| PartIClinical analysis of surgery for hilar cholangiocarcinomaBackground and purposeCholangiocarcinoma,a malignant cancer from biliary epithelial cells,accounts for about 3%of the digestive system neoplasms with a predisposition of male compared to the female[1]It is divided into intra-and extra-hepatic bile duct carcinoma according to the anatomic sites.There are three parts for the intra-hepatic bile duct carcinoma,including hilar bile duct(upper segment),confluence of cystic duct to the upper margin of the pancreas(middle segment)and the superior border of pancreas to the bile duct near the duodenal wall(lower segment).Hilar cholangiocarcinoma(HCC),also known as Klatskin tumor,is a type of bile duct cancer that occurs in the bile ducts that lead out of the liver(hepatic ducts)and join with the gallbladder.The causes for HCC are still not well defined.In a recent survey,the prevalence of HCC is on an raising trend in China mainland,but there is no effective method for the early diagnosis.In the early onset,patients are usually symptom free except upper abdomen pain and jaundice.Unfortunately,most of the patients are at the middle and advance stages[2-5].Nowadays,most of the studies in this field have been focusing on the selection of treatment options and post-treatment prognosis.Up to now,different treatment options have been utilized.In 1965,Klatskin et al defined the HCC,and summarized the clinical pathology in a detailed manner.With the advances of scientific and technical improvements,several options are available for treating HCC,such as palliative treatment,surgery and the partial resection of lung tissues.Surgery is considered as an effective option for treating HCC.In a previous study including 230 HCC cases of various Bismuth-Corlette typing,hepatectomy and caudate lobectomy conutibuted to the R0 resection[6].Meanwhile,a total effective rate of-25%was reported in a previous study[7].For the patients with early onset,liver transplantation combined with neoadjuvant chemotherapy is the ideal option for the treatment[8]Moreover,for the patients with local advanced stage w:ith no metastasis,liver transplantation may be the only method of cure,with the 5-year survival rate of 25%-42%191.Nowadays,the basic chemotherapy regimen is relied on the combination of cisplatin and Gemcitabine.However,extensive studies revealed that HCC cells were not susceptible to the chemotherapy.Besides,chemotherapy is also considered as a type of palliative therapy,which can only extend the life span by 3 months[10].Therefore,the treatment of HCC is still a challenge in clinical practice despite great advances have been made in the past decades.In this study,we retrospectively analyzed the association between surgical treatment and the survival of HCC patients,with an aim to provide guidance to the evidence-based medicine.MethodsPatients admitted to the Affiliated Hospital of Qingdao University for surgery between Aug.2006 to Aug.2016 diagnosed with HCC were included in this study.The patients were followed by through telephone or Out-Patient Department visiting,as well as email box.The survival duration was calculated from the date of surgery or biliary drainage to.death or the time of last visiting.The follow-up deadline was Jan.31,2018.The collected information was subject to statistical analysis.Besides,we tried to confirm the type of disease,and analyze the association between surgery and survival.Results1.A total of 188 cases(male:127;female:61;mean age,61.43 yrs.)were included,and all received surgery for treating HCC including 99 R0,49 R1,and 40 R2.2.The median survival of patients of Bismuth grade Ⅰ,Ⅱ,Ⅲ and IV was 45.1±8.1,30.3 ± 1.9,12.0±0.6,and 9.5 ± 0.4 months,respectively.3.The median survival of patients of AJCC(TNM,8 th edition)stage Ⅰ,Ⅱ,Ⅲ andⅣ was 48.8 ± 5.4,21.012.8,13.6± 1.5,and 11.4±1.7 months,respectively.4.The median survival for the patients received radical surgery(R0)was 21.7±3.3 months,while those for the palliative surgery(R1 and R2)were 14.0±1.1 and 13.0±0.6 months,respectively.5.The ninety-nine patients of R0 resection with complete clinical data were divided into three groups according to the type of surgery,including extrahepatic bile duct resection plus biliary-intestine anastomosis group,right-sided hemihepatectomy plus biliary-intestine anastomosis group,and left-sided hemihepatectomy plus biliary-intestine anastomosis group.The median survival of the right-sided hemihepatectomy plus biliary-intestine anastomosis group,left-sided hemihepatectomy plus biliary-intestine anastomosis group and extrahepatic bile duct resection plus biliary-intestine anastomosis group was 11.0±0.6,11.4±1.3,and 34.0±4.7 months,respectively.Conclusions:1.The typing of the Bismuth is even,with no obvious aggregation.This is not in line with the fact that most of the patients in big centers in China are of type III and IV that involving complex surgery.2.Precise surgery for the HCC contributed to the long-term treatment efficiency.Peri-hilar radical resection is beneficial to the type I and II patients,together with achieving no cancer cells in the cutting margin.For those of type III and IV,it is necessary to receive caudate lobectomy combined with liver lobectomy,in order to improve the possibility of no cancer cells in the margin and the treatment efficiency.3.Based on the standard procedures and the precise treatment,the long-term efficiency of the patients in this study is longer than the previous efficiency.In future,we believe there might be potential increase in the treatment efficiency.PartⅡEffects of FBP1 on the proliferation and invasion of cholangiocarcinomaCholangiocarcinoma(CC)ranks as the second common primary malignant hepatic cancer,however,its early diagnosis is still a challenge and the prognosis is usually poor with higher mortality.The treatment of CC is mainly based on the surgery combined with chemoradiotherapy.Nevertheless,its prognosis is rather poor.In order to improve the clinical diagnosis and treatment efficiency,it is necessary to pay much attention to the pathogenesis and molecular marking of CC.therefore,extensive studies have been made on the pathogenesis and development of HCC,together with the valuable markers,which may contribute to the research of CC.In mammals,two major methods are utilized to maintain glucose homeosatsis including the method based on the glycolysis and degradation of oxidative phosphorylation,in addition to the synthesis based on the gluconeogenesis pathway.In the glucometabolism process,gluconeogenesis pathway involved in the transmission of non-sugar substances(e.g.lactic acid,glycerol,and glycogenic amino acid)into glucose or glycogen,which played crucial roles in the maintenance of glucose homeostasis.Besides,it contributed to the balance of blood glucose.The differential features of normal cells and cancer cells were the metabolic means,which was also considered as a typical feature for the cancer development[5].To date,Warburg effect has been extensively presented in the cancer cells,and glucometabolic in cancer cells has been considered as a new hot.FBP1,a regulatory enzyme in the gluconeogenesis pathway,is reported to play crucial roles in the pathogenesis and development of cancer.In this study,we firstly investigated the effects of FBP1 on the malignant typing of CC cells.MethodsThis section included three sections.Section one:This part was designed to investigate the expression of FBP1 in the HCC tissue samples,in order to approve the differential expression of FBP1 in the tissues with carcinoma of bile duct and analyze the clinical significance of various variables.HCC samples were obtained from 67 cases received surgery in the Affiliated Hospital of Qingdao University between May 2014 and August 2016 for surgery.All cases received no radiotherapy or chemotherapy before surgery.Immunohistochemisty:The HCC tissues were fixed using formalin,followed by embedded using paraffin.The sections(5 μm)were dried at 60 ℃,and then incubated with xylene and ethanol.Afterwards,the mixture was incubated with antigen recovery solution for 10 min.the sections were blocked for 10 min using 3%H2O2,and then incubated with goat serum for 10 min.Subsequently,the sections were incubated with FBP1 antibody(1:200)overnight at 4℃.After washing with PBS,the sections were incubated with biotin labeled IgG(Beyotime)for 20 min at 37℃,and then incubated with DAB.After staining with haematoxylin for 3 min,the sections were dehydrated using ethanol and xylene.Finally,the sections were blocked using gum and observedunder a magnification of 400×.For the cliniopathological analysis of FBP1 in the patients with carcinoma of bile duct,there were no statistical differences in the low and high expression of FBP1 based on the grouping using age,gender,histological differentiation,tumor size and TNM stage.Nevertheless,statistical differences were noticed in the low and high expression of FBP1 based on the grouping according to the metastasis(P<0.05).Section two:To analyze the effects of FBP1 overexpression on the viability of cell linesCell culture and transfectionHuman RBE and HCCC-9810 cell lines were cultured in DMEM medium containing 10%fetal bovine serum.FBP1 over-expressing plasmid pCMV3-FBP1 was established using Lipofectamine,according to the manufacturer’s instructions.The monoclonal cells were obtained after cell culture.Real-Time PCRTotal RNA was extracted using Trizol reagent,followed by purification using isopropyl alcohol and ethanol.M-MLV reverse transcriptase was used,and the cDNA synthesis was performed using the Oligo(dT)and random primers.The FBP1 mRNA expression was determined using Real-Time PCR amplification.Western blotRIPA lysis was extracted using total protein.After protein denaturation,the protein was separated and then transferred to the PVDF membrane.Finally,the membrane was incubated using ECL agent,and analyzed using Gel-Pro Analyzer software.MTT assayRBE or HCCC-9810 cells were then subject to MTT assay for the determination of cellular proliferation.Clone formationRBE or HCCC-9810 cells were inoculated in petri dish(35 mm).After fixation and staining,the number of clone formation was counted under the microscopy.Positive findings were defined as presence of 50 cells.The colony forming efficiency was calculated using the following formula:colony forming efficiency=clone number/total inoculated number × 100%.Flow cytometryFlow cytometry was used for the determination of cell cycle and apoptosis.Hoechst stainingRBE or HCCC-9810 cells were treated by antifade mounting medium,and then observed under a fluorescence microscope under a magnification of 400x.Scratch assayRBE or HCCC-9810 cells were scratched on the medium using a transferpettor(200 μl).Then the cells were observed under a microscope at 0 h,12 h and 24 h,respectively.The scratch width was measured respectively.Transwell assayThe migration and invasion of RBE or HCCC-9810 cells were measured using the Transwell assay.Results:FBP1 was over-expressed in RBE and HCCC-9810 cells,and inhibited the proliferation and clone formation of RBE and HCCC-9810.Besides,it arrested the cell cycle.Upon over-expression of FBP1,it contributed to the apoptosis of RBE and HCCC-9810 cells through up-regulating the cleaved caspase-3 and cleaved PARP,and down,regulating the expression of Bcl-2.Moreover,RBP1 over-expression decreased the migration and invasion of RBE and HCCC-9810 cells,and inhibited the Wnt/β-catenin signaling pathway.Section three:This section was designed to construct the FBP1 over-expressing vector and a transplanted tumor model.We aim to investigate the expression of FBP1 gene on the oncogenicity in animals and the apoptosis and Wnt/β-catenin signaling pathway in HCCC-9810 cells.BALB/c mice(5 g,2 months)were randomly divided into two groups(6 per group).The mice were subject to adaptive feeding for 1 week,followed by tumor transplantation.HCCC-9810 cells(1×106)transfected with blank vector and FBP1 overexpressing vector were injected via subcutaneous way in both groups(Group A and B).The diameter and volume of tumor were observed every 3 days.All the mice were sacrificed 30 days after injection,followed by weighing the tumor mass.Since day 6,the tumor volume(short and long diameters)after transplantation was determined every 3 days,using a caliper,followed by establishing of the volume growth curve.Thirty days later,the mice were sacrificed to separate the transplanted tumor and determine the weight.On day 6,the tumor growth showed significant increase in the blank vector group compared with that of the FBP1 overexpressing group(P<0.05).The tumor weight in the FBP1 overexpressing group was significantly lower compared with that of the blank control group(112.67±26.07 mg vs.258±69.55 mg,P<0.01).For the histological findings,HE staining findings were observed under a light microscope.Compared with the blank control,the cellular space was enlarged in the FBP1 over-expressing group,together with dispersed arrangement,shallow staining,as well as less atypia and karyokinesis.TUNEL assay showed the positive cells were those with nucleus stained in a buffy color in both groups.Few positive cells were observed in the blank control group.Compared with control group,the number of positive cells showed significant increase in FBP1 overexpressing group.It was featured by nuclear atrophy,chromatic agglutination,and shrinkage of volume.Immunohistochemistry findings indicated that FBP1 was mainly expressed in the cytoplasm of cells,and those with buffy granules with high density staining than the background was defined as positive cells.The number of positive cells in the FBP1 overexpressing group was significantly higher than that of the blank control group(P<0.05).In cases of FBP1 overexpression,expression of β-catenin protein in the cancer cells was significantly higher than that of the blank control.Compared with the control,the expression of FBP1 protein showed significant up-regulation in FBP1 overexpressing group,while the expression of β-catenin,Wnt 1 and c-myc showed significant down-regulation(P<0.05).Conclusions:FBP1 inhibited the proliferation,migration and invasion of HCC cells through deactivating the Wnt/β-catenin signaling pathway.It may serve as a potential candidate for the early diagnosis and treatment target for FBP1. |
PDF Full Text Request