Research On The Abnormal Expression Of LOXL1and CBLC In Hepatocellular Carcinoma

Primary liver cancer (PLC) is one of the common malignant tumors. The incidence of PLC has risen in recent years. It is in fifth place, every year the new patient is about622,000and the death rate in third place. In our country, the new patients are about340,000every year, nearly110,000die from PLC, which in second place.This disease is more common in male of middle age, which the proportion of man and women is2～5:1. PLC has three pathological type, that is Hepatocellular Carcinoma(HCC), Intrahepatic cholangiocarcinoma(ICC), mixed type of HCC and ICC. Among them the HCC is in the majority, taking more than90%.The main cause of PLC has litter difference according to different region. About80%of the HCC suffer combines HBV or HCV infection in Asia or Africa, while in Europe and America the HCV and alcoholic cirrhosis is the common reason. The area distribution of HCC is relate to infected food of edible aflatoxin and drink pond water with alga poison,besides, heredity, drug, parasite (Clonorchis sinensis, schistosome and so on) bacillosis infection may be bound up with HCC. These factors can direct or indirect stimulate cell growth, resulting in unusual grow of cellular and causing HCC. Because of the development of HCC is hiding, early stage lack of special clinical symptoms, most patients already enter into middle and late age when the disease was diagnosed, only10%-30%can receive a treat of cutting tumor, which after5years the survival of operation rate about30%-60%,recurrence rate about80%. Therefore early diagnosis and efficient treat is very important. In the clinical, AFP is extensively applied to diagnosis of HCC as serum tumour marker, which positive rate only60%-70%. AFP is also increase in pregnancy, gestation, hepatopathy active stage, germen embryonal carcinoma.Lysyl oxidase-like1(LOXL1) is located in human’s chromosome I5q22. It is strictly distributed in the tissues containing elastic fiber, involves in the synthetization of elastin in partial tissue, and is also the key enzyme to promote the elastic fiber mature and to maintain homeostasis. The rising of the LOXL1expression and the abnormal rising of enzymatic activity may cause the excessive accumulation of indissolvable collagen and lead to the further development of fibrosis disease.Cas-Br-M (murine) ecotropic retroviral transforming sequence c (CBLC) belongs to RING ubiquitin ligase E3,which is the key factor in autophagy or lysosomal pathway and ubiquitin-proteasome pathway, gene translation and expression, modulates cell cycle, and downstream signaling molecules, then negative control cell signal transduction.This research apply RT-PCR, IHC and Western blot to test the genetic and protein expression condition of LOXL1and CBLC in normal liver, Cirrhosis and HCC, and further discuss the effect of LOXL1in the forming and developing of HCC and the relations between LOXL1and clinicopathologic features.1. Materials and Methods1.1Materials:All of the tissues were obtained from surgical liver specimens in Henan Provincial Hospital from March2010to May2010. The diagnosises are verified by pathology. The HBsAg in Cirrhosis tissues and HCC tissues all shows positive, while in the normal liver shows negative. The cases of Normal Liver were twenty-three, and it is taken from the surrounding tissues after cutting the pathologic position of hemangionma; The cases of Cirrhosis tissue were twenty-two, and it is taken from the patient with Portal Hypertension (PHT) who has the operation of pericardial revascularization and wants to have the pathological examination. The cases of HCC tissue were forty-three, and it is taken from specimen of patients with HCC operation. The tissues chosen are fresh and they were informed consent by the patients.1.2Methods1.2.1RT-PCR:Respectively extract the RNA of normal liver, cirrosis and HCC tissues, calculate the absorbance A260/A280value of total RNA, measure the purity and integrity. Reverse transcription according to the request of RT-PCR kit. The amplified product is identified by the2%agarose gel electrophoresis(AGE). Then, analyse the amplified product of LOXL1and CBLC through the scanning and semi-quantitative.1.2.2Western blot:Extract total protein(TP). All the protein sample is used with equidensity. After the protein is extracted by the Tricine-SDS-PAGE electrophoresis, transfer the protein molecule to PVDF membrane, and then put it into the primary antibody diluent (LOXL11:750; CBLC1:600) under the room temperature, incubate the KPL diluent(1:1000) marked by HRP under the room temperature. ECL chemiluminescence and X-ray pictures to expose and appear, object to do the Semi-quantitative analysis.1.2.3Immunohistochemistry:All of the tissues were stained with HE, through the pathologic grade. Adopt S-P methods of IHC, according to the instructions of dyeing steps, take pictures and store the photos.1.3Statistic methods:The statistic software SPSS17.0is applied to do the analysis. The single factor analysis variance is adopted to compare the multigroup quantitate data. LSD test is applied to compare two inner the group and qualitative date is tested by chi-quare test. All the data are previously tested by normality and homogeneity of variance, and a=0.05is set as the testing standard.2. Results2.1RT-PCR:The extraction result of total RNA in normal liver, cirrhosis and HCC is ideal results, and the absorbance A260/A280value of total RNA are all between1.8to2.0, the bands of purpose genes and reference gene are clear.The positive expression rate of LOXL1in normal liver, cirrcious and HCC tissues raised gradually,39.1%,68.2%and88.4%,respectively. So the differences among the three groups all have statistical significance (χ2=17.528, P<0.05) Multiple comparisons we can found that each diversity between normal liver and HCC, cirrhosis and HCC have statistical significance (P<0.05). The positive expression rate of CBLC in normal liver, cirrhosis and HCC tissues reduced gradually,that is82.6%、54.5%and27.9%, respectively, so the differences among the three groups all have statistical significance (χ2=18.324, P<0.05); multiple comparison we can found that each diversity between normal liver and HCC, cirrhosis and HCC, normal liver and cirrhosis, have statistical significance (P<0.05).2.2Western blot:The positive expression rate of LOXL1in normal liver, cirrhosis and HCC tissues raised gradually,26.1%,54.5%and79.1%, respectively. So the differences among the three groups all have statistical significance (χ2=17.652, P<0.05). The positive expression rate of LOXL1in normal liver, cirrhosis and HCC tissues raised gradually,78.3%、45.5%and20.9%, respectively. So the differences among the three groups all have statistical significance (χ2=20.352, P<0.05)2.3Immunohistochemistry:The positive expression rate of LOXL1in normal liver, cirrhosis and HCC tissues is13.3%,45.5%and88.4%, respectively, and the expression intensity among the three groups has obviously differences (χ2=36.78, P<0.0l).A statistical significance differences in the intensity of LOXLl protein expression was also observed in different differentiation (χ2=10.001,P=0.007) and pathological grades of HCC (χ2=17.423,P=0.001). The analysis by IHC shows that the poorer differentiated level and the higher nuclear grade, the stronger expression of LOXL1protein in the HCC tissue.3. Conclusion1. Compared to the expression in liver cirrhosis and healthy liver, LOXL1is highly expressed in HCC tissues. The intensity of LOXL1expression is associated with clinical pathological characteristics, the poorer differentiated level and the higher nuclear grade, the stronger expression of LOXLl protein in the HCC tissue.The high expression of LOXL1in HCC may play the important role in the forming and developing process of HCC.2. The expression of CBLC in HCC tissues is significantly lower than in normal liver. This may play an important part in the development of HCC. It may become a drug of molecular target therapy.