CircCRIM1 Regulates TCF12 By Interacting MiR-342-3p To Promote Epithelial-mesenchymal Transition Of Esophageal Squamous Cell Carcinoma

Circular RNA(circ RNA)is a new class of endogenous non-coding RNA,which is an important molecular type involved in cell epiregulation and plays a crucial role in the occurrence and development of malignant tumors.Studies have revealed that circCRIM1 can play as an oncogene or tumor suppressor in different malignant tumors.However,circCRIM1 has not been reported in esophageal squamous cell carcinoma(ESCC).In this study,the different expression of circCRIM1 in ESCC tissues and para-cancerous tissues was discovered.The effects of circCRIM1 on proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of ESCC cells were analyzed,and the possible mechanism of circCRIM1 in ESCC was preliminarily explored.Part One The expression and clinical significance of circCRIM1 in esophageal squamous cell carcinoma tissuesObjective: To investigate the expression of circular RNA cysteine-rich transmembrane BMP regulator 1(circCRIM1)in ESCC tissues,and to examine its relationship with clinicopathological characteristics and prognosis of ESCC patients.Methods: 1.Fifty pairs of ESCC tissues and para-cancerous tissues were collected from patients who underwent surgical resection in the Department of Thoracic Surgery of the Fourth Hospital of Hebei Medical University between January 2020 and February 2021.The expression of circCRIM1 in ESCC tissues and para-cancerous tissues was detected by quantitative real-time polymerase chain(q RT-PCR).2.According to the expression level of circCRIM1 in ESCC tissues,a Chi-square test was performed to determine the relationships between the expression of circCRIM1 in ESCC tissue and clinicopathological parameters.3.The expression of circCRIM1 and the survival of ESCC patients were analyzed using Kaplan-Meier analysis.4.Univariate and multivariate Cox proportional hazard regression models were utilized to examine the potential factors affecting the postoperative prognosis of ESCC patients.Results: 1.The results of q RT-PCR showed that circCRIM1 expression wassignificantly higher in ESCC tissues than that in para-cancerous tissues(P<0.001).2.An analysis of the relationship between circCRIM1 expression andclinical pathological parameters was carried out in ESCC patients.Circ CRIM1 expression level was significantly related to tumor invasion range,TNM stageand lymph node metastasis in ESCC patients(all P<0.05).3.High expression of circCRIM1 was significantly associated with poorprognosis in patients of ESCC analyzed by Kaplan-Meier(P<0.01).4.Univariate analysis found that high expression of circCRIM1,TNMstage,and lymph node metastasis(P<0.05)were important factors for theprognosis of patients with ESCC.Moreover,the expression of circCRIM1 andTNM stage were independent prognostic factors for patients with ESCCestimated by multivariate Cox proportional risk regression analysis(P<0.05).Summary: 1.The expression of circCRIM1 in ESCC tissues is higher than that in para-cancerous tissues.2.Circ CRIM1 expression is closely associated with tumor invasion range,TNM stage,and lymph node metastasis of patients with ESCC,and its high expression is an independent risk factor for poor prognosis of ESCC patients.Part Two Effects of circCRIM1 on the biological function of esophageal squamous cell carcinoma cellsObjective: To study the effects of knockdown of circCRIM1 on malignant biological behaviors such as proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of ESCC cells.Methods: 1.The expression of circCRIM1 in normal esophageal epithelial cell(Het-1A)and ESCC cell lines(TE1,KYSE30,KYSE410 and KYSE150)was detected using q RT-PCR.TE1 and KYSE30 cell lines with higher circCRIM1 expression were chosen for further experiments.2.The cyclized splicing junction sequence of circCRIM1 was determined by Sanger sequencing.3.Divergent and convergent primers were designed respectively.Complementary DNA(c DNA)and genomic DNA(g DNA)were amplified by PCR,and an agarose gel electrophoresis was performed to separate the products.4.q RT-PCR was used to detect the changes in the expression level of circCRIM1 and its parent m RNA in ESCC cells after the addition of RNase R.5.The nuclear-plasma separation experiment and fluorescence in situ hybridization(FISH)experiment were utilized to detect the subcellular localization of circCRIM1 in ESCC cells.6.The ESCC cells with circCRIM1 knockdown were successfully constructed by small interfering RNA(siRNA),and q RT-PCR was utilized to examine the transfection efficiency.7.CCK-8 and colony formation assays were utilized to determine the effect of knockdown of circCRIM1 on the proliferation of TE1 and KYSE30 cells.8.Wound healing and transwell assays were used to detect the effect of knockdown of circCRIM1 on the migration and invasion of TE1 and KYSE30 cells.9.Western-blot assay was performed to examine the expression of EMT- related proteins of TE1 and KYSE30 cells with circCRIM1 knockdown.Results: 1.Compared with normal esophageal epithelial cell(Het-1A),the expression levels of circCRIM1 in ESCC cells TE1,KYSE30,KYSE410,KYSE150 were significantly upregulated(P<0.01).TE1 and KYSE30 cells with relatively higher expression levels were selected for further study.2.Sanger sequencing confirmed the cyclic splicing junction sequence of circCRIM1.3.The results of agarose gel electrophoresis experiments showed that c DNA and g DNA of TE1 and KYSE30 cells were used as templates,and the circular transcript could only be amplified in c DNA by divergent primers,while the linear transcript could be amplified in both c DNA and g DNA by convergent primers.4.q RT-PCR results showed that after RNase R digestion,the expression level of the parent m RNA was significantly decreased(P<0.001),while the expression level of circCRIM1 did not change significantly(P>0.05).5.The nuclear-plasma separation experiment and fluorescence in situ hybridization(FISH)experiment confirmed that circCRIM1 was primarily found in the cytoplasm.6.q RT-PCR analysis indicated that circCRIM1 expression in si-circCRIM1 group was signally lower than that in the control group after knockdown of circCRIM1(P<0.001),suggesting that the ESCC cell lines with knockdown of circCRIM1 were successfully constructed.7.CCK-8 and plate colony formation assays revealed that knockdown of circCRIM1 could inhibit the proliferation of TE1 and KYSE30 cells(P<0.01).8.Wound healing and transwell assays found that knockdown of circCRIM1 could inhibit the migratotry and invasive capabilities of TE1 and KYSE30 cells(P<0.05).9.Western-blot results demonstrated that when circCRIM1 was knocked down in TE1 and KYSE30 cells,the expression of epithelial cell marker(Ecadherin protein)was up-regulated,while the expression of mesenchymal cellmarkers(N-cadherin protein and Vimentin protein)was down-regulated.Summary: 1.Circ CRIM1 is highly expressed in ESCC cells.2.Circ CRIM1 knockdown can suppress the proliferation,migration,invasion and EMT of ESCC cells.Part Three circCRIM1 promotes epithelial-mesenchymal transformation of esophageal squamous cell carcinoma through miR-342-3p/TCF12 axisObjective: To explore the possible mechanism that circCRIM1 plays an oncogene in ESCC by absorbing miR-342-3p to regulate its downstream target gene transcription factor 12(TCF12)and then promoting the EMT process.Methods: 1.miRanda,RNAhybrid and star Base online databases were used to predict miRNAs that might interact with circCRIM1.The miRNA with the highest binding potential was screened out according to the binding force score.2.q RT-PCR was performed to quantify the expression of miR-342-3p in ESCC tissues,and the correlation between miR-342-3p and circCRIM1 expression level was analyzed.3.The subcellular localization of circCRIM1 and miR-342-3p in TE1 and KYSE30 cells was determined by FISH.4.We utilized dual luciferase reporter gene assay and RNA Binding Protein Immunoprecipitation(RIP)assay to confirm the interaction between circCRIM1 and miR-342-3p.5.q RT-PCR was performed to quantify the expression of miR-342-3p in ESCC cells.6.We utilized CCK-8 and plate colony formation assays to estimate the effect of miR-342-3p mimic on the proliferation of TE1 and KYSE30 cells.7.We performed wound healing and transwell assays for detecting the effect of miR-342-3p mimic on the migration and invasion of TE1 and KYSE30 cells.8.We employed miRWALK,miRDB,star Base and Target Scan databases to predict the downstream target genes that could bind to miR-342-3p.9.We made a dual luciferase reporter gene assay to verify whether miR-342-3p could bind to TCF12.10.q RT-PCR was used to detect the changes of TCF12 m RNA expression levels in TE1 and KYSE30 cells after transfection of miR-342-3p mimic.11.Western-blot method was performed to examine the changes of TCF12 protein expression level in TE1 and KYSE30 cells after transfection of miR-342-3p mimic.12.Rescue experiments were performed in TE1 and KYSE30 cells,respectively.They were divided into si-NC+miRNA inhibitor NC,si-circCRIM1+miRNA inhibitor NC and si-circCRIM1+miR-342-3p inhibitor groups.The effects of circCRIM1 knockdown and transfection of miR-342-3p inhibitor on the proliferation,migration and invasion of ESCC cells were detected by rescue experiments.13.Western-blot method was used to examine the proteins expression changes of TCF12 and EMT-related factors E-cadherin,N-cadherin and Vimentin in si-NC+miRNA inhibitor NC,si-circCRIM1+miRNA inhibitor NC and si-circCRIM1+miR-342-3p inhibitor group of ESCC cells.Results: 1.Through miRanda,RNAhybrid and star Base database predictions,miR-342-3p was finally selected as our study object based on the binding capacity scores and references to relevant literature.2.q RT-PCR results showed that a significant reduction in miR-342-3p level was observed in ESCC tissues(P<0.001)and the expression of miR-342-3p negatively correlated with the expression of circCRIM1(P<0.001).3.FISH assay results suggested that circCRIM1 and miR-342-3p were colocated in the cytoplasm of TE1 and KYSE30 cells.4.Dual-luciferase reporter gene assay and RNA binding protein immunoprecipitation assay results revealed that circCRIM1 could interact with miR-342-3p as a ce RNA in TE1 and KYSE30 cells(P<0.001).5.q RT-PCR results demonstrated that compared with normal esophageal epithelial cell Het-1A,miR-342-3p expression levels in ESCC cells TE1,KYSE30,KYSE410 and KYSE150 were significantly reduced(P<0.001).6.CCK-8 and plate colony formation assays results revealed that miR-342-3p mimic inhibited the proliferation of TE1 and KYSE30 cells(P<0.01).7.The results of wound healing and transwell assays showed that miR-342-3p mimic suppressed the migration and invasion of TE1 and KYSE30 cells(P<0.05).8.miRWALK,miRDB,star Base and Target Scan online databases predicted the target genes that can bind to miR-342-3p.By overlapping and references to relevant literature,we finally confirmed TCF12 as a downstream target gene for miR-342-3p.9.Dual luciferase reporter gene assay revealed that miR-342-3p could interact with TCF12(P<0.001).10.q RT-PCR results found that miR-342-3p mimic could decrease TCF12 m RNA expression in TE1 and KYSE30 cells(P<0.001).11.Western-blot results revealed that miR-342-3p mimic could downregulate the expression of TCF12 protein in TE1 and KYSE30 cells.12.It was demonstrated by rescue experiments that circCRIM1 knockdown could suppress the proliferation,migration and invasion of ESCC cells,while transfection of miR-342-3p inhibitor at the same time could partially reverse these phenomenons(P<0.01).13.Western-blot analysis showed that compared with the control group,the expression of TCF12 and N-cadherin and Vimentin protein were significantly downregulated and E-cadherin expression was upregulated after knocking down circCRIM1.Knockdown of circCRIM1 and transfection of miR-342-3p inhibitor at the same time partially restored TCF12,E-cadherin,Ncadherin and Vimentin expression levels in TE1 and KYSE30 cells.Summary: 1.miR-342-3p is circCRIM1’s target miRNA and it is significantly downregulated in ESCC tissues.miR-342-3p can inhibit the proliferation,migration and invasion abilities of ESCC cells and act as a tumor suppressor in ESCC.2.Circ CRIM1 can act as a "molecular sponge" for miR-342-3p and indirectly regulate the expression of its downstream target gene TCF12.The circCRIM1/miR-342-3p/TCF12 axis may take part in regulating epithelialmesenchymal transformation in ESCC cells.Conclusions: 1.Circ CRIM1 expression is increased in ESCC tissues,and its high expression is closely correlated with a poor prognosis in ESCC patients.2.Circ CRIM1 promotes the EMT process of ESCC by upregulating TCF12 through sponging miR-342-3p,which promotes the malignant progression of ESCC.