| Objective To investigate the expression of long non-coding RNA CCAL in gastric cancer(GC)tissues and its underlying clinical value,to study the effect of CCAL on the proliferation,apoptosis,invasion and migration of GC cells,and to explore its potential molecular mechanism.Methods The expression level of CCAL in 48 cases of GC and adjacent normal tissues was detected by quantitative real-time PCR(qRT-PCR).Receiver operating characteristics(ROC)curve was constructed to investigate the possibility of CCAL as a potential tumor marker for diagnosing GC.The CCAL interference vector,overexpression vector and the respective control were transfected into HGC-27 and SGC-7901 cell lines.The effect of CCAL on cell proliferation was detected by CCK-8 and colony formation assay.The effect of CCAL on cell cycle distribution and apoptosis was detected by flow cytometry.The effect of CCAL on the invasion and migration of GC cells was detected by wound healing assay and transwell assay.The effect of CCAL on the expression of vimentin and E-cadherin was detected by Western blot.The luciferase reporter gene was used to confirm the target gene miR-149 of CCAL in GC based the bioinformatics prediction.The wound healing assay and transwell assay were used to analyze the effect of CCAL via miR-149 on regulating the invasion and migration of GC cells.The effect of CCAL on the expression of vimentin and E-cadherin was detected by Western blot.Combined with previous studies and in vitro cell function experiments,we confirmed that FOXM1 was a target of miR-149 in GC.The relationship between CCAL/miR-149/FOXM1 was studied by correlation analysis and Western blot.Results The expression of CCAL in gastric cancer patients was significantly higher than that in adjacent normal tissues(P<0.05).ROC curve analysis showed that CCAL had better diagnostic marker value(AUC=0.8563).Proliferation assay showed that the growth rate of GC cells was significantly slowed down after knockdown of CCAL(P<0.05),and the growth was significantly accelerated when CCAL was overexpressed(P<0.05).Flow cytometry showed that the percentage cells of S phase was significantly decreased when CCAL was knocked down(P<0.05),while CCAL was overexpressed had the opposite effect(P<0.05).Meanwhile,knockdown of CCAL promoted apoptosis in GC cells(P<0.05).The transwell and wound healing assay showed that knockdown CCAL could largely impaired the invasion and migration ability of GC cells(P<0.05).When CCAL was overexpressed,the ability would enhanced(P<0.05).Western blot showed that knocked down CCAL could downregulated vimentin and upregulated E-cadherin expressions(P<0.05),respectively.While CCAL was overexpressed,the expression of vimentin was increased and E-cadherin was decreased at protein level(P<0.05).Predicted by bioinformatics software and reported by luciferase reporter gene,miR-149 is a target gene for CCAL in GC.The transwell and wound healing assay showed that miR-149 mimic could largely impaired the invasion and migration ability of GC cells(P<0.05).When pcDNA-CCAL was added,the ability would restored(P<0.05).Western blot showed that transfected miR-149 mimic downregulated vimentin and upregulated E-cadherin expressions(P<0.05),respectively.When pcDNA-CCAL was added,the expression of vimentin was increased and E-cadherin was decreased at protein level(P<0.05).Western bolt had the similar result.Previous studies had demonstrated that FOXM1 was a downstream target of miR-149 in other cancer cells,and our study confirmed this relationship in gastric cancer.Western blot showed that compared with control group,the expression of FOXM1 was significantly lowerer in transfected with the mi R-149 mimic group.Compared with the vector control,the expression level of FOXM1 was significantly increased in the miR-149 inhibitor group.The expression of FOXM1 in gastric cancer tissues was significantly higher than that in adjacent normal tissues(P<0.05).FOXM1 mRNA levels were negatively correlated with miR-149 and positively correlated with CCAL.Western blot showed that knockdown of CCAL resulted in a decrease of FOXM1 expression,while CCAL was overexpressed,FOXM1 expression was increased.In addition,transfected miR-149 mimic downregulated FOXM1 expression(P<0.05),when pcDNA-CCAL was added,the expression of FOXM1 was restored(P<0.05).Conclusions CCAL was highly up-regulated in GC tissues and had better diagnostic marker value.CCAL promoted cell growth via increasing the percentage of S phase cells and inhibited cell apoptosis.Meanwhile,CCAL positively regulated FOXM1 expression by inhibiting miR-149,thereby promoted GC cell invasion and migration.All these suggesting that CCAL can serve as a potential new target for GC treatment. |
PDF Full Text Request