Molecular Mechanisms Of LncRNA GUARDIN Involvement In Proliferation And Apoptosis Of Colorectal Cancer

Objective: The aim of the study was to explore the impact of the expression of long non-coding RNA GUARDIN(lnc RNA GUARDIN)in human colorectal cancer(CRC)cell lines and plasma on the proliferation and apoptosis of CRC cells.Furthermore,the molecular mechanism of lnc RNA GUARDIN regulating CRC cell proliferation and apoptosis will be further clarified.Methods: 1.Quantitative real-time PCR(q RT-PCR)was used to quantitatively calculate the expression level of GUARDIN in the plasma of 100 previously untreated CRC patients and 100 healthy volunteers.2.Similarly,q RT-PCR was used to quantitatively detect the expression level of GUARDIN in HCT116,SW48 cell lines and normal colorectal epithelial cells NCM460.3.The above cell lines were transfected with pc DNA-GUARDIN to increase the expression level of GUARDIN in each cell line.Subsequently,plate clone formation assay,CCK-8 cell proliferation assay and flow cytometry assay were performed.4.The miRNA bound with GUARDIN and its downstream target genes were predicted by bioinformatics software such as miRDB,Star Base3.0-V and Target Scan.5.The expression level of miR-325-3p in the plasma of 100 previously untreated CRC patients and 100 healthy volunteers was quantitatively detected by q RT-PCR.6.The relationship between GUARDIN and miR-325-3p,as well as the relationship between miR-325-3p and its downstream target gene ATG12 were verified by dual luciferase reporter gene assay.7.The co-transfer assay verified the conjecture that whether the mechanism of the proliferation and apoptosis of CRC regulated by the GUARDIN--miR-325-3p--ATG12 axis is the ce RNA mechanism.Results: 1.The expression level of GUARDIN in plasma of CRC patients was significantly lower than that of healthy volunteers,and the expression of GUARDIN was significantly correlated with CRC TNM stage.This correlation is reflected in the expression of GUARDIN in plasma,which is lower in patients with advanced CRC than in patients with early CRC.In addition,the expression level of GUARDIN in patients’ plasma after surgery was significantly higher than that before surgery.2.Comparing the expression levels of GUARDIN in three cell lines,it was found that the expression levels of GUARDIN in human cell lines HCT116 and SW48 were significantly lower than those in NCM460 cells.3.The number of living cells and the ability to form clones after pc DNA-GUARDIN transfection(increased the expression level of GUARDIN in cells)was significantly reduced,and cell proliferation activity was significantly inhibited.Same as the results of the plate clone formation assay,the proliferation experiment of CCK-8 cells showed that the cell survival rate after the increased expression of GUARDIN was lower than that of the control group.In addition,flow cytometry showed that when the level of GUARDIN was overexpressed,the apoptosis rate of CRC cells increased.4.Bioinformatics software prediction showed that there was a binding site between miRNA-325-3p and GUARDIN.The level of miRNA-325-3p in plasma of CRC patients and CRC cell lines was significantly higher than that of healthy people and NCM460 cells,respectively.5.The dual luciferase reporter gene experiment results showed that GUARDIN could bind miRNA-325-3p,which in turn could directly bind to ATG12,and the three had exactly the same binding site.6.The co-transfer experiment confirmed that GUARDIN competitively bind miRNA-325-3p through the ce RNA mechanism,thus increasing the expression of free ATG12.Conclusion: The study found that lnc RNA GUARDIN was low expressed in plasma of CRC patients and human CRC cells compared with healthy people.As a tumor suppressor gene of CRC,GUARDIN will competitively bind the site of ATG12,the target gene of miRNA-325-3p,through direct binding with miRNA-325-3p.Therefore,when the expression of GUARDIN is low,the number of free miRNA-325-3p increases,resulting in the decrease of free ATG12.LncRNA GUARDIN played a role in the development of CRC through GUARDIN--miR-325-3p--ATG12 axis.Therefore,this study shows that the low expression of GUARDIN is closely related to the development of CRC.Detection of the expression of GUARDIN in plasma and cancer tissue can be used as a marker for early diagnosis of CRC and a clinical indicator to predict the prognosis of CRC,which has a broad clinical application prospect.