| Over 100 members of the small G protein superfamily of monomeric 20~30 kDa GTP-binding proteins have been found so for. Based on both sequence homology and function, they have been subdivided into at least six families: Ras, Rho, Rab, Arf, sar1 and Ran, which have complicated and important function. Rho(Ras homologue) family play an important role in the organization of the actin cytoskeleton and regulate transcription. Not until recently have they been linked to the tumorigenesis and metastasis. But there is no report about the relation of Rho family and neoplasms in our country. In this article, we would study the potential effect of Rho GTPases on the colorectal carcinogenesis and metastasis.PART â… RhoA gene expression in colorectal carcinomaFirst of all, we investigated the expression of the RhoA in colorectal carcinoma. After establishing an optimal reverse transcription polymerase chain reaction(RT-PCR) condition, the mRNA expression levels of the RhoA gene in 42 surgically resected colorectal carcinoma and correspondingnormal mucosa were examined semi-quantitatively and the expression of RhoA gene mRNA in 42 colorectal carcinoma samples was compared with that in corresponding normal mucosa tissue and the relation between these expression levels and clinic-pathological markers were compared. The statistics analysis suggested the expression of RhoA gene mRNA in carcinoma were significantly higher than that of normal mucosa (P<0.01). The average value of RhoA expression of neoplasms VS corresponding mucosa was 2.02 and all 42 patients were divided into two groups by the average value: the overexpression group(19 cases) and high-expression group(23 cases). In the overexpression group, the cases cataloged in Dukes C and Dukes D stage accounted for 14/19, with a similar proportion of the lymph-node metastases (13/19). However, such patients accounted for only a small minority in the high-expression group (9/23 and 8/23 respectively). The chi-squared test analysis demonstrated that the cases in advanced stage (Dukes C and Dukes D) and with lymph-node metastases in the overexpression group were significantly higher than those in the high-expression group (P<0.05). Moreover, the difference in metastatic nodes between the two groups was statistically significant (P<0.01). However, Mutation was not found in the RhoA gene sequence examined. These data suggested the RhoA gene overexpression, but not the gene mutation, might play an important role in colorectal carcinogenesis and colorectal carcinoma metastasis. PART â…¡: Establishment of human colon cancer cell line LoVo-DNstably expressing RhoA-DN geneTo further clarify the possible effect of RhoA gene on the colorectal carcinogenesis in the clutured cell, a human colon cancer cell line stably expressing the dominant negative mutation of RhoA gene------RhoA(T19N), namely RhoA-DN, was established. The dominant negative activity of RhoA-DN results from competition with the endogenous wild-type RhoA GTPase for RhoGEF binding and activation, then the function of the wild-type RhoA are inhibited. Firstly, the full length encoding sequence of RhoA-DN following the enhanced green fluorescence protein (EGFP) encoding sequence was inserted into the eukaryotic expression vector pcDNA3, harboring the neomycin gene, in the sense orientation (pcDNA3-RhoA-DN). And the plasmid pcDNA3-RhoA-DN was confirmed to be in the correct orientation by DNA sequencing and then was transfected into the human colon cancer cell line LoVo by lipofectamine method. The stable transfectants (LoVo-DN) were screened by G418 and identified by the expression of EGFP. At the same time, the empty plasmid pcDNA3 was transfected into the LoVo cell and the stable transfectants (LoVo-PC) were screened by G418 for the control. Then the mRNA expression levels of the RhoA gene including the wild-type RhoA and the dominant negative mutation RhoA-DN were examined by RT-PCR analysis and werecompared with those of the control cell line which was transfected wi...
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