The Molecular Mechanism Of MiR-450a-5p-SOX2 Axis Regulating Stemness Properties In Colorectal Cancer

Lots of evidence strongly suggests that human cancer can be considered as a stem cell disease.As“cancer stem cell”theory postulates,tumor growth is driven by a small number of cancer stem cells which possess self-renewal and infinity replication capabilities,as well as multi-directional differentiation potential and tumorigenicity,and even produce chemore si stance and radioresistance.The discovery of cancer stem cells provides a theoretical basis for elucidating the mechanisms of tumorigenesis,growth and metastasis.Stemness-related genes,including SOX2,REX1,NANOG,KLF4 and Oct4,regulate the occurrence and development of tumors and maintain stem cell properties.We used western blot to verify these important stem cell transcription factors in SW480 and SW620 cell lines and found that the expression of SOX2 in SW620 was significantly increased compared to SW480.SOX2(Sex determining region Y-box 2)is a transcription factor belonging to the SOX gene family and closely related to the self-renewal of embryonic cells and is an important factor to induce and maintain stemness properties.SOX2 induces and maintains the characteristics of cancer stem cells.GANGEMI et al demonstrated that Sox2 knockdown in TICs derived from adult human glioblastoma led to inhibition of proliferation and loss of tumorigenicity in immunodeficient mice,indicating that Sox2 was also fundamental for maintaining the self-renewal capacity of TICs.Basu-Roy et al also drew a conclusion that Sox2 maintained self-renewal of TICs in osteosarcomas because depletion of Sox2 couldn’t form osteospheres and differentiate into mature osteoblasts any longer.In gastric cancer,the inhibition of SOX2 expression can reduce sphere formation and chemoresistance.Similarly,SOX2 also maintains stemness properties in other tumors,such as non-small cell lung cancer,glioma,colorectal cancer,and breast cancer.Studies have shown that the methylation of SOX2 significantly decreased the expression of SOX2 in gastric cancer tissues,which in turn enhanced the cell proliferation and migration.However,SOX2 expression promotes malignant development in tumors such as pancreatic cancer,esophageal cancer,colorectal cancer,liver cancer,and breast cancer.We tested the ability of spheres formation,clonies formation and chemoresistance in SW480 and SW620 by using the sphrere formation assay,soft agar assay and cell activity,respectively.The results showed that the ability of spheres formation,colonies formation and chemoresistance in SW620 were significantly enhanced compared to SW480.Similarly,we used spheres formation assay,cell viability experiments,flow cytometry,CCK8 and transwell migration experiments to further verify the effect of exogenous SOX2 on the stemness properties in colorectal cancer.Stably overexpression of SOX2 in SW480 cells can promote the formation of tumor spheres,produce chemotherapy resistance and enhance cell proliferation and migration ability.Conversely,stable knockdown of SOX2 expression in SW620 cells is not conducive to the formation of tumor spheres and chemoresistance and attenuates cell proliferation and migration.Thus we are further confirmed that SOX2 promotes stemness in colorectal cancer cells.Recent studies have shown that miRNAs are closely related to the biological behavior of cancer stem cells.According to miRNA sequencing results and bioinformatics knowledge,miR-450a-5p,miR-126-3p,miR-509-3p and miR-200-3p were found to bind directly to SOX2.miR-450a-5p has the most obvious diffenence and has not yet been reported.Dual luciferase report assay results show that miR-450a-5p binds to SOX2 3’UTR directly and inhibits its expression.Therefore,we next mainly explored the effect of miR-450a-5p on stemness properties in colorectal cancer by using sphrere formation assay,cell viability experiments,flow cytometry,CCK8 and transwell migration experiments.We transfected miR-450a-5p inhibitor into SW480 and found the ability of spheres formation,chemoresistance,cell proliferation and migration were enhanced.Conversely,we transfected miR-450a-5p mimic into SW620 and found the ability of spheres formation,chemoresistance,cell proliferation and migration were reduced.Finally,the clinical data of 90 patients carried by tissue microarrays revealed a negative correlation between SOX2 and miR-450a-5p.The lower expression of miR-450a-5p or the higher expression of SOX2 leads to the lower differentiation of tumor,the more lymph node metastasis and the later AJCC stage.Low miR-450a-5p+High SOX2 expression group had a shorter survival time and worse prognosis than High miR-450a-5p+Low SOX2 expression group.At present,there is no report on how miR-450a-5p and SOX2 regulate the stemness properties in colorectal cancer.Therefore,the main purpose of this article is to explore the molecular mechanism of miR-450a-5p-SOX2 axis regulating stemness properties in colorectal cancer.We report for the first time that miR-450a-5p can inhibit the stemness properties in colorectal cancer and provide a new molecular therapeutic target for the treatment of colorectal cancer.