 in cancer tissues between different differentiation degree ,invasion depth ,TNM stage and lymph node metastasis (all P< 0 .05) .The expression of KLF4 was negatively correlated with CD44 expression ,either at protein level or mRNA level (r= -0 .284、-0 .518 ,both P< 0 .01) .The median survival time of patients with positive KLF4 expression in cancer tissues was 33 months ,which was longer than that of patients with negative KLF4 expression (20 months) ,and the difference was statistically significant (χ2 =4 .021 , P= 0 .019) .The median survival time of patients with positive CD44 expression in cancer tissues was 24 months ,which was shorter than that of patients with negative CD44 expression (37 months) , and the difference was statistically significant (χ2 = 4 .379 , P= 0 .016) .The results of univariate analysis showed that TNM stage ,KLF4 expression and CD44 expression were correlated with overall survival time (all P<0 .05) . The results of multivariate analysis indicated that TNM stage ,lower KLF4 expression and higher CD44 expression were the independent risk factors for survival (all P<0 .05) .Conclusion Lower expression of KLF4 and higher expression of CD44 in ESCC may be closely correlated with its occurrence ,development and prognosis .
ABSTRACT
Objective@#To investigate the impact of lincRNA-ROR, a ceRNA by binding miR-145 on the expression of the downstream genes Oct4, Sox2 and Nanog, and related biological characteristics of colon cancer stem cells, and to elucidate the clinical significance of this molecular regulatory network.@*Methods@#Fifty-two cases of colorectal cancer tissue and adjacent tissue were collected at Nanyang City Central Hospital and Nanyang Second Hospital, Henan Province, from 2014 to 2016. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of lincRNA-ROR and miR-145 in colorectal cancer tissue and isolated colon cancer cells. The correlation between the expression of lincRNA-ROR, miR-145 and the clinicopathologic features of colon cancer was performed. CD44-CD133- and CD44+ CD133+ cells were isolated from SW1116 by using flow cytometry. The expression of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR and miR-145 in cells were detected by qPCR. The relationship between lincRNA-ROR, miR-145, Oct4, Sox2 and Nanog was analyzed by bioinformatics, dual luciferase reporter assay, qPCR and Western blot. The effects of silencing lincRNA-ROR on the proliferation and chemosensitivity of colon cancer stem cells were detected by MTT, colony formation.@*Results@#LincRNA-ROR was frequently up-regulated and inversely correlated with miR-145 down-regulation in the colon cancer specimens(P<0.05). LincRNA-ROR was related to tumor size, lymph node involvement and distant metastasis(P<0.05), and miR-145 was found related to tumor size and tumor location(P<0.05). CD44+ CD133+ cells were successfully isolated from SW1116 by flow cytometry. The levels of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR in CD44+ CD133+ cells were significantly increased, while miR-145 was decreased compared with CD44-CD133-cells(P<0.05). The levels of CD44, CD133, lnc-ROR in CD44+ CD133+ cells were significantly reduced upon cell adherence, while miR-145 was significantly increased(P<0.05). Bioinformatics analysis revealed that lincRNA-ROR shared miRNA response elements with core transcription factors Oct4, Sox2 and Nanog. MiR-145 significantly inhibited the expression of lincRNA-ROR, Oct4, Sox2 and Nanog. Silencing lincRNA-ROR significantly inhibited colon cancer stem cells proliferation and increased the sensitivity to chemotherapy.@*Conclusions@#Linc-ROR functions as a key ceRNA to prevent core TFs, e. g., Oct4, Sox2, Nanog, from miR-145-mediated suppression in colon cancer stem cells and regulates cell proliferation and chemosensitivity.The data may provide insights into the pathophysiological interactions of the components of genetic networks in the development of colon cancer and may lead to new therapies in the future.