The Role And Molecular Sponge Mechanism Of Hsa＿circ＿0001707 Involved In The Development Of Esophageal Squamous Cell Carcinoma

Background and Objective:Esophageal cancer(EC)is a disease with high morbidity and poor prognosis,which ranks 6th in the cancer-related death and threatens human health seriously worldwide.It is formed by the abnormal hyperplasia of esophageal squamous epithelium or glandular epithelium,including two clinical subtypes of esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EA).China is the region of high ESCC incidence,the number of new cases per year accounts for more than 50% of the world.The symptom of ESCC is not obvious in the early stage,resulting in that it is difficult to be found and there are few effective markers for early screening and diagnosis,the overall survival rate is only 15% for the past five years.Consequently,it is urgent to seek potential epidemic biomarkers,which are not only to contribute to promote the early identification and intervention for ESCC,reduce the canceration rate,but also accelerate the development of targeted therapies and improve the overall prognosis.CircularRNAs(circRNAs)is a novel class of non-codingRNAs(ncRNAs),with the characteristics of covalent closed structure,resistance to Rnase R,high stability,conversation and specificity.Previous studies have illustrated that circRNAs could play a significant role in gene regulatory network,which was involved in mediating the progression of various kinds of cancers.Furthermore,with the deepening of research,it is reported that circRNAs was closely related to the biological processes of tumor abnormal proliferation,differentiation and metastasis,acted as the role of oncogene or tumor suppressor gene.Therefore,as a novel biomarker,circRNAs is expected to exert biological functions in the malignant phenotype,molecular mechanism,diagnosis and treatment of ESCC.Based on the above understanding,our research group has applied the high throughput sequencing technology and screened a representative circRNA,hsa＿circ＿0001707,which is upregulated in ESCC tissues.The objective of this study is to investigate the regulation function and molecular mechanism of hsa＿circ＿0001707 played in ESCC.Detect the expression level of hsa＿circ＿0001707 in ESCC tumor samples and explore the relationship between hsa＿circ＿0001707 expression level and the risk of ESCC,which is expected to provide theoretical basis for achieving early screening and diagnosis,prognosis evaluation of patients and discovering new treatment targets of ESCC.Methods:1.qRT-PCR was applied to detect the expression level of hsa＿circ＿0001707 in ESCC tissues and the corresponding non-cancer tissues,analyzed and compared the differences statistically.The correlation between hsa＿circ＿0001707 expression and the risk of ESCC was explored by using conditional logistic regression analysis.The diagnostic value of hsa＿circ＿0001707 as a biomarker was evaluated by drawing ROC curves.Furthermore,to detect the general characteristics of this circularRNA,Rnase R,actinomycin D and agarose gel electrophoresis test were applied and analyzed the differences from linearRNA.2.Detect the biological function of hsa＿circ＿0001707 in ESCC cell lines: Lentiviral vectors were constructed and transfected into EC109 and EC9706 cells respectively,hsa＿circ＿0001707 stably over-expression and interference cell lines were established.The transfection efficiency was measured by qRT-PCR.CCK-8,Ed U,clone formation test and transwell analysis were applied to determine the effect of hsa＿circ＿0001707on the cell viability,proliferation,colony-formation ability,invasion and metastasis of ESCC cell lines.3.Analyze the subcellular localization of hsa＿circ＿0001707 in ESCC: The fluorescence in situ hybridization(FISH)and nuclear and cytoplasmic separation test were applied to determine the localization of hsa＿circ＿0001707 in ESCC cells.Bioinformatics analysis was utilized to forecast the potential miRNAs that could combine with hsa＿circ＿0001707.Dual luciferase reporter(DLR)assay was applied to further screen and define the candidate miRNA.4.Determine the interaction between hsa＿circ＿0001707 and miRNA: The expression level of miRNA with the changes of hsa＿circ＿0001707 expression was detected by qRT-PCR.In combination with the DLR assay to fully determine a direct interaction between the circRNA and miRNA.Rescue assay was designed to detect the biological function of miRNA regulated by circRNA,miRNA inhibitor was transfected into ESCC cells,and the cell viability,colony-formation ability,invasion and metastasis were analyzed by CCK-8,clone formation assay and transwell analysis respectively.5.Furthermore,bioinformatics,DLR assay,qRT-PCR and western blot were utilized to explore the molecular mechanism of hsa＿circ＿0001707 as a “miRNA sponge” to regulate the downstream gene and signaling pathway.Results:1.The expression level of hsa＿circ＿0001707 in ESCC tissues and verification of the general characteristics as a circularRNA1)This study has collected 96 pairs of ESCC tissues and their adjacent normal tissues,the expression level of hsa＿circ＿0001707 in samples was detected by RT-q PCR.The results showed that compared with the normal tissue,hsa＿circ＿0001707 was upregulated in cancer tissues,the difference was significant(p<0.001).2)The results of conditional logistic regression analysis showed that the upregulated hsa＿circ＿0001707 in tissues could increase the risk of ESCC as a hazard factor,the OR value is 2.323.The results of ROC curve suggested that hsa＿circ＿0001707 was of great value in diagnosis of ESCC,the AUC was 0.793(95%CI: 0.730～0.855),sensitivity was0.735,specificity was 0.724,the difference was significant(p<0.001).3)The results of Rnase R assay and actinomycin D inhibition test suggested that hsa＿circ＿0001707 had higher stability than its linearRNA,ABCA13.4)Agarose gel electrophoresis results,in combination with sanger sequencing,confirmed that hsa＿circ＿0001707 was an intronic circRNA derived from its maternal gene ABCA13.2.The effects of hsa＿circ＿0001707 on ESCC cell lines1)The expression level of hsa＿circ＿0001707 was further detected in various kinds of ESCC cell lines,including EC109,EC9706 and KYSE510,as well as the normal esophageal epithelial cell,Het-1A.qRT-PCR results indicated that hsa＿circ＿0001707was statistically expressed higher in tumor cells than that in normal cell(p<0.05).2)Lentivirus vectors have been constructed and transfected into EC109 and EC9706 cells respectively,the transfection efficiency was detect by qRT-PCR.Results showed that in EC109 and EC9706 cells,the hsa＿circ＿0001707 expression level in overexpression group(lv-circ-0001707)was 10 and 40 times than that in control group(lv-NC).Meanwhile,the expression level of hsa＿circ＿0001707 in interference group(sh-circ-0001707)was significantly knocked down 65% and 75%,compared to the negative control(sh-NC)(p<0.05).3)Hsa＿circ＿0001707 overexpression could enhance the cell viability and proliferative capacity,promote cell invasion and metastasis,increase the colony-formation ability.As the results of CCK-8 showed,the OD value in lv-circ-0001707 group was consistently higher than that in lv-NC group at the time of 24 h,48h,72 h,96h(p<0.05,n=6).Ed U results suggested that hsa＿circ＿0001707 overexpression could enhance cell proliferative capacity.Transwell results indicated that hsa＿circ＿0001707 upregulation could significantly promote cell migration and invasion(p<0.05).Compared with the negative control,numbers of colony-formation were obviously increased in lv-circ-0001707 group(p<0.05).4)When the expression level of hsa＿circ＿0001707 was interfered,cell viability,proliferation,colony-formation ability,invasion and metastasis were inhibited respectively(p<0.05),which suggested that hsa＿circ＿0001707 may play a role in promoting tumor progression.3.Study of molecular mechanism of hsa＿circ＿0001707 as a “miRNA sponge”1)The results of FISH and nuclear cytoplasmic separation test showed that hsa＿circ＿0001707 was mainly distributed in cytoplasm,less in nuclear,which suggested that cytoplasm was the main site for it to function.2)Hsa＿circ＿0001707 could competitively interact with miR-203a-3p in a ceRNA manner: qRT-PCR results showed that interfered the expression level of hsa＿circ＿0001707 could significantly upregulate the expression of miR-203a-3p(p<0.05).To detect the direct interaction between two molecules,the wild type(WT)and mutant type(MUT)reporter gene vectors of hsa＿circ＿0001707 were constructed.DLR results showed that the fluorescence signal of group hsa＿circ＿0001707 WT+miR-203a-3p was significantly lower than that of other groups.Compared with the miR-NC group,it was reduced 37%;compared with the hsa＿circ＿0001707 Mut+miR-203a-3p group,it was reduced 41%,the differences were significant(p<0.001).3)Rescue assays were conducted and the results of CCK-8,colony-formation assay and transwell analysis manifested that the effects of hsa＿circ＿0001707 on ESCC could be partly attenuated by miR-203a-3p.4)Hsa＿circ＿0001707/miR-203a-3p/Snail2 axis is involved in regulating the process of epithelial-mesenchymal transition in ESCC: Firstly,the public bioinformatics databases,combined with data of differentially expressed proteins from TCGA database,were predicted and screened,Snail2 was discovered as the downstream gene of miR-203a-3p.Furthermore,miR-203a-3p mimic/NC were transfected into EC109 and EC9706 cells,western blot and qRT-PCR results indicated that the expression levels of Snail2 gene were inhibited when the miR-203a-3p expression was downregulated(p<0.05).Therefore,DLR assay was conducted to further verify the binding relationship,results showed that the fluorescence value was significantly decreased in miR-203a-3p+Snail2(WT)group,compared with miR-NC group,it reduced 40%;compared with miR-203a-3p+Snail2(Mut)group,it reduced 48%(p<0.001).These results verified that miR-203a-3p could directly interact with Snail2 through the 3’UTR binding sequences.Based on these results,in order to further verify that hsa＿circ＿0001707 could “sponge”miR-203a-3p with the competition of Snail2,two ESCC cells were applied to perform DLR assay.The experiment was devided into two groups:(1)lv-circ-0001707+Snail2(WT)+miR-203a-3p;(2)lv-NC+Snail2(WT)+miR-203a-3p.Results suggested that compared with the first group,the fluorescence value in group(2)in EC109 was significantly reduced 31% and was decreased 27% in EC9706(p<0.001).Meanwhile,western blot showed that hsa＿circ＿0001707 downregulation could result in the inhibition of Snail2 expression,the protein expression levels in EC109 and EC9706 were reduced by 40% and 48% respectively(p<0.05).miR-203a-3p mimic/NC were transfected into two kinds of ESCC cell lines,western blot was applied to detect the expression of EMT marker proteins.Results suggested that compared with the miR-NC group,miR-203a-3p mimic in EC109 significantly increased E-cadherin expression,with the protein expression level increased 30%.While reduced N-cadherin and Vimentin expression,the protein expression level was decreased 40%,32% respectively(p<0.05).In EC9706,the protein expression level of E-cadherin in miR-203a-3p mimic group was upregulated 31%,N-cadherin and Vimentin was downregulated 35%,44% respectively(p<0.05).Inhibitors of miR-203a-3p were transfected into hsa＿circ＿0001707 stable interference cells,results of western blot manifested when the expression of hsa＿circ＿0001707 was restrained,the expression levels of N-cadherin and Vimentin were decreased,compared with the negative control,the protein expression level in EC109 was decreased 55%,63%,in EC9706 was decreased 57%,60%(p<0.05).While the E-cadherin expression levels were increased 30%,35% respectively,which suggested that hsa＿circ＿0001707downregulation could inhibit the progression of EMT in ESCC.These phenomenon could partly reversed by miR-203a-3p inhibitors,as the results showed,in miR-203a-3p inhibitor group,the protein level of E-cadherin expression in EC109 and EC9706 was reduced by 20%,17%(p<0.05).By comparison,the protein levels of N-cadherin and Vimentin were recovered slightly,it was increased 27%,29% in EC109 and 20%,17% in EC9706 respectively(p<0.05).Conclusions:1.Hsa＿circ＿0001707 is upregulated in ESCC tissues,the high expression level of hsa＿circ＿0001707 could increase the risk of ESCC,as well as improve the diagnostic accuracy,which is regarded as a potential biomarker.2.Hsa＿circ＿0001707 overexpression increases cell viability,cell proliferation ability,enhances colony-formation ability and promotes cell invasion and metastasis,acting as an oncogene in esophageal carcinoma cells.3.Hsa＿circ＿0001707 could serve as a “molecular sponge” of miR-203a-3p,regulate downstream gene Snail2 indirectly.The hsa＿circ＿0001707/miR-203a-3p/Snail2 signal axis participates in the regulation of EMT process in ESCC.