Expression of exon 13 from the Ki-67 gene in human cells and tissues by digoxigenin-labelled mRNA in situ hybridization

OBJECTIVE: To get insight on the regulatory mechanism of Ki-67 gene expression in malignant cell cycle. METHODS: Non-radioactive in situ hybridization (ISH) was undertaken, combined with immunohistochemistry to study the Ki-67 gene transcription and translation in various human cells and tissues. HeLa cells and fresh colon cancer cells, tonsil, normal pancreas and pancreatic cancer tissues were used in this study. A 435 bp cDNA fragment located in exon 13 of the Ki-67 antigen gene was amplified by polymerase chain reaction (PCR). Digoxigenin-labelled antisense and sense RNA probes were prepared for detecting Ki-67 mRNA, combined with MIB-1 immunohistochemistry. RESULTS: Successful localization of Ki-67 mRNA in human HeLa cells, colon cancer cells, tissues specimen of the tonsil and pancreatic cancer tissue sections was accomplished by digoxigenin-labelling in situ hybridization technique. ISH to colon cancer cells and pancreatic cancer tissue slides showed that much stronger cytoplasm and perinuclear mRNA signals of the Ki-67 gene were present in malignant cells than in normal cells, which was in accordance with MIB-1 nuclear protein signals. CONCLUSIONS: A sensitive and practical in situ hybridization method for the analysis of Ki-67 antigen mRNA in human cell and tissue was developed. Abnormal transcription of exon 13 of Ki-67 gene might be responsible for malignant cell proliferation in colon and pancreatic cancer.

get insight on the regulatory mechanism of Ki 67 gene expression in malignant cell cycle MethodsNon radioactive in situ hybridization (ISH) was undertaken, combined with immunohistochemistry to study the Ki 67 gene transcription and translation in various human cells and tissues HeLa cells and fresh colon cancer cells, tonsil, normal pancreas and pancreatic cancer tissues were used in this study A 435?bp cDNA fragment located in exon 13 of the Ki 67 antigen gene was amplified by polymerase chain reaction (PCR) Digoxigenin labelled antisense and sense RNA probes were prepared for detecting Ki 67 mRNA, combined with MIB 1 immuno^histochemistry Results Successful localization of Ki 67 mRNA in human HeLa cells, colon cancer cells, tissues specimen of the tonsil and pancreatic cancer tissue sections was accomplished by digoxigenin labelling in situ hybridization technique ISH to colon cancer cells and pancreatic cancer tissue slides showed that much stronger cytoplasm and perinuclear mRNA signals of the Ki 67 gene were present in malignant cells than in normal cells, which was in accordance with MIB 1 nuclear protein signals Conclusions A sensitive and practical in situ hybridization method for the analysis of Ki 67 antigen mRNA in human cell and tissue was developed Abnormal transcription of exon 13 of Ki 67 gene might be responsible for malignant cell proliferation in colon and pancreatic