Prognostic Value Of PTEN On Gallbladder Carcinoma And Its Regulatory Role In Chemosensitivity

Research background and aim:Gallbladder carcinoma(GBC)is a malignant tumor of the biliary system with a high incidence,ranking first in the malignant tumors of the biliary system and fifth in the malignant tumors of the digestive system.In recent years,there is also a tendency of rising year by year.Gallbladder cancer has a long incubation period with no obvious clinical manifestations,making early diagnosis difficult.Gallbladder cancer often occurs in gallstone caused by "unexpected gallbladder cancer" Coupled with the surgical approach not yet fixed,a lot of unexpected gallbladder cancer through laparoscopic cholecystectomy led to extensive early abdominal metastasis.Moreover,drug chemotherapy and radiation therapy is not satisfactory to patients who cannot receive surgery or patients with postoperative recurrence or metastasis.Therefore,this disease has been attracting everyone's attention.Gallstones,cholecystitis to gallbladder cancer have been called "inflammation of the cancer".The molecular pathogenesis of gallbladder carcinoma is not clear,and the characteristics of strong resistance,a variety of genes,signal transduction pathways are widely studied.As reported by Nakamura,EGFR,ERBB3 and PTEN in gallbladder cancer are among those genes.A mutation has occurred as well as the experimental implementation of a drug or inhibitor of HER2,VEGF,PI3 k / PTEN AKT / mTOR,FGFR,IDH,MEK / ERK and multikinase pathways.However,there are still no specific molecular mechanisms and targeted therapies for the development of gallbladder cancer.Phosphatase and tensin homolog PTEN is a very important cancer surveillance gene and also the second largest tumor suppressor gene next to P53.PTEN-encoded proteins can inhibit PI3 K / Akt,MAPK,FRAP / mTOR,NF-?B and other signaling pathways,participating in a variety of life activities such as cell growth,metabolism,homeostasis maintenance,and its expression and function abnormalities are closely related to the occurrence and development of a variety of tumor.PTEN exerts its strong anti-cancer effect by inhibiting cell cycle,inducing apoptosis,inhibiting tumor angiogenesis,inhibiting tumor invasion and metastasis,and maintaining immune stability.In gallbladder cancer,PTEN also plays a very important role in the development of tumors.The lack of PTEN is closely related to the clinical,pathological and prognosis of gallbladder carcer,especially gallbladder adenocarcinoma,but the molecular mechanism is still unclear.Further exploration of the role of PTEN in the occurrence and development of gallbladder cancer and the specific molecular mechanism of function can provide a new idea and potential intervention target for clinical targeted therapy of gallbladder cancer.Research methods:1.This study collected patients with gallbladder cancer in Eastern Hepatobiliary Surgery hospital from January 2010 to December 2016 after radical treatment and postoperative pathology confirming gallbladder cancer from clinical and pathological data.A total of 350 patients were included in this study.All patients underwent preoperative examination,all underwent radical resection.The clinical stage of gallbladder cancer is based on the American Joint Committee on Cancer(AJCC)Eighth Edition TNM staging(2017).The patients were followed up according to the standard follow-up procedure.The overall survival time is the end of this study.The categorical variables were expressed as the number of cases(percentage),and the groups were compared using ? 2 test,Yates' test or Fisher's test.Continuous variables were expressed as median(quartiles),with t-test or Mann-Whitney U test for differences between groups.Kaplan-Meier method was used to estimate the overall postoperative survival rate.Comparison of survival differences using Log-Rank test.To explore the independent risk factors of postoperative recurrence and overall survival in patients with GBC by using the proportional hazard regression model of COX.Data analysis was performed using SPSS 19.0 and R software 2.10.1(R Foundation for Statistical Computing,Vienna,Austria;www.r-project.org).2.The mRNA extracted from gallbladder and gallbladder cancer tissues collected in our hospital was detected by RT-PCR in real time and the expression level of PTEN was detected at the transcriptional level.Meanwhile,sample proteins were extracted for Western blot analysis.Subsequently,these samples were paraffin-embedded and immunohistochemically examined for PTEN expression.3.From patients with gallbladder cancer from the Oriental Hepatobiliary Surgery Hospital Professor Zhang Yongjie operation group during 2014.1-2016.12,a total of 157 cases of paraffin wax pathological tissue specimens underwent immunohistochemical staining to detect PTEN expression.Using Kaplan-Meier method,we compared the prognosis of patients with high and low expression of PTEN in the test samples.4.The protein levels of PTEN,p-AKT,AKT and ?-actin in GBC-SD,EHGB1,NOZ,NZ,and the extracted protein of gallbladder carcinoma cell line were detected by western blotting.Primers designed for exons of the PTEN gene were designed to perform PCR assays on four cell lines.Targeted PTEN probes were designed for deep targeting sequencing analysis.5.To construct stable PTEN cell line,detect the mRNA and protein level of PTEN by Western blotting.Plate cloning experiments were performed with this cell line and CCK-8 assay was used to detect cell proliferation.6.The low expression of PTEN and control cells were soft agar colony formation experiments to explore the expression of PTEN malignant gallbladder cancer cell growth.Subsequently,Matrigel-coated Boyden chamber was used to examine the effect of PTEN on metastasis and invasion of gallbladder cancer cells.7.Western blotting was used to detect the protein levels of PARP,p53 and?H2AX in response to DNA damage.Quantitative PCR and Western Blotting were used to detect the phenotype of EMT in PTEN-differentiated cancer cells.8.Select a variety of pathway inhibitors and commonly used chemotherapy drugs CCK-8 test method for drug screening of gallbladder cancer.9.Interfering and control groups were treated with gemcitabine(GEM),5-Fu,EPB and Bortezomib,respectively,using the NOZ,GBC-SD cell line stably interfered with PTEN,still using the CCK-8 assay.The treated cells were then tested for Caspase 3/7 activity.Followed by the light microscope to observe the interference of PTEN gallbladder cancer cell death.PI staining was performed on the drug-treated control and interfering cells,which were confirmed under light microscopy by counting under a fluorescence microscope.Flow cytometry was performed on Bortezomib and GEM-treated apoptotic cells.10.In vitro nude mice control and interfere PTEN cell lines in nude mice bearing tumors.11.Establishment of a patient-derived tumor tissue xenograft model(PDX).After successful modeling,immunohistochemistry CK19 confirmed that at the same time PTEN protein staining,divided into positive and negative / low expression group.Success again tumor tissue expansion in vitro,and susceptibility testing.12.The relationship between PTEN expression and proteasome chymotrypsin,trypsin,and Caspase-like activity was examined by simultaneous quantitative PCR with overexpression of PTEN in adenocarcinoma cells in GBC-SD cells along with PTEN-disrupted cells.Western blotting was used to analyze the protein levels of PSMB1,PSMB5,PSMD10 and PSMD11 in PTEN and overexpression PTEN cells.Research result:1.The study included 350 patients,univariate analysis showed that jaundice,the tumor is located in the liver side,lymph node N2,N1 metastasis,radical resection,tumor differentiation,and TNM staging III and IV postoperative patients with total survival The impact of factors.Multivariate analysis showed that the tumor located on the liver side,lymph node N2,N1 metastasis,non-radical resection and TNM stage III & IV were independent risk factors of overall postoperative survival.2.The expression of PTEN in gallbladder carcinoma tissues was lower than that in normal gallbladder tissues.Analysis of frozen gallbladder cancer and gallbladder tissue samples revealed that PTEN was low-protein expressed in 5 of 6 tumor tissues and high expression of p-AKT in 4 of gallbladder cancer tissues.PTEN was detected in both groups,PTEN was highly expressed in normal gallbladder tissues,and about 50% in gallbladder cancer tissues was low-level expression.3.In 157 cases of gallbladder carcinoma of paraffin tissue,PTEN expression is divided into two groups,67 cases of high expression group,90 cases of low expression group.Patients with low-expression PTEN had shorter survival and poorer prognosis than those with high-expression PTEN(P =0.006).And low expression of PTEN is an independent risk factor for overall survival.4.The expression of PTEN,p-AKT,AKT and ?-actin in the four kinds of gallbladder carcinoma cell lines showed that PTEN was low level in GBC-SD cells,while the other three showed the higher level.It suggests that the low expression of PTEN promotes the activation of downstream signaling pathway.The number of exon 9 in PTEN gene was detected.Exon 9 of GBC-SD cells was single copy,and the other three cells were double copies.The designed targeting PTEN probe was subjected to deep targeting sequencing analysis.The loss of heterozygosity of PTEN was observed in GBC-SD cells,and the rest of cells were double-copy.No mutation of PTEN was detected in all four cells.5.The establishment of PTEN stable interference cell lines,clonal formation and CCK-8 cell proliferation,suggesting that PTEN on normal cultured gallbladder cancer cells may have little effect on proliferation.Using soft agar colony formation assay,interference PTEN very significantly inhibited the malignant proliferation of tumor cells,but also inhibited the migration and invasion of gallbladder cancer cells.6.Determination of DNA damage related protein levels,suggesting that PTEN interference caused by genomic DNA damage caused by the instability of the genome.PTEN has a protective effect on gallbladder carcinoma interstitial phenotype.7.Single-copy PTEN GBC-SD gallbladder cancer cells show a higher sensitivity to multiple drugs than wild-type NOZ.8.Interfering with PTEN expression enhances the drug sensitivity of NOZ-shPTEN to GEM,whereas EPB is resistant to drugs and is highly sensitive to Bortezomib.Bortezomib treatment caused more PTEN knockdown cells to undergo apoptosis.This was also confirmed by light microscopy and PI staining,post-treatment cell-cleaved PARP and Caspase 3 levels,and flow cytometry of apoptotic cells.9.Stable interference with PTEN GBC-SD cell line,interfere with PTEN further enhance the drug sensitivity of GBC-SD cells to Bortezomib.Bortezomib treatment caused more apoptosis in GBC-SD PTEN knockdown cells.10.In vitro NOZ control and interference PTEN cell lines,nude mice subcutaneous tumor-bearing test and drug sensitivity test,shPTEN + BZ group the smallest tumor.In the shPTEN group,the Ki67 protein level in Bortezomib treatment group was significantly lower than that in control group and saline treatment group,and the up-regulation of Caspase3 was the most significant in Bortezomib treatment shPTEN group.11.PDX mice in vivo tumor expression by PTEN levels,were administered,the results confirmed that Bortezomib alone PTEN-negative PDX tumor cells have a good therapeutic effect.12.The expression of PTEN significantly inhibited the proteasome activity and inhibited the intracellular proteasome activity of PTEN significantly up-regulated by overexpression and interference of PTEN in GBC-SD cells.Interfering PTEN significantly increased the protein levels of proteasome components,whereas overexpression of PTEN significantly reduced their protein levels.Conclusion:Based on the above experimental results,we can clearly find out that PTEN has a high percentage of deletions or low expression in gallbladder cancer patients,but normal expression in normal gallbladders.Patients with missing or low PTEN expression have a short overall survival and high degree of malignancy.In gallbladder cancer cell lines,we found that PTEN has a regulatory effect on the genomic stability of tumor cells and DNA damage repair,and the loss of PTEN will affect the stability of the genome and DNA damage,resulting in a poor prognosis.We selected a variety of inhibitors of signal pathways and commonly used tumor chemotherapy drugs in clinical screening for gallbladder cancer treatment.Whether it is PTEN-deleted tumor cells,in vivo tumor-bearing experiments in nude mice,or in vivo tumor-bearing experiments in PDX mice,it is suggested that BTZ alone can achieve good therapeutic effects when administered alone.Finally,we further confirmed in cell lines that the absence or low expression of PTEN up-regulates the expression of proteasome components and the protease activity,making tumor cells highly dependent on high proteasomal activity.