Studies On The Effects Of N-glycans In The Course Of Malignant Transformation Of MDCK Cell Line With Overexpression Of HpIgR

Polymeric Ig receptor (pIgR) is a member of immunoglobulin superfamily, which transport polymeric immunoglobulins (IgA and to a lesser extent IgM) across mucosal epithelial cells. Polymeric Ig (pIg) receptor located mainly at the basolateral surface of many types of glandular epitheial cells and at the sinusoidal surface of hepatocytes.Studies indicated that Polymeric Ig (pIg) receptor expresses abnormally in cancer tissues such as gastric carcinoma, endometrial cancer, nasopharyngeal carcinoma, colon carcinoma, and adenocarcinoma of lung. which shows that there are close relations between pIgR and tumor.Carbohydrates play an key physiological role in embryonal growth and development, intercellular signal transduction and molecular recognition process as signaling molecules.which involves in the development of serious diseases such as cancer and neurodegenerative diseases. pIgR is highly glycosylated, which has seven N-glycan sites, and sugars contribute up to 25% of its molecular mass. Previous studies have demonstrated that obvious epithelial mesenchy-mal transformation (EMT) which plays key role in tumour's invasion and metastasis, occurred in MDCK cell line with overexpression of pIgR. It is unclear whether up to 25% N-glycans play key role in the development of tumour's invasion and metastasis. In the present study, N-glycan deficient mutants gene had been constructed by site-specific mutagenesis, and had been stably transfected on MDCK cells. The effects of N-glycans on proliferation, migration and invasion of MDCK cells were then investigated to elucidate the mechanism of influence of N-glycans on malignant transformation of MDCK cell line with overexpression of hpIgR.1.Construction and identification of N-glycan-deficient mutants highly expressing cell line.1) Seven single N-glycan-deficient mutants pcDNA3.1-cmyc-hpIgR have been successfully constructed by site-specific mutagenesis.And then three double N-glycan-deficient mutants pcDNA3.1-cmyc-hpIgR also have been successfully constructed on the basic of single N-glycan-deficient mutants by site-specific mutagenesis. Which can be used in the next step?2) N-glycan-deficient mutants pcDNA3.1-cmyc-hpIgR plasmids were stably transfected to MDCK with lipofectamine2000, and the model cell lines were obtained by G418 selecting. Western-blot and immunocytochemicalassay analysis showed that there was a great expression of target proteins.2.Studies on the effects of N-glycan-deficient mutants pcDNA3.1-cmyc-hpIgR highly expressing MDCK cell lineThe influence of N-glycans on actions of mutant MDCK cell clones including proliferation, migration, invasion, F-actin and E-cadherin was further investigated using the four cell models mentioned above. Results show that N-glycans of hpIgR can not affect the proliferation of MDCK cells; N-glycans in Domain4 and Domain5 can obviously enhance cell migration and invasion. Moreover, it can changed the form of F-actin and reduced E-cadherin.The results suggested that N-glycans in Domain 4 and Domain 5 play a key role in the course of malignant transformation of MDCK cell line with overexpression of hpIgR.In a word, we successfully constructed N-glycan deficient mutant MDCK cell line with overexpression of hpIgR by site-specific mutagenesis and liposome technique. Based on that, it was found that N-glycans in Domain 4 and Domain 5 of hpIgR play a key role in the course of malignant transformation of MDCK cell line with overexpression of hpIgR.