CircRNA3356 Promoted The Invasion And Migration Of Colorectal Cancer Cells Via Regulating The MiR-296-5p/PDPN Axis

BackgroundColorectal cancer（CRC）is one of the most common malignant tumors.Studies have reported that the incidence of CRC is relatively high.In China,the incidence of CRC ranks among the top five.The prognosis of patients with early stage CRC is good,but once the CRC metastasizes,the prognosis of the patient will decrease significantly.The ability of tumor cells to gain metastasis is an important sign of malignant tumors,and invasion is the first and most important step in the formation of metastasis.The ability of tumor cells to invade and migrate is the basis for tumor metastasis.The metastasis of CRC is directly related to the survival period of patients after surgery,and it is also a difficult problem in the current clinical treatment of CRC.Therefore,finding the key factors that regulate the invasion and migration of CRC cells and conducting in-depth research on them are of great significance for the treatment of CRC.Non-coding RNA（ncRNA）refers to RNA that does not encode protein,and circular RNA（circRNA）is one of them.Due to its stage specificity,site specificity,structural stability,strong abundance,and high degree of conservation,circRNA plays an important role in the progression of tumors.One of the ways in which circRNA functions is to combine with miRNA through the mechanism of competitive endogenous RNAs（ceRNA）,and then affect the regulation of miRNAs on target molecules.More and more studies have reported that circRNA can play a role in tumorigenesis and development through the ceRNA mechanism.However,at present,the functional mechanism of circRNA in the invasion and migration of CRC cells is not fully understood,and more in-depth research is needed.ObjectiveIn this study,high-throughput sequencing technology was used to screen the circRNA that was significantly highly expressed in tumor tissues of metastatic CRC patients,and to explore the mechanism of its effect on the invasion and migration of CRC cells.The results of this study are helpful to reveal the role of circRNA in the process of CRC metastasis.Methods1.Collected 5 cases of metastatic CRC tumor tissues and 4 cases of CRC tumor that have not metastasized for high-throughput sequencing,and sorted the sequencing data.2.Bioinformatics analyzed the difference in sequencing data,used P value and logFC to screen out the target circRNA3356 that was highly expressed in metastatic CRC patients,and verified the expression of circRNA3356 in 30 pairs of metastatic CRC tissues and non-metastatic CRC tissues..3.The RNA of HCT116 and SW480 cells was extracted and reverse transcribed into cDNA,and the gDNA of HCT116 and SW480 cells was extracted at the same time.According to the sequence of circRNA3356,designed its convergent and divergent primers.Using these two primers,qRT-PCR was performed on the cDNA and gDNA of HCT116 and SW480 cells,then agarose gel electrophoresis experiment was performed to verify the circular structure of circRNA3356.A certain concentration of actinomycin D was added to HCT116 and SW480 cells,RNA of HCT116 and SW480 cells was extracted at different periods and reversed transcribed into cDNA,and the expression of circRNA3356 and its parent gene PI4KB was detected by qRT-PCR.The nucleus and cytoplasmic RNA of HCT116 and SW480 cells were extracted and reverse transcribed into cDNA.The expression of circRNA3356 in the nucleus and cytoplasm was detected by qRT-PCR technology to verify the location of circRNA3356 in the cell.4.Overexpress/interfere circRNA3356 in HCT116 and SW480 cell lines,established stable cell lines overexpressing circRNA3356 and transient cell lines interfering with circRNA3356.Transwell experiments verified the effect of circRNA3356 on the invasion and migration of HCT116 and SW480 cells.Wound healing experiments verified the effect of circRNA3356 on the migration ability of HCT116 and SW480 cells.5.RegRNA2.0 bioinformatics online database predicted the miRNAs that circRNA3356 may target,and used dbDEMC bioinformatics online database to screen low-expressed miRNAs in CRC,and verified them by qRT-PCR,the miRNA most likely targeted by circRNA3356 was screened out.Then by transfecting miRNA mimics/inhibitor,Transwell and Wound healing experiments were used to verify the effect of miRNA on the invasion and migration of CRC cells,so as to verify its role in the migration and invasion of HCT116 and SW480 cells.Then,through miRWalk2.0 bioinformatics online database to predict the mRNAs that miRNA may target,and the most likely targeted mRNAs of the miRNAs were screened through qRT-PCR and Western blot technology.6.Used the Targetscan7.2 bioinformatics online database to find the seed sequence of miRNA,and designed circRNA3356 and mRNA wild-type and mutant plasmids.In 293T cells,wild-type and mutant plasmids were transfected respectively,and the targeting relationship between circRNA3356 and miRNA,and the targeting relationship between miRNA and mRNA were verified by dual-luciferase reporter gene experiments.7.Through rescue experiments,transfected miRNA inhibitor/mimics in the stable cell line overexpressing circRNA3356 and the transient cell line interfering with circRNA3356,and verified the expression of mRNA by qRT-PCR and Western blot experiments.At the same time,miRNA inhibitor/mimics was transfected into the stable cell line overexpressing circRNA3356 and the transient cell line interfering with circRNA3356.Transwell experiments and wound healing experiments verified whether circRNA3356 can regulate the invasion and migration of HCT116 and SW480 cells through miRNA..Results1.The high expression of circRNA3356 in the metastatic CRC tissues was screened from the sequencing data,and the high expression of circRNA3356 in CRC tissues was identified in 30 pairs of metastatic CRC tissues and non-metastatic CRC tissues..This result laid the foundation for subsequent experiments.2.Convergent and divergent primer experiments found that in the gDNA of HCT116 and S W480 cells,the convergent primer had obvious bands,while the divergent primer had no bands,indicating that circRNA3356 has a good circular structure.The results of the actinomycin D experiment showed that with the accumulation of time,the expression of circRNA3356 was stable in HCT116 and SW480 cells,but the expression of its linear parent gene PI4KB decreased significantly,indicating that circRNA3356 has a long half-life and stable expression.Nuclear and cytoplasmic isolation experiments found that circRNA3356 was expressed in the nucleus and cytoplasm of HCT116 and SW480,but mainly in the cytoplasm.3.Successfully established a stable transduction cell line overexpressing circRNA3356 and a transient cell line interfering with circRNA3356.Trans well experiments and Wound healing experiments found that after interfering with the expression of circRNA3356,the invasion and migration ability of HCT116 and SW480 cells decreased.At the same time,after overexpression of circRNA3356,the invasion and migration ability of HCT116 and SW480 cells increased.The results showed that circRNA3356 promoted the invasion and migration ability of HCT116 and SW480 cells.4.The RegRNA2.0 and the dbDEMC bioinformatics online website successfully predicted the candidate miRNAs targeted by circRNA3356:miR-132-3p,miR-296-5p,and miR-449a.The results of qRT-PCR showed that after interference with circRNA3356,only the expression of miR-296-5p increased in both HCT116 and SW480 cell lines;while after overexpression of circRNA3356,only the expression of miR-296-5p decreased in both HCT116 and SW480 cell lines.At the same time,after transfection of miR-296-5p mimics/inhibitor in HCT116 and SW480 cell lines,Transwell experiment and Wound healing experiment were performed.It was found that miR-296-5p inhibited the invasion and migration ability of HCT116 and SW480 cells.This result indicates that miR-296-5p is the most potential target of circRNA3356.5.Next,the miRWalk bioinformatics online website successfully predicted the mRNAs targeted by miR-296₅p,and selected the mRNAs related to metastasis:LYVE-1,PROX-1,VEGFC,PDPN.The results of qRT-PCR and Western blot showed that after interference with circRNA3356,the expression of PDPN decreased in both HCT116 and SW480 cell lines,while after overexpression of circRNA3356,the expression of PDPN increased in both HCT116 and SW480 cell lines.At the same time,after transfection of miR-296-5p mimics,the expression of PDPN decreased in both HCT116 and SW480 cell lines.After transfection with miR-296-5p inhibitor,the expression of PDPN increased in both HCT116 and SW480 cell lines.This result indicates that PDPN was the most potential target of miR-296-5p.6.The double-luciferase reporter gene experiment found that after transfection of miR296-5p mimics,the fluorescence values of wild-type circRNA3356 and PDPN decreased,while the mutant type did not change.This result showed that circRNA3356 and miR-296-5p,and miR-296-5p and PDPN can all directly bind,thus speculating that circRNA3356 can directly target miR-296-5p,and miR-296-5p can directly target PDPN.7.The results of rescue experiments showed that in HCT116 and SW480 cells,overexpression of circRNA3356 can promote the expression of PDPN,but after simultaneous transfection of miR-296-5p mimics,the expression of PDPN was reversed.Interfering with the expression of circRNA3356 can inhibit the expression of PDPN,but after transfection with miR-296-5p inhibitor at the same time,the expression of PDPN was reversed.In addition,cell functional rescue experiments showed that overexpression of circRNA3356 in HCT116 and SW480 cells can promote the invasion and migration ability of HCT116 and SW480 cells.However,after transfection of miR-296-5p mimics at the same time,the invasion and migration ability of HCT116 and SW480 cells was reversed.After interfering with the expression of circRNA3356,it can inhibit the invasion and migration ability of HCT116 and SW480 cells,while the simultaneous transfection of miR-296-5p inhibitor reversed the invasion and migration ability of HCT116 and SW480 cells.This result indicated that circRNA3356 can regulate the invasion and migration of HCT116 and SW480 cells through miR-296-5p.Conclusions1.The circular RNA circRNA3356 can promote the invasion and migration of CRC cells.2.CircRNA3356 indirectly enhances PDPN expression by targeting miR-296-5p,thereby promoting the invasion and migration of CRC cells.