Study On The Function Of TRIM36 In Esophageal Cancer And Its Related Molecular Mechanism

BackgroudEsophageal cancer is a common malignant tumor of the digestive tract,which is more common in men and is one of the main causes of cancer-related deaths.According to histological classification,esophageal cancer can be divided into two major subtypes,esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC).EAC is mainly distributed in Northern and Western Europe,and is common in the lower digestive tract.ESCC is more common in South and Central Asia,and mostly appear in the upper and middle esophagus.Most of the ESCA cases in China are esophageal squamous cell carcinoma,and the distribution of cases has significant regional characteristics.The original symptoms of ESCA are not obvious,so most patients missed the best treatment period when they receive diagnosis,and some patients have even developed distant metastases.The current treatments for esophageal cancer include surgery,radiotherapy and chemotherapy.In recent years,immunotherapy and targeted therapy have gradually been applied to the clinical treatment.However,the prognosis of patients with esophageal cancer is still poor,with a low 5-year survival rate.Therefore,there is an urgent need to explore the marker molecules that can assist early diagnosis and prognosis evaluation of ESCA,and to further explore the relevant molecular mechanisms of the occurrence and malignant development of esophageal cancer,in order to find more effective therapeutic targets and provide references for clinical strategy.There are many members of the Tripartite motif(TRIM)protein family.Most of the proteins are composed of an N-terminal RING finger domain,one or two zinc-binding motifs called B-boxes,and a coiled-coil motif.Due to the particularity of the N-terminal RING domain,most TRIM family members with RING domain have the function of E3 ubiquitin ligase.Up to now,80 TRIM family protein members have been found,which play a key regulatory role in intracellular signal transduction,apoptosis,embryonic development,immune response,genetic diseases,and cancer development.Most of TRIM family members participate in the occurrence and progression of tumors.In hematological tumors,some members of the TRIM family regulate cancer development through chromosomal translocation or abnormal expression.In solid tumors,more members of the TRIM family are involved in the occurrence and malignancy of cancer.The Wnt signaling pathway is composed of the Wnt/?-catenin,Wnt/PCP pathway,and the Wnt/Ca2+pathway.The classic Wnt/?-catenin signaling pathway has a key regulatory role in cell growth,embryonic development,and cancer development.When the Wnt signal is not activated,the intracellular ?-catenin concentration is low.Once the Wnt signal is activated,through a series of signal transduction,the intracellular inhibitory signal of ?-catenin is lifted,and the accumulation of ?-catenin in the cytoplasm is gradually increased,resulting in the transferring into nucleus.After entering the nucleus,?-catenin interacts with transcription factors to initiate the expression of a series of molecules related to cell cycle and apoptosis.The abnormal activation of Wnt//?-catenin signaling pathway is related to abnormal cell proliferation and the malignant development of various cancers.The activated Wnt/?-catenin signaling pathway may also promote the occurrence and malignant development of esophageal cancer.The activation of Wnt/?-catenin pathway is regulated by the triple motif(TRIM)family of proteins.Some members of the TRIM protein family play an important role in the occurrence and development of cancer by interacting with the Wnt/?-catenin signaling pathway,such as TRIM29,TRIM28,TRIM32,TRIM44.TRIM36 is a microtubule-binding protein that plays a role in embryogenesis,acrosome reaction,chromosome segregation and cell cycle progression.TRIM36 has been identified as an androgen response gene,and also exerts its tumor suppressor effect in prostate cancer.Moreover,TRIM36 expression decreased in non-small cell lung cancer cell lines.Among gastric cancer patients,patients with high expression of TRIM36 have a higher rate of receiving radiotherapy OS.Therefore,TRIM36 is also considered to be an important factor affecting the clinical prognosis of gastric cancer patients with radiotherapy,and may be regarded as a potential radiosensitivity gene marker.However,there is no report to study the relationship between TRIM36 and?-catenin in esophageal cancer and its clinical significance.This study first analyzed the expression of TRIM36 through the esophageal cancer RNA sequencing dataset on the TCGA website.Iimmunohistochemical staining was adopted to clarify the expression of TRIM36 and ?-catenin in clinical samples.The relationship between the expression levels of TRIM36 and ?-catenin and the pathological indicators and prognosis of patients with ESCA was analyzed.Then,the effects of TRIM36 on the malignant proliferation and apoptosis of esophageal cancer cell lines were preliminarily explored in the in vitro cell line system.We used biochemical methods and cell function tests to explore the function of TRIM36 interacting with ?-catenin in esophageal cancer cells and the regulatory mechanism between the two.The effect of TRIM36 and ?-catenin on the proliferation of esophageal cancer cells was verified in a xenograft mouse model.Finally,the expression of TRIM36 in clinical samples was detected,and the relationship between TRIM36 and the malignant development of esophageal cancer was further established,which provides references for future molecular mechanism research and clinical treatment.Part I The expression of TRIM36 and ?-catenin in esophageal cancer and their relationship with prognosisObjectTo explore the expression level of TRIM36 in the RNA sequencing results of esophageal cancer tissues from the TCGA,and detect TRIM36 and ?-catenin in clinical esophageal cancer lesions,and analyze the relationship between the expression levels of TRIM36 and ?-catenin and clinicopathological features,and the prognosis.Methods1.RNA sequencing information of ESCA and normal tissue from the TCGA website was downloaded,and R language DESeq program package was used to analyze gene expression.Initial data screening and data standardization preprocessing were performed based on the download dataset,and the difference in expression of TRIM36 in ESCA and normal tissues was analyzed.2.Clinical ESCA samples from 80 patients who underwent esophageal cancer tissue resection between February 2009 and May 2010.All patients received esophageal cancer tissue resection for the first time without other primary tumors.None of the patients underwent anti-tumor treatment including systemic chemotherapy and radiotherapy before surgery.Clinical characteristics such as patient age,gender,tumor size,TNM staging,lymph node metastasis,and life status were collected and analyzed,and the TNM staging of the intraoperative tissue was performed.3.The mRNA level of TRIM36 in clinical ESCA and normal tissues were quantified by qRT-PCR method.4.Immunohistochemical staining method was used to detect TRIM36 and ?-catenin in isolated ESCA tissues and normal tissues.5.Fisher's Exact Test was used to analyze the correlation between TRIM36 expression and clinical features of ESCA.6.Kaplan-Meier analysis was adopted to draw survival curves to compare the prognosis of patients with ESCA at different expression levels of TRIM36 and?-catenin.Results1.Among the 152 esophageal cancer samples and 21 control samples downloaded from the TCGA website,the expression of TRIM36 in ESCA samples was significantly reduced(P<0.05).In 27 pairs of randomly selected clinical ESCA tissues,the expression of TRIM36 in ESCA tissues was significantly decreased(P<0.001).2.Immunohistochemistry experiments showed that in 80 patients with ESCA,53 patients showed low expression of TRIM36,accounting for 66.25%of the total number of patients;27 patients had high expression of TRIM36,accounting for 33.75%of the total number of patients,and it was mainly expressed in the cytoplasm.?-catenin is expressed in the cytoplasm and nucleus.Among the enrolled ESCA patients,39 patients showed low expression of ?-catenin in ESCA tissue,accounting for 48.75%of the total patients,and 41 patients had high expression of ?-catenin in ESCA tissue,accounting for 51.25%of the total patients.3.GSEA analysis revealed that TRIM36 expression negatively correlated with?-catenin signaling pathway.4.Fisher's exact test displayed that there was a significant correlation between the expression of TRIM36 in ESCA tissues and tumor size(P=0.0104),tumor stage(P=0.0169),lymph node metastasis(P=0.0021),patient survival status(P=0.0443)and ?-catenin expression(P=0.0329)(P<0.05).5.The survival curve drawn by the Kaplan-Meier method showed that the survival rate of patients with ESCA with low TRIM36 expression was lower than that of patients with ESCA with high TRIM36 expression(P=0.0235).The survival rate of ESCA patients with high ?-catenin expression is lower than that of ESCA patients with low ?-catenin expression(P=0.0088).Patients with ESCA with low TRIM36 expression and high ?-catenin expression have the shortest overall survival time.Patients with ESCA with high TRIM36 expression and low ?-catenin expression had the longest overall survival time(P=0.0028)Conclusion1.The expression level of TRIM36 was significant decreased in ESCA tissues.2.TRIM36 level negatively correlated with ?-catenin expression in ESCA tissues.3.The expression of TRIM36 in ESCA tissue is significantly correlated with tumor size,tumor stage,lymph node metastasis,patient survival status,and ?-catenin.4.The expression of TRIM36 and ?-catenin in ESCA patients is related to overall survival time.Part ? The effect of TRIM36 on the growth of esophageal cancer cells and its related mechanismObject1.To explore the effect of TRIM36 on the proliferation and apoptosis of ESCA cell lines.2.To explore the interaction and regulation mechanism between TRIM36 and?-catenin.3.To investigate the effect of the interaction between TRIM36 and ?-catenin on proliferation,apoptosis of ESCA cells.4.To verify the effect of TRIM36 on the proliferation and apoptosis of ESCA cells in vivo.Methods1.Lentiviral transduction was used to establish ESCA cell lines that continuously overexpress TRIM36,and shTRIM36 cell lines.2.CCK-8 assays were performed to detect the proliferation of ESCA cells stably expressing TRIM36 or shTRIM36.3.Flow cytometry experiments were adaopted to detect the apoptosis of ESCA cells stably expressing TRIM36 or shTRIM36.4.The effect of overexpression of TRIM36 or knockdown of TRIM36 on the expression of ?-catenin was detected by western blotting.5.The interaction between TRIM36 and ?-catenin and its underlying mechanism was investigated using immunoprecipitation method.6.The effect of TRIM36 interacting with ?-catenin on cell proliferation was studied using ?-catenin inhibitors and cell lines overexpressing ?-catenin.7.The effects of TRIM36 and ?-catenin on the proliferation of ESCA cells was further explored in xenograft mouse model.8.The expression of TRIM36 and ?-catenin in clinical ESCA tissues was detected and quantified by WB.Results1.Compared with control group,TRIM36 expression markedly decreased in ESCA cells.2.Ectopic expression of TRIM36 inhibited proliferation of ESCA cells while knockdown of TRIM36 promoted ESCA cells growth.3.TRIM36 overexpression made ESCA cells stagnate in G0/G1 phase,and also promoted the apoptosis of ESCA cells,inhibited the expression of ?-catenin in the nucleus,and reduced the expression of Srvivin,Cyclin D1 and c-Myc activated by?-catenin.4.Co-immunoprecipitation results showed that TRIM36 interacted with ?-catenin,and TRIM36 could ubiquitinate ?-catenin at the K625 site of ?-catenin.5.Addition of ?-catenin inhibitor XAV939 could partially offset the proliferation of ESCA cells inhibited by knockdown of TRIM36.Overexpression of ?-catenin also partially offset the inhibitory effect of overexpression of TRIM36 on the proliferation of ESCA cells.6.In a xenograft mouse model,overexpression of TRIM36 significantly inhibited the growth of ESCA cells and promoted apoptosis of ESCA cells,while overexpression of?-catenin partially restored the inhibitory effect of TRIM367.In clinical ESCA tissue samples,compared with control tissues,TRIM36 was less expressed in tumor tissues,while ?-catenin was highly expressed in tumor cell nuclei.Conclusion1.TRIM36 induced ESCA cells to arrest in G0/G1 phase in vitro,inhibited the proliferation of ESCA cells,and promoted ESCA cell apoptosis.2.TRIM36 promoted the ubiquitination of ?-catenin in human ESCA cells.3.TRIM36 inhibited the proliferation of ESCA cells and promoted the apoptosis of ESCA cells in xenograft mouse model.4.In ESCA tissues of patients,the expression of TRIM36 was inhibited and the expression of ?-catenin was up-regulated.