Construction of eukaryotic plasmid expressing human TGFBI and its influence on human corneal epithelial cells

TGFBI(transforming growth factor beta-induced gene) is a transforming growth factor(TGF-beta) induced extracellular matrix (ECM) protein. Its mutations have been proven to be responsible for majority of an inherited sight-threatening disorders, namely corneal dystrophy, but its function in cornea is unknown.
Objective In the study we detect TGFBI protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI.
Methods Immunohistochemistry(IHC) detect the expression of TGFBI in the cornea tissue from the corneal transplant donors. TGFBI cDNA was obtained by reverse transcription-PCR from total RNA extracted from corneal tissue that donors supplied and cloned into pCMV-N-HA vector. The recombinant pCMV-N-HA-TGFBI plasmid transfected human corneal epithelial cells. Forty eight hours later, CCK-8 detect the cell proliferation , mRNA and proteins were harvested from cells for real-time PCR analysis and western blot assay respectively.
Results CCK-8 assay shown there was no obvious changes after transfection in cell proliferation. IHC indicated TGFBI exist below the corneal epithelial layer. Transfection of recombinant pCMV-N-HA-TGFBI into human corneal epithelial cells resulted in effective expression of TGFBI in human corneal epithelial cells, as shown by increased mRNA level detected by real-time PCR as well as increased protein level detected by western blot. Meanwhile the result of real-time PCR and western blot also shown the expression of MMP1、MMP3 (matrix metalloproteinases MMP) increased while the expressin of TIMP1 (tissue inhibitors of matrix metalloproteinases TIMP) decreased.
Conclusion TGFBI exists below the corneal epithelial layer, recombinant eukaryotic expression vector harboring human TGFBI cDNA was obtained and efficiently overexpressed in human corneal epithelial cells. TGFBI overexpression in human corneal epithelial cells result in MMP1、MMP3 increasing and TIMP1 decreasing. The result might be helpful for studying the function and role of TGFBI in pathogenesis of corneal dystrophy. [Supported by GuangDong Natural Science Fund (10151063201000044)]