Expression Of LSD1 In Esophageal Squamous Cell Carcinoma And Its Influences On Some Of Tumor Biological Behaviors

BackgroundCovalent modifications of histone proteins are important epigenetic patterns, including acetylation, methylation, phosphorylation, ubiquitination and so on, in which acetylation and methylation are studied more at present. Lysine specific demethylase 1(LSD1) was first discovered in 2004 by Shi and his partners, which broke the view that histone methylation was irreversible.In the demethylation reaction catalyzed by LSDl, different molecular chaperone can mediate the action of LSD1 on different substrates and result in different biological effects. When LSD1 combined with target genes thought the CoREST (Co-repressor of the repressor element silencing transcription factor) complex, it can demethylate H3K4me1/ me2, which associated with the chromatin condensation and transcription inhibition. At this point, LSD1 can inhibit gene transcription as a transcriptional inhibitor. When LSD1 combined with Androgen Receptor (AR) or Estrogen Receptor (ER), it can act on H3K9me1/2 specifically. Demethylation of H3K9 was related to the chromatin conformation opening and gene expression, induced to the up-regulation of AR/ER dependent genes. LSD1 can also promote transcriptional activation. Recent years, a number of functional studies have found that LSD1 regulate the expression of target genes and play an important role in the tumorigenesis, embryonic differentiation, heterochromatin formation, the reasonable maintenance of intracellular DNA methylation status, the formation of induced pluripotent stem cells and some other aspects.Previous research found that LSD1 was high expressed in prostate cancer, breast cancer and blood system tumors generally, and they can inhibit the proliferation of tumor cells significantly by LSD1 knock-down or LSD1 function inhibiting. In addition, there are a variety of histone modification enzyme inhibitors had been used for treatment of tumors, such as HDACi, while the LSD1 inhibitor is expected to be a new target for development of anti-cancer drugs. It is of great significance to the tumor prevention and treatment that get profound understanding the LSD1 action mechanism and revealing the dynamic balance mechanism of histone methyl and demethylation.Esophageal cancer is the eighth common cancers worldwide. The occurrence of esophageal cancer had a obvious regionality that Asia is one of the high incidence areas. Patients with early esophageal carcinoma had a relatively high 5 years survival rate after operation. But along with the progressing of esophageal cancer, the prognosis of patients get worse and worse, especially in patients with metastasis. With regard to esophageal cancer, the 5-year survival rate of esophageal squamous cell carcinoma(ESCC) is only 5%-12.3% in Europe. At the same time, as the low detection rate of early esophageal cancer, the therapeutic effect of the disease is serious impacted. Previous researches found that, like most other tumors, the occurrence and development of esophageal cancer underwent both genetic and epigenetic modification mutations, while the reversible of epigenetic modification also shows the possible of prevention and treatment of ESCC. LSD1 was high expressed in many tumors, which demonstrated that LSD1 may be associated with the pathogenesis of ESCC, but the expression and significance of LSD 1 in ESCC is obscure. Our study try to explore the contribution of LSD 1 in the tumorigenesis and development of ESCC, lay a preliminary foundation for the in-depth study of the mechanism, and find novel molecular targets that reduce postoperative recurrence and metastasis in ESCC.objectivesIn this study, we examined and compared the expression of LSD1 in ESCC and its corresponding microarray tissues, esophageal precancerous lesions and normal esophageal epithelium mucosa, to reveal the association of LSD1 expression with the differentiation, infiltration, metastasis and some other biological properties, and conjecture the possible relationship between LSD1 and the prognosis of ESCC patients. Meanwhile, we tested and disturbed the expression and function of LSD 1 in ESCC cell lines, to observe the changes of cell invasion and migration, to further explore the possible roles of LSD 1 played in the occurrence and development of ESCC, and to provide a novel molecular target for the prevention and treatment for ESCC.MethodsA. Tissue specimens experiment:to investigate the expression and significance of LSD1 in different clinical specimens.Experimental groups:Group A. Human esophageal cancer tissues; Group B. Esophageal precancerous lesions (including intraepithelial neoplasia and atypical hyperplasia); Group C. Normal esophageal mucosas.Experimental procedure:make tissue micro-assay, HE staining was performed to reconfirm the diagnosis of ESCC. Immunohistochemical was performed to measure the expression of LSD1 and Ki67 in these tissues, meanwhile, cell apoptosis was detected by TUNEL(TdT-mediated dUTP nick end labeling). Immunohistochemical staining was evaluated by two pathologists according to the IRS(semi-quantitative Remmele scoring system), which links the SI(IHC staining intensity) with the PP(percentage of positive cells). SI was scored between 0 and 3 as following:0:no positive cells were observed,1:all positive nuclei display a weak staining,2:the most stained nuclei show a moderately positive,3:the most stained nuclei display a intensive staining. PP was scored according to the following criteria:0:no positive cells were observed,1:only less than 10% nuclei display intense staining,2:11%-50% nuclei were observed,3:51%-80%,4:more than 80%. Finally the IHC score was calculated by SI × PP. Then, in order to analysis the relationship between LSD1 expression and some clinicopathological characteristics, patients were classified into three groups according to the IHC scores:negative (IHC score = 0), low expression(0<IHC scores<4) and high expression(4<IHC scores<12).Tracing and follow-up of ESCC patients, to obtain the basic informations, data related to the clinical treatment and prognosis. Analysis the association between LSD1 and the informations, and to explore that wether LSD1 was related to the ESCC proliferation and apoptosis.B. Cellular experiment:to explore the effect of LSD1 on parts of the ESCC cell lines biological behaviors.Experimental groups:according to the different ESCC cell line, divided into three groups:KYSE150, KYSE450 and EC109.Experimental procedure:Western Blot and RT-PCR were performed to detect the RNA and protein level of LSD1 in different ESCC cell lines. Transwell and scratch assays were used to test the invasion and mobility in these cell lines with different LSD1 expression level.C. Interference experiment:knockdown the expression of LSD1 or inhibit the function of LSD1 using pharmacological inhibitor, and then observe the changes of the biological behaviors in ESCC cell lines.1. Knockdown the LSD1 expression in KTSE450 cells using lentivirus designed specifically for LSD1. At the same time, negative virus without functional shRNA was used as a control. The green fluorescent protein(GFP) expression was the sigh of the success of stable transfected by lentivirus.Experimental groups:According the disparate viruses with different effects on LSD1 knockdown, we divided the cells into three groups:Group A. Knockdown 1(KD1); Group B. Knockdown 2(KD2); Group C. Negative control(NC).Experimental procedure:pack the lentivirus and knockdown LSD1 expression in KYSE450 cells, RT-PCR and Western Blot were used to detect the knockdown effects. Investigated the changes of invasion and mobility in KYSE450 cells after LSD1 knockdown and analyzed the relationship between LSD1 expression and cell biological behavior.2. Treated KYSE450 cells with tranylcypromine, which acts as a LSD1 inhibitor, in different concentrations to inhibit the function of LSD1, and then observed the changes of cell biological behavior.Experimental groups:according to the concentration of tranylcypromine, divided the groups into:Group A. OmM(control); Group B.50uM; and Group C.250uM.Experimental procedure:cultured KTSE450 cells with or without treatment of tranylcypromine and extracted the protein. Detected the expression of LSD1, H3K4mel/2 and H3K9mel/2 with RT-PCR or Western Blot, to determine the possible site of LSD1 acted on in ESCC. At the same time, examined the motion and invasion of ESCC when cells treated with tranylcypromine.Results1. A total of 29 normal esophageal mucosas,23 cases of esophageal precancerous tissue lesions and 134 cases of histopathology conformed ESCC were involved in our study. Examined and compared the expression of LSD1, we found that only 51.7% of the normal esophageal epithelial mucosas demonstrated the slightly positive LSD1 expression at the nuclei, while precancerous lesions presented a higher expression level of LSD1(73.9%). The location of LSD1 at the nuclei can be observed in 75.4% of ESCC tissues, of which, 43.7% of LSD1-positive cells were high expression, indicating that LSD1 was expressed at a higher level in ESCC relative to normal or precancerous tissues. It suggested that LSD1 may have some contributions to tumorigenesis. In addition, ESCC patients with lymphatic metastasis expressed LSD1 (56.8%) at a higher level than that in the non-metastatic cases (32.6%).2. LSD1 expression was not correlated with of the following clinicopathologic characteristics:sex, age, differentiation, infiltration, and histological type (p>0.05). However, we detected a correlation between LSD1 expression and lymph node metastasis in ESCC patients. Meanwhile, LSD1 expression was significantly associated with the prognosis of patients after operation, patients with a higher expression of LSD1 were significantly inferior to overall survival as compared to patients with lower LSD1 expression (p<0.05).3. LSD1 and Ki67 were both expressed in the basal part of normal esophageal mucosas, which displayed a strong proliferating ability. Meanwhile, the expression of LSD1 and Ki67 in ESCC tissues was significantly correlated(p< 0.01) that high expression of LSD1 was accompanied with a high expression of Ki67. But expression of LSD1 was not correlated with cell apoptosis in ESCC that apoptotic cells in ESCC tissues were rarely observed, regardless of high or low LSD1 expression.4. Different ESCC cell lines, including KYSE450,KYSE150 and EC 109, expressed LSD1 in a discrepant level. The expression of LSD1 in KYSE450 cells and KYSE150 cells was significantly higher than that in CE109 cells.5. The scratch and Transwell assay confirmed that the motility and invasion ability of KYSE450 and KYSE150 cells were much stronger than that of EC 109 cells.6. We got two cell lines with LSD1 knockdown in different level by lentiviral transfection. Knockdown of LSD1 significantly inhibited migration and invasion of LSD1-positive KYSE450 cells as compared with cells transfected with control shRNA.7. Treated KYSE450 cells with LSD1 inhibitor tranylcypromine, and we found that with increasing concentrations of tranylcypromine, H3K4mel expression decreased, while HeK4me2 expression increased. However, there was not a significant change in levels of H3K9mel or H3K9me2.8. Tranylcypromine suppressed the migration and invasion of KYSE450 cells in a dose-dependent manner.Conclusions1. LSD1 expression was relatively higher in ESCC tissue compared with normal esophageal epithelial or atypical epithelial hyperplastic tissues. Moreover, among the clinicopathological parameters, LSD1 expression was tightly correlated with lymph node metastasis and suggested that LSD1 may be predictive of poorer prognosis in ESCC patients. The expression of LSD1 was related to the Ki67, implying that LSD1 may be associated with ESCC proliferation, but uncorrelated with cell apoptosis.2. A discrepancy in LSD1 expression between three ESCC cells lines was observed, in which, KYSE150 and KYSE450 cells with a stronger motion and invasion abilities expressed LSD1 higher than the EC 109 cells. LSD1 knockdown lead to the sharp decrease in the motility and invasion of KYSE450 compared to the control shRNA treated cells.3. Treated KYSE450 cells with tranylcypromine can suppress the migration and invasion of KYSE450 cells significantly. Meanwhile, we uncovered an increase in H3K4me2 methylation and a decrease in H3K4mel methylation, tranylcypromine reducing the chance of transformation from H3K4me2 to H3K4mel in a dose-dependent manner. These results implied that LSD1 may regulate downstream genes via demethylating H3K4me2.