Analysis On Methylation Status Of Gene DLC1、ASC、P16 And DLK1 In Induced Liver Cancer Of Rat

Objective To study relevance among the methylation of lack of liver cancer gene 1 (deleted in liver cancer-1, DLC1), apoptosis related mottled protein (apoptosisi-associated speck-like protein containing a CARD , ASC), p16 also called many tumor suppressor genes (multiple tumor suppressor1, MTS) and fat precursor cells factor 1 (Delta-like lhomologue, DLK1) the four candidate genes promoter region and Aflatoxin B1 induced rat liver cancer.Methods 1.experimental specimens:50 male Wistar rats were randomly divided into by weight aflatoxin B1 (AFB1, AflatoxinB1) group (35) and blank control group (15). Hepatocellular carcinoma(HCC) was induced in 35 Wistar rats in the experimental group by intraperitoneal injection of AFB1 in Regularly, and to establish experimental animal model of liver cancer in rats. Another group of 15 rats received no drugs and served as a negative control. 2.specimen collection:In the fifty-second week, all rats were killed and tissue samples were harvested to observe changes of pathology.3.Using Methylation-Specific Polymerase Chain Reaction(MS-PCR)、agarose gel electrophoresis and gene sequencing methods to detect 4 kinds of candidate genes in rat liver tissue DLC1, ASC, p16, and DLK1 promoter region methylation, analysis of the four genes methylation status and the relationship between the AFB1 induced hepatocellular carcinoma.Results 1、In 52 weeks to see the typical pathological changes of liver cancer:experimental rat liver surface uneven, liver tissue in various abnormal hyperplasia focal and hyperplastic nodule, spherical or cotyledons, some for a single nodule, some are dispersed in the liver, some giant block type carcinoma nodules with scattered satellite nodules, pale, qualitative hard, hemorrhage, necrosis are common center, with the surrounding tissue boundary is not clear;Histologic findings of liver cells in sizes, as polygon, mostly in eosinophilic, increases than normal liver cell nucleus, chromatin hyperchromatic, dual-core or multi-core phenomenon, increased nuclear/cytoplasmic ratios, and the phenomenon of atypical hyperplasia and cancer of the liver cells. Abundant cytoplasm, granular, basophilic, some produce bile, cells arranged in funicular or thin beam shape, has the rich blood sinus between cell line, no other interstitial typical pathological changes of liver cancer.experimental rats induced liver cancer model is successful. And the control group in the rat liver tissue surface is smooth, bright color, palm red, texture is soft and fragile, under a microscope to observe the liver tissue did not see abnormalities.2、MS-PCR technique to detect according to four kinds of candidate genes in rat liver tissue DLC1, ASC, p16 and DLK1 methylation in the promoter region of the rate of 83.3%(25/30),93.3%(28/30) and 86.7% (26/30),10%(3/30), in normal liver tissues in rats with the four candidate genes methylation rate was 14.3%(2/14),35.7%(5/14),21.4%(3/14),85.7%(12/14), with statistical method to calculate P< 0.05, the comparative differences are statistically significant. Agarose gel electrophoresis tests revealed four candidate genes methylation are all positive.3、In 30 cases of rat liver cancer tissues, there are 18 cases at the same time the four genes abnormal methylation, there are 28 cases at the same time there are three or more genes abnormal methylation,30 cases all at the same time there are two or more genes abnormal methylation, no 1 cases only one abnormal gene methylation. By the statistical methods, the four genes in rat liver abnormal methylation rate consistent.Conclusion Methylation rate of DLC1, ASC, p16 genes were higher than normal liver tissue of AFB1 induced liver cancer of rat, but DLK1 showed a lower level of methylation. The results suggested that the aberrant tmethylation of DLC1, ASC, p16 and DLK1 could play an important role in liver cancer of rat.