The Role And Mechanism Of Hsa＿circ＿0062682 In The Occurrence And Progression Of Colorectal Cancer

Objective:This project aims to explore the role and mechanism of hsa＿circ0062682(circ＿0062682)in the occurrence and progression of colorectal cancer(CRC).Methods:Firstly,data mining was performed in the GEO databases based on bioinformatics methods,and differentially expressed genes were analyzed by edge R package to screen circ RNA that were significantly highly expressed in CRC.Secondly,using bioinformatics methods combined with databases(Circ Interactome,Circ Bank,Targetscan,mi RDB and etc.)to predict the micro RNAs that may bind to circ＿0062682 and micro RNAs’target genes.Quantitative real-time PCR(q PCR)was used to detect the expression levels of circ＿0062682,mi R-940 and phosphoglycerate dehydrogenase(PHGDH)in CRC cells and tissues.We also analyzed the correlations of circ＿0062682 to clinicopathological parameters and prognosis of CRC patients.The effect of circ＿0062682 on the proliferation of CRC cells in vitro was verified by CCK-8 cell proliferation assay,clone formation assay,immunofluorescence staining and cell cycle assay.Nude mice were subcutaneously implanted with CRC cells to detect the effect of circ＿0062682 on the tumorigenesis.Finally,functional rescue experiments were performed to verify the regulatory axis of circ＿0062682/mi R-940/PHGDH.Meanwhile,the physiological significance of this signaling pathway in cellular stress was explored using serum starvation experiments and serine deprivation experiments.Results:For the first time,we identified that circ＿0062682 is a circular RNA molecule.Circ＿0062682 was significantly up-regulated in CRC tumor tissues compared with adjacent normal tissues,and was significantly associated with poor prognosis.Functional experiments showed that knockdown of circ＿0062682 reduced the proportion of cells in the S phase of the cell cycle and blocked the G2/M phase,and could significantly inhibit the proliferation of CRC cell lines HCT8 and DLD1.Bioinformatics analyses showed that circ＿0062682 could bind to mi R-940,and PHGDH might be a new target gene of mi R-940.Overexpression of mi R-940promoted CRC cell proliferation,while knockdown of mi R-940 inhibited CRC cell growth.Dual luciferase experiments demonstrated the direct binding relationship between circ＿0062682 and mi R-940,as well as mi R-940 and PHGDH.PHGDH was significantly highly expressed in CRC tissues and was positively correlated with the expression of circ＿0062682.In the CRC cell lines with stable knockdown of circ＿0062682,we noticed a significant dcrease of PHGDH,serine,glycine levels,the ratio of NADPH/NADP~+,and glutathione GSH,and an accumulation of reactive oxygen species(ROS).The results of animal experiments showed that knockdown of circ＿0062682 inhibited tumor growth in vivo,which corroborated with the results of in vitro experiments.Serine deprivation and serum starvation induced up-regulation of circ＿0062682 expression in CRC cells and promoted serine metabolism and tumor proliferation through the mi R-940/PHGDH regulatory axis.Conclusion:Circ＿0062682 could sponge mi R-940,then restore the expression of PHGDH,at last promote serine metabolism and cell proliferation of CRC cells.A new regulatory axis—circ＿0062682/mi R-940/PHGDH was identified in CRC,and both serum starvation and serine deprivation were found to increase the expression of circ＿0062682 and activate this signaling axis,promoting the survival of cancer cells under metabolic stress conditions.In summary,all these data suggest that circ＿0062682 is a novel tumor metabolism regulator and may serve as a candidate therapeutic target for CRC.