Effect Of FMR1 On Colorectal Cancer Proliferation And Its Molecular Mechanism

BackgroundFragile X Mental Retardation 1(FMR1),a gene encoding a protein responsible for encoding the Fragile X Mental Retardation 1 protein(FMRP),could regulate variable RNA shearing,m RNA stability and translation.It is also involved in important biological processes such as RNA transport or RNA translocation by binding to the m RNA coding region or 3 ’UTR of its target.Studies have shown that FMR1 protein plays a very important role in the growth and progression of many tumours,including breast cancer.However,whether FMR1 could regulate tumorigenesis of colorectal cancer and its underlying mechanism are still unknown.ObjectiveTo analyze the expression of FMR1 in colorectal cancer,and explore the effect and underlying mechanism of FMR1 on the proliferation of colorectal cancer cells.Methods1.FMR1 expression was analyzed using data from the Cancer Genome Atlas(TCGA)and GEO(GSE41258 and GSE 32323)public databases.2.Real-time PCR(RT-q PCR),Western blot and Immunohistochemistry were used to analyze the expression of FMR1 in colorectal tissues.3.RT-q PCR and Western blot were used to test the expression of FMR1 in colorectal cancer cells(DLD1,SW480,HT29,HCT116,CACO2,Lo Vo,HCT8)and the normal intestinal cell epithelial SW460.4.Lentivirus vectors were used to upregulate the expression of FMR1 stably in SW480 and HCT116,and knock down the expression FMR1 stably in CACO2 and RKO.And the efficiency was verified by using Western blot.5.CCK8 and plate colony formation assays were used to detect the proliferation of colorectal cancer cells after FMR1 overexpression in SW480 and HCT116,or after FMR1 knock down in CACO2 and RKO.6.Cell apoptosis was detected by flow cytometry,and the apoptosis rate was analyzed.Western blot was used to detect the expression of key moleculars for apoptosis,such as PARP,Cleaved PARP,Bax,and Bcl2.7.The cell cycle of the indicated CRC cells was analyzed by flow cytometry.Western blot was used to detect the expression of of key moleculars for cell cycle progression,such as p53,p21,p27 and Cyclin D1.8.The subcutaneous xenograft model was established by using colorectal cancer cells.The tumor weight was measured,H&E or IHC staining were performed and the Ki67 index was calculated.Result1.TCGA and GEO data showed that compared with normal intestinal mucosal epithelial tissues,the expression of FMR1 in colorectal cancer was significantly increased.FMR1 expression was further up-regulated in liver metastases compared with primary lesions.2.The results of RT-q PCR,Western blot and Immunohistochemistry(IHC)revealed that the expression of FMR1 m RNA and protein were both up-regulated in colorectal cancer tissues compared with normal adjacent intestinal mucosal tissues.3.RT-q PCR and Western blot were used to detect the expression of FMR1 in 8 CRC cell lines,including DLD1,SW480,HT29,HCT116,CACO2,Lo Vo,HCT8 and normal intestinal mucosal epithelial cells NCM460.The results showed that FMR1 was up-regulated in CRC cells.4.CCK8 and plate colony formation assays showed that overexpression of FMR1 could promote the proliferation of colorectal cancer cells,and knockdown of FMR1 could inhibit the proliferation of colorectal cancer cells.5.The results of Flow cytometry showed that the apoptosis rate was decreased when FMR1 was overexpressed,while the apoptosis rate was higher when FMR1 was inhibited.And the results of Western blot revealed that the anti-apoptotic protein BCL2 increased and the pro-apoptotic proteins Bax and lyse-PARP decreased when FMR1 was overexpressed,and vice versa.6.The distribution of colorectal cancer cells in each phase of the cell cycle was detected by flow cytometry.The percentage of G1/G0 peak cells decreased and the percentage of S peak cells increased in FMR1 overexpression cells.However,the percentage of G1/G0 peak cells increased and the percentage of S peak cells decreased in CRC cells treated with FMR1 inhibition.Western blot results also showed that the expression of Cyclin D1 was upregulated when FMR1 was overexpressed,while the expression of p53,p21 and p27 was down-regulated,and vice versa.7.The results of in vivo experiment showed that the volume and growth rate of FMR1 overexpression tumors were faster than those of vector group,and the percentage of Ki67-positive cells in subcutaneous tumors with FMR1 overexpression was higher.ConclusionsFMR1 is overexpressed in colorectal cancer,and it can promote the proliferation of colorectal cancer.Preliminary studies revealed that FMR1 may promote the proliferation of colorectal cancer cells by regulating cell apoptosis and cell cycle progression.