| Part One Expression of long non-coding RNA UCA1 in esophageal squamous cell carcinoma(ESCC)patients in zinc deficiency areasObjectives: To detect the expression of long non-coding RNA UCA1 in ESCC tissues in zinc deficiency areas,and analyze its relationship with the clinicopathological features of ESCC patients.Methods:1 High-throughput sequencing was used to screen differentially expressed lncRNAs in 3 ESCC patients' tissues in zinc deficiency areas.UCA1 with large differential expression ratio was selected as the target gene.2 The expression of UCA1 was detected in 55 ESCC patients' tissues and corresponding adjacent noncancerous tissues by polymerase chain reaction(PCR),and analyzed the relationship between UCA1 expression and clinicopathological features of ESCC patients in zinc deficiency areas.Results:1.High-throughput sequencing showed that UCA1 expression was significantly higher in 3 ESCC patients' tissues(0.86±0.13)than in corresponding noncancerous tissues(0.36±0.02)in zinc deficiency areas,and its expression was upregulated by 2.39 times(t=7.735,P<0.05).2.The results of PCR showed that UCA1 expression was significantly higher in 55 ESCC patients' tissues(1.48±0.58)than in corresponding adjacent noncancerous tissues(0.96±0.37)in zinc deficiency areas,and its expression was upregulated by 1.54 times(t=12.627,P<0.05).3.The expression of UCA1 was closely related to lymph node metastasis and TNM stage in ESCC patients in zinc deficiency areas(lymph node metastasis: ?2=5.770,P=0.016;TNM stage: ?2=9.888,P=0.002).Part Two Relationship between UCA1 expression and intracellular zinc levels in esophageal cancer cellsObjectives: To study the relationship between UCA1 expression and different levels of intracellular zinc deficiency in esophageal cancer cell lines.Methods:1 Different concentrations of TPEN were placed in normal cell culture fluid to prepare a complete medium with different zinc concentrations.Esophageal cancer cells were cultured with different concentrations of zinc and the levels of intracellular zinc in esophageal cancer cells were detected.2 PCR was used to detect the expression of UCA1 in esophageal cancer cells under different concentrations of TPEN.Results:1.Within a certain range,the expression levels of intracellular zinc decreased in esophageal cancer cells as TPEN increased in the medium.Compared with the normal control group(TPEN=0),the relative expression of intracellular zinc decreased to 0.97-fold and 0.96-fold in Eca109 and KYSE170 esophageal cancer cells when the concentration of TPEN was 0.001umol/ml,respectively(P>0.05).As the TPEN concentration increased to 0.002,0.004 and 0.006umol/ml,the relative expression of intracellular zinc decreased to 0.41,0.40,0.25-fold and 0.42,0.40,0.22-fold in Eca109 and KYSE170 esophageal cancer cells,respectively(P<0.05).As the TPEN concentration increased to 0.008 umol/ml,the cells are unable to tolerate and die.2.The expression of UCA1 in esophageal cancer cells was correlated with the level of intracellular zinc.Within a certain range,as the level of zinc decreased,the expression of UCA1 gradually increased in esophageal cancer cells.PCR results showed that compared with the normal control group(TPEN=0),the relative expression of UCA1 increased to 1.03-fold and 1.02-fold in the Eca109 and KYSE170 esophageal cancer cell when the TPEN concentration was 0.001,respectively(P>0.05).As the TPEN concentration increased to 0.002,0.004 and 0.006umol/ml,the relative expression of UCA1 increased to 1.67,1.75,2.49-fold and 1.60,1.70,2.35-fold in Eca109 and KYSE170 esophageal cancer cells,respectively(P<0.05).However,as the TPEN concentration increased to 0.008,the UCA1 expression reduced to 0 as the cells have died.Part Three Effects of long non-coding RNA UCA1 on the biological characteristics of esophageal cancer cells and mechanism researchObjectives:1.To detect the effects of long non-coding RNA UCA1 on proliferation,migration,invasion,cell cycle and apoptosis of different esophageal cancer cell lines.2.To investigate the possible molecular mechanism of UCA1 affecting the biological characteristics of esophageal cancer cells.Methods:1 PCR method was applied to detect the mRNA expression levels of UCA1 in esophageal cancer cell lines and immortalized esophageal epithelial cell line NE1.2 The Eca109 and KYSE170 esophageal cancer cell lines with low UCA1 expression were constructed by transfecting plasmid siUCA1.TE1 and TE13 esophageal cancer cell lines with high UCA1 expression were constructed by transfecting plasmid pcDNAUCA1.The transfection efficiency was detected by PCR.3 MTS and clone formation assay,scratch test,transwell chamber invasion assay and flow cytometry were applied to detect the effect of low/overexpression UCA1 on esophageal cancer cell proliferation,migration,invasion ability,cell cycle and cell apoptosis.4 The mRNA and protein expression levels of EMT related genes(E-cadherin?N-cadherin?Snail and Vimentin)of esophageal cancer cells with UCA1 low/over-expression were detected by PCR and western blot.Results:1.UCA1 expression in esophageal cancer cell lines and immortalized esophageal epithelial cell line: The relative expression levels of UCA1 in esophageal cancer cell lines were significantly higher than that in immortalized esophageal epithelial cell line NE1(Eca109: 0.58±0.09,KYSE170: 0.53±0.10,KYSE150: 0.30±0.06,KYSE30: 0.28±0.15,KYSE9706: 0.29±0.09,TE1: 0.23±0.13,TE13: 0.22±0.13 vs NE1: 0.06±0.03,P<0.05).2.Successfully constructed Eca109 and KYSE170 esophageal cancer cell lines with low expression of UCA1,and TE1 and TE13 esophageal cancer cell lines with over expression of UCA1.3.The effect of UCA1 low/over expression on cell proliferation abilities of esophageal cancer cell lines.The results of MTS assay demonstrated that low expression of UCA1 could inhibit the proliferation ability of esophageal cancer cells Eca109 and KYSE170(Eca109: 0.78±0.16 vs 1.33±0.12 and 1.50±0.12,P<0.05;KYSE170: 1.54±0.14 vs 1.95±0.20 and 2.03±0.13,P<0.05).Over expression of UCA1 could promote the proliferation ability of esophageal cancer cells TE1 and TE13(TE1: 1.17±0.10 vs 1.04±0.06 and 1.05±0.07,P<0.05;TE13: 1.21±0.10 vs 0.89±0.14 and 0.96±0.13,P<0.05).Clone formation assay results are consistent with MTS results(Eca109: 1.45%±0.19% vs 3.82%±0.34% and 4.02%±0.26%,P<0.05;KYSE170: 1.81%±0.36% vs 5.17%±0.31% and 5.35%±0.23%,P<0.05;TE1: 16.01%±0.60% vs 7.41%±0.74% and 8.29%±0.38%,P<0.05;TE13: 25.07%±0.33% vs 12.81%±0.18% and 13.10%±0.22%,P<0.05).4.The effect of UCA1 low/over expression on cell migration abilities of esophageal cancer cell lines.Scratch test demonstrated that low expression of UCA1 could inhibit the migration ability of esophageal cancer cells Eca109 and KYSE170(Eca109: 64.00%±4.97% vs 44.75%±2.63% and 44.00%±10.03%,P<0.05;KYSE170: 42.01%±3.31% vs 22.73%±1.92% and 21.39%±4.82%,P<0.05).Over expression of UCA1 could promote the migration ability of esophageal cancer cells TE1 and TE13(TE1: 43.66%±3.83% vs 58.33%±4.32% and 53.35%±6.32%,P<0.05;TE13: 50.19%±8.35% vs 66.88%±2.91% and 65.50%±2.55%,P<0.05).5.The effect of UCA1 low/over expression on cell invasion abilities of esophageal cancer cell lines.Transwell chamber invasion assay showed that low expression of UCA1 could inhibit the invasion ability of esophageal cancer cells Eca109 and KYSE170(Eca109: 224.75±17.02 vs 301.25±11.62 and 309.00±17.05,P<0.05;KYSE170: 208.75±24.14 vs 289.50±20.89 and 319.75±12.89,P<0.05).Over expression of UCA1 could promote the invasion ability of esophageal cancer cells TE1 and TE13(TE1: 434.00±13.34 vs 267.25±11.93 and 291.25±19.14,P<0.05;TE13: 393.75±16.72 vs 218.00±14.94 and 214.25±14.38,P<0.05).6.The effect of low/over expression of UCA1 gene on cell cycle of esophageal cancer cells.Flow cytometry analysis showed that low expression of UCA1 could arrest the cell cycle in the S phase(Eca109: 42.26±3.68 vs 33.43±1.62 and 35.64±1.53,P<0.05;KYSE170: 38.29±1.60 vs 32.29±3.12 and 33.69±1.52,P<0.05)and affect cell proliferation.Over expression of UCA1 could accelerate the cell cycle in G0/G1 phase(36.72±0.80 vs 52.85±2.29 and 52.83±1.51,P<0.05)and affect the TE1 cell proliferation.Over expression of UCA1 could accelerate the cell cycle in S phase(30.70±2.72 vs 38.48±1.90 and 38.00±1.67,P<0.05)and affect the proliferation of TE13 cells.7.The effect of low/over expression of UCA1 gene on cell apoptosis of esophageal cancer cells.Flow cytometry analysis showed that low expression of UCA1 could promote the apoptosis of esophageal cancer cells Eca109 and KYSE170(Eca109: 10.78±2.92 vs 1.85±0.83 and 1.91±0.91,P<0.05;KYSE170: 9.75±1.74 vs 1.98±0.64 and 2.08±0.46,P<0.05).Over expression of UCA1 could inhibit the apoptosis of esophageal cancer cells TE1 and TE13(TE1: 1.10±0.12 vs 2.72±0.69 and 2.72±0.77,P<0.05;TE13: 1.03±0.22 vs 3.46±0.58 ? 3.73±1.01,P<0.05).8.The effects of low/over expression of UCA1 on expression of EMT related genes in esophageal cancer cells.PCR analysis demonstrated that the expression of E-cadherin in siUCA1 transfection group increased to 1.55-fold and 1.32-fold in Eca109 and KYSE170 esophageal cancer cells compared with the si NC transfection group,respectively.And the expression of N-cadherin,Snail and Vimentin decreased to 0.69,0.63,0.64-fold and 0.65,0.74,0.70-fold in Eca109 and KYSE170 esophageal cancer cells,respectively(P<0.05).Compared with the pcDNANC transfection group,the expression of E-cadherin in pcDNAUCA1 transfection group decreased to 0.71-fold and 0.70-fold in TE1 and TE13 esophageal cancer cells,respectively.And the expression of N-cadherin,Snail and Vimentin increased to 1.33,1.26,1.42-fold and 1.23,1.20,1.37-fold in TE1 and TE13 esophageal cancer cells,respectively.Western blot results demonstrated that low expression of UCA1 upregulated E-cadherin expression(Eca109: 0.97±0.09 vs 0.48±0.07 and 0.49±0.08,P<0.05;KYSE170: 0.90±0.10 vs 0.42±0.06 and 0.40±0.10,P<0.05),but downregulated N-cadherin(Eca109: 0.42±0.07 vs 0.95±0.05 and 0.94±0.05,P<0.05;KYSE170: 0.27±0.06 vs 0.65±0.11 and 0.70±0.10,P<0.05),Snail(Eca109: 0.33±0.06 vs 0.93±0.05 and 0.93±0.06,P<0.05;KYSE170: 0.28±0.04 vs 0.69±0.08 and 0.75±0.08,P<0.05)and Vimentin(Eca109: 0.36±0.03 vs 0.85±0.03 and 0.87±0.13,P<0.05;KYSE170: 0.13±0.03 vs 0.77±0.04 and 0.80±0.05,P<0.05)expression in esophageal cancer cells Eca109 and KYSE170.Over expression of UCA1 downregulated E-cadherin expression(TE1: 0.31±0.09 vs 0.89±0.07 and 0.89±0.03,P<0.05;TE13: 0.32±0.05 vs 0.74±0.07 and 0.79±0.07,P<0.05),but upregulated N-cadherin(TE1: 0.84±0.07 vs 0.54±0.04 and 0.56±0.15,P<0.05;TE13: 0.84±0.06 vs 0.44±0.08 and 0.49±0.05,P<0.05),Snail(TE1: 0.84±0.02 vs 0.26±0.06 and 0.28±0.02,P<0.05;TE13: 0.90±0.05 vs 0.50±0.06 and 0.51±0.03,P<0.05)and Vimentin expression(TE1: 0.80±0.05 vs 0.40±0.11 and 0.41±0.13,P<0.05;TE13: 0.85±0.04 vs 0.42±0.06 and 0.44±0.05,P<0.05)in esophageal cancer cells TE1 and TE13.Conclusions:1.UCA1 was upregulated in ESCC tissues and esophageal cancer cell lines in zinc deficiency areas,and its expression level was closely related to lymph node metastasis and TNM stage in ESCC patients.2.The expression of UCA1 in esophageal cancer cells was correlated with intracellular zinc levels.Within a certain range,as the level of zinc decreased,the expression of UCA1 gradually increased in esophageal cancer cells.3.UCA1 could significantly promote the proliferation,migration and invasion and inhibit the apoptosis of esophageal cancer cells,and affect the proliferation of esophageal cancer cells by affecting the cell cycle.4.The effect of UCA1 on invasion and migration of esophageal cancer cells may be related to the biological process of EMT. |
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