Studies On The Growth Inhibitary Effect Of Wild-type KRAS2 Gene On Colon Adenocarcinoma Cells And Loss Of Heterozygosity In Chinese Colon Carcinoma

Object:The wild-type KRAS2 gene, located in the region of 12p12-13, is thought to be a tumor suppressor gene, and loss-of-function mutation is involved in colon carcinogenesis. In this study, the loss of heterozygosity (LOH) in the 12p12-13 region was investigated in 10 colon carcinoma cases. Meanwhile, the growth inhibitary effect of wild-type KRAS2 gene on colon adenocarcinoma cell line Caco-2 was observed. Methods:1. Extract DNA from 10 specimens of cancer tissue, 10 specimens of adjacent tissue, 10 specimens of normal tissue. Analyze LOH of KRAS2 gene by polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis using 11 microsatellite markers in the 12p12-13 region.2. Clone wild-type KRAS2 gene, and construct pCI-neo vector consisting of wild-type KRAS2 gene.3. Transfect the colon adenocarcinoma cell line Caco-2 with the wild-type KRAS2 gene, and select stable expression clones.4. Detect the growth of the colon adenocarcinoma cell by monotetrazolium (MTT), analyze the cell cycle by flow cytometry (FCM), and investigate the expression of P21^ras by Western Blot.Results:1. LOH in at least one of the loci in 12pl2-13 region was detected in 30% (3/10) of adjacent tissues, the highest frequency of LOH was identified at the loci of D12S1034 in 28.57% (2/7) of adjacent tissues. 60% (6/10) carcinoma tissues were detected for LOH in at least one loci in the same region, the most frequentLOH was founded at the loci of D12S1034 and D12S1591, both about 42.88% (3/7). Among all samples, 3 cases were detected for LOH in both adjacent and tumor tissues, and 3 cases were showed with LOH only in tumor tissues.2. Occurrence of LOH was not correlated with sex, age, tumor size and lymph node metastasis.3. No mutations were found in the clone of KRAS2.4. Compared with the control group, the growth speed of human colon cancer cell line Caco-2 transfected with wild-type KRAS2 was slower(P<0.05). The proportion of cells in GO Gl phase was higher (50.22% and 35.79% separatively), the proportion of cells inG2—M phase was lower (16.90% and 24.17% separatively), the proportion of cells in S phase was lower (32.88% and 40.04% separatively).Conclusion:1. Allelic loss in 12pl2-13 region would influence the Kras2 expression by reducing the gene-dosage in colon carcinogenesis.2. The wide-type KRAS2 gene effectively inhibited the growth of human colon cancer cell line Caco-2, mainly arrested cell cycle.in the G0/G1 phase.