| Part one the expression and bioinformatics analysis of mi R-193a-5p in esophageal cancerObjectives: To apply bioinformatics technology and clinical samples to identify the role of micro RNA(micro RNA,mi RNA,mi R)-193a-5p in esophageal cancer in bioinformatics,expression and analysis of expression levels and clinical pathological characteristics of esophageal cancer patients.Correlation level in order to provide valuable molecular biological markers for early prevention,accurate diagnosis and treatment of patients with esophageal squamous cell carcinoma.Materials and Methods:1.Retrieve microarray data of 4 mi RNA sequencing data(GSE59856,GSE59973,GSE97049,and GSE114110)and 2 m RNA sequencing data(GSE17351 and GSE20347)from the Gene Expression Omnibus(GEO)of the National Center for Biotechnology Information Center.The R software performs probe annotation and standardization based on platform information,and analyzes differentially expressed mi RNAs and m RNAs.2.Apply the prediction results of the online prediction sites of mi RNA target genes Targetscan and mi Rwalk to intersect the differentially expressed genes of the two m RNA chip sequencing data,and use genes that exist in at least three sets of data sets as candidate target genes for mi R-193a-5p Perform downstream analysis.3.Use the STRING database to construct a mi R-193a-5p candidate target gene protein interaction network.The DAVID online database and the R package Clusterprofile were used to perform gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis on the obtained target genes.4.Detection of mi R-193a-5p in 42 cases of esophageal cancer and its corresponding using quantitative real-time reverse transcript polymerase chain reaction(q RT-PCR)The expression in normal tissues adjacent to the cancer,and the correlation between the expression level and the clinicopathological data of patients.5.Draw ROC working characteristic curve to analyze the value of mi R-193a-5p expression level in diagnosis of esophageal cancer.6.Online survival database analysis of the relationship between mi R-193a-5p and survival of patients with esophageal cancer.Results:1.mi RNA chip analysis found that the expression level of mi R-193a-5p in GSE59856,GSE59973,GSE97049 and GSE114110 esophageal cancer tissues was significantly lower than that in non-tumor tissues.2.Through the mi RNA target gene online prediction website and the differentially expressed gene analysis results of 2 m RNA chip sequencing data,the protein interaction network analysis was used to finally determine BUB1,STIL and KIF14 and so on were used as the main target genes of mi R-193a-5p.3.Functional enrichment analysis found that mi R-193a-5p is mainly involved in biological processes and molecular functions such as extracellular matrix composition,nuclear fission,organelle fission,and affecting metal endopeptidase activity;mainly involved in interleukin-17 IL-17),transcriptional disorders,cell cycle,and nuclear factor-kappa B(NF-κB)signaling pathways.4.The expression level of mi R-193a-5p in 42 cases of esophageal cancer was significantly lower than that in normal tissues adjacent to the corresponding cancer.And the expression level of mi R-193a-5p was significantly correlated with lymph node metastasis and TNM stage in patients with esophageal cancer(all P <0.05),but it was not statistically significant with gender,age,smoking history,tumor size and family history(P > 0.05).5.Working characteristic curve(Receiver Operating Characteristic)analysis revealed that the area under the curve of mi R-193a-5p to distinguish esophageal cancer and adjacent cancer was 0.759(P <0.01).6.KM survival analysis showed that mi R-193a-5p expression level was not significantly correlated with the prognosis of patients with esophageal cancer(P> 0.05).Part Two The mechanism of mi R-193a-5p of esophageal Cancer in ZincDeficiency AreasObjectives: Zn(zinc)homeostasis plays an important role in the occurrence and development of esophageal cancer,and ZIP5 is an important zinc transporter.Previous studies have found that ZIP5 plays an important role in the occurrence and development of esophageal cancer,and mi RNAs are deficient in Zn.It is an important way to affect the occurrence of esophageal cancer.Therefore,this article aims to analyze the mechanism of mi R-193a-5p regulating ZIP5 expression and promote the occurrence of esophageal cancer during zinc deficiency,in order to provide a reference for the diagnosis and treatment of esophageal cancer in zinc-deficient areas.Materials and Methods:1.Detection of mi R-193a-5p expression in esophageal cancer cell line and immortalized esophageal epithelial cell line NE1 by q RT-PCR.2.Transfection plasmids mi R-193a-5p mimics and mi R-193a-5p inhibitor were used to construct over-/low-expressing mi R-193a-5p esophageal cancer cell lines,respectively,and the transfection efficiency was detected by q RT-PCR.3.The effects of low/overexpressed mi R-193a-5p on the proliferation,migration,invasion,and cell cycle of esophageal cancer cells were measured using MTS and clone formation experiments,scratch experiments,Transwell chamber invasion experiments,and flow cytometry.4.The expression level of mi R-193a-5p detected by q RT-PCR in low zinc cultured esophageal cancer cell lines.5.After mi R-193a-5p expression was down/up-regulated,q RT-PCR and Western blot were used to detect changes in ZIP5 m RNA and protein expression levels.6.Using immunoluciferase experiments to verify the relationship between mi R-193a-5p and ZIP5.7.q RT-PCR and Western blot were used to detect the effects of low/ overexpressed mi R-193a-5p on the expression of EMT-associated molecules(E-cadherin,N-cadherin,and Vimentin)in esophageal cancer cells.Results:1.The relative expression of mi R-193a-5p in six esophageal cancer cell lines were: Eca109(0.46 ± 0.03),KYSE150(0.31 ± 0.02),KYSE170(0.34 ± 0.02),KYSE9706(0.46 ± 0.03),and TE1(0.37 ± 0.03)were lower than NE1(0.63 ± 0.03)in immortalized esophageal epithelial cells,and the difference was statistically significant(P<0.05).2.q RT-PCR detection of low/over-expressed mi R-193a-5p esophageal cancer cells showed that the relative expression of mi R-193a-5p in the transfection group was significantly lower / higher than that in the NC group and the normal culture untreated group.Significance(P<0.05).3.Effect of down/over-expressed mi R-193a-5p on the proliferation of esophageal cancer cells: MTS and clone formation experiments showed that low expression of mi R-193a-5p significantly promoted the in vitro proliferation of KYSE150 and KYSE170(P<0.05);Expression of mi R-193a-5p significantly inhibited the proliferation of Eca109 and KYSE9706 in vitro(P<0.05).4.Effect of down/over-expressed mi R-193a-5p on the migration ability of esophageal cancer cells: The results of scratch experiments showed that the low expression of mi R-193a-5p gene significantly promoted the migration ability of esophageal cancer cell lines Eca109 and KYSE9706(P<0.05);Overexpression of mi R-193a-5p gene significantly inhibited the migration ability of esophageal cancer cell lines KYSE150 and KYSE170(P<0.05).5.Effect of down/over-expressed mi R-193a-5p on the invasion ability of esophageal cancer cells: Transwell chamber invasion experiments showed that low expression of mi R-193a-5p can promote the invasion ability of esophageal cancer cell lines Eca109 and KYSE9706(P<0.05);Overexpression of mi R-193a-5p can inhibit the invasion ability of esophageal cancer cell lines KYSE150 and KYSE170 in vitro(P <0.05).6.Effects of down/over-expressed mi R-193a-5p on the esophageal cancer cell cycle: Flow cytometry results show that down-regulating the mi R-193a-5p gene can promote cell division at G0 / G1 and G2 / M phases and block S Phase division affects the cell cycle of esophageal cancer cell lines Eca109 and KYSE9706(P<0.05).Overexpression of mi R-193a-5p gene can affect the cell cycle of esophageal cancer cell lines KYSE150 and KYSE170 by inducing cell arrest in G0 / G1 phase and shortening S phase(P<0.05).7.In low zinc cultured esophageal cancer cell lines,q RT-PCR detection of mi R-193a-5p expression level was found to be within a certain range.As TPEN concentration increased,mi R-193a-5p expression level significantly decreased.8.RT-PCR and Western blot results showed that: low expression of mi R-193a-5p gene can promote the expression level of ZIP5 m RNA and protein;high expression of mi R-193a-5p gene can inhibit the expression level of ZIP5 m RNA and protein.9.Immunoluciferase experiments show that mi R-193a-5p directly binds to ZIP5 and regulates its expression.10.RT-PCR and Western blot results showed that overexpression of mi R-193a-5p gene can inhibit the expression of N-cadherin and Vimentin m RNA and protein,and promote the expression of E-cadherin m RNA and protein(P <0.05);low expression of mi R-193a-5p gene can promote the expression of N-cadherin and Vimentin m RNA and protein,and inhibit the expression of E-cadherin m RNA and protein(P<0.05).Conclusions:1.The expression level of mi R-193a-5p in esophageal cancer tissues and cell lines was significantly down-regulated,and its expression level was significantly correlated with lymph node metastasis and TNM staging in patients with esophageal cancer.2.mi R-193a-5p has a strong ability to diagnose esophageal cancer,and its expression level has no significant correlation with the prognosis of patients with esophageal cancer.3.Abnormal expression of mi R-193a-5p significantly affects esophageal cancer cell proliferation,invasion and migration ability,and cell cycle,and it may play tumor suppressor role in EC.4.mi R-193a-5p and low Zn cultured esophageal cancer cells and immunoluciferase experiments suggest that mi R-193a-5p may be related to Zn ions,and ZIP5 is a direct target of mi R-193a-5p.5.The expression level of mi R-193a-5p can significantly affect the expression of EMT-related molecules(E-cadherin,N-cadherin,and Vimentin)and may affect the development of esophageal cancer by targeting ZIP5 to regulate the EMT process under low zinc conditions. |
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