The Function And Mechanism Of Circular RNA Circ4953 In Promoting The Proliferation And Metastasis Of Colorectal Cancer

BackgroundColorectal cancer(CRC)is one of the most common malignant tumours,the incidence and mortality rate of CRC are the third and second in all malignant tumours,respectively.At present,the treatment of CRC includes surgical resection,chemotherapy,radiotherapy,etc.However,the survival rate of CRC was not significantly improved due to the lack of efficient therapeutic targets,so it is urgent to explore accurate therapeutic targets.The rapid proliferation and metastasis of CRC are the main factors leading to the poor prognosis of patients.Circular RNA(circRNA)plays an essential role in the process of tumour proliferation and metastasis.The closed loop structure of circRNA makes it more stable.In addition,circRNAs are widely and diversely present in eukaryotic cells,with tissue specificity,disease specificity as well as spatiotemporal specificity.Hence,circRNA has the characteristics to be an ideal therapeutic target.However,the clinical significance,regulatory roles and potential mechanisms of circRNAs were still largely unknown.Exploring the roles and mechanisms of key circRNAs in CRC is important for the treatment of tumour-related diseases.PurposeTo explore new circRNAs and detecting their expression in CRC tissue and serum,which provides clues for mining potential diagnostic markers of CRC.Furthermore,the rapid proliferation and metastasis of CRC seriously affecting the prognosis of patients,it is urgent to explore complex regulatory mechanisms of CRC.To provide a theoretical basis and precise therapeutic targets for the treatment of CRC and improve patient prognosis by excavating new circRNAs and revealing functions and mechanisms of circRNAs that regulate CRC progression.Part Ⅰ The screening and expression of circ4953Methods1.The circRNA expression profiles in 4 pairs of CRC tissues and paracancer control tissues were explored using high-throughput sequencing.2.The circRNAs with significant differential expression in high-throughput sequencing were screened via absolute value of fold change>2.0 and P<0.05.3.Screen target molecules based on the expression of circRNAs in 28 pairs of CRC and paracancerous control tissues.4.qRT-PCR was used to detect the expression of circRNA in 40 pairs of CRC and adjacent control,as well as the expression in serum of 19 CRC and 19 healthy controls.5.In situ hybridization(ISH)was used to detect the expression of circRNA in CRC tissue sections.According to ISH to analyze the effect of circRNA expression on the survival and lymph node metastasis of patients.Results1.High-throughput sequencing results showed differences in circRNA expression profiles between CRC tissues and paracancer control tissues.2.Using absolute value of fold change>2.0 and P<0.05 as the screening criteria,33 significantly up-regulated and 47 significantly down-regulated circRNAs were screened out.According to the literature and NCBI gene annotation,8 circRNAs were selected whose parental genes promote CRC proliferation and metastasis.3.The expression of circRNAs was verified in 28 pairs of CRC tissues,and the significantly up-regulated circ4953 was selected.4.Circ4953 was significantly up-regulated in CRC cells and tissues,and was significantly up-regulated in the serum of early CRC patients.5.Survival analysis based on ISH-score showed that high expression of circ4953 was not conducive to the prognosis of CRC patients,and patients with high expression of circ4953 had a higher proportion of lymph node metastasis.Part Ⅱ The structure identification and cell localization of circ4953Methods1.Divergent primers were used to amplify circ4953 in HCT116 cells.The amplified products were used to verify the closed-loop structure of circ4953 by agarose gel electrophoresis,band recovery and Sanger sequencing.2.Amplify circ4953 at the gDNA level and cDNA level using convergent primers and divergent primers,respectively.The amplified products were subjected to agarose gel electrophoresis to verify the closed circular structure of circ4953.3.The tolerance of circ4953 and its linear transcripts to exonuclease(RNase R)was detected by qRT-PCR and nucleic acid electrophoresis.4.The stability of circ4953 was detected by actinomycin D assay.5.RNA in nucleus and cytoplasm was extracted by nucleocytoplasmic separation experiment,respectively.The extracted RNA was used to compare the distribution of circ4953 in the nucleus and cytoplasm via reverse transcription and qRT-PCR.6.The cellular localization of circ4953 was observed by fluorescence in situ hybridization(FISH).SW480 cells were incubated with fluorescently labeled circ4953specific probe and imaged by confocal microscopy.Results1.Sanger sequencing confirmed that the sequence of circ4953 was consistent with RNAseq and the annotation of exoRBase database,indicating that circ4953 was a closed loop structure.2.Convergent primers were successfully amplified at both gDNA and cDNA levels.Divergent primers can only successfully amplify at the cDNA level,but not at the gDNA level,confirming that circ4953 was the closed and circular structure.3.Total RNA extracted from SW480 cells was digested by RNase R,circ4953 decreased slowly,while its corresponding linear transcript AURKA decreased rapidly,indicating that circ4953 was highly tolerant to RNase R.4.After incubation of SW480 cells with actinomycin D,the linear transcript AURKA was degraded rapidly over time,while circ4953 showed good stability.5.The results of RNA nucleoplasm isolation experiments showed that circ4953 was distributed in the cytoplasmic and nucleus in HCT116,SW480 and DLD-1 cells,with a preference in the cytoplasm.6.FISH experiments showed that circ4953 was distributed in both cytoplasm and nucleus in SW480 cells,with a preference in the cytoplasm.Part Ⅲ Biological function of circ4953Methods1.The effect of interference or overexpression of circ4953 on the clone formation ability of HCT116 and SW480 cells was measured by clone formation assay.2.The EdU experiment was used to observe the effect of circ4953 on the proliferation ability of HCT116 and SW480 cells.3.Transwell experiments were used to observe the effect of circ4953 on the migration and invasion abilities of HCT116 and SW480 cells.4.HCT116 cells were transfected with lentivirus to construct cell lines stably overexpressing circ4953 and control cell lines.The nude mouse xenograft tumour model was constructed to compare the volume of subcutaneously transplanted tumours in two groups of nude mice.5.The nude mouse tail vein metastasis model was established by stably overexpressing circ4953 cells and control cells to observe the metastasis of CRC in nude mice.Results1.The clone formation experiments showed that interference or overexpression of circ4953 significantly reduced or increased the clone formation ability of HCT116 and SW480 cells,respectively.2.EdU experiments showed that interference or overexpression of circ4953 significantly decreased or increased the EdU-positive cell rates of HCT116 and SW480 cells,respectively.3.Transwell experiments showed that interference or overexpression of circ4953 inhibited or promoted the migration and invasion abilities of HCT116 and SW480 cells,respectively.4.The nude mouse xenograft tumour model showed that the tumour volume in the overexpression circ4953 group was significantly higher than that in the control group,indicating that circ4953 promotes CRC growth.5.The nude mouse tail vein metastasis model showed that circ4953 promotes CRC metastasis.Part Ⅳ Molecular mechanism exploration of circ4953 functionMethods1.The downstream effector molecules of circ4953 was excavated by transcrip to me sequencing.2.The effect of circ4953 on CRC cell cycle was detected by flow cytometry.3.The RNA binding protein of circ4953 was explored by pull down,mass spectrometry and RNA immunoprecipitation(RIP)experiments.4.The effect of circ4953 on ACLY protein levels and catalytic activity was detected using siRNA,overexpression plasmid,ATP and acetyl coenzyme A kits.5.The effect of circ4953 on CTNNB1 protein level was detected by siRNA and Western blot.6.HCT116 cells were treated with cycloheximide(CHX)to observe the effect of circ4953 on CTNNB1 protein stability.7.IP experiment was used to detect the effect of circ4953 on the ubiquitination level of CTNNB1 protein.8.The effect of circ4953 on the interaction between ACLY and CTNNB1 were examined using CO-IP assay.9.Rescue experiments combined with IP experiments were used to explore whether circ4953 inhibits the degradation of CTNNB 1 by promoting the association of ACLY with CTNNB 1.Results1.Transcriptome sequencing verified by qRT-PCR confirmed that interference with circ4953 caused a significant decrease in CCND1 levels.2.Cell cycle assay results indicated that interference with circ4953 leads to G1 phase block in CRC cells,which may be related to the decrease in CCND1 caused by interference with circ4953.3.Pull down,mass spectrometry and RIP experiments showed that circ4953 can bind to ACLY and CTNNB1 proteins,suggesting that ACLY and CTNNB1 may be the downstream molecules of circ4953.4.Circ4953 has no significant effect on ACLY protein levels and catalytic activity.5.Interference with circ4953 resulted in a significant decrease in the protein level of CTNNB 1.6.CTNNB 1 protein degradation was faster in the interfering circ4953 group after CHX inhibited protein synthesis.7.Overexpression of circ4953 caused a decrease in the level of ubiquitination of CTNNB 1 protein,indicating that circ4953 inhibits the degradation of CTNNB 1.8.CO-IP experiments showed that circ4953 promoted the interaction between ACLY and CTNNB 1 proteins.9.The results of rescue and IP experiments showed that circ4953 prevented the degradation of CTNNB 1 protein by promoting the association of ACLY with CTNNB 1.Part Ⅴ Circ4953 promotes CRC progression dependent on CTNNB1Methods1.Three subgroups of rescue experiments were constructed by siRNA and overexpression plasmid:control group,overexpression circ4953 group and CTNNB 1 interference group based on overexpression circ4953.2.The proliferation ability of the three groups of cells was observed by clone formation experiment and EdU experiment.3.The migration and invasion ability of the three groups of cells were measured by Transwell experiment.4.Three stably transfected cell lines for rescue experiments were constructed by lentiviral transfection of HCT116 cells:control cell,overexpressing circ4953 cell,overexpression of circ4953 with interference of CTNNB 1.Nude mouse xenograft tumour models were constructed by using the above three cell lines to compare the volume of subcutaneously transplanted tumours in three groups of nude mice.And qRT-PCR,Western blot,IHC were used to detect the expression of downstream key molecules in each group.5.The above three cell lines were used to construct the nude mouse tail vein metastasis model.The fluorescence intensity of lung metastases in three groups of nude mice was compared by in vivo imaging technology.HE staining was used to observe the lung metastatic nodules in three groups of nude mice.Results1.The expression level of circ4953 was significantly increased after transfection of the overexpression plasmid,and the expression level of CTNNB1 was significantly decreased after transfection of siRNA.2.The enhanced cell proliferation induced by overexpression of circ4953 could be reversed by interference with CTNNB1 in HCT116 and SW480 cell.It indicated that the function of circ4953 to promote proliferation of CRC cells was dependent on CTNNB1.3.The increase in the number of migration and invasion cells caused by overexpression of circ4953 could be reversed by interference with CTNNB1 in HCT116 and SW480 cell,indicating that the ability of circ4953 to promote migration and invasion of CRC cells was dependent on CTNNB1.4.Interference with CTNNB1 reversed circ4953-induced CRC tumour growth in nude mice.Overexpression of circ4953 induced upregulation of downstream effector molecules(CCND1,c-myc)dependent on CTNNB1.5.Circ4953 enhanced the ability of CRC cells to develop lung metastases in nude mice and was able to be reversed by interference with CTNNB1.In addition,HE staining and panoramic scan images showed that overexpression of circ4953 caused an increase in metastases in the lungs of nude mice,which was significantly reduced by interference with CTNNB1.Conclusions1.Circ4953 was significantly up-regulated in CRC cells,CRC tissues and serum of CRC patients.2.Circ4953 promoted CRC proliferation and metastasis in vitro and in vivo.3.Circ4953 promoted CRC progression by inhibiting CTNNB1 protein degradation.