The Mechanisms Underlying Regulation Of Myosin X In The Proliferation,Invasion And Metastasis Of Colorectal Cancer

Objective According to the data of the National Cancer Center in China and the world,colorectal cancer(CRC)is one of the five frequent cancer and the five leading cause of cancer death.The incidence and mortality of CRC can not be underestimated.Invasion and metastasis of cancer cells are the main characteristics of malignant tumors.Metastasis of colorectal cancer is very common in clinic,and it is also the main cause of death.To study the metastasis mechanism of CRC and find the key target molecule for regulating the metastasis of CRC is of great significance for the prevention and treatment of CRC metastasis.Filopodia and lamellopodia are the sensors of cells for the extracellular environment.Many studies have shown that Filopodia and lamellopodia play a key role in the initial stage,intermediate stage,and the final stage of metastasis.Myosin X(Myo 10)is an unconventional myosin located at the tip of filopodia,which was found in our previous studies.It can enhance the migration and invasiveness of tumor cells by promoting the growth of filopodia.In this study,Myo10 was knocked out by CRISPR/Cas9 technology to examine the effects of Myo10 on proliferation and migration of CRC in vitro and in vivo and explore the underlying mechanisms.Methods Design the CRISPR/Cas9 knockout sg RNA sequence according to the human and mouse Myo10 gene sequences obtained from NCBI,and connect the sg RNA sequence to the vector lenti-CRISPR v2,and use the 293 T cells to make lentivirus invasion.The HCT116,SW620 and CT26 cell lines were screened by limiting dilution method,and the expression of Myo10 protein in monoclonal cell line was verified by Western blotting.The HCT116 cell line was selected,and the cell proliferation activity of Myo10 knockout was compared by CCK8 cell proliferation assay;cell cycle changes were detected by flow cytometry;rhodamine phalloidin staining was used to compare cell morphology changes before and after knockout;cell scratch test and Transwell migration assay The changes of migration ability were compared.The real-time PCR assay was used to investigate the changes of m RNA levels before and after the knockdown of Myo10.At the same time,HCT116-NC and HCT116-KO cells were inoculated into 2×106 cells/cells and subcutaneously into the right hind limb of Balb/cnu mice(n=5)to construct a subcutaneous xenograft model in nude mice.The differences in tumor formation ability between the two groups were compared.Results According to the primer design website,5 pairs of human sg RNA and 5 pairs of mouse sg RNA sequences were designed.The sequencing showed that they were successfully linked to the lenti-CRISPR v2 vector.The human cell line of colorectal cancer successfully knocked out by Myo10 was verified by Western Blot.2 species,1 mouse cell line;The morphology of HCT116 cells was changed after knocking out Myo10.Rhodamine phalloidin staining showed that the morphology of HCT116 cells changed from sharp to large,and the number of F-actin decreased.The cell proliferation rate slowed down,and CCK-8 showed HCT116 cell proliferation.The viability decreased;flow cytometry found that the cell cycle changed after knocking out Myo10,and the cell cycle-associated protein changed accordingly.The migration ability of HCT116 cells changed after knocking out Myo10.The cell scratch test and Transwell migration assay showed that the cell migration ability decreased after knocking out Myo10.The q PCR results showed that the m RNA level of a sequence and cell invasion and metastasis related genes changed,HCT116-KO The m RNA levels of LRFN4,CAPN2,CD44,RAC1 and CDC42 decreased.At the same time,the subcutaneous transplantation model of HCT116 cells in nude mice was successfully constructed.After 5 days of cell inoculation,similar nodular processes were formed at the injection site,and mouse formation was measured.The long diameter(L)and short diameter(W)of the tumor were calculated and the tumor volume was calculated.The tumor volume formed by the HCT116-NC group was 114.66±54.50 mm3,and the tumor volume formed by the HCT116-KO group was 73.25±38.84 mm3.After the next day,the nude mice were sacrificed after 12 days.The tumor volume formed by HCT116-NC group was 5492.47±1781.23 mm3,and the tumor volume formed by HCT116-KO group was 1794.87 ± 786.27 mm3.The difference was statistically significant by One-way ANONA.,the tumor growth rate decreased after knocking out Myo10;Conclusions Knockout of Myo10 reduced the proliferation and cell migration ability of HCT116 cells and decreased the expression of cell cycle-associated proteins.Knockout of Myo10 may reduce the invasion and migration of colorectal cancer by affecting cell mitosis and reducing the expression level of genes involved in invasion and migration.