| Esophageal squamous cell carcinoma(ESCC)is a common malignant tumor of digestive tract.Due to its insidious onset and poor prognosis,the existing treatment methods can’t significantly improve the prognosis of esophageal cancer.Therefore,new treatment methods are urgently needed.Long non-coding RNA plays a regulatory role in the occurrence and development of various tumors,and long non-coding RNA00114 can promote the occurrence and development of colorectal cancer.In hepatocellular carcinoma,zeste promoter homolog 2/ deleted in liver cancer 1(EZH2/DLC1)can mediate the proliferation,invasion and apoptosis pathways of hepatocellular carcinoma cells.Meanwhile,a variety of long non-coding RNAs are involved in ESCC cell proliferation and glycolysis,but the expression and mechanism of long non-coding RNA00114 in ESCC are rarely studied.Therefore,in this study,we first detected the expression differences of LINC00114,EZH2 and DLC1 in ESCC tumor tissues,normal tissues,esophageal cancer cell lines and epithelial cells.Then we investigated the relationship between the expression differences and EC cell proliferation,migration,invasion,apoptosis and glycolysis.In the second part,we used Sh-RNA and Si-RNA techniques to intervene the expressions of LINC00114,EZH2 and DLC1 respectively to explore the changes of cell proliferation,migration,invasion,apoptosis and glycolysis of EC cells after intervention in vitro.In part III,we verified the mechanism of LINC00114 and DLC1 in the xenograft tumor model of immunodeficient mice.Part Ⅰ The expression of LINC00114 in ESCC and its effect on the biological function of EC cells.Objective: To study the expression of LINC00114 in ESCC and its effect on biological function of EC cells.Methods: The expression of LINC00114 in ESCC tissues,non-tumor tissues,tumor cell lines and esophageal epithelial cells was detected by RT-q PCR and Western blot.The changes of EC cell proliferation,migration,invasion,apoptosis and glycolysis before and after LINC00114 knockdown were detected by CCK-8 assay,colony formation assay,Transwell assay and flow cytometry.Results: 1.RT-q PCR was used to detect the expression of LINC00114 in ESCC and non-tumor tissues.The expression of LINC00114 in ESCC was significantly higher than that in non-tumor tissues.Compared with HEEC cell lines,the expression of LINC00114 was significantly up-regulate in TE1,Eca109,KYSE150 and EC9706.And LINC00114 increased more significantly in Eca109 and TE1 cells.2.The sh RNAs,sh-LINC00114-1 and sh-LINC00114-2,were transfected into Eca109 and TE1 cells by lipo2000,respectively.Both plasmids could down regulate the expression of LINC00114 in EC,and the down-regulation of sh-LINC00114-1 was more obvious.CCK-8 and colony formation assay confirmed that knockdown of LINC00114 inhibited the proliferation of EC.3.The transwell assay indicated that knockdown of LINC00114 inhibited migration and invasion of EC cells.Flow cytometry showed that shLINC00114-1 down-regulated LINC00114 significantly promoted apoptosis of Eca109 and TE1 cells.4.Detection of glycolysis in tumor cells indicated that down-regulation of LINC00114 inhibits the growth and glycolysis of EC cells.Conclusions: The expression of LINC00114 in ESCC tissues was higher than that in non-tumor tissues and EC cells was higher than that in esophageal epithelial cells.Knockdown of LINC00114 in EC cells inhibited the growth and glycolysis of esophageal carcinoma cells.Part Ⅱ The experiment on the relationship among LINC00114,EZH2 and DLC1 and its molecular mechanism in ESCC.Objective: To investigate the relationship among LINC00114,EZH2 and DLC1 and its molecular mechanism in ESCC.Methods: The expressions of EZH2 and DLC1 in ESCC tissues and cell lines were detected by RT-q PCR and Western blot,and the effects of EZH2 and DLC1 on the proliferation,migration and glycolysis of esophageal cancer cells were evaluated respectively.The mechanism between LINC00114 and EZH2 and DLC1 was verified by Ch IP.LINC00114,EZH2 and DLC1 of EC cells were down-regulated in vitro,and the proliferation,migration,invasion,apoptosis and glycolysis of EC cells were detected by CCK-8 assay,colony formation assay,Transwell assay and flow cytometry before and after the intervention.Results: 1.RT-q PCR and Western blot indicated that the expression of EZH2 in ESCC was significantly higher than that in non-tumor tissues,and its expression in Eca109 and TE1 cells was higher than that in esophageal epithelial cells.The expression of EZH2 was down-regulated by transfecting sh-EZH2-2 targeting EZH2.CCK-8 and colony formation assay confirmed that knockdown of EZH2 inhibited the proliferation of EC cells.Transwell assay showed that knockdown of EZH2 significantly inhibited migration of EC cells.Detection of glycolysis confirmed that down-regulation of EZH2 inhibited glucose intake in EC cells.The above conclusions were consistent with the first part,indicating that there may be a correlation between EZH2 and LINC00114.2.RNA pull-down assay showed that LINC00114 could bind to EZH2,and LINC00114 could be enriched in immunoprecipitation.Further research indicated that LINC00114 could combine with EZH2 and affect the expression of DLC1 through H3K27me3 pathway,thereby affecting the activity of EC cells.3.The expression of DLC1 in ESCC,non-tumor tissues,EC cell lines and esophageal epithelial cells was detected by RT-q PCR.The results indicated that the expression of DLC1 in ESCC and EC cell lines was significantly lower than that in non-tumor tissues and esophageal epithelial cells.Down-regulation of DLC1 enhances the proliferation of EC cells.Down-regulation of DLC1 enhanced migration and invasion,glucose intake,lactate production and ATP production in Eca109 and TE1 cells.In summary,DLC1 might be a tumor suppressor gene of EC.4.After the specific knockout of DLC1 in EC cells,the proliferation and invasion ability of EC cells and the glucose intake ability were enhanced,that is,the knockout of DLC1 could reverse the role of sh-LINC00114 in EC cells.Conclusions: Silencing EZH2 inhibited the proliferation of EC cells and affected glycolysis of EC cells.LINC00114 could affect DCL1 expression by binding to the EZH2 and accumulating H3K27me3 in DLC1 promoter locus.Knockdown of DLC1 promoted EC cell proliferation and glycolysis and reversed the inhibitory effects of sh-LINC00114 on EC cell proliferation and glycolysis.Part Ⅲ The experiment on the role of LINC00114 and DLC1 in ESCC in xenograft.Objective: To evaluate the role of LINC00114 and DLC1 in ESCC in xenograft.Methods: Eca109 cells transfected with sh-LINC00114 or si-DLC1 were subcutaneously transplanted into BALB/C nude mice to evaluate tumor growth rate and detect the expression of DLC1 in tumor tissues.Results: 1.The growth rate of sh-LINC00114 transfected Eca109 cells was significantly decreased in BALB/c nude mice.The growth rate of Eca109 cells interfered with DLC1 was significantly increased in vivo.2.The expression of DLC1 in Eca109 cells transfected with sh-LINC 00114 was significantly increased.The tumor volume was inversely correlated with the expression of DLC1.Conclusions: Sh-LINC00114 significantly reduced the tumorigenic ability of Eca109 cells,while si-DLC1 significantly accelerated the tumor growth rate.Immunohistochemistry showed that DLC1 expression was elevated in the sh-LINC00114 group,while si-DLC1 inhibited DLC1 expression. |
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