Expression Of MLL2 In Kazakh's ESCC And Its Mechanism In Tumor Cell Invasion And Metastasis

Objective: In the previous study,we found that MLL2 is one of the differentially mutant genes in Kazak's esophageal cancer,but there is no report on the relationship between MLL2 and esophageal cancer.Based on previous studies,we further studied the expression of MLL2 gene in Kazakh esophageal squamous cell carcinoma(ESCC)and adjacent normal tissues,and analyzed the relationship between MLL2 expression and clinicopathological characteristics and prognosis of esophageal carcinoma.At the cellular and animal level,we further explored the effect of MLL2 on the proliferation,invasion and migration of esophageal cancer cells,and preliminary explored its molecular mechanism of promoting cancer in esophageal squamous cell carcinoma.Methods:(1)qRT-PCR and immunohistochemistry(IHC)were used to detect the expression of MLL2 mRNA and protein in cancer tissues and adjacent tissues of ESCC patients,including 42 cases for qRT-PCR and 67 cases for IHC.The correlation of the MLL2 expression with clinicopathologic parameters and prognosis of ESCC was analyzed.(2)CRISPR/Cas9 genome editing technique was used to knockout MLL2 in esophageal cancer cell line Eca109.Three sgRNAs were designed according to the MLL2 sequence,and the sgRNA with the highest knockout efficiency was selected for the following experiments.MTT and colony formation assay was used to detect the effect of MLL2 knockout on cell proliferation;flow cytometry(FCM)was used to investigate cell apoptosis and cell cycle;Scratch wound-healing and Transwell assays was used to investigate the effect of MLL2 on cell migration and invasion.(3)The effect of MLL2 on the proliferation and metastasis of esophageal cancer cells in vivo were studied by subcutaneous transplantation of tumors and tail vein-lung metastasis assays in nude mice.(4)Western blot was used to investigate the effect of MLL2 on the expression of EMT and TGF-? related proteins in Eca109 cells and nude mice subcutaneous transplanted tumors.The correlation between MLL2 and EMT and its possible mechanism were analyzed.The expression of MLL2 and EMT related proteins were detected by IHC in tissue level to further confirm the results of the in vitro and in vivo study.Results:(1)PCR and IHC showed that the expression of MLL2 in esophageal cancer tissues was significantly higher than adjacent tissues,and further analysis of the relationship between MLL2 protein expression and clinicopathological features,high expression of MLL2 was associated with TNM stage(P=0.037),the degree of differentiation(P=0.032)and tumor size(P=0.035).Kaplan-Meier survival analysis showed that MLL2 significantly correlated with the prognosis of patients with esophageal cancer,the survival rate of patients with MLL2 high expression was significantly lower than that of patients with MLL2 low expression(P<0.05,Log-rank).Cox multivariate analysis showed that the lymph node metastasis and differentiation was an independent risk factor for the prognosis of esophageal cancer.(2)MTT and colony formation assay results showed that knockout of MLL2 decreased the cell proliferation ability.FCM assay showed that knockout of MLL2 induced cell cycle arrest at the G1 phase,but it has no significant effect on cell apoptosis.Scratch wound-healing and Transwell assays showed that knockout of MLL2 inhibited cell proliferation and migration ability.(3)In the subcutaneous transplantation assay of nude mice,the growth rate of MLL2 overexpression group was significantly higher than that of control group,while that of MLL2 knockout group was significantly lower than that of control group(P < 0.05).The weight of tumors in MLL2 overexpression group was significantly higher than that in control group,and the weight of tumors in knockout group was significantly lower than that in control group(P < 0.05).The tumorigenicity of esophageal cancer cells in vivo was increased with the increase of MLL2 expression.The results of tail vein-lung metastasis assay showed that the number of lung metastasis nodules in MLL2 overexpression group was significantly more than that in control group(P<0.05).Overexpression of MLL2 could significantly enhance the distant metastasis ability of esophageal cancer cells in vivo.(4)Western blot showed that knockout of MLL2 could inhibit EMT by up-regulation of E-Cadherin and Smad7 as well as down-regulation of Vimentin and p-Smad2/3 in ESCC cells in vitro.The expression of E-cadherin and Smad 7 decreased while the expression of Vimentin and p-Smad 2/3 increased with the increase of MLL2 expression in nude mice subcutaneous transplantation model.These results suggest that MLL2 is associated with the occurrence of EMT in esophageal cancer.It may induce EMT through TGF-?/Smad signaling pathway,and then promote tumor invasion and metastasis.The tissue IHC results showed that compared with the adjacent tissues,the expression of E-cadherin and Smad7 protein were decreased in cancer tissues,and the expression of Vimentin was increased in cancer tissue.Analysis of their association with the expression of MLL2 showed that the expression of E-cadherin in MLL2 high expressed patients was significantly lower than those with MLL2 low expression,and the expression of Vimentin was significantly higher in MLL2 high expressed patients than those with MLL2 low expression,which is consistent with previous study.There was no significant difference in the expression of Smad7 between the two groups.However,the IHC score of Smad7 protein in patients with MLL2 low expression was higher than that in patients with MLL2 high expression.This trend was also consistent with previous Western blot results.Conclusion: MLL2 is highly expressed in Kazakh esophageal cancer,and its expression is related to TNM stage,differentiation degree and tumor size,and is significantly correlated with prognosis of patients.The prognosis of patients with high MLL2 expression is poorer.MLL2 could promote esophageal cancer cell proliferation,invasion and metastasis both in vitro and in vivo.MLL2 is associated with EMT in ESCC.MLL2 could promote the occurrence of EMT,its mechanism may be through inhibiting the expression of Smad7 which could not play its inhibitory role on TGF-? signaling pathway,and promote the occurrence of EMT.