Molecular Cloning And Identification Of A New Full Length CDNA Gene Relate To Hepatocellular Carcinoma

Background: Human primary hepatocellular carcinoma (HCC) is one of the highly prevalent malignant diseases worldwide which carries a very poor prognosis, and the 5-year survival rate is limited to 5% after surgery. HCC, like other solid tumors, occurs in the context of various genetic and epigenetic changed and grows more and more malignant with the increase of altered molecules. Although a number of cellular genes have been described as involved during the HCC carcinogenesis, little is reported unique for HCC. Therefore, it is critical for HCC to seek more HCC-associated genes. Identification of novel HCC-associated genes will greatly improve our understanding of this disease and will encourage us to develop new targets for diagnosis and biotherapy. In this paper, we reported the identification and characterization of a novel human HCC-associated gene. This gene was initially identified as a candidate gene that is overexpressed in HCC tissues (HCCs) but undetectable in nontumorous or normal liver tissues as determined by a differential display experiment. It was verified in vivo that gene is expressed preferentially in HCCs but not in the adjacent liver tissues at mRNA levels. Objectives: To clone and identify novel human HCC-associated susceptible gene for early diagnosis and treatment of HCC. Methods: 1.Previously, an EST fragment differentially expressed between HCC and normal tissues was cloned using suppression subtractive hybridization (SSH). To search for the EST homologous genes in HCC tissue, we designed a primer for 3'RACE from highly conserved region among the above EST. Using this 3'primer and another 5'primer provided by 3 RACE kit, we do rapid amplification of cDNA 3'end .The cDNA bands got from 3'RACE were cloned into PMD18-T Vector, and the positive clones were confirmed by EcoR I and Hind III enzyme digestion. Then the sequences of inserted fragments were analyzed. The bioinformatics source was utilized to get more information of these cDNAs, and submitted them to Genbank. 2.Testifying cDNAs expression in different HCC tissues. Northern blot and technology are applied to detect the expression of these bands in HCC and normal tissues, combination of virtual Northern and multiple tissues Northern blot, expression of cDNAs in multiple normal and carcinoma tissues were analyzed. Image analysis sofeware was used to quantitate the expression of cDNA bands, and mean OD was used as the quantitative index. 3 . The full-length cDNA fragment of gene were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and identified by sequencing. The amino acid sequence coded by it was postulated by using DNA Tool software. The position of gene on chromosome was located by biologic information analysis. 4.Combination of RT-PCR and multiple tissues Northern blot and virtual Northern, expression of the cDNA in multiple normal and carcinoma tissues were analyzed. Results: 1.Five EST fragments were cloned, including two EST fragments with ploy(A) tail(694 447-3, 724 447-3).They were named 697 447-3,694 447-3,724 447-3,692 447-3 and 711 447-3, respectively. These EST fragments were submitted to Genbank dbEST database and already admitted, and Accession Number were CK730344,CK730345,CK730346,CK730347,CK730348. 2.Compared with ESTs in GenBank, the two EST fragments with ploy (A) tail (694 447-3, 724 447-3) represented novel genes with a common sequence. Northern blot revealed that 694 447-3, 724 447-3 presented higher expression in HCC tissue than in normal tissue (P<0.01). 3.One of the important cDNA fragments with ploy (A) tail was cloned. The fragment was named 694 447-3. Sequence analysis showed that 694 447-3 consists of 550bp. Basic local aligment search tool analysis revealed that 694 447-3has 63% homology with the gene BC047440. 4.The open reading frame of gene BC047440 is 603bp, which encodes a protein of 200 amino acids. Gene BC047440 maps to chromosome 20q11.22. 5.RT-PCR revealed that BC047440 presented higher expression in HCC tissue than in normal tissue (P<0.01). Multiple tissue Northern blot revealed that gene BC047440 presented a higher expression in HL-60,Burkitt's lymphoma Raji,A549 and normal heart tissues, and a lower expression in Melanoma G361 and normal muscel tissues (P<0.01). Virtual Northern revealed that gene BC047440 presented higher expression in tumor series analysis of gene expression libraries than in normal. Conclusion: 1.Five EST fragments were cloned by RACE technology, among these fragments, two novel human HCC-associated EST fragments two with ploy(A) tail was identified. These all EST fragments were submitted to Genbank dbEST database and already admitted, and Accession Number were CK730344 ,CK730345,CK730346,CK730347,CK730348. 2 . (694 447-3, 724 447-3) represented novel genes with a common sequence. Northern blot revealed that 694 447-3, 724 447-3 presented higher expression in HCC tissue than in normal tissue. 3.A novel full-length cDNA fragment (gene BC047440) which might be involvded in HCC was identified. RT-PCR revealed that BC047440 presented higher expression in HCC tissue than in normal tissue. Multiple tissue Northern blot and Virtual Northern revealed that gene BC047440 presented higher expression in tumor series analysis of gene expression (libraries) than in normal. BC047440 may be a novel gene relate to hepatocellular carcinoma.