CircASS1 Suppressed The Invasion And Metastasis Ability Of Breast Cancer Cell Line MDA-MB-231 By Targeting Gene ASS1 And Harboring MiR-4443

Objective:The invasion and metastasis of breast cancer is a major problem in the treatment of advanced breast cancer,and the potential molecular mechanism of metastasis is still unclear.The purpose of this study is to explore the role of circ ASS1(hsa＿circ＿0089105)in breast cancer,which may become a new therapeutic target for breast cancer and a potential diagnostic biomarker.Methods:1.The circular RNA in breast cancer cell line MCF-7 and MDA-MB-231 was analyzed using the chip of circ RNAs.2.The difference expression levels of circ RNAs in breast cancer cell lines: Mc F-7,mda-mb-231 were verified by real-time fluorescence quantitative PCR(rt-qpcr).Fluorescence in situ hybridization(FISH)was used to determine the localization of circ RNA in cells and the joint sequence of circ ASS1 was verified by sanger sequencing.3.Overexpression circ ASS1 plasmid was build,and transfection was carried out to overexpress circ ASS1 in breast cancer cell line MCF-7 and MDA-MB-231,the RT-q PCR was carried to test the expression of circ ASS1.After 24 h plasmid transfection,function experiment were launched to explored the biological effect of overexpression of circ ASS1 in MCF-7 and MDA-MB-231 cells.Meanwhile,RT-q PCR was used to explore the influence of circ ASS1 on the expression level of its parent gene argininosuccinate synthase 1(ASS1).4.Downstream potential mi RNAs of circ ASS1 was predicted by using bioinformatics software.Double Luciferase reporter system was used to confirm adsorption relationship between circ ASS1 and mir-4443.The transfection of mi R-4443 analogue(mimics)was performed on MCF-7 cells,and mi R-4443 function was confirmed by the experiment and transwell migration experiment.Finally,the biological function of mi R-4443 could be inhibited by the expression of circ ASS1.Results:1.The circular RNA gene chip detected tens of thousands of circrnas in the MCF-7and MDA-MB-231 cell lines.Based on the expression level of circrnas in MCF-7,there were 916 up-regulated circ RNAs and 221 down-regulated circ RNAs in MDA-MB-231.We note that the circrnas has＿circ＿0089105 is reduced by about0.067 times,and its parent gene is argininosuccinate synthase 1(ASS1),so we named it circ ASS1.2.Real time fluorescence quantitative PCR(RT-q PCR)results confirmed that,compared with MCF-7 cell line,circ ASS1 was down-regulated in MDA-MB-231 cell lines by 0.07 times.circ Base database shows that circ ASS1 was cyclized by chromosome 9q34.11 area of pure ASS1 exon 9,10 and 11.Sanger sequencing results show that the joint sequenceof circ ASS1.In situ hybridization confirmed that circ ASS1 was present in both the nucleus and cytoplasm.3.The expression of circ ASS1 in MDA-MB-231 cell lines transfected circ ASS1 overexpression plasmid(p-circ ASS1)increased about 600 times,and wound healing and transwell migration and invasion assays show that compared with the control group,after overexpressing circ ASS1,the invasion and migration ability of MDA-MB-231 cells were weakened significantly.on the other hand,the expression level of circ ASS1 in the MCF-7 cell line of circ ASS1 was decreased by 0.188 times after transfected with si-circ ASS1.At the same time,wound healing and transwell migration and invasion assays showed that the invasion and metastasis ability of the experimental group were significantly enhanced,suggesting that circ ASS1 could inhibit the invasion and metastasis of breast cancer cells.Circ ASS1 can affect the expression level of its parent gene,argininosuccinate synthase 1(ASS1),which is positively correlated.Western blot also showed that the protein expression of ASS1 was increased in the mda-mb-231 cell lines after transfected with p-circ ASS1 compared with the control group.4.Mi Randa bioinformatics prediction indicates that mi R-4443 contains a binding site matching with circ ASS1 and has a strong binding possibility.The results of the double luciferase reporting system suggested that circ ASS1 could harbor mi R-4443.At the same time,both wound healing and transwell migration and invasion assays indicated that mi R-4443 could promote the invasion and migration of MCF-7 cell lines.The opposite biological function of circ ASS1 and mi R-4443 suggests that circ ASS1 could be one of mi RNA "sponges".Conclusion:1.The expression level of circ ASS1 was negatively correlated with the invasion and metastasis ability of breast cancer cells.2.Bioinformatics shows that circ ASS1 can harbor mi R-4443,and its expression can negatively regulate the biological function of mi R-4443.3.Circ ASS1 may positively regulate the expression level of its parent gene ASS1.4.Overexpression of circ ASS1 is likely to improve the prognosis of breast cancer patients.