The Study Of Loss Of Heterozygosity On Hepatocellular Carcinoma And Nodulars Of Altered Hepatocytes

Objective:To study the features of loss of heterozygosity(LOH) on hepatocellular carcinoma(HCC) and focal nodular hyperplasia(FNH), by nest-PCR, denatured polyacrylamide gel electrophoresis and silver staining method,and analysised the frequency of LOH on above loci of HCC and FNH. Furthermore, we analysised their relationship between LOH and clinicopathological characteristics of HCC, and further discussed the relationship between LOH at special locus with occurrence and development of HCC,and it might help to supply new molecular markers for early diagnose and prognosis warning of HCC.Methods:1 Samples collection:44 HCC samples were taken from the paraffin block tissues filed during January of 2006 to October of 2008 by the Department of Pathology of Tangdu Hospital. 42 nodulars of altered hepatocytes (NAH) were collected in FNH samples from the same department as well. Every sample had undergone the examination of histology and had been confirmed by two experienced doctors.2 Microsatellite loci:The sequence of primors were from data Genbank supplied.Eleven microsatellite loci as following:D1S199(1p36.13),D1S507(1p36.1),D1S2843(1p36.1),D6S1008 (6q26),D8S261(8p22-p21.3),D8S277(8p23.3-p22),D8S298 (8p21),D8S1819(8p23.1),D10S215(10q23),D11S1301 (11p13),D16S402(16q24-2-q24-3).3 DNA abstraction :Each paraffin embedded sample was cut into 8 pieces in 15μm to collect tissues with the use of microdissection.Abstracted DNA with equal volume phenol-chloroform method with a final concentration of DNA as 50ng/mL.4 Microsatellite polymorphism analysis :Do nest-PCR on eleven selected microsatellite loci.Total PCR reaction volume should be 50μl and make use of Bio Rad thermal cycler. The mixture of 4μl PCR production and equal volume essay buffer were added to 8% denatured-polyacrylamide gel, and electrophoresised at 120V for 4h.Visulized the gel after silver staining and record data with UVP gel analysing system and optical picturing.Results:LOH was found in 35 of 44 HCCs at least one locus, the total LOH frequency is 79.55% (35/44);top three loci were D11S1301 (64.29%,27/42),D1S2843(62.50%,25/40), and D16S402(54.76%,23/42).Totally, LOH had no apparent correlation with sex, age, serum AFP, HBsAg, the size of tumor, Edmondson grading, cirrhosis of liver, metastasis. But LOH at some particular loci were relevant with certain clinicopathological parameters of HCC.LOH on D8S261 and D1S2843 were significantly higher in the cases with positive serum HBsAg than in those with negative HBsAg(P <0.05 and P <0.01, respectively);LOH on D1S507 and D11S1301 were more frequent in tumors larger than 3 cm in size (P <0.05 and P <0.01, respectively) ;LOH frequency of D11S1301 was significantly higher in poorly or moderately differentiated cases than in well-differentiated cases(P <0.01);LOH on D16S402 was more frequently detected in tumors with intrahepatic metastasis than in those without metastasis(P <0.05).LOH was found in 2 of 42 NAHs at D11S1301, LOH frequency was 4.76%, No LOH was found at any loci else.Conclusion:LOH at some particular loci are associated with occurrence,development and clinicopathological parameters of HCC. This study find out that locus of D11S1301 of the district of 11p13 in chromosome has higher LOH frequency. This discovery implies the potential existance of a new tumor-suppressor at D11S1301 which related to the development of HCC, which supplies a significent foudation and clues to further study the tumor suppressor gene of HCC. It was discovered that LOH occurred at specific locus is associated with such prognosis factors as the occurence of positive HBsAg HCC, differentiated degrees of HCC and the extention or metastasis of it, which enjoys profoundly clinical importance to supply a new theorical foundation to the individualized diagnose, treatment and the prognosis warning of HCC.Moreover, LOH was found in 2 of 42 NAHs at the locus of D11S1301,with a frequency of 4.76%.This result shows that FNH exsists LOH that sharing the same locus with HCC, it points that FNH may be kind of precancerous lesions in the level of molecular genetics,and implies new theorical foundation to the early diagnosis of HCC.