| Background and objective:Esophageal squamous cell carcinoma(ESCC)is one of the prevalent and deadly cancers worldwide,especially in Eastern Asia(including China),and advanced ESCC frequently presents with extensive local invasion or regional lymph node metastasis It is imperative to seek more effective biomarkers for the early diagnosis of ESCC.Recent studies showed that long noncoding RNAs(lnc RNAs)have critical roles in diverse biological processes,including tumorigenesis.One example of such an oncogenic lnc RNA is linc RNA-ROR(linc-ROR,ROR),which is highly expressed in several cancers such as breast cancer,hepatocellular carcinoma,endometrial cancer,and pancreatic cancer.However,the relationship between ROR and ESCC remains largely unknown.In the present study,we investigated the expression of ROR in ESCC tissues and cells,examined the relationship between ROR expression levels in tumor tissues and the risk features of ESCC,identified the effect of ROR on the malignant phenotype of ESCC cells,and explored the signal pathway mechanism of the regulatory role of ROR in ESCC.Methods and results:1.Study on the relationship between ROR and the risk of ESCCincidence and poor prognosis.91 pairs of ESCC tumor tissues and matched adjacent tissues were collected from the First People's Hospital of Huai'an City in 2010.The expression of ROR was detected by q RT-PCR.The results showed that the expression level of ROR in cancer tissues was significantly higher than that in adjacent tissues of ESCC,which was 1.717 times that of adjacent tissues(OR=2.138,p<0.001).The expression of ROR was also detected in three kinds of ESCC cell lines including KYSE510,EC109,EC9706 and one immortalized esophageal epithelial cell H5E46.It was found that the expression of ROR in ESCC cells was significantly higher than that of normal esophageal cells,which was 2.5 times,10.8 times and 29.5 times of that(p<0.001).It can be suggested that the increase of ROR expression was significantly associated with the risk of ESCC and may be one of the risk factors for ESCC.RNA sequencing microarray data of 96 ESCC patients were also collected from the TCGA database for survival analysis of ROR and ESCC.The results showed that patients with higher ROR expression had shorter survival time than those with lower ROR(p<0.001).It can be suggested that high expression of ROR is associated with poor prognosis of ESCC patients.2.Effect of ROR on malignant phenotype of ESCC cellsEC109 cell line stably over-expressing ROR was constructed by lentivirus transfection for subsequent cell function assays.The transfection efficiency was measured by q RT-PCR.The ROR level of cells in the overexpression group(treatment group)was approximately 16,000 times that of the negative control group.The viability,proliferation,cell cycle,apoptosis,migration,and invasiveness of cells in treatment and control groups were examined.The results showed that the viability,proliferative ability,migration and invasion ability of treatment group cells were stronger than those of the control group,but the apoptosis rate was lower,and the cells in the S phase were more than the control group.(1)CCK-8 assay was conducted to detect cell viability.The OD was measured at 0.5h,1h,2.5h and 4h after CCK-8 treatment.The OD of the treatment group was higher than the control group and had a time-response relationship.After administration for 4 hours,the OD value of the treatment group increased by 34.1% compared with the control group(p<0.001).The cell viability of the treatment group was higher than that of the control group.(2)EdU cell proliferation assay was used to detect cell proliferation.The results showed that the number of cells entering proliferative phase in the treatment group was(35.77±3.83)%,and the proportion of cells entering proliferative phase in the control group was(30.70±5.11)%.Compared with the control group,the number of cells entered into proliferative phase of the treatment group was increased by 16.7%(p<0.001).(3)Using PI staining with flow cytometry to detect cell cycle.The percentages of cells in G0/G1 phase of the treatment group and control group were(62.28±2.32)% and(65.56±1.14)%,respectively,and the G2/M phase were(13.83±0.91)% and(13.17±0.58)%,respectively,S phase were(23.90±1.53)% and(21.27±0.44)%,respectively(p<0.001,n = 3).In the treatment group,the cells entering the S phase increased by 12.4% compared with the control group.Correspondingly,the G0/G1 phase ratio of the treatment group was lower than that of the control group.(4)Using Annexin V-APC/7-AAD staining with flow cytometry to detect cell apoptosis.The apoptosis rateof the treatment group and the control group was respectively(8.13±0.76)% and(11.97±2.05)%.The apoptosis rate of the treatment group was lower than that of the control group(p<0.05).Compared with the control group,the apoptosis rate in the treatment group was reduced by 32.0%.(5)Cell migration was detected using Transwell chamber.The number of cells passed through membrane in the treatment group and the control group was respectively 50.71±6.35 and 23.60±5.37.The number of cells passed through membrane in the treatment group was significantly higher than that in the control group(p<0.001).(6)Cell invasion was detected using Transwell chamber.The number of in the treatment group was 38.75±6.05 and 32.19±6.42,respectively.The number of cells passed through membrane in the treatment group was significantly higher than that in the control group(p<0.01).3.Analysis of the signaling pathways ROR involved in the regulation of ESCCIn order to further study the signaling pathway of ROR as a competitive endogenous RNA(ce RNA)involved in the regulation of ESCC,the data of 161 esophageal cancer tissues and 11 adjacent tissues in the TCGA database were collected and screened for differentially expressed genes,including 82 mi RNAs and 1398 m RNAs.Screening criteria were p<0.05,false discovery rate(FDR)< 0.1,and differential expression fold change(FC)?2.Among these 82 mi RNAs,11 of them have been predicted binding sites with ROR(predicted by Target Scan and mi Randa)and are down-regulated in esophageal cancer tissue.In combination with dual luciferase reporter(DLR)assay and q RT-PCR results,mi R-145 and mi R-204 were selected as the mi RNAs to be studied.This study investigated separately the mechanisms of ROR targeting mi R-145 / FSCN1 and mi R-204 / MDM2 involved in the regulation of ESCC.(1)Pathway analysis of ROR targeting mi R-145 / FSCN1 involved in regulating migration and invasion ability of ESCC.1)The effect of ROR on the expression of mi R-145 was analyzed by q RT-PCR.The results showed that the expression level of mi R-145 in the cells overexpressing ROR was lower than that of the control group,which was about 0.50 times that of the control group(p<0.01).2)Dual-luciferase reporter system was used to analyze the binding sites of ROR and mi R-145.Three predicted binding sites with the highest scores were selected for mutation.Using pmir GLO reporter gene vector,5 groups were set up:(1)ROR-WT+NC mimic,(2)ROR-WT+mi R-145 mimic,(3)ROR-MUT1+mi R-145 mimic,(4)ROR-MUT2+mi R-145 mimic,(5)ROR-MUT3+mi R-145 mimic(WT: Wild type,MUT: Mutant type).The results showed that the fluorescence signal of group 2 was significantly lower than that of group 1(0.52 times,p<0.001);the fluorescence signal of group 3,4,5 were higher than that of group 2.Compared with group 2,group 3(MUT1)and 4(MUT2)were statistically significantly higher,respectively 1.45-fold(p<0.01)and 1.36-fold(p<0.05).3)RNA binding protein immunoprecipitation(RIP)assay was used to verify the binding relationship between ROR and mi R-145.Two groups were established,namely the treatment group and the control group.Each group was separately immunoprecipitated with Argonaute2(AGO2)antibody and Ig G,and the product was subjected to q RT-PCR to detect the content of mi R-145.The results showed that the amount of mi R-145 in the treatment group was 1.81 times that of the control group(p<0.01).4)Western blotting(WB)was used to verify the regulation of mi R-145 on FSCN1 and the indirect regulation of ROR on FSCN1(with GAPDH as an internal reference).The results showed that the grayscale value of the FSCN1 protein band of cells transfected with mi R-145 mimic was 0.57 times that of the NC mimic group(p<0.01),and the grayscale value of the FSCN1 protein band of ROR-overexpressing treatment group was 1.41 times that of the control group(p<0.05).5)Overexpression of mi R-145 reverses the function of ROR promoting ESCC cell migration and invasion.The results showed that the enhanced migratory ability and invasive ability of ROR-overexpressing cells transfected with mi R-145 mimic was significantly reduced(p<0.001,p<0.05).(2)Pathway analysis of ROR targeting mi R-204 / MDM2 involved in regulating P53 in ESCC.1)The effect of ROR on mi R-204 expression was analyzed by q RT-PCR.The results showed that the expression level of mi R-204 in the cells overexpressing ROR was lower than that of the control group,which was about 0.27 times that of the control group(p<0.01).2)Dual-luciferase reporter system was used to analyze the binding sites of mi R-204 and MDM2.Two predicted binding sites to mi R-204 of identical sequences on MDM2 were selected for mutation.Using reporter gene vector pmir GLO,four groups were set up:(1)MDM2-WT+NC mimic,(2)MDM2-WT+mi R-204 mimic,(3)MDM2-MUT+ NC mimic,(4)MDM2-MUT+ mi R-204 mimic.The results showed that the fluorescence signal of group 2 was significantly lower than that of group 1(0.53 times,p<0.01);and the fluorescence signal of group 4 was significantly higher than that of group 2,which was approximately 1.75 times(p< 0.01).3)Dual-luciferase reporter gene system was used to verify the relationship of competitively binding to mi R-204 between ROR and MDM2.To verify their direct binding relationship,the predicted binding site of ROR and mi R-204 was selected for mutation.Using the pmir GLO reporter gene vector,4 groups of cells were set up:(1)ROR-204WT+NC mimic,(2)ROR-204WT+mi R-204 mimic,(3)ROR-204MUT+NC mimic,(4)ROR-204MUT+mi R-204 mimic.The results showed that the fluorescence signal of group 2 was significantly lower than that of group 1(0.70-fold,p<0.001);and the fluorescence signal of group 2 was significantly lower than that of group 4(0.72-fold,p<0.001).Based on this result,two more groups were set to verify ROR competitively binding to mi R-204:(5)ROR overexpressing cells+MDM2-WT plasmid + mi R-204 mimic,(6)negative control cells+MDM2-WT plasmid+mi R-204 mimic.The results showed that the fluorescence signal of group 6 was lower than that of group 5,which was about 0.87-fold(p<0.05).4)RIP assay was used to verify the binding relationship between ROR and mi R-204.Two groups were established,namely the treatment group and the control group.Each group was separately immunoprecipitated with AGO2 antibody and Ig G,and the product was subjected to q RT-PCR to detect the content of mi R-204.The results showed that the amount of mi R-204 in the treated group was 1.25 times that of the control group(p<0.05).5)WB was used to verify the regulation of mi R-204 on MDM2 and the indirect regulation of ROR on P53.The results showed that the grayscale value of the MDM2 protein band of cells transfected with mi R-204 mimic was 0.83 times that of the NC mimic group(p<0.05);the grayscale value of the P53 protein band of the treatment group with high expression of ROR was 0.64 times that of the control group(p<0.05).6)Overexpression of mi R-204 reverses the function of ROR promoting ESCC cell entering S phase from G1 phase.The results showed that cell cycle arrest in G0/G1 phase occurred in ROR overexpressing cells transfected with mi R-204 mimic(p<0.01).Conclusion:1.ROR is highly expressed in ESCC tissues and cells,and its high expression may increase the risk of ESCC.It can be indicated that high expression of ROR is a risk factor for ESCC.2.ROR overexpression can increase ESCC cell viability,proliferation,migration,invasion,the number of cells to entering the S phase and inhibit apoptosis.It has a function similar to an oncogene.3.ROR can act as a mi RNA molecular sponge to competitively bind mi R-145,mi R-204 and other mi RNAs to inhibit their function,which reduce the target m RNA degradation such as FSCN1 and MDM2.4.ROR can enhance the migration and invasion of ESCC cells through ROR/mi R-145/FSCN1 pathway.It can also inhibit the P53 pathway through ROR/mi R-204/MDM2 pathway,which promotes cells to enter S phase from G1 phase. |
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