Mechanism Of Silencing HDAC6 Gene To Inhibit Proliferation And Metastasis Of Esophageal Cancer Through HSP90

Background:Esophageal cancer is the sixth most common malignant tumor in China,which is the second of the gastrointestinal cancer.Esophageal cancer's incidence rate and fatality rate in China are high,especially in the high incidence areas,such as in the central and western rural areas where the health resources are short.With the improvement of medical level,the prevention and treatment of esophageal cancer have made remarkable progress.Surgical surgery is still the most important treatment for esophageal cancer,but the prognosis of patients with locally advanced esophageal cancer is not satisfactory.The 5 year survival rate of simple surgical resection for esophageal squamous carcinoma in stage? to stage ? is still low.Because esophageal cancer is less sensitive to chemotherapy and radiotherapy,postoperative chemotherapy or radiotherapy can not effectively improve the prognosis of patients with esophageal cancer.Early diagnosis and treatment is the key to improve the prognosis of patients with esophageal cancer.However,we have not yet fully understood the etiology and pathogenesis of esophageal cancer.Genetic mutations such as gene mutation,chromosome rearrangement,transcriptional regulation and modification are playing an extremely important role in esophageal cancer.Therefore,searching for specific genes related to esophageal cancer may provide a new theoretical basis for the diagnosis and treatment of esophageal cancer,and become the key for the prevention and treatment of esophageal cancer.Epigenetic regulation does not involve DNA sequence change.It is a dynamic,reversible and hereditary process,which is closely related to many diseases.Histone deacetylases(histone deacetylases,HDACs)and histone acetyltransferases(histone acetylases,HATs)are dynamic regulatory mechanisms of the level of core histone acetylation and deacetylation.Hence,acetylation regulation is one of the most important ways of epigenetic regulation.HDAC6,one of the members of the HDACs protein family,is a relatively unique histone deacetylase containing two independent functional domains of HDAC catalysis.It mainly catalyzes the deacylation of non histone,such as alpha microtubule and heat shock protein HSP90,and the specific inhibition of HDAC6 activity has no obvious effects on histone acylation.HDAC6 can participate in tumorigenesis and progression by removing acetyl and other transcriptional regulation of histone.Improper deacetylation in cell division cycle and redistribution from cytoplasm to nucleus may cause transcriptional inhibition,abnormal gene expression and induce tumor.More and more studies have shown that inhibiting the activity or expression of HDACs has become a very useful molecular target for the treatment of malignant tumors.A large number of studies have shown that many malignant tumors,including breast cancer,oral squamous cell carcinoma,leukemia,lung cancer and other tissues,have abnormal expression of HDAC6,indicating that it is closely interrelated to the oncogenesis and metastasis of tumor.As a molecular chaperone,HSP90 plays an important role in the stability and function of many proteins to maintain cell protein homeostasis and cell survival.Similarly,HSP90 plays an important role in the stability and function of various carcinogenic proteins,essential for tumor development.In esophageal cancer,HSP90 is over-expessed in the epithelium of squamous cell carcinoma of the esophagus compared with normal epithelium,and the inhibitory effect of HSP90 inhibitor 17-AAG on the proliferation of esophageal cancer cells.HYSP90 is the substrate of HDAC6,and the inactivation or knockout of HDAC6 leads to the loss of HSP90 acetylation and HSP90 HPAON activity,and the joint inhibition of HDAC6 and HSP90 shows synergism.In human leukemia cells,the combined inhibition of HDAC6 and HSP90 plays a synergistic role in anticancer activity.HSP90-HDAC6's drug therapy may be a promising strategy for esophageal cancer.However,up to now,there are few reports about the mechanism of HDAC6 gene inhibiting proliferation and metastasis of esophageal cancer through HSP90.Objective:1.To investigate the expression and clinical significance of HDAC6 gene in esophageal carcinoma.2.To construction and preparation of the lentivirus vectors target silenced HDAC6 shRNA to provide basic experimental tools for the subsequent verification of the effectiveness of the gene therapy for esophageal cancer in vivo and in vitro.3.To investigate the effect of lentivirus mediated targeting HDAC6 gene silencing combined with HSP90 inhibition on the biological characteristics of esophageal cancer cells,and provide scientific theoretical and experimental basis for the final application of this system to the clinical treatment of esophageal cancer.Methods:1.The expression and clinical significance of HDAC6 in esophageal cancer10 cases of esophageal cancer and paracancerous tissues were collected,and qRT-PCR and Western blot methods were performed to detecte the expressions of HDAC6 mRNA and HDAC6 protein in both tissues.In addition,the expression of HDAC6 was detected by IHC in 90 esophageal cancer specimens,and the relationship between the expression of HDAC6 and the clinicopathology of ovarian cancer patients was analyzed.2.Construction and preparation of lentiviral vector targeting HDAC6 gene silencing2.1 According to the HDAC6 gene(NM₀001130416.1)sequence as the target,through BLAST homology analysis,the oligonucleotides sequence of two segments of 21nt was designed and selected as the shRNA fragment of the target HDAC6 gene,and then two pairs of long primer single strand DNA fragments were synthesized.After annealing,double enzyme digestion,the plasmid pLKO.1-EGFP was connected and transformed.Two plasmids were obtained.2.2 The plasmids obtained were transfected into squamous cell carcinoma of the esophagus lines KYSE140 and KYSE180,respectively.QRT-PCR and Western blot were used to screen the most obvious plasmids for the expression of HDAC6 gene expression,and then,based on the plasmid,the lentivirus package plasmid containing the targeted silent HDAC6 gene shRNA fragment,pLKO.1-shRNA(HDAC6)-EGFP,was constructed on the basis of the gene cloning technique.2.3 The Lenti-shRNA(HDAC6)-EGFP lentivirus expression vector system was prepared by the co-transfection system of Invitrogen Company.The expression of HDAC6 gene in HEEC-1,KYSE140,KYSE170 and KYSE180 cells was detected by qRT-PCR and Western blot,and the stable transfected cells were screened.The expressions of HDAC6 mRNA and HDAC6 protein in KYSE140 and KYSE180 cell lines were detected by qRT-PCR and Western blot,respectively.3.Effects of lentivirus mediated HDAC6 gene silencing combined with HSP90 suppression on biological characteristics of esophageal cancer cells3.1 Targeting HSP90-HDAC6 inhibits the biological characteristics of esophageal cancer cells in vitroThe Lenti-shRNA(HDAC6)-EGFP lentivirus expression vector system was used to infect esophageal cancer cell lines KYSE140 and KYSE1803.1.1 Effects of silencing HDAC6 expression on proliferation of esophageal cancer cells were tested by CCK-8.3.1.2 Effects of silencing HDAC6 expression on migration of esophageal cancer cells by were tested Transwell assay.3.1.3 Effects of different concentrations of HDAC6 specific inhibitor Tuabasin A on proliferation of esophageal cancer cells were tested by CCK-8 assay.3.1.4 Effects of different concentrations of Tuabasin A on migration of esophageal cancer cells were tested by Transwell assays.3.1.5 Effects of HDAC6 siRNA and Tubastatin A on acetylation of alpha tubulin by were tested by Western blot and immunofluorescence assays.3.1.6 Effects of HDAC6siRNA and Tubastatin A on acetylation of HSP90 protein by were tested immunoprecipitation assays3.1.7 Effects of different concentrations of HSP90 specific inhibitor PU-H71 and Tubastatin A on downstream protein expression were detected by Western blot assays.3.1.8 Effects of HSP90-HDAC6 inhibitor on proliferation of esophageal squamous cell carcinoma cells were detected by CCK-8 assay3.1.9 Effects of HSP90-HDAC6 inhibitor on migration of esophageal cancer cells were detected by Transwell assay.3.1.10 Effects of HSP90-HDAC6 inhibitor on downstream protein expressions were detected by Western blot assay.3.2 Inhibitory effects of HSP90-HDAC6 i on growth of transplanted esophageal cancer cells in nude miceAfter 2 weeks of inoculation,nude mice were divided into PU-H71 group,Tubastatin A group,PU-H71+Tubastatin A group and excipients as control group.The effects of excipients on the growth of transplanted tumors,gross specimens of tumors,survival and downstream protein expression in nude mice bearing tumors were observedResults:1.The expression and clinical significance of HDAC6 in esophageal cancer1.1 The expression of HDAC6 mRNA in the tissues of squamous cell carcinoma of the esophagus and paracancerous tissue tested by qRT-PCR method was(5.13±3.78)VS(2.12±2.04)respectively.The difference between the two groups was statistically significant(P<0.05).In esophageal squamous cell carcinoma(ESCC),the expression of HDAC6 mRNA was upregulated.1.2 The respectively expression of HDAC6 protein in the tissues of squamous cell carcinoma of the esophagus and paracancerous tissue tested by Western blot was(2.35±1.64)VS(1.21 ±0.57).The difference between the two groups was statistically significant(P<0.05).The expression of HDAC6 protein was up-regulated in ESCC.1.3 Immunohistochemical examination showed that the positive expression of HDAC6 was located in the nucleus of cancer cells.Of the 90 esophageal squamous cell carcinoma specimens,62 were positive for HDAC6 and the expression rate was 68.9%.1.4 HDAC6 was related to the clinicopathological stage and lymph node metastasis(P<0.05)in patients with esophageal cancer(P<0.05),but there was no relation betweenHDAC6 and gender,age,neoplasm location,neoplasm diameter,neoplasm differentiation,and depth of neoplasm invasion(P>0.05).2.Construction and preparation of lentiviral vector targeting HDAC6 gene silencing2.1 Results of interference target design Human HDAC6 specific shRNA-1 sequence(positive sequence:CCGGGCAGUUAAAUGAAUUCCAUTTCTCGAGAAATGGAATTCACCCAACT GCTTTTTC,reverse sequence:AATTGAAAAAGCAGUUAAAUGAAUUCCAUTTCTCGAGAAATGGAATTCAC CCAACTGC);human HDAC6 specific shRNA-2 sequence(positive sequence:CCGGGGAGUUAACUGGCAGGCAUTTCTCGAGAAATGCCTGCCAGTTAACT CCTTTTTC,reverse sequence:AATTGAAAAAGGAGUUAACUGGCAGGCAUTTCTCGAGAAATGCCTGCCAG TTAACTCC).2.2 sequencing and identification of recombinant plasmid pLKO.1-shRNA(HDAC6)-EGFPThe results of recombinant plasmid pLKO.1-HDAC6-shRNA sequencing were analyzed by BLAST software,and the sequencing results were compared with the original sequence,without the deletion,mutation and code shift of the base.It proved that the design sequence was correctly inserted into the carrier.It showed that the 2 target points and negative controls were successfully constructed into recombinant plasmids.In line with the expected design,the construction of the carrier is successful.2.3 The high titer Lentivirus Expression Vector System Lenti-shRNA(HDAC6)-EGFP,which was prepared by CO transfection strategy of Invitrogen Company,was identified by PCR to confirm that the lentivirus was constructed correctly,with high titer and high infection activity.3.Effects of HDAC6 siRNA and HDAC6-HSP90 i on biological characteristics of esophageal cancer cells.3.1 Biological characteristics of HDAC6 siRNA and HDAC6-HSP90 I against esophageal cancer cells in vitro3.1.1 CCK-8 was used to detect the proliferation of esophageal squamous cell carcinoma cell lines KYSE140 and KYSE180 in negative control group,iRNA 1 and iRNA 2 groups.In the KYSE140 and KYSE180 cell lines,the cell proliferation rate in the iRNA 1 and iRNA 2 group were lower than the negative control group,There were statistically significant difference between the groups(P<0.05).3.1.2 Transwell assayed the migration and motility of esophageal squamous cell carcinomaCompared to the negative control group,the number of transmembrane metastases of iRNA 1 and iRNA 2 group was less,and the difference was statistically significant(P<0.05).3.1.3 The HDAC6 specific inhibitor,Tuabasin A,also inhibited the proliferation of KYSE140 and KYSE180 cells in esophageal cancer,and was dose-dependent.When the concentration reached 500 nM,there was a significant difference between the negative control group(P<0.05).3.1.4 In addition,Tuabasin A also inhibited the migration of KYSE140 and KYSE180 cells in esophageal cancer,and was dose-dependent.When the concentration reached 500 nM,the difference was statistically significant compared with the negative control group,and was more obvious in the KYSE 140 cells of the esophageal carcinoma(P<0.05).3.1.5 Alpha tubulin is a target of HDAC6,which is related to cell motility.The results of Western blot assay showed that in KYSE140 and KYSE180 cells,iRNA 1 and iRNA 2 groups was all higher than those in the negative control group,and the difference was statistically significant(P<0.05).In KYSE140 and KYSE180 cells,the relative expression of the negative control group,the Tuabasin A(100 nM)group,the Tuabasin A(500 nM)group and the Tuabasin A(1 ?M)acetylation alpha microtubule increased gradually,and there was a significant difference between the groups(P<0.05).Immunofluorescence assay showed that microtubules were decomposed after cold-induced microtubule destruction,and almost no microtubules were observed in KYSE140 and KYSE180 cells.Compared with the negative control group,more microtubules were detected in the cells treated with Tuabasin A(P<0.05).3.1.6 The results of immunoprecipitation assay showed that in KYSE140 and KYSE180 cells,compared with the negative control group,the expression of acetylated HSP90 protein in iRNA 1 and iRNA 2 groups was higher,and the difference was statistically significant(P<0.05).In KYSE140 and KYSE180 cells,the relative expression of the acetylated HSP90 protein in the negative control group,the Tuabasin A(100 nM)group,the Tuabasin A(500 nM)group and the Tuabasin A(1 ?M)increased gradually,the difference between the negative control group were statistically significant differences(P<0.05).3.1.7 HSP90 inhibitors PU-H71 and Tubastatin A regulate protein expression.In KYSE140 and KYSE180 cells,the levels of EGFR,Akt,PK and phosphorylated ERK in PU-H71 treated cells were lower than those in the control group.Tubastatin A treatment also decreased the levels of EGFR,AKT,phosphorylated AKT and phosphorylated ERK in KESE140 and KYSE180 cell lines,but the degree was lower(P<0.05).3.1.8 The results of CCK-8 detection showed that in the KYSE140 and KYSE180 cell lines,the average cell proliferation rates of the negative control group,the individual Tubastatin A(100 nM),the individual PU-H71(100 nM)and the Tubastatin A(100 nM)group were 100%,91.45%,78.26%,and 62.03%respectively.Combined Tubastatin A+PU-H71 significantly reduced cell viability compared with either inhibitor alone(P<0.05).3.1.9 The results of Transwell detection showed that in the KYSE140 and KYSE180 cell lines,the number of cell transference in the negative control group,Tubastatin A(100 nM),PU-H71(100 nM)and the Tubastatin A(100 nM)+PU-H71(100 nM)group decreased gradually,and the difference was statistically significant(P<0.05).Tubastatin A+PU-H71 combined therapy significantly decreased cell migration and motility compared with either inhibitor alone(P<0.05).3.1.10 The results of Western blot detection showed that in the KYSE140 and KYSE180 cell lines,the expression of EGFR,p-AKT,p-ERK in the negative control group,Tubastatin A(100 nM),PU-H71(100 nM),Tubastatin A(100 nM)+PU-H71(100 nM)group were gradually decreasing,there were statistically significant difference among the groups(P<0.05).In the KYSE140 and KYSE180 cell lines,the experimental results showed that the protein expression levels of EGFR,p-AKT and p-ERK were the lowest in the combination of Tuabasin A and PU-H71,compared with the individual Tuabasin A or PU-H71 treatment.3.2 Effects of HDAC6-HSP90i on transplanted tumor of esophageal cancer KYSE140 cells in nude mice3.2.1 Establishment of subcutaneous transplantation tumor model in nude miceThe human esophageal squamous cell carcinoma KYSE140 cell line was transplanted subcutaneously in nude mice to observe the tumor size at different time points.On average about 14 days,the transplanted tumor of the groin in nude mice was about 0.2-0.6 cm in diameter 3 weeks later.All the inoculated animals survived and had tumor formation.There was no inflammation and swelling in the inoculation point.At the beginning of the third week,the diameter of transplanted tumor in nude mice of PU-H71+Tubastatin A injection group was significantly lower than that of the other three control groups.The difference was significant(P<0.05).At the beginning of sixth weeks,the diameter of transplanted tumor in nude mice of PU-H71 injection group was lower than that of the vehicle injection group and the Tubastatin A injection group.The difference was statistically significant(P<0.05).Significant tumor suppression was observed only in Tuabasin A(10 mg/kg)and PU-H71(50 mg/kg)Co injected nude mice group.3.2.2 Observed the survival of nude mice and the gross specimen of subcutaneous tumorIn the control group,the Tuabasin A group and the PU-H71 group,the nude mice were less active,and the mental state,poor appetite and lethargy were poor,while the PU-H71+Tuabasin A group had more activity,good mental state and good appetite.Combined PU-H71+Tuabasin A injection significantly inhibited tumor growth and improved the quality of life in nude mice.The combined injection group of Tuabasin A and PU-H71 had smaller size of transplanted tumor,a clear and complete envelope around the tissue,a clear boundary between the surrounding tissue,no obvious adhesion and infiltration,and the tumor was completely stripped.The tumor in the nude mice injected with vihicle and Tuabasin A the tumor was larger,not clear with the surrounding tissue,and the membrane was incomplete and involved the surface of the skin.3.2.3 Effects of HSP90-HDAC6 inhibitor on downstream protein expression in transplanted tumor bearing nude miceIn the tumor bearing nude mice,the expression of EGFR,p-AKT and p-ERK in the tumor of the control group,the Tubastatin A(100 nM)group,the PU-H71(100 nM)group and the Tubastatin A(100 nM)+PU-H71(100 nM)group showed a gradual decreasing trend.There were statistically significant differences among the groups(P<0.05).Compared with the other three groups,Tuabasin A(10 mg/kg)and PU-H71(50 mg/kg)combined injection group had the lowest levels of EGFR,p-AKT and p-ERK expression.In addition,IHC analysis also showed that Tuabasin A(10 mg/kg)+PU-H71(50 mg/kg)combined injection group could significantly inhibit the expression of EGFR protein in transplanted tumors of nude mice.Conclusions:1.The high expression of HDAC6 in esophageal cancer tissue promotes the occurrence and development of esophageal cancer,and is expected to be an important target for the treatment of esophageal cancer.2.Using the successfully constructed lentivirus shuttle plasmid vector Lenti-shRNA(HDAC6)-EGFP,the high titer recombinant lentivirus vector Lenti-shRNA(HDAC6)-EGFP was prepared by plasmid co transfection strategy.Through PCR identification and Western blot detection,it was confirmed that the lentivirus carrier system was constructed correctly,with high titer,with high infection activity,which provides a necessary experimental tool for further research on the preclinical research of HDAC6 gene in the treatment of esophageal cancer.3.HSP90 was the target of HDAC6,and the ligand activity of HSP90 wass partly activated by HDAC6 mediated deacetylation.The target silencing HDAC6 gene lentivirus vector RNAi can effectively inhibit the proliferation and cell migration of KYSE140 and KYSE180 in the esophageal cancer cell lines in vitro.The silence of HDAC6 led to the excessive acetylation of HSP90 and ligand activity loss.The combination of HDAC6 specific inhibitors(Tubastatin A)and HSP90 inhibitor(PU-H71)significantly reduced the proliferation of KYSE140 and KYSE180 in esophageal cancer cell lines,the mobility of cells and the protein levels of EGFR,p-AKT,and p-ERK,compared with the treatment of any of the individual inhibitors4.Successful modeling of esophageal cancer cell KYSE140 xenografts in nude miceHSP90-HDAC6 inhibitors significantly inhibited tumor growth and improve the survival quality of nude mice.HSP90-HDAC6 inhibitors significantly reduced the levels of EGFR,p-AKT and p-ERK protein expression in nude mice transplanted tumor.5.The target silencing HDAC6 gene may effectively block the changes of EGFR,p-AKT and p-ERK expression profiles of cell signal transduction pathway in esophageal cancer through overacetylation of HSP90,which provides a new molecular biological mechanism for the pathogenesis of esophageal cancer,and has great potential for application.