| Background:Hepatocellular carcinoma(HCC)accounts for 90%of liver cancers,and the remaining is hepatobiliary cancer.Hepatocellular carcinoma is a common cancer worldwidely,and the mortality raises year by year.In Europe,alcohol abuse may cause about half of HCC patients,and viral infection in China is the main cause of HCC.As the hepatitis virus is gradually controlled,alcohol-induced HCC patients increase year by year.Hepatocellular carcinoma often begins with cirrhosis and gradually becomes the second high motality cancer in the world.It often arises insidiously and it is difficult to diagnose early.Therefore,the treatment method has a strong dependence on the discovery period.Currently,the main methods for treating hepatocellular carcinoma include surgery,local interventional therapy,and radiotherapy or chemotherapy.Despite advances in detection and treatment technology,approximately 60%of patients are already advanced at the time of discovery,so the mortality is very high all the time.With the development of molecular biology,people have discovered that the occurrence and development of HCC are closely related to gene expression.Abnormalities in all aspects of gene transcription and translation play an important role in the occurrence and development of hepatocellular carcinoma.For example,SIRT2 from histone deacetylase(HDAC)family significantly elevates in hepatocellular carcinoma and plays an important role in the development of hepatocellular carcinoma.Therefore,the study of the molecular mechanism of the occurrence and development of hepatocellular carcinoma may provide an important way to overcome this disease.Gene expression refers to the process of protein synthesis according to the DNA sequence.It contains gene transcription and translation processes.During the transcription process,the splicing process of pre-mRNA also plays a key role in gene expression.It is well-known that genes include exons and introns,and RNA formed by transcription of one DNA strand is called pre-mRNA,also called heterogenous nuclear RNA(hnRNA).The pre-mRNA is composed of exons and introns,and the splicing process is to remove the introns and connect the exons.The site of cleavage locates at the 3' AG end of the exon and at the 3' AG end of the intron which connects the next exon.Alternative splicing refers to the selective binding or removal of exons in a pre-mRNA to form different mature RNAs.Alternative splicing is an important method of post-transcriptional regulation and plays an important role in protein diversity and genomic research.Alternative splicing often occurs continuously in the biological activity of the organism and responds to external stimuli to determine the expression of the genome.The realization of alternative splicing requires the participation of cis-and trans-acting splicing factors in variable exons and flanking introns.Changes in alternative splicing can lead to many diseases,and alternative splicing is also used in many fields.Now,we have studied that alternative splicing is associated with the development of tumors,neurological diseases,hematologic diseases,and the others.Alternative splicing basically affects all aspects of protein function,so it is a core part of gene expression.Because it affects almost all gene expression,the current study is only the tip of its iceberg.Currently,changes of alternative splicing in tumors have been extensively studied,and the abnormality of alternative splicing is considered to be hallmarkers of cancer development.Studies have shown that the splicing factor SF2/ASF controls exons associated with epithelial-mesenchymal transition(EMT),and EMT plays an important role in the invasion of tumor cells.Since most of the tumors are anaerobically glycometabolic,studies have shown that alternative splicing is involved in the expression of enzymes that control the metabolism in tumors,such as pyruvate kinase.Most of the tumor cells have lower pH,and the acidic environment can promote the transport of splicing factors from the nucleus into the cytoplasm.For example,in hypoxic or artificially acidic conditions,the splicing factor tra2/betal will accumulate in the cytoplasm,which may also be related to the development of tumors.Therefore,changes in the microenvironment of tumor cells may cause the disorder of alternative splicing,which may cause the development of tumors by affecting the expression of certain proteins.In summary,studies have shown that alternative splicing occurs in all aspects of tumor development,such as cell proliferation,apoptosis,cell invasion,loss of methylation,and resistance to chemotherapeutic drugs.Therefore,alternative splicing is a hot spot in recent research,but its role in the development of hepatocellular carcinoma remains unclear.Alternative splicing is controlled by the binding between sequence-specific RNA-binding proteins(RBPs)and pre-mRNA.The process of each alternative splicing is influenced by many RBPs proteins,and its role is mutual.In eukaryotic cells,the SR protein is considered to be the most characteristic splicing protein.They act both on the basic pre-mRNA splicing and on the alternative splicing process,so their function is more unique than the other RBPs.The RNA recognition motif(RRM)of the SR protein includes an RNA binding protein site and an arginine/serine rich RS region.Both of these structures are important components of the spliceosome,in which RRM can play a role in RNA recognition and binding,while the RS region is the site of protein-protein interaction.However,some studies have shown that the role of RRM and RS is not absolute,and there may be crossovers.It is generally accepted that the alternative role of the SR protein is achieved by combining exon splicing enhancers(ESEs).This mechanism is considered to be able to selectively splice some weak splicing sites.HnRNP A/B is thought to enhance this effect of the SR protein.The splicing factor SRSF2 is a member of the SR protein family.It is also one of the most studied SR proteins.It also has the general characteristics of the SR protein with RRM and RS regions and plays a positive role in regulating alternative splicing by binding to ESE.In addition,SRSF2 also plays a key role in transcriptional activation,RNA stabilization,mRNA transportation,and mRNA translation.Studies have shown that specific knock-out of SRSF2 causes acute liver failure and early death in mice.The splicing factor SRSF2 plays an important role in liver metabolism.In addition,they also found that the splicing factor SRSF2 was also involved in cholic acid metabolism and energy homeostasis.This study shows that splicing factors play an important role in liver function,but it has not been clearly studied in the development of liver tumors.Moreover,there is no study on the clinical manifestations of splicing factor SRSF2 in hepatocellular carcinoma.We first studied the relationship between the splicing factor SRSF2 and the clinical stage,tumor differentiation and alpha-fetoprotein levels of hepatocellular carcinoma.This study will play an important role in the cure of liver tumors.Objective:1.The expression of splicing factor SRSF2 in human hepatocellular carcinoma and corresponding adjacent normal liver tissues.2.To analyse the relationship between splicing factor SRSF2 protein expression and clinicopathological features in human hepatocellular carcinoma.3.To analyse the relationship between splicing factor SRSF2 protein expression and the clinical stage of human hepatocellular carcinoma and serum alpha-fetoprotein levels.4.To analyse the relationship between splicing factor SRSF2 protein expression and survival time in patients after operation with hepatocellular carcinoma.Methods:Part one:The expression of SRSF2 in human hepatocellular carcinoma and corresponding adjacent normal liver tissues.1.1 Specimen Collection:I collected a total of 153 patients with hepatocellular carcinoma who had undergone surgical treatment from January 2010 to March 2013 in Qilu Hospital.None of the patients were treated with chemoradiotherapy or biological therapy before the resection,and all of them were diagnosed as HCC by pathological examination.After surgical resection of the diseased tissues,an appropriate tissue was sent to the pathology department for paraffin-embedded pathological diagnosis,and the soy-sized tissue was immediately frozen in liquid nitrogen.There are no ethical controversies in this selection process and patients and their families were notified and they agreed with the study.1.2 Expression of splicing protein SRSF2 in clinical specimens.1.2.1 The clinical specimens were stained by immunohistochemistry(IHC).The paraffin sections were subjected for immunohistochemical detection of SRSF2 expressions according to the instructions in the commercial kit.PBS was used as the negative control antibody.At least 5 visual fields were taken for observation in every formalin section,and the percentage of positive cells and staining conditions were recorded(splicing factor SRSF2 was only expressed in the nucleus).The following criteria was used for the immunohistochemical scoring:Immunohistochemically stained sections are scored according to the staining conditions:Final score for each visual field = the score of the proportion of positive cells score X the score of dyeing condition.The final score of each specimen is the average of five vivual fields.And the final criteria for being positive or negative as following:1.2.2 Western blot was used to detect the splicing protein SRSF2 level in selected tumor tissues and paraneoplastic tissues.Take the tumor tissues and paraneoplastic tissues stored in liquid nitrogen to extract the total protein according to the instructions of the commercial kit.We took 100 u g total protein extracion to have protein electrophoresis.When the strips were clearly separated,the target strips were transferred to wet NC membrane by wet transfer.Then the membrane was blocked by 5%fat free milk,and incubated with antibody of SRSF2 over night in 4?.Then add the secondary antibody for 1 hour in room temprature.The band density was analyzed by Image-J software.Part two:Analysis of the relationship between splicing factor SRSF2 protein expression and clinicopathological features of human hepatocellular carcinoma.SPSS20.0 statistical software was used to analyze the relationship between SRSF2 protein expression and clinicopathological features of human hepatocellular carcinoma.2.1.Analysis of the relationship between splicing factor SRSF2 protein expression and clinicopathological features of human hepatocellular carcinoma.2.2.Analysis of the relationship between splicing factor SRSF2 protein expression and the clinical stage of human hepatocellular carcinoma and serum alpha-fetoprotein levels.2.3.Analysis of the relationship between splicing factor SRSF2 protein expression and mortality in patients with hepatocellular carcinoma.Results:1.The splicing factor SRSF2 highly expressed in human hepatocellular carcinoma.2.The expression of splicing factor SRSF2 increased with the degree of tumor differentiation.3.The expression of splicing factor SRSF2 was related to the TNM staging of human hepatocellular carcinoma.4.The expression of splicing factor SRSF2 was related to lymph node metastasis and distant metastasis of human hepatocellular carcinoma.5.The expression of splicing factor SRSF2 was positively correlated with the level of alpha fetoprotein in patients with hepatocellular carcinoma.6.The expression level of splicing factor SRSF2 could influence the survival time of human hepatocellular carcinoma patients after operation.Conclusion:1.Higher expression of SRSF2 was detected in the HCC tissues compared with the normal paratumoral tissues.2.There was relationship between the high expression of SRSF2 and clinic pathological features.3.Patients with higher expression of SRSF2 had lower suvival time afer operation.Research background:Our aim is to research the role and mechanism of SIRT2 protein played in the development of cholangiocarcinoma.Cancer is one of the major diseases affecting people's health.Cholangiocarcinoma(CCA)is always a tricky disease for hepatobiliary surgeons because of its insidious onset,untypical symptoms,easy relaps.Cholangiocarcinoma is the second most common primary hepatic malignancy originating from the biliary epithelium which arises either within the liver(intrahepatic cholangiocarcinoma,ICC)or in the extrahepatic bile ducts(extrahepatic cholangiocarcinoma,ECC).Despite advances in early detection and standardized treatment,the postoperative recurrence rate is very high,so it is still necessary to study the prognostic factors after surgical resection and promising molecular target,which affects tumor invasion and metastasis.Epigenetics is one of the hot spots in molecular biology research.It includes DNA methylation,RNA interference,histone modification,chromatin reorganization,and so on.Histone acetylation and deacetylation is an important part of histone modification.Histone acetylase(HAT)and histone deacetylase(HDAC)are involved in this process.HDAC protein family currently contains 18 subtypes,but they have different tissue distribution and different target proteins.They regulate the basic cell biological processes by adjusting the target protein acetylation status,including gene expression,genome stability,mitosis,nutrient metabolism,aging,mitochondrial function and cell death.Sirtuin proteins are atypical type III HDAC proteins having NAD+dependency.Seven Sirtuin proteins have been found for now,and they share the same catalytic domain containing conserved 275 amino acids.SIRT proteins have different subcellular distribution,such as SIRT6 / 7 are in the nucleus,SIRT3-5 locate in mitochondria,and SIRT1-2 locate in the nucleus and cytoplasm.SIRT2 protein is one Sirtuin protein,which plays an important role in the regulation of cell metabolism,oxidative stress and cell migration,apoptosis.Although the research of Sirtuin proteins develops,people still know least about SIRT2 protein.Especially the role of SIRT2 protein played in tumorogenesis and development is still a relatively controversial issue.SIRT2 protein deficiency mice are more susceptible to have tumors.In addition,it was reported that SIRT2 protein expression in human glioma reduced,and over-expression of SIRT2 reduced the colony forming ability of glioma cell lines.However,it was also reported that SIRT2 protein expression increased in hepatocellular carcinoma,and this overexpression might mediate epithelial-mesenchymal transition by Akt/GSK-3?/?-Catenin signaling pathway.And it elevated in 6 of 11 cases of human pancreatic adenocarcinoma,and it also elevated in breast cancer.Histone deacetylase inhibitors can increase the anticancer effect of gemcitabine against cholangiocarcinoma cell line HUCCT1.Therefore,the role of SIRT2 protein in tumor development may be related to tumor type.But the role of SIRT2 protein played in the development of cholangiocarcinoma has not been studied,ffe studied the role and the mechanism of SIRT2 played in the cholangiocarcinoma for the first time.Objective:1.To investigate the expression of SIRT2 protein in human intrahepatic cholangiocarcinoma.2.To investigate The effect of SIRT2 protein on the proliferation, migration and invasion of intrahepatic cholangiocarcinoma cells by blocking the expression of SIRT2 protein in cholangiocarcinoma cell lines with siRNA transfection.3.To explore the molecular mechanism of SIRT2 protein in promoting the occurrence and development of cholangiocarcinoma.Methods and Results:1.We chose 8 cases of intrahepatic cholangiocarcinoma operated in Qilu Hospital,and examined the SIRT2 expression in the tumor and the paratumor tissues by Western blot method.The tissues were snap-frozen in liquid nitrogen after tumor resection.Appropriate tissue was lysated on ice with RIPA lysis to get the total protein extraction.And we measured the concentrations by BCA method.Take 20 u g total protein on the 10% SDS polyacrylamide gel for electrophoresis separation.Proteins were then transferred to a PVDF membrane,after blocking with 5% skim milk,and primary antibody was added to incubate overnight at 4 ?.HRP-labeled secondary antibodies were added to incubate at room temperature after washing the membrane with TBST solution.ECL was added to light the HRP,and we get grayscale analysis with software.We found SIRT2 protein significantly elevated in seven cases of eight in tumor tissues compared with the paratumoral tissues.This suggests that SIRT2 protein may be overexpressed in cholangiocarcinoma tissues.2.And we chose 8 cases of intrahepatic cholangiocarcinoma patients operated in Qilu Hospital to have immunohistochemical analysis according to the manufacture's protocols.And at last we found SIRT2 elevated significantly in the tumor tissues compared with the paratumoral tissues.3.Two intrahepatic cholangiocarcinoma cell lines(FRH0201 and QBC939)were chosen to research the role of SIRT2 played in cholangiocarcinoma.Cells were cultured with 1640 medium under 37 ?,5% CO2 conditions.To reduce the SIRT2 expression in cholangiocarcinoma cell lines,we transfected the cells with siRNA of SIRT2 protein according to the manufacture' s protocols.Then the transfection rate was evaluated with Western blotting method.And we found SIRT2 expression significantly reduced after transfection.3.1We evaluated the proliferation ability of cholangiocarcinoma cell lines affected by SIRT2 protein by MTT assay.After the test we found the proliferation ability of cholangiocarcinoma cell lines was inhibited significantly by SIRT2 down-regulation.3.2Wound healing test was processed to evaluate the effect of SIRT2down-regulation on the migration ability of cholangiocarcinoma cell lines.And we found the migration ability of cholangiocarcinoma cell lines reduced significantly after SIRT2 down-regulation.3.3The invasion assay was processed in a Transwell chamber model.The invasion ability of cholangiocarcinoma cell lines reduced significantly after down-regulation of SIRT2 protein.3.4Flow cytometry was used to detect the effect of SIRT2 deficiency on cell cycle arrest and apoptosis.We found SIRT2 down-regulation could induce apoptosis of cholangiocarcinoma cell lines,while the cell cycles did not have significant differences.3.5 At last,we detected the acetylation status of p53 protein.As we all know,p53 is a famous tumor suppressor gene.SIRT2 can reduce the acetylation status and activity of p53.So we found the acetyl-p53 protein elevated significantly after SIRT2 down-regulation.Conclusion:1.This study for the first time found that SIRT2 elevated in the intrahepatic cholangiocarcinoma tissues2.We found SIRT2 down-regulation could inhibit the cholangiocarcinoma cell proliferation,invasion,migration,and induced apoptosis of cholangiocarcinoma cell lines for the first time.3.Because of the elevation of acetyl-P53 protein,we think this process may be related to the acetylation status of p53 protein.Significance:Recently SIRT2 inhibitor is considered to be an effective method for the treatment of age-related diseases such as neurological diseases and cancer-The role of SIRT2 in cholangiocarcinoma is unknown.While the role of SIRT2 in many tumors has been studied,but its mechanism is not clear.Herein,we for the first time found SIRT2 elevated in the intrahepatic cholangiocarcinoma tissues compared with the paratumoral tissues.This suggested SIRT2 might be related to the development of cholangiocarcinoma.And SIRT2 down-regulation could inhibit the proliferation,invasion,migration ability of cholangiocarcinoma cells and induced apoptosis via p53 pathway for the first time.These results suggest that SIRT2 inhibitors and their analogs may be a novel strategy for targeted therapy of cholangiocarcinoma overexpressing SIRT2 protein. 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