RNA Binding Protein Musashi2 Promotes The Acquisition Of Stemness In Colorectal Cancer Cells

Background:Colorectal cancer is the third most common cancer and the second leading cause of cancer-related death worldwide.The 5-year relative survival rate of Colorectal cancer varies from more than 90%in patients with stage Ⅰ disease to more than 10%in patients with stage Ⅳ disease.With the deepening of research,a new cell called cancer stem cells(cancer stem cells,CSCs)has received more and more attention.CSCs constitute only 0.1-1%of tumor cells,are considered tumorigenic and express a variety of tumor cell stemness-related genes,such as KLF 4 and SOX 2,as well as surface markers including CD44 and CD 133.Most patients with Colorectal cancer often have recurrence and poor prognosis,which is thought to be closely related to the presence of CSCs.Therefore,further understanding of how CSCs maintain their stem-like properties and seek CSCs related targets and therapies to improve Colorectal cancer prognosis is needed.Musashi 1(MSI 1)is a member of the Musashi family of RNA binding proteins,which has been widely studied.It is closely related to the stem cell characteristics of cancer cells,and is considered to serve as a key reguLator in maintaining the stemness of pluripotent neural progenitors and affecting cell differentiation.Meanwhile,another member of the Musashi family,Musashi 2(MSI2),has a high degree of homology with the amino acid sequence of MSI 1,with 85%to 95%identity.MSI2 was initially found to be highly expressed in neural stem cell/neural progenitor cells.Later studies showed that MSI2 is also distributed in hepatocytes and is widely seen in a variety of cancers,including acute and chronic leukemia,pancreatic cancer,non-small cell lung cancer,prostate cancer,and Colorectal cancer.Our previous study showed that MSI2 is a potent biomarker for the regulation of TGF β/Smad2/3 signaling pathway and can be used for the precise diagnosis and treatment of CRC.Previous studies have suggested that MSI2 may also play an important role in the liver metastasis of Colorectal cancer.MSI2 has also been shown to maintain the stemness of HCC cells through the notch 1 signaling pathway.But little is known about the role of MSI2 in promoting the stem cell properties of CRC cells.Objective:This study intends to elucidate the role of MSI2 in Colorectal cancer cell tumor stem cells through cyological,zoological and histological approaches,so as to provide new targets for Colorectal cancer development and progression.Method:1.Colorectal cancer dataset from bioinformatics and TCGA database Correlation of MSI2 with cancer stem cell markers CD44 and CD133,the expression level of MSI2 protein in Colorectal cancer cancer stem cells using RT-qPCR and other techniques.2.MSI2 overexpression and knockdown stable transfer cell lines were constructed to detect the expression level of tumor stem cell marker protein after interfering MSI2 protein expression by Western blotting method,and tumor cell pellet assay was used to detect the stemness changes of Colorectal cancer cells after interfering MSI2 expression.3.After changes in MSI expression,changes in proliferation capacity of gastric cancer cells were observed by CCK-8 and EdU experiments.Next,cell migration ability was observed by cell scratch assay and transwell assay.Finally,cytotoxicity experiments were used to test the chemoresistance ability of stable Colorectal cancer cells overexpressing MSI2.4.After the change of MSI2 expression,the effect of plate cloning experiments on the self-renewal ability of Colorectal cancer cells,Colorectal cancer spheroid cells and monolayer cells were used to construct subcutaneous tumor model and immunohistochemistry to evaluate the role of MSI2 in tumor stem cells.Result:1.showed by bioinformatics that MSI2 is associated with CSC as markers CD44 and CD 133,and RT-qPCR resuLts indicated that MSI2 expression is elevated in CSCs.2.changing the level of MSI2 affects the sternness changes of Colorectal cancer tumor cells.3.After upregulation of MSI2,EdU and CCK-8 demonstrated enhanced proliferation;migration;after down reguLation of MSI 2,the results were contrary to the appeal.Its chemoresistance was also enhanced after upregulating MSI2 expression.4.The plate cloning experiment showed that the self-renewal ability and immunohistochemistry in nude mice showed that the sphere cells were larger than the monolayers,and the MSI2 expression in the tumor tissue increased.Conclusion:Overexpression of the RNA binding protein MSI2 promotes the generation of a stem cell-like popuLation in CRC cells.These cells exhibited increased CSC-associated characteristics in vitro,including the expression of stem cell factors,sphere formation ability,invasion ability,chemoresistance,as well as in vivo tumor growth.