| Part One Expression and clinical significance of circCYP24A1 in esophageal squamous cell carcinomaObjective: Esophageal Squamous Cell Carcinoma(ESCC)is a common digestive system malignant tumor,whose molecular mechanism of carcinogenesis and progression has not been fully elucidated.Circular RNAs(circRNAs)participate in the occurrence and development of malignant tumors through a variety of molecular mechanisms,while the clinical significance and functional mechanism of circRNAs involved in the malignant progression of ESCC are still unclear.In this study,the high-expressed circCYP24A1 in ESCC was screened by transcriptome high-throughput sequencing,and its expression and clinical significance in ESCC tissues and cells were explored.Methods:1.Five pairs of fresh ESCC tissues and their corresponding normal esophageal tissues were screened for highly expressed circRNAs by transcriptome high-throughput sequencing.2.Convergent and divergent primers were designed,and the expression and cyclization sites of circCYP24A1 in ESCC cells were identified by RT-PCR and Sanger sequencing.3.The cyclization characteristics of circCYP24A1 were identified by treated with RNA exonuclease R and actinomycin D.4.RNA FISH assay was used to detect the expression of circCYP24A1 in paraffined tissue microarray containing 114 ESCC tissues and corresponding66 paracancerous tissues.The relationship between circCYP24A1 expression and clinical parameters with prognosis of ESCC patients was analyzed.Results:1.The transcriptome high-throughput sequencing results showed that compared with normal tissues,5248 circRNAs were up-regulated and 5385 circRNAs were down-regulated in ESCC group;the significantly up-regulated circCYP24A1(log2Fold Change=5.8451,P<0.0001)was selected for further research.2.Sanger sequencing was carried out on the fragment sequence amplified by the circCYP24A1 convergent primer,and the result was compared with that in circbase database,which confirmed the existence of circCYP24A1.3.Genomic DNA amplification,RNA exonuclease RNase R and actinomycin D treatment were used to confirme the circular characteristic of circCYP24A1.4.The results of RNA FISH showed that circCYP24A1 was highly expressed in ESCC tissues and correlated with poor prognosis,which was an independent prognostic factor of ESCC patients(P<0.05).Summary:1.The differentially expressed circRNA in ESCC was identified by high-throughput sequencing technology,and the highest expressed circCYP24A1 was selected for the study.2.The cyclization characteristics of circCYP24A1 were confirmed by Sanger sequencing,RNase R digestion and actinomycin D treatment.3.Circ CYP24A1 was highly expressed in ESCC tissues and associated with poor prognosis of ESCC patients.Part Two Effect of circCYP24A1 on biological behavior of esophageal squamous carcinoma cellsObjective: To explore the effect of circCYP24A1 on the proliferation,colony formation,migration and invasion abilities of ESCC cells.Methods:1.KYSE30 and KYSE150 with relatively high-expressed circCYP24A1 in ESCC cell lines were selected to transfecting with si-NC and si-circCYP24A1.RT-qPCR was used to detect knockdown efficiency of circCYP24A1.2.KYSE170 and TE1 with relatively low-expressed of circCYP24A1 in ESCC cell line were selected to transfecting with empty vector p LC5-ci R and the overexpression vector p LC5-ci R-circCYP24A1,respectively.After screening with purinomycin,the overexpression efficiency was detected by RT-qPCR.3.CCK-8 assay,clonoe formation assay,scratch healing assay,Transwell assay and reverse invasion assay were used to detect the effects of circCYP24A1 knockdown or overexpression on ESCC cell proliferation,colony formation,migration and invasion abilities.Results:1.The results of RT-qPCR showed that the expression level of circCYP24A1 in KYSE30 and KYSE150 cells was significantly reduced after transfecting with si-circCYP24A1(P<0.05),while the expression level of parental gene CYP24A1 was not affected(P>0.05).2.The results of RT-qPCR showed that the expression level of circCYP24A1 in KYSE170 and TE1 cells was significantly increased after transfecting with circCYP24A1 and screening with purinomycin(P<0.05),while the expression level of parental gene CYP24A1 was not affected(P>0.05).3.The results of CCK-8 assay showed that circCYP24A1 knockdown could inhibit the proliferation ability of KYSE30 and KYSE150 cells(P<0.05);Overexpression of circCYP24A1 could enhance the proliferation ability of KYSE170 and TE1 cells(P<0.05).4.The results of colony formation assay showed that knockdown of circCYP24A1 could inhibit the colony formation ability of KYSE30 and KYSE150 cells(P<0.05);Overexpression of circCYP24A1 could enhance the colony formation ability of KYSE170 and TE1 cells(P<0.05).5.The results of scratch healing assay,Transwell assay and reverse invasion assay showed that knockdown of circCYP24A1 could inhibit the migration and invasion ability of KYSE30 and KYSE150 cells(P<0.05);overexpression of circCYP24A1 could enhance the migration and invasion ability of KYSE170 and TE1 cells(P<0.05).Summary:1.ESCC cells transfected with si RNA or overexpression vector targeting circCYP24A1 could significantly inhibit or promote the expression of circCYP24A1,while the expression level of parental gene CYP24A1 was not affected.2.Knockdown of circCYP24A1 can significantly inhibit the proliferation,colony formation,migration and invasion abilities of ESCC cells.3.Overexpression of circCYP24A1 can significantly promote the proliferation,colony formation,migration and invasion abilities of ESCC cells.Part Three circCYP24A1 affects the progression of esophageal squamous cell carcinoma by promoting the autocrine of CCL5Objective: The m RNA expression profile microarray was used to screen the downstream target genes and signal pathways of circCYP24A1,which could explore the molecular mechanism of circCYP24A1 promoting the progress of ESCC.Methods:1.After transfecting with si-NC and si-circCYP24A1 in KYSE150 cells,Agilent expression microarray was used to screen downstream genes and signal pathways related to circCYP24A1.The genes(ATF3,KLF6,DUSP1,BCOR,RSAD2,CCL5,CCL19)with higher multiple of variation were selected for validation by RT-qPCR.2.The effects of knockdown or overexpression of circCYP24A1 on the secretion of CCL5 in ESCC cells was detected by ELISA assay.3.CCK-8 assay,colony formation assay,scratch healing assay,Transwell assay and reverse invasion assay were used to detected the effects of exogenous addition of recombinant protein CCL5 on the proliferation,colony formation,migration and invasion abilities of KYSE150 and TE1 cells.4.Recombinant protein CCL5 was exogenously added in ESCC cells with knockdown of circCYP24A1.CCK-8 assay,colony formation assay,scratch healing assay,Transwell assay and reverse invasion assay were used to detected the biological functions of ESCC cells.Results:1.The results of Agilent expression profiling microarray showed that 265 genes were up-regulated and 219 genes were down-regulated in si-circCYP24A1 group compared with si-NC group.The regulatory pathways involved in circCYP24A1 mainly focused on IL-17 signaling pathway,Jak-STAT signaling pathway,MAPK signaling pathway,TGF-β signaling pathway,NF-κ B signaling pathway and so on.2.Genes with higher fold changes were selected for validation by RT-qPCR and some results were consistent with the above results,and the most significantly changed CCL5 was selected as the target gene of circCYP24A1 for further studies.3.The results of ELISA assay showed that the amount of CCL5 protein expression in the supernatant of KYSE30 and KYSE150 cells was significantly decreased after knockdown of circCYP24A1(P<0.05),whereas it was significantly increased in the supernatant of KYSE170 and TE1 cells after overexpression of circCYP24A1(P<0.05).4.The results of CCK-8 assay,colony formation assay,scratch healing assay,Transwell assay and reverse invasion assay showed that KYSE150 and TE1 cell proliferation,colony formation,migration and invasion abilities were significantly increased after exogenous addition of CCL5 compared with control group(P<0.05).5.The results of rescue experiments targeting circCYP24A1 and CCL5 showed that the proliferation,colony formation,migration and invasion abilities in si-circCYP24A1 group were significantly reduced compared with control group and si-circCYP24A1 while added with CCL5(P<0.05).Summary:1.CCL5 may serve as a downstream target gene of circCYP24A1 involved in ESCC malignant progression.2.Exogenous addition of CCL5 promoted proliferation,colony formation,migration and invasion abilities of ESCC cells.3.The addition of CCL5 partly reversed the inhibition of cell proliferation and colony-forming ability induced by circCYP24A1 knockdown in ESCC cells.Part Four Mechanism of circCYP24A1 promotes esophageal squamous cell carcinoma progression by binding PKM2Objective: To explore the mechanism of circCYP24A1 binding with PKM2 to regulate ESCC progression.Methods:1.The subcellular localization of circCYP24A1 was examined in ESCC cells by RNA FISH.2.The binding proteins of circCYP24A1 was analyzed by RNA pulldown combined with protein mass spectrometry assay.Western blot assay was used to identify the candidate proteins.3.RNA FISH-IF assay was used to detecte the subcellular localization of circCYP24A1 and PKM2 in KYSE30 and TE1.4.The effects of circCYP24A1 and PKM2 on NF-κB pathway were determined by Western blot assay.5.RT-qPCR and ELISA assay were used to detect the effect of overexpression of circCYP24A1 while knocking down PKM2 on CCL5 secretion in ESCC cells.6.PKM2 was knocked down simultaneously in ESCC cells overexpressed with circCYP24A1,and changes in the biological function of ESCC cells were detected by CCK-8 assay,colony formation assay,scratch healing assay,Transwell assay and reverse invasion assay.7.The NOD-SCID mice xenograft tumor model was constructed by using KYSE170 cell line that stably overexpressed circCYP24A1.Transplanted tumors from NOD-SCID mice of each group were measured for the growth.IHC staining mothed was used to detect the expression of p65/NF-κB,p-p65/NF-κB and CCL5 in each group of transplanted tumors.Results:1.The results of RNA FISH showed that circCYP24A1 is mainly localized in the cytoplasm of ESCCs.2.The results of RNA pulldown combined mass spectrometry and Western blot assays showed that circCYP24A1 could bind to PKM23.The results of RNA FISH-IF assay showed that circCYP24A1 and PKM2 were both localized in cytoplasm.4.Western blot analysis showed that,compared with the control group,the expression of p-p65/NF-κB was decreased after knocking down PKM2alone;the expression of p-p65/NF-κB was increased after overexpression of circCYP24A1;overexpression of circCYP24A1 while knocking down PKM2 could partially reverse this change.5.The results of RT-qPCR and ELISA showed that after overexpression of circCYP24A1,the expression of CCL5 in KYSE170 and TE1 cells was significantly increased compared with control group(P<0.05).After knocking down PKM2,the expression of CCL5 in KYSE170 and TE1 cells was significantly reduced compared with control group(P<0.05).Compared with knockdown of PKM2,overexpression of circCYP24A1 while knocking down PKM2 could partially reverse the expression of CCL5(P<0.05).6.The results of CCK-8 assay,colony formation assay,scratch healing assay,Transwell assay and reverse invasion assay showed that compared with control group and overexpression of circCYP24A1 while si-PKM2 group,the proliferation,clonal formation,migration and invasion ability of KYSE170 and TE1 cells in the overexpression of circCYP24A1 group were significantly increased(P<0.05).The proliferation,clonal formation,migration and invasion capabilities of KYSE170 and TE1 cells in the si-PKM2 group were significantly reduced(P<0.05).7.The results of in vivo experiments showed that compared with control group and overexpression of circCYP24A1 while si-PKM2 group,the volume,weight,p-p-p65/NF-κB protein level and CCL5 protein level of graft tumors were increased significantly in overexpression of circCYP24A1 group(P<0.05).The volume,weight,p-p65/NF-κB protein level and CCL5 protein level of transplanted tumors were significantly reduced in si-PKM2 group(P<0.05).Summary:1.cric CYP24A1 was interacted with PKM2 and co-located in the cytoplasm of ESCC cells.2.Knockdown PKM2 significantly inhibited xenograft tumor growth promoted by circCYP24A1 overexpression.3.In vitro and in vivo assays validated that circCYP24A1 interacting with PKM2 to activate NF-κB pathway,which promotes the secretion of CCL5.Conclusions:In the process of ESCC occurrence and development,the high expression of circCYP24A1 is associated with poor prognosis of ESCC patients,and can promote the proliferation,clonal formation,migration and invasion ablities of ESCC cells.The study revealed that up-regulated circCYP24A1 could activate NF-κB pathway by binding PKM2,which promotes the autocrine of CCL5 and accelerate malignant progression of ESCC. |
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