| Background and objectivesHepatocellular carcinoma (HCC) is the main type of primary liver cancer and the third most common cause of cancer mortality worldwide. Every year more than a half new-onset HCC cases are in China. Recently 20 years, the incidence of HCC is rising, in China about 110,000 people die from HCC every year, which is accounting for 45% of HCC mortality and 18.8% of all the malignant tumor mortality in the world. In recent years, with the rapid development of the molecular biology,virology and genetics, the results of related studies generally indicate that the carcinogenesis of HCC is a multi-factor, multi-channel(such as the path of p53, the path of Rb, the path of Wnt, the path of ras and so on)and multi-step long-term complex process. At present, the high risk factors for HCC includes external environmental factors such as chronic viral hepatitis (mainly the infection of HBV and HCV), cirrhosis, alcoholism, nonalcoholic fatty liver disease, biliary diseases, parasites and bacteria, the intake of aflatoxin B1, water pollution and smoking, drugs and toxins, and some inherited metabolic diseases such as hemochromatosis and the syndrome of alpha-1-antitrypsin deficiency. Many alterations which are genetic or epigenetic lead to the changes about the cellular signal transduction passageway which is involved in regulation of cell growth, differentiation, apoptosis and mitotic. These changes could cause chronic inflammation or regeneration of hepatocytes, so they directly or indirectly induce the carcinogenesis of HCC. HCC has many characteristics, such as occult onset, short natural cause, rapid development, fast infiltrating growth. So it severely threatens the health and life of human. So current focus is finding out more tumor markers which have higher sensitivity and specificity and potential target therapies based on the molecular pathogenesis of HCC.AFP(AFP, a-fetoprotein) is serological marker for diagnosis of HCC,which is the first discovered and extensively applicated in the clinic.But the positive rate with AFP level in diagnosis of HCC is probably 60%, and its positive rate is lower for its early diagnosis or the finding of small HCC. At present, there is no tumor marker of any kind could diagnose all the HCC, mostly through the joint detection to improve the detection rate of HCC. Therefore, the search for new molecular markers for diagnosis and targeted therapy of HCC, especially in the early stage is significant important.Human WEE1 (WEE1A; WEElhu) encodes a nuclear protein, which is a tyrosine kinase belonging to the Ser/Thr family of protein kinases. It is the key components to regulation of cell cycle G2 arrest which is involved in DNA replication and DNA damage repair prior to entering mitosis. MCM6 (minichromosome maintenance protein 6) is first found in the mutant yeast in the 1980s by Holthoff HP, and then separated and extracted by the technology of molecule and biochemistry. MCM6 belongs to the family of minichromosome maintenance protein which exists in all eukaryotic cells and includes other 5 MCMS (MCM2, MCM3, MCM4, MCM5, MCM7). MCM6 is the key protein of DNA replication and extension and it is considered to be a specific factor related to cell proliferation.In the study, Reverse transcriptional polymerase chain reaction (RT-PCR) was used to measure the expression of WEE1, MCM6 mRNA in normal liver tissue, cirrhosis and HCC; Western blot and immunohistochemistry were respectively used to detect the differential expression of WEE1, MCM6 protein. Then the relationship between WEE1, MCM6 expression and the clinicopathological characteristics of HCC was analyzed.Materials and MethodsDuring 2010.3-2010.5 we got total 85 specimens from surgical operation in Henan Provincal People's Hospital. Normal human liver tissue 23 cases, cirrhosis 20 cases, HCC 42 cases, both cirrhosis and HCC were infected by HBV virus, but negativity in normal human liver tissue. All the specimens were confirmed by routine pathology evaluation and diagnosis confirmation, and with consent of the patients. Tissue specimens were immediately cut into small pieces and frozen in liquid nitrogen for later using.1. RT-PCR According to the Bori company's recommended protocol the total RNA was extracted. To estimate the total RNA integrity, the absorbance was examined at 260/280nm by spectrophotometer. And then the intensity ratio of 28S to 18S rRNA in total RNA samples was examined by 1% agarose gel electrophoresis (180V, 10min). RT-PCR was performed according to the protocol provided by Bori company. Aim gene and reference gene were amplificated in one PCR tube. PCR amplification products were tested by 2% agarose gel electrophoresis(90V,35min), scanned, photoed and semi-quantitative analyzed by BIO-RAD Gel Doc XR Imaging System (BIO-RAD Com, Amercia).2. Western blot According to conventional method extracted the total protein of the liver tissue, then measured the density of protein samples, SDS-PAGE Electrophoresis, Transfered total protein to membrance of PVDF(WEE1 half dry Transfer 20V,30min; MCM6 wet Transfer 300mA,2h), Blockng, incubation of the first antibody, inucubation of the second antibody, ECL chemiluminescence, exposured with X-ray films, at last analysised the results using Quantity One software.3. Routine HE was used to staine all the specimens, and then classifity the specimens by pathology. Then immunohistochemistry was performed in S-P method, and the results were judged by at least three different observers. The pathological section was scanned and photo-taken by Scan Scope Vitural Microscopy System (America, Aperio Co.).4. Statistic text the data was analyzed statistically by SPSS 17.0. The statistical methods included LSD-t test,χ2 test, Fisher's exact test and Kruskal-Wallis rank sum test. The test criteria was 0.05 (a=0.05).Results1. All the total RNA were extracted with good results. Electrophoresis results confirmed the high purity of the extracted RNA. The ratio of absorbance of total RNA at OD260/280nm was between 1.5-2.0. The band of PCR amplificated product was clear.2. RT-PCR mRNA expression In normal liver, cirrhosis and HCC, the positive rate of WEE1 was increased and they were 21.7%,55% and 90.5%. Normal liver tissue and HCC, cirrhosis and HCC, normal liver tissue and cirrhosis (,P<0.05), There were statistically significant differences among the three groups (P<0.01). The positive rate of MCM6mRNA was respectively 21.73%,20.00%and 83.33%. The difference among the three group were statistically significant(χ2=32.76, P<0.01), normal liver tissue and HCC, cirrhosis and HCC (P<0.05), but there was no statistically significant difference between normal liver tissue and cirrhosis (P>0.05).3. Western blot and immunohistochemistry were respectively used to detect the different expression, the positive rates of WEE1 protein expression in three groups were 13.04%/17.4%,40%/60% and 78.6%/83.3%. There were statistically significant differences among the three groups. The up-regulated expression of WEE1 was significantly correlated with the pathological grade of HCC(P<0.01).4. Western blot was used to detect the different expression, the positive rates of MCM6 protein expression in these three groups were 13.04%,20% and 71.43%. There were statistically significant differences among the three groups (P<0.01). The over expression of MCM6 was significantly correlated with HCC(P<0.01). Conclusions1. On the level of mRNA and protein, WEE1 was significant up expressed in HCC. And the gene up-regulated expression was closely concerned with tumor differentiation in HCC. MCM6 was up expressed in HCC. It suggested that the participation of DNA replication maybe associated with the occurrence of HCC.2. WEE1 may be used as an ideal candidate gene for the early diagnosis of HCC and it could be provided the basis of molecular biology for a potential molecular therapeutic approaches. MCM6 was related to cell proliferation, MCM6 could be as a sensitive biomarker for diagnosis HCC. |
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