The Expression Of MiR-193b And Function Related To ZIP5(SLC39A5) In Esophageal Cancer In Zinc Deficiency Areas

Objectives: Studies showed that Zn(zinc)deficiency was involved in the development of esophageal cancer,miRNAs were important pathways to affect the occurrence of esophageal cancer,and ZIP5(SLC39A5)regulated the level of Zn in the body.At present,there was no research on the relationship between the expression of miRNAs and ZIP5.Therefore,this study aims to study the molecular mechanisms of miRNAs involved in the regulation of ZIP5 expression in the high-incidence area of esophageal cancer,in order to elucidate the role of miRNAs in esophageal cancer,and provide experimental basis and direction in the effective diagnosis and precise treatment of patients with high incidence of esophageal cancer.Methods: 1 Objects: 1.1 Cell lines: human normal esophageal cancer cell line NE1,human esophageal cancer cell KYSE150,KYSE170,Eca109,KYSE9706,TE1,tool cell 293 T.1.2 Tumor tissues: 48 cases of cancer and esophageal non-tumor tissue in the esophageal cancer patients in the high incidence area of esophageal cancer provided by the Department of Thoracic Surgery,Fourth Hospital of Hebei Medical University from 2016 to 2017.2 Experiment methods 2.1 Databases were applied to screen for miRNAs that might be associated with ZIP5 and Zinc.2.2 KYSE150 and KYSE170 esophageal cancer cells were cultured in gradient with TPEN to simulate low-zinc environment in high-incidence areas of esophageal cancer,and cell growth was observed.The expression of miRNAs in esophageal cancer cells under different concentrations of TPEN was further detected by qRT-PCR.2.3 The relationship between miR-193 b and ZIP5 was verified by immunofluorescence enzyme assay.2.4 qRT-PCR was used to detect the expression of miR-193 b in the immortalized esophageal epithelial cell line NE1 and esophageal cancer cell lines.2.5 Over/low expression of miR-193 b esophageal cancer cell lines were constructed using transfection plasmids miR-193 b mimics and inhibitor.2.6 The effects of miR-193 b on proliferation,migration,invasion,cell cycle and apoptosis of esophageal carcinoma cells were detected by MTS and clone formation experiments,scratch assays,transwell chamber invasion assay and flow cytometry.2.7 RT-PCR and western blot were used to detect the expression of ZIP5,CyclinD1 and E-cadherin mRNA and protein after over/low expression of miR-193 b gene.2.8 HE staining was used to detect the histological types of cancer and adjacent non-tumor tissues in patients with esophageal cancer.2.9 The expression of miR-193 b in tumor and paracancerous non-tumor tissues of 48 patients with ESCC was detected by qRT-PCR.The relationship between miR-193 b expression and clinical pathological data of 48 patients with ESCC was analyzed.And correlation analysis was used to analyze the correlation between miR-193 b and ZIP5 in patients with ESCC.3.Statistical analysis: All data were statistically analyzed using SPSS 21.0 software.The measurement data were analyzed by t test or one-way ANOVA.The count data were analyzed by ?2 or corrected ?2 test,and the correlation analysis was performed by Spearman correlation.Results: 1 Screening of miR-193 b and verification of its relationship with ZIP5 1.1 Using the mirDPI,Sanger,Pictar databases to screen for miRNAs associated with ZIP5 and Zinc,results showed that miR-193 b,miR-328,and miR-137 might be associated with ZIP5 and Zinc.1.2 The results of qRT-PCR showed that the expression level of miR-193 b in esophageal cancer cells decreased gradually with the increasing of TPEN concentration in the medium,and the difference was statistically significant(P<0.05).However,with the increasing of TPEN concentration,there was no significant difference in the expression of miR-328 and miR-137(P>0.05),suggesting that miR-193 b might be related to Zn level.Therefore,miR-193 b was selected for subsequent research.1.3 Targetscan database showed that primer sequences of miR-193 b and ZIP5 were complementary to each other.The immunofluoresceinase results showed that the fluorescence activity of the ZIP5 3'UTR WT group(0.72±0.03)was significantly weaker than that of the ZIP5 3'UTR NC(1.00±0.00,P<0.05),while the fluorescent activity expression of the ZIP5 3'UTR MUT group(0.96±0.02)was not statistically significant compared with the ZIP5 3'UTR NC(1.00±0.00,P>0.05),when adding miR-193 b mimics.These suggesting that ZIP5 was the target gene of miR-193 b.2 Functional mechanisms of miR-193 b in esophageal cancer in vivo 2.1 Expression of MiR-193 b in normal esophageal epithelial and esophageal cancer cell lines and construction of over/low expressed miR-193 b esophageal cancer cell lines: the expression level of miR-193 b in normal esophageal epithelial cells was 0.71±0.03 which was higher than in Eca109(0.45±0.03),KYSE150(0.23±0.02),KYSE170(0.27±0.01),KYSE9706(0.43±0.02)and TE1(0.30±0.03)esophageal cancer cells(P<0.05).Therefore,KYSE150 and KYSE170 esophageal cancer cells were selected to do the overexpression of miR-193 b gene and Eca109 and KYSE9706 esophageal cancer cells were selected to do the downregulated expression of miR-193 b gene.2.2 Effect of over/low expression of miR-193 b on cell proliferation ability: MTS results: proliferation of KYSE150 and KYSE170 cells were inhibited after overexpression of miR-193 b gene(KYSE150: 0.68±0.04 vs 0.84±0.07 and 0.89±0.04,P<0.05;KYSE170: 0.66±0.07 vs 0.76±0.06 and 0.79±0.07,P<0.05),low expression of miR-193 b gene could promote the proliferation of Eca109 and KYSE9706 esophageal cancer cells(Eca109: 0.58±0.05 vs 0.44±0.03 and 0.44±0.04,P<0.05;KYSE9706: 0.72±0.06 vs 0.64±0.03 and 0.64±0.03,P<0.05).Cell clone formation assay further verified the proliferation ability of miR-193 b on esophageal cancer cells,and the results were consistent with MTS.Overexpression of miR-193 b inhibited the number of clonal cells of esophageal cancer cells(KYSE150: 104.67±2.52 vs 247.67±2.52 and 251.67±2.08,P<0.05;KYSE170: 115.33±6.51 vs 251.67±4.04 and 257.67±2.52,P<0.05),low expression of miR-193 b could increase the number of cloned cell clusters(Eca109: 342.00±4.00 vs 250.67±3.79 and 255.67±4.51,P<0.05;KYSE9706: 329.67±2.52 vs 256.33±3.51 and 261.00±1.00,P<0.05).2.3 Effect of over/low expression of miR-193 b on cell migration ability: the results showed that overexpression of miR-193 b inhibited the migration of esophageal cancer cells(KYSE150: 53.70%±4.90% vs 30.31%±13.48% and 26.08±9.88%,P<0.05;KYSE170: 60.15%±4.00% vs 28.26%±8.18% and 19.11%±2.38%,P<0.05),low expression of miR-193 b could promote the migration ability of esophageal cancer cells(Eca109:14.02%±2.79% vs 28.92%±3.62% and 22.51%±4.63%,P<0.05;KYSE9706: 22.20%±3.41% vs 43.43%±4.46% and 39.10%±0.05%,P<0.05).2.4 Over/low expression of miR-193 b on cell invasion ability: overexpression of miR-193 b gene could inhibit the invasion of esophageal cancer cells(KYSE150: 89.67±7.51 vs 278.33±7.64 and 288.33±12.58,P<0.05;KYSE170: 106.00±6.56 vs 284.33±10.79 and 300.00±10.00,P<0.05),low expression of miR-193 b gene could promote the invasion ability of esophageal cancer(Eca109: 315.33±5.51 vs 285.67±14.01 and 295.67±4.04,P<0.05;KYSE9706: 361.67±12.58 vs 283.67±7.77 and 290.33±1.53,P<05).2.5 Over/low expression of miR-193 b on cell cycle: overexpression of miR-193 b gene could inhibit cell cycle by acting on G1 phase of esophageal cancer cell cycle(KYSE150: 64.03±2.31 vs 53.15±3.70 and 53.96±5.36,P<0.05;KYSE170: 60.96±1.57 vs 50.08±2.66 and 52.25±4.17,P<0.05),low expression of miR-193 b gene could promote cell by promoting cell transformation from G1 to S phase(Eca109: 44.53±3.51 vs 54.27±4.90 and 54.54±3.90,P<0.05;KYSE9706: 48.59±0.06 vs 52.02±0.68 and 51.42±1.05,P<0.05).2.6 Over/low expression of miR-193 b on apoptosis:The ratio of apoptosis of esophageal cancer cells was both less than 5% after over/low expression of miR-193 b,miR-193 b was not considered to influence apoptosis of esophageal cancer.2.7 Over/low expression of miR-193 b on the mRNA levels of ZIP5,CyclinD1 and E-cadherin genes: overexpression of miR-193 b gene could inhibit the expression of ZIP5 and CyclinD1 mRNA(KYSE150: ZIP5: 0.14±0.01 vs 0.20±0.04 and 0.19±0.04,P<0.05;CyclinD1: 0.46±0.17 vs 0.88±0.13 and 1.01±0.16,P<0.05;KYSE170: ZIP5: 0.26±0.01 vs 0.38±0.03 and 0.35±0.02,P<0.05;CyclinD1: 0.63±0.06 vs 0.95±0.18 and 0.96±0.14,P<0.05),promoted the expression of E-cadherin gene(KYSE150: 0.11±0.02 vs 0.05±0.02 and 0.05±0.03,P<0.05;KYSE170: 0.94±0.16 vs 0.56±0.08 and 0.57±0.09,P<0.05),low expression of miR-193 b gene could promote the expression of ZIP5 and CyclinD1 mRNA(Eca109: ZIP5: 0.25±0.04 vs 0.15±0.01 and 0.15±0.02,P<0.05;CyclinD1: 1.08±0.05 vs 0.89±0.11 and 0.86±0.02,P<0.05;KYSE9706: ZIP5: 1.02±0.17 vs 0.25±0.01 and 0.25±0.03,P<0.05;CyclinD1: 1.26±0.11 vs 0.97±0.07 and 0.96±0.09,P<0.05),could reduce the expression of E-cadherin gene(Eca109: 0.07±0.03 vs 0.12±0.01 and 0.14±0.02,P<0.05;KYSE9706: 0.21±0.02 vs 0.85±0.29 and 0.86±0.22,P<0.05).2.8 Over/low expression of miR-193 b on the protein levels of ZIP5,CyclinD1 and E-cadherin genes:overexpression of miR-193 b gene could inhibit the expression of ZIP5 and CyclinD1 protein(KYSE150: ZIP5: 0.44±0.02 vs 0.82±0.01 and 0.78±0.05,P<0.05;CyclinD1: 0.31±0.01 vs 1.39±0.03 and 1.35±0.04,P<0.05;KYSE170: ZIP5: 0.30±0.01 vs 0.73±0.04 and 0.68±0.05,P<0.05;CyclinD1: 0.52±0.03 vs 1.03±0.11 and 1.05±0.10,P<0.05),promoted the expression of E-cadherin gene(KYSE150: 0.72±0.02 vs 0.65±0.02 and 0.65±0.01,P<0.05;KYSE170: 0.86±0.03 vs 0.43±0.01 and 0.42±0.06,P<0.05),low expression of miR-193 b gene could promote the expression of ZIP5 and CyclinD1 gene proteins(Eca109: ZIP5: 1.03±0.05 vs 0.63±0.07 and 0.63±0.09,P<0.05;Cyclin D1: 0.79±0.01 vs 0.35±0.02 and 0.39±0.03,P<0.05;KYSE9706: ZIP5: 0.81±0.01 vs 0.33±0.01 and 0.32±0.01,P<0.05;Cyclin D1: 0.91±0.01 vs 0.34±0.02 and 0.33±0.01,P<0.05),could reduce the expression of E-cadherin gene(Eca109: 0.45±0.03 vs 0.98±0.07 and 0.97±0.09,P<0.05;KYSE9706: 0.36±0.02 vs 0.80±0.02 and 0.78±0.05,P<0.05).3 Expression of miR-193 b in ESCC patients in areas with zinc deficiency and its correlation with ZIP5 3.1 The expression of miR-193 b was decreased in the cancer tissues of 48 ESCC patients(0.45±0.19)with high incidence of esophageal cancer,and the expression level was decreased by 2 times compared with non-tumor tissues adjacent to the cancer(0.90±0.32)(P<0.05).3.2 The expression of miR-193 b was associated with lymph node metastasis and TNM stage in ESCC patients with high incidence of esophageal cancer(lymph node metastasis: ?2=15.965,P=0.000;TNM stage: ?2=14.106,P=0.000).3.3 The relative expression levels of miR-193 b and ZIP5 in ESCC patients were negatively correlated(r=-0.509,P=0.000).Conclusions:1.Culturing of esophageal cancer cells with low Zn and immunofluoresceinase experiments suggested that miR-193 b was related to Zn,and ZIP5 was a direct target of miR-193 b.2.MiR-193 b affected the proliferation,migration and invasion of esophageal cancer by affecting the expression levels of ZIP5,ClylinD1 and E-Cadherin.3.The expression of miR-193 b was decreased in esophageal cancer tissues and correlated with lymph node metastasis and TNM staging.The expression levels of miR-193 b and ZIP5 in tissues of esophageal cancer patients were negatively correlated.