Transduction of an optimized recombinant lentivirus expressing E-cadherin shRNA resulted in stable downregulation of CDH1 gene and obvious cell morphological change in the human colorectal cancer cell line HT29
Journal Title: International Journal of Medical Research & Health Sciences (IJMRHS) - Year 2016, Vol 5, Issue 11
Abstract
BACKGROUND: E-cadherin (encoded by CDH1 gene) is a protein associated with invasiveness, metastasis, and poor prognosis of tumors and a critical protein in maintaining the structural integrity of epithelial sheets. The inhibition of E-cadherin expression has been reported in several types of cancers, including colorectal cancer, esophageal adenocarcinoma, gastric cancer, and pancreatic cancer. The aim of this study was to prepare an optimized recombinant lentivirus expressing short hairpin RNAs to stable downregulation of E-cadherin and evaluation of its transduction effects on cell morphology. METHODS: Human pGIPZ lentiviral shRNA vector was used to downregulation of the E-cadherin in the HT29 human colorectal cancer cell line. The expression level of mRNA was assessed using qRT-PCR. The changes in protein expression were confirmed by western blotting and ICC. Non-transduced HT29 and transduced HT29 with pGIPZ non-silencing lentiviral shRNA vector were used as controls. Furthermore, morphology of the transduced cells was monitored for 40 days using light and fluorescent microscopy. RESULTS: While the morphology of HT29 cells was epithelial-like, they changed gradually into fibroblast-like appearance after transduction. Our results showed that these molecular and morphological changes were stable in our monitoring time. CONCLUSION: It can be concluded that the suppression of CDH1 gene by shRNA method leads to depletion of E-cadherin protein expression and morphological changes in the human colorectal cancer cell line HT29.
Authors and Affiliations
Zohreh Saltanatpour| Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran, Mehdi Kadivar| Department of Biochemistry, Pasteur institute of Iran, Tehran, Iran, Behrooz Johari| Department of Biochemistry, Pasteur institute of Iran, Tehran, Iran, Majid Lotfinia| Department of Biochemistry, Pasteur institute of Iran, Tehran, Iran, Faezeh Maghsood| Department of Biochemistry, Pasteur institute of Iran, Tehran, Iran, Sirous Zeinali| Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran, Leila Nasehi| Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran, Ali Fallah| Mede Bioeconomy Company, Tehran, Iran, Aslan Fanni| Cell Therapy Center, Royan Institute, Behrooz Nikbin| Department of Immunology, Tehran University of Medical Sciences, Tehran, Iran, Corresponding Email: nikbinbe@sina.tums.ac.ir
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