Effects Of Mucin-type O-glycans On The Biological Behavior In Colorectal Cancer

Background and PurposeColorectal cancer(CRC) is one of the common malignant tumors of digestive tract. With the improvements of people’s living standards and the changes happening in daily diets, the incidence of colorectal cancer is increasing year by year in China.Although the current kinds of therapeutic methods are increasing and the skills of treatments are continuously improving, CRC is difficult to cure and easy to recurrent and metastasize, which are still serious threats to patients’ lives. Therefore, it is of great significance to study the molecular mechanism of the occurrence,development and metastasis of CRC, and to search for the effective methods of diagnosis and treatment of CRC,which is important to individualized treatment.Mucin-type O-glycans are a kind of carbohydrate complex which are catalyzed by polypeptide α-N-acetylgalactosaminyltransferase(pp Gal NAc T) to modify glycoproteins.They participate in many important biological processes such as to maintain the structure of proteins,to take part in the cell adhesion,cell aggregation and signal transduction.A large study showed that the structure and quantity of Mucin-type O-glycans were changing during the canceration and development of cells,and they might be related to invasion,adhesion,proliferation,apoptosis and migration of cancer cells,and some of which has been demonstrated in pancreatic cancer.Mucin-type O-glycans are key components of mucous layer of intestinal epithelial cells.There are truncated O-glycans emerging in many patients suffered from colorectal cancer,whose tumor-associated antigen Tn is exposed,but we haven’tknown the effects of Mucin-type O-glycans on the biological behavior in colorectal cancer. During the synthesis of Mucin-type O-glycans,T-synthase(core1β3-galactosytransferase) is the key enzyme.And the formation of active T-synthase requires a molecular chaperone named Cosmc in human and other vertebrates.The dysfunction of Cosmc can result in inactive T-synthase,which leads to truncated O-glycans and the expression of Tn antigen.In this research, we study the dysfunction of Cosmc and the consequent truncated O-glycans in vitro,thus studying the effects of truncated O-glycans on the biological behaviors in CRC.Methods1. Establishment of CRC cell lines stably not expressing Tn antigen(1)Cosmc was cloned into the lentivirus vectors GV367 labeled with EGFP.(2)We infect CRC cell lines LS174T(Tn+) with constructed GV367-EGFPCosmc and GV367-EGFP-blank vector as control.Then we culture the two cell lines above with media contain puromycin to screen the cell groups which stably express Cosmc with EGFP.(3)Cell lines stably contain GV367-EGFP-Cosmc/GV367-EGFP-control are observed through fluorescent microscope to make sure the efficiency of infection.(4)Real-time PCR is used to examine the expression of Cosmc from the level of transcription.(5)Western blot is uses to examine the expression of Cosmc from the level of protein expression.(6)Expression of cell surface antigen Tn was detected by flow cytometry.2. Effects of Mucin-type O-glycans on the biological behavior of human CRC(1)Detect the differences of proliferation ability of the infected cell lines using CCK8.(2)Detect the differences of apoptosis of the infected cell lines using Annexin V/7-AAD.(3)Detect the differences of migratory ability of the infected cell lines through the experiments of wound-healing and Transwell.(4)Detect the differences of invasive ability of the infected cell lines through the experiments of Transwell.Results1. Establishment of CRC cell lines stably not expressing Tn antigen Almost all the infected cells can be stimulated green fluorescence under fluorescent microscope.The expression of Cosmc from the level of transcription between the cell lines LS174T(Tn+)+Cosmc and LS174T(Tn+)+control or LS174T(Tn+) shows significant differences by the statistical method of 2(-△△Ct)with the approach of Real-time PCR(P=0.0018).The results of western blot are consistent with the results of Real-time PCR.The expression of Tn antigen turn negative obviously through the infection of Cosmc on the basis of the statistics of flow cytometry.2. Effects of Mucin-type O-glycans on the biological behavior of human CRC a.The CCK8 assays showed that compared to LS174T(Tn+)+control group,the proliferation of LS174T(Tn+) cell was significantly decreased in Cosmc stable expression group from the second day of culture(P<0.05).b.Apoptosis assays demonstrated that the total apoptosis of LS174T(Tn+)+Cosmc group was markedly increased compared to the LS174T(Tn+)+control group by flow cytometry(P=0.0010).c.The effects of Cosmc on cell migration: Wound-healing assays showed that cells infected with Cosmc had decreased migration ability compared to the controlgroup(24h: P=0.0215, 48h:P=0.0028), and the Transwell experiment showed the same way(P=0.0008).d.The effects of Cosmc on cell invasion:the total number of cells through the membrane between the LS174T(Tn+)+Cosmc group and the LS174T(Tn+)+control group has no obvious difference(P=0.9192).Conclusion Molecular chaperone Cosmc of T-synthase can repair the normal synthesis of O-glycans of cell lines LS174T(Tn +),which can make the Tn antigen overcast and inhibit the ability of proliferation and migration but promote the process of apoptosis.