Identification Of Novel Liver Cancer Metastatic Marker On Chromosome 8p And Preliminary Functional Analysis Of HTPAP Gene

Surgical resection is a main approach for the therapy of hepatocellular carcinoma (HCC). However, the 5-year recurrent rate after curative resection remains about 50%. Metastasis and recurrence is a major obstacle to further improvement on the long-term survival after curative resection.The abnormality of chromosome 8p exists in various tumors. Previous study showed that the LOH of chromosome 8p might contribute to the development of HCC metastasis, and more obvious allelic imbalances were found on 8pll. 2 and 8p23. 3 in metastatic lesion than in primary tumors by whole genome microsatellite analysis. The main purpose of our study was to identify candidate metastatic suppress genes of HCC on chromosome 8p by comparing the differential expression between two kinds of established cell strains with various metastatic potential.To provide a resource for cloning of chromosome 8p EST, a HCC cDNA library was first constructed and 18 cDNA clones located on chromosome 8p were obtained. Together with the cDNA clones on 8p commercially available , we finally construct a specialized cDNA microarray for chromosome 8p. This cDNA microarray was composed of 100 ESTs of chromosome 8p which covered all the regions of chromosome 8p and 1100 genes related to cell signaling , and it provided a base for identifying HCC metastatic-associated genes.We compared the expression profile of high-metastatic cell strain MHCC97H and low-metastatic cell strain MHCC97L using the specialized chromosome 8p cDNA microarray. It was found that (1 )Total 384 genes changed with 2-fold up-regulation or down-regulation. Among these 384 genes, 82 genes were found to be up-regulated in MHCC97H comparing with MHCC97L, whereas 302 genes down-regulated. (2)With the comparison of MHCC97L, total 12 differentially expressed chromosome 8p ESTs were identified, and they consisted 12% of chromosome 8p ESTs. Among these 12 differentially expressed ESTs, 6 located on 8p22-23 and 3 located on 8pll. 2 ESTs showed up-regulated expression and 10 ESTs down-regulated in MHCC97H. (3) Amongthe most significantly down-regulated 30 genes, 8 have relationship with NF-кB signal transduction pathway and this implied that the change of NF-кB signal transduction pathway may related to the metastatic potential of cultured HCC cell strains. (4) As a measure of the validity of data obtained from microarray, we examined the expression of randomly selected 4 genes by RT-PCR. The expression status of these 4 genes was similar to the results obtained by cDNA microarray.We selected an EST of chromosome 8p (EST46), named HTPAP, which was down-regulated in MHCC97H comparing with MHCC97L and acquired the full length of it. After that the expression vector containing full length of HTPAP cDNA and its related antisense RNA were constructed respectively. We further observe its function on apoptosis and invasion. Our results indicated that apoptosis rate increased in the cells transfected with HTPAP gene, while decreased treated with antisense RNA. HTPAP can inhibit the invasion of MHCC97H cells, in contrast, antisense RNA can release from this kind of inhibition. We speculated that apoptosis rate may contribute to the change of invasion. All the results imply that HTPAP gene may be a new hopeful liver cancer metastatic suppressor gene.For conclusion, there exists differentially expressed genes between high-metastatic cell strain MHCC97H and low-metastatic cell strain MHCC97L. HTPAP gene is a new candidate liver cancer metastatic suppressor gene located on chromosome 8p.