The Mechanism Of CircEMB＿003 Inhibiting The Proliferation And Migration Of Colorectal Cancer

BackgroundColorectal cancer(CRC)is a common gastrointestinal malignancy with high morbidity and mortality.Therefore,it is scientifically and clinically important to investigate the molecular mechanism of colorectal cancer development and evolution,and to search for key molecular markers and drug targets.Circular RNAs(circ RNAs)are a new star in the field of non-coding RNA in recent years.It has been demonstrated that circ RNAs are closely related to the occurrence and development of tumors.CircEMB＿003(also known as circ＿0001481)is a candidate circ RNA for inhibiting the growth and metastasis of colorectal cancer.This study will explore the role and molecular mechanism of circ EMB＿003 in colorectal cancer,in order to provide a new theoretical basis for the investigation of colorectal cancer.ObjectiveTo clarify the role of circ EMB＿003 in the proliferation and metastasis of colorectal cancer,and to explore the specific molecular mechanism of circ EMB＿003 in inhibiting the proliferation and metastasis of colorectal cancer,in order to provide a new theoretical basis for the study of the molecular mechanism of the occurrence and progression of colorectal cancer.Methods1.The expression of circ EMB＿003 in colorectal cancer tissues was detected by Fluorescence in situ hybridization(FISH).2.The circ EMB＿003 stably interfered colorectal cancer cell lines SW480 and RKO were constructed and also validated by RT-q PCR,and the interfering virus that stably interfered with circ EMB＿003 but had no effect on its linear parent gene EMB was selected for subsequent experiments.3.CCK8 cell proliferation assay,plate clone formation assay,Transwell cell migration assay and scratch healing assay were used to confirm whether interfering with circ EMB＿003 could promote the proliferation and migration of CRC cells in vitro.4.RNA pull-down combined with mass spectrometry and bioinformatics analysis were used to study the protein PTBP3 that may bind to circ EMB＿003.RNA binding protein immunoprecipitation(RIP)was used to verify it.5.Transcriptome sequencing and Gene Set Enrichment Analysis(GSEA)were used to explore PLD signaling pathway,which may be regulated by circ EMB＿003,and verified by Western blotting and RT-q PCR.6.Bioinformatics was used to analyze the correlation between the expression of PTBP3 and key molecules in PLD signaling pathway,and Western blotting was used to detect the effect of PTBP3 on the expression of key molecules in PLD signaling pathway.Results1.The results of Fluorescence in situ hybridization confirmed that circ EMB＿003 was down-regulated in CRC tissues and mainly localized in the cytoplasm.2.Colorectal cancer cell lines SW480 and RKO with circ EMB＿003 stably knockdown were constructed and verified by RT-q PCR.It showed that stable cells with circ EMB＿003 knockdown were successfully constructed.The interference virus that stably interfered with circ EMB＿003 but had no effect on its linear parent gene EMB was selected for subsequent experiments.3.The results of CCK8 cell proliferation assay,plate clone formation assay,Transwell cell migration assay and scratch healing assay revealed that inhibition of circ EMB＿003could promote the proliferation and migration of CRC cells in vitro.4.RNA pull-down combined with mass spectrometry,bioinformatics analysis and RIP experiment confirmed that circ EMB＿003 could bind to PTBP3.5.Transcriptome sequencing,gene enrichment analysis,Western blotting and RT-q PCR experiments confirmed that circ EMB＿003 could regulate PLD signaling pathway.6.The results Bioinformatics analysis and Western blotting showed that PTBP3 could regulate PLD signaling pathway.ConclusionCircEMB＿003 may regulate the PLD signaling pathway through PTBP3,which consequently inhibits the proliferation and migration of colorectal cancer.