The Expression And Regulation Mechanism Of Long Non-Coding RNA XLOC₀05009 In Esophageal Squamous Cell Carcinoma

In recent years,long non-coding RNAs?lnc RNAs?have become a hot-spot.Lnc RNAs may play important roles in both normal development and diseases including cancer.A link between XLOC₀05009 and cancer was first established in pancreatic cancer,with XLOC₀05009 showing reduced expression in pancreatic cancer cells.Further analysis showed that abnormal methylation of the XLOC₀05009 gene results in breast invasive cancer and lung squamous cell cancer.Although XLOC₀05009 DNA methylation was observed to correlate in the progression of some cancer,little is known about the molecular mechanisms underlying esophageal squamous cell carcinoma?ESCC?.Therefore present study aimed to investigate the expression levels and methylation status of long non-coding RNA XLOC₀05009 in human esophageal cancer cell lines and esophageal squamous cell carcinoma?ESCC?tissues,and to evaluate the clinicopathologic and prognostic significance of it in ESCC.Furthermore,the effect of over-expression of XLOC₀05009 on proliferation,migration,invasion,cell cycle and apoptosis of esophageal cancer cells was studied to explore the possible molecular mechanism of XLOC₀05009 involved in biological characteristics of esophageal cancer cells.The main research contents and results were shown as follows:Part one Expression and methylation status of long non-coding RNA XLOC₀05009 in esophageal squamous cell carcinomaObjectives: To detect the expression and methylation status of long non-coding RNA XLOC₀05009 in human esophageal cancer cell lines and esophageal squamous cell carcinoma?ESCC?tissues,and to discuss clinicopathologic and prognostic significance of it in ESCC.Methods:1 RT-PCR method was applied to detect the m RNA expression levels of XLOC₀05009 in esophageal cancer cell lines?TE1,TE13,T.Tn,YES2,Eca109,Kyse170?treated or untreated with DNA methylation transferase inhibitor 5-Aza-d C and 57 esophageal squamous cell carcinomas?ESCC?tissues and corresponding noncancerous tissues.2 MSP method was applied to detect the methylation status of XLOC₀05009 in esophageal cancer cell lines?TE1,TE13,T.Tn,YES2,Eca109,Kyse170?treated and untreated with 5-Aza-d C and esophageal squamous cell carcinomas?ESCC?tissues and corresponding noncancerous tissues.Result:1 The m RNA expression and methylation status of XLOC₀05009 in esophageal cancer cell lines.The expression of XLOC₀05009 in six treated cell lines was increased.Each site of XLOC₀05009 gene showed hypermethylation in six untreated cell lines,after 5-Aza-d C treatment,the methylation status of XLOC₀05009 was decreased.2 The m RNA expression of XLOC₀05009 in ESCC tissues and corresponding noncancerous tissues.The expression of XLOC₀05009 in ESCC tissues was obviously lower than that in corresponding noncancerous tissues?P<0.05?.The expression of XLOC₀05009 was closely correlated with lymph node metastasis,pathological differentiation and TNM stages?P<0.05?.3 The methylation status of XLOC₀05009 in ESCC and corresponding noncancerous tissues.Methylation frequency of XLOC₀05009 in distal Cp G island [77.19%?44/57?vs 68.42%?39/57?,P=0.29] and the exon 2 region[66.67%?38/57?vs 54.39%?31/57?,P=0.18] were not significantly different between ESCC tissue and corresponding noncancerous tissues,and were not associated with lymph node metastasis,pathological differentiation,TNM stages,age and gender?P>0.05?.Methylation frequency of XLOC₀05009 in the exon 1 region was significantly higher in ESCC tissues than in corresponding noncancerous tissues,and was closely correlated with lymph node metastasis,pathological differentiation and TNM stages?P<0.05?.4 The association of XLOC₀05009 with the methylation status of different sites in ESCC tissues.Methylation frequency of XLOC₀05009 in the exon 1 region was significantly lower than that in the distal Cp G island and exon 2 region [45.61%?26/57?vs 77.19%?44/57?,P<0.05;45.61%?26/57?vs 66.67%?38/57?,P<0.05].Methylation frequency of XLOC₀05009was not significantly different between the distal Cp G island and exon 2region [77.19%?44/57?vs 66.67%?38/57?,P>0.05].5 The association between the different sites methylation status of XLOC₀05009 and its expression in ESCC tissues.The expression of XLOC₀05009 in ESCC tissues with XLOC₀05009 methylation in the exon1 was significantly lower than that in ESCC tissues without methylation of this region?0.06±0.06 vs 0.33±0.23,P<0.05?.The expression of XLOC₀05009 in ESCC tissues with XLOC₀05009 methylation in the distal Cp G island and the exon 2 was not significantly different from that in ESCC tissues without methylation of both region?0.18±0.20 vs 0.29±0.25,P>0.05;0.17±0.21 vs 0.27±0.23,P>0.05?.Part Two Effect of long non-coding RNA XLOC₀05009 on the biological characteristics of esophageal cancer cellsObjectives: To detect the effect of long non-coding RNA XLOC₀05009on proliferation,migration,invasion ability,cell cycle and apoptosis of human esophageal cancer cell lines.Methods:1 The pc DNA3.1-XLOC₀05009 expression vector was transfected into human esophageal cancer cell lines.Transfection efficiency was evaluated by RT-PCR.2 MTS and clone formation experiment were respectively applied to detect the proliferation ability of esophageal cancer cell lines with XLOC₀05009 over-expression.3 Scratch test was applied to detect the migration ability of esophageal cancer cell lines with XLOC₀05009 over-expression.4 Transwell chamber invasion assay was applied to detect the invasion ability of esophageal cancer cell lines with XLOC₀05009 over-expression.5 Flow cytometry was applied to detect the cell cycle and cell apoptosis of esophageal cancer cell lines with XLOC₀05009 over-expression.Result:1 Successfully established XLOC₀05009 over-expression plasmid pc DNA3.1-XLOC₀05009 transient transfected esophageal cancer cell lines.RT-PCR analysis revealed that m RNA expression levels of XLOC₀05009were increased significantly in transfected Eca109,Kyse170 cells than that in negative control cells?Eca109: 0.39±0.17 vs 0.02±0.00,P<0.05;Kyse170:0.35±0.08 vs 0.01±0.01,P<0.05?.The transfection efficiency was sufficient to continue the further experiment.2 The effect of XLOC₀05009 gene over-expression on cell proliferation abilities of esophageal cancer cell lines.MTS and clone formation experiment demonstrated that the over-expression of XLOC₀05009 gene could inhibit the proliferation ability of esophageal cancer cells in vitro?P<0.05?.3 The effect of XLOC₀05009 gene over-expression on cell migration abilities of esophageal cancer cell lines.Scratch test demonstrated that the over-expression of XLOC₀05009 gene could inhibit the migration ability of esophageal cancer Kyse170 cell in vitro?P<0.05?,but over-expression of XLOC₀05009 had no effect on migration ability of esophageal cancer Eca109 cell in vitro?P>0.05?.4 The effect of XLOC₀05009 gene over-expression on cell invasion abilities of esophageal cancer cell lines.Transwell chamber invasion assay demonstrated that the over-expression of XLOC₀05009 gene could inhibit the invasion ability of esophageal cancer cells in vitro?P>0.05?.5 The effect of XLOC₀05009 gene over-expression on cell cycle and apoptosis of esophageal cancer cell lines.Flow cytometry assay demonstrated that the over-expression of XLOC₀05009 increased relative proportion in S phase.In addition,over-expression of XLOC₀05009 inhibited esophageal cancer cells proliferation probably through the induction of S phase cell cyclearrest.No significant effect of XLOC₀05009 over-expression on cell apoptosis of esophageal cancer cells was found.Part Three Study on the mechanism of long non-coding RNA XLOC₀05009 influences biological characteristics of esophageal cancer cellsObjectives: To explore the mechanism of XLOC₀05009 on inhibiting the proliferation,migration and invasion abilities of esophageal cancer cells.Methods:1 The expression of proliferation-related molecules?Ki-67,PCNA?and metastasis-related molecules?MMP-2,MMP-9,E-cadherin,N-cadherin and Vimentin?of esophageal cancer cells with XLOC₀05009 over-expression were detected by RT-PCR.2 The expression levels of EMT related genes?Snail1,Twist1,zeb1,?-catenin?of esophageal cancer cells with XLOC₀05009 over-expression were detected by RT-PCR.Result:1 The effect of XLOC₀05009 gene over-expression on proliferation-related and metastasis-related molecules expression levels.RT-PCR analysis revealed that the over-expression of XLOC₀05009down-regulated the m RNA expression levels of Ki-67,PCNA,N-cadherin and Vimentin?P<0.05?,while up-regulated the m RNA expression levels of E-cadherin?P<0.05?.No significant effect of XLOC₀05009 over-expression on the m RNA expression levels of MMP-2 and MMP-9 was detected?P>0.05?.2 The effect of XLOC₀05009 gene over-expression on EMT related genes expression levels.RT-PCR analysis revealed that the over-expression of XLOC₀05009 down-regulated the m RNA expression levels of Twist1 and zeb1?P<0.05?.No significant effect of XLOC₀05009 over-expression on m RNA expression levels of Snail1 and ?-catenin was detected?P>0.05?.Conclusion:1 The decreased expression of XLOC₀05009 is closely related to theoccurrence and development of ESCC and its exon 1 region methylation may be one of the mechanisms to lead to the silence of XLOC₀05009 in ESCC.2 The over-expression of XLOC₀05009 could inhibit the proliferation,invasion and migration ability of esophageal cancer cells in vitro.XLOC₀05009 over-expression inhibited esophageal cancer cells proliferation probably through the induction of S phase cell cycle arrest.3 XLOC₀05009 could affect the proliferation of esophageal cancer cells in vitro,it may be related to the proliferation-related molecules Ki-67 and PCNA.XLOC₀05009 could also affect the invasion abilities of esophageal cancer cells in vitro,this effect may be related to the biological process of EMT.