Effect Of CircRPL13 On Malignant Biological Behavior Of Esophageal Squamous Cell Carcinoma And Its Molecular Mechanism

Esophageal squamous cell carcinoma（ESCC）is the most common malignant tumor of digestive system in China.Due to its high incidence and poor prognosis,it has been regarded as a major public health problem.There are obvious geographical differences in the histological types of esophageal cancer.Most esophageal adenocarcinomas occur in developed countries,while the incidence of ESCC in China accounts for more than 90%.Owing to the lack of early specific symptoms,a large number of patients with ESCC are diagnosed with late stage or distant metastasis,and the prognosis is poor.Despite the application of new tumor markers and advanced therapeutic strategies such as targeted therapy and immunotherapy,the 5-year survival rate for ESCC remains low.Local recurrence and distant metastasis are common even after surgical resection.Therefore,there is an urgent need to clarify the molecular mechanism of the occurrence and metastasis of ESCC,and determine more specific and effective biomarkers for targeted therapy of the maliganancy.Circular RNA（circRNA）is a class of endogenous non-coding RNAs with covalently closed circular structures formed by end-end splicing of the exons of precursor m RNA.In recent years,with the rapid development of bioinformatics and high-throughput sequencing technologies,expression profiles of circRNAs associated with a variety of diseases have been reported.Through bioinformatics analysis and functional studies,it has been found that many differentially expressed circRNAs are closely related to the occurrence and development of diseases,especially cancer.In this study,through the analysis of GEO database,it was found that circRPL13（hsa_circ₀092337）was underexpressed in both blood samples and tumor tissue samples of ESCC.However,whether it plays a role in the occurrence and progression of ESCC and its potential molecular mechanism remain unclear.In this study,a series of in vitro and in vivo experiments were conducted to investigate the effects of circRPL13 on the proliferation,apoptosis,migration and invasion of ESCC cells in vitro and their growth in vivo,and to reveal its potential molecular mechanism of action,in order to provide experimental reference for finding new clinical therapeutic targets for ESCC.Part One Expression and clinical significance of circRPL13 in ESCC Objective: This study was conducted to explore the differentially expressed circRNAs in ESCC by analyzing GEO database,and investigate the relationship between circRPL13 expression and overall survival rate and clinicopathologic features of ESCC.Methods:1.Circ RNAs differentially expressed in ESCC were screened by analyzing the circRNA chip data（GSE131969 and GSE112496）related to ESCC in GEO database.2.ESCC cells were treated with RNase R and actino-mycin D,and the expression of circRPL13 and linear RPL13 in the cells was detected by real-time quantitative PCR（qRT-PCR）to analyze the expression stability of circRPL13.3.qRT-PCR was used to detect the expression of circRPL13 in serum and tumor tissue of 50 patients with ESCC.K-M survival curve was used to analyze the relationship between circRPL13 expression in serum and tissue and overall survival rate of patients with ESCC,and the relationship between circRPL13 expression and clinicopathologic features of patients with ESCC.Results:1.Circ RPL13 is down-regulated circRNA in the blood and tumor tissues of patients with ESCC.2.Compared with linear RPL13,the expression of circRPL13 in esophageal squamous cell cells showed better stability.3.Patients with lower expression of circRPL13 ESCC had lower overall survival rate.4.The low expression of circRPL13 is significantly correlated with lymph node metastasis and TNM stage in patients with ESCC.Compared with paracancer normal tissue,circRPL13 expression in ESCC tumor tissue was significantly decreased,and its decreased expression was associated with more lymph node metastasis.Summurys:The expression of circRPL13 is decreased in the blood and tumor tissues of patients with ESCC,which may be involved in the malignant progression of ESCC and act as a biomarker for prognosis prediction of ESCC.Part Two Effects of circRPL13 on malignant biological behavior of ESCC cells Objective: To investigate the effects of circRPL13 on proliferation,clonogenesis,apoptosis,cell cycle,cell migration and invasion of ESCC cells by function loss and function gain experiments.Methods:1.The expression of circRPL13 in human esophageal epithelial cell line HET-1A and ESCC cell line TE-1,KYSE-150,ECA-109 and KYSE-410 was detected by qRT-PCR.2.TE-1 cells were transfected with sh-circRPL13 to knock down the expression of circRPL13.Circ RPL13 overexpression plasmid was transfected into KYSE-150 cells to induce circRPL13 expression in the cells.3.The effect of circRPL13 on cell proliferation was detected by Ed U assay and clonal formation assay.4.Flow cytometry was used to evaluate the effect of circRPL13 on apoptosis and cell cycle of ESCC cells.5.Transwell assay was performed to evaluate the effect of circRPL13 on the migration and invasion of ESCC cells.Results:1.Compared with HET-1A cells,the expression levels of circRPL13 in TE-1,KYSE-150,ECA-109 and KYSE-410 were significantly decreased.2.Knockdown of circRPL13 significantly promoted the proliferating ability of TE-1 cells,while overexpression of circRPL13 significantly inhibited the proliferating ability of KYSE-150 cells.3.Circ RPL13 knockdown significantly inhibited the apoptosis of TE-1cells and promoted the cell cycle from G0/G1 phase to S phase.Overexpression of circRPL13 promoted the apoptosis of KYSE-150 cells and blocked the cell cycle in G0/G1 phase.4.The number of migration and invasion of TE-1 cells increased significantly after circRPL13 knockdown.After overex-pression of circRPL13,the number of migration and invasion of KYSE-150 cells was significantly reduced.Summurys:1.There is a reduced expression of circRPL13 in ESCC cells.2.Knockdown of circRPL13 promotes the proliferation and cell cycle progression of cancer cells,inhibits cell apoptosis,and promots cell migration and invasion.3.Overexpression of circRPL13 inhibits the proliferation of cancer cells,blocks the cell cycle,induces cell apoptosis,and inhibits cell migration and invasion.4.It is suggested that circRPL13 is involved in the malignant progression of ESCC and may be a potential therapeutic target for ESCC.Part Three Molecular mechanism of circRPL13 regulating malignant biological behavior of ESCC Objective: To explore the molecular mechanism of circRPL13 regulating malignant biological behavior of ESCC cells.Methods:1.The expression and localization of circRPL13 in ESCC cells were analyzed by cytoplasmic localization experiment.2.The online databases circMine and circ Bank were used to analyze the miRNAs with complementary binding sites of circRPL13,and the online databases Target Scan and miRDB were used to analyze the miRNAs with complementary binding sites of CDKN2 A 3’UTR,and the two parts of data were analyzed by Wayne diagram.Screening miRNAs that can bind to both circRPL13 and CDKN2 A 3’UTR.3.The target miRNA was further screened by RNA pull down,and the targeted binding of circRPL13 and miR-9-5p/miR-9-5p and CDKN2 A 3’UTR was verified by RIP and double luciferase reporter assay.4.CDKN2 A m RNA and protein expression in ESCC tissues and cells were detected by qRT-PCR and Western blot.The relationship between CDKN2 A expression in tumor tissues and overall survival rate was analyzed by K-M survival curve.5.Pearson correlation analysis was performed to determine the relationship between circRPL13 expression and CDKN2 A expression in tumor tissues.The effect of circRPL13 on CDKN2 A expression in TE-1 and KYSE-150 cells was determined by qRT-PCR and Western blot.Sh-CDKN2 A and CDKN2 A overexpression plasmid were transfected into Circrpl13-downregulated and Circrpl13-overexpressed cells,respectively,to perform functional rescue experiments.Cell proliferation,clonogenesis,apoptosis,cell cycle,cell migration and invasion were determined by Ed U,clonogenesis,flow cytometry and Transwell assay.6.Dual luciferase reporter gene assay verified the targeted binding of miR-9-5p to CDKN2 A 3’UTR,and qRT-PCR and western blot were used to detect the effect of miR-9-5p on CDKN2 A gene expression in ESCC cells.7.In ESCC cells,circRPL13 acts as a sponge of miR-9-5p to positively regulate CDKN2 A gene expression.8.Inhibition of miR-9-5p could attenuate the promoting effect of circRPL13 knockdown on the proliferation,cycle,migration and invasion of TE-1cells;meanwhile,overexpression of miR-9-5p could attenuate the inhibiting effect of circRPL13 overexpression on the proliferation,cycle,migration and invasion of KYSE-150 cells.9.The effects of circRPL13/miR-9-5p axis on proliferation,clonogenesis,apoptosis,cell cycle,cell migration and invasion of esophageal squamous cell cells were detected by Ed U,clonogenesis,flow cytometry and Transwell assay.Results:1.Circ RPL13 was mainly expressed in the cytoplasm of ESCC cells,and miR-9-5p can bind to circRPL13 and CDKN2 A 3’UTR simultaneously.2.The decreased expression of CDKN2 A in ESCC tissues and cells was associated with poor overall survival of patients.3.There was a significant positive correlation between circRPL13 expression and CDKN2 A expression in ESCC.In ESCC cells,knockdown of circRPL13 inhibited CDKN2 A gene expression,while overexpression of circRPL13 up-regulated CDKN2 A gene expression.4.Overexpression of CDKN2 A can reversed the promoting effect of circRPL13 knockdown on the proliferation,cycle,migration and invasion of TE-1 cells;meanwhile,inhibition of CDKN2 A reversed the inhibiting effect of circRPL13 overexpression on the proliferation,cycle,migration and invasion of KYSE-150 cells.5.In ESCC cells,circRPL13 acted as a sponge of miR-9-5p to positively regulate CDKN2 A gene expression.6.The high expression of miR-9-5p in the tissues and cells of ESCC was associated with poor overall survival.In addition,the expression of miR-9-5p in tumor tissues was negatively correlated with the expression of circRPL13 and CDKN2A.7.Inhibition of miR-9-5p attenuated the promoting effect of circ-RPL13 knockdown on the proliferation,cycle,migration and invasion of TE-1 cells;meanwhile,overexpression of miR-9-5p attenuated the inhibiting effect of circRPL13 overexpression on the proliferation,cycle,migration and invasion of KYSE-150 cells.8.circRPL13/miR-9-5/CDKN2 A axis regulated the expression of proliferation,migration,invasion and apoptosis related proteins in ESCC cells Summurys:1.Circ RPL13 can positively regulate the expression of CDKN2 A in ESCC cells.Interference of CDKN2 A or overexpression of CDKN2 A reverses the effect of circRPL13 on the malignant biological behavior of ESCC.It is suggested that CDKN2 A is one of the downstream effector factors of circRPL13 exerting biological functions.2.Mi R-9-5p can be targeted to circRPL13 and CDKN2 A 3’UTR.Mi R-9-5p can negatively regulate the expression of CDKN2 A in ESCC cells.In addition,the effect of circRPL13 on CDKN2 A expression in cells can be reversed by miR-9-5p,suggesting that circRPL13 can act as a sponge of miR-9-5p and regulate the expression of CDKN2 A gene.3.Circ RPL13 regulates the malignant biological behavior of ESCC cells by targeting miR-9-5p.Part Four Circ RPL13 can inhibit tumorigenicity of ESCC cells in a mouse model Objective: To investigate the effect of circRPL13 on the growth of ESCC cells in vivo.Methods:1.The xenograft tumor mouse model was established by subcutaneous inoculation of TE-1 cells transfected with sh-circRPL13 or KYSE-150 cells transfected with OE-circRPL13.The tumor growth volume in mice was measured every 7 days from the beginning of cell inoculation.On the 28 th day,the mice were euthanized,the transplanted tumors were stripped,photographed,and weighed.2.The expressions of circRPL13,miR-9-5p and CDKN2 A in transplanted tumors were detected by qRT-PCR.3.Expressions of CDKN2 A,Cyclin D1,E-cadherin and Cleaved caspase3 were detected by immunohistochemical staining（IHC）.Results:1.Compared with the control group,the volume growth and weight of the transplanted tumor in the circRPL13 knockdown group were significantly accelerated.In the circRPL13 overexpression group,the tumor volume growth slowed down significantly and the tumor weight decreased.2.The expressions of circRPL13 and CDKN2 A were decreased and the expressions of miR-9-5p were increased in the circRPL13 knockdown group.However,circRPL13 and CDKN2 A expressions were increased and miR-9-5p expression was decreased in the circ-RPL13 overexpression group.3.Positive expressions of CDKN2 A,E-cadherin and Cleaved caspase3 decreased in circRPL13 knockdown group,and positive expression of Cyclin D1 increased.In the circRPL13 overexpression group,positive expressions of CDKN2 A,E-cadherin and Cleaved caspase3 were increased,while positive expression of Cyclin D1 was decreased.Summurys:Knockdown of circRPL13 promotes the growth of ESCC cells in vivo,while overexpression of circRPL13 inhibits the growth of ESCC in vivo.Conclusions:Circ RPL13 may be involved in the malignant progression of esophageal squamous cell carcinoma and is a biomarker for prognosis prediction of esophageal squamous cell carcinoma.Circ RPL13 could act as a sponge for miR-9-5p to regulate CDKN2 A gene expression.Circ RPL13 regulates the malignant biological behavior of esophageal squamous cell carcinoma cells by targeting miR-9-5p.