| ?.Research Background and PurposeStudies have found that abnormal expression of miRNAs is associated with the occurrence and development of many types of tumors,suggesting that miRNAs may become biomarkers for tumor diagnosis and prognosis.Based on the results of the previous Solexa high-throughput sequencing and miRDeep2 software predictions,we performed the secondary structure predictions and experimental validation of candidate novel miRNAs,then we evaluated the risk of candidate novel miRNA and esophageal cancer.Further,we preliminary explored the biological functions and possible regulatory mechanisms.Our research provides basic data support for finding potential tumor markers and their clinical applications.?.Research Methods and Results(1)Identification of candidate novel miRNA related to esophageal cancer and their risk with esophageal cancerBased on the result of the Solexa high-throughput sequencing of esophageal cancer RNA and predictions from miRDeep2 software,we selected four novel miRNAs with the highest miRDeep2 score and used the sflod online prediction website to analyze the secondary structure of candidate novel miRNA precursor sequences.The results showed that all the precursor sequences of the four candidate novel miRNAs can form stem-loop structures with low free energy,and the mature sequences were located on the 3' and 5' arm of the stem loop,respectively.Fluorescent quantitative PCR was used to detect the expression of candidate novel miRNAs in esophageal cancer cells and esophageal epithelial cells.The results showed that novel-miR-4885,novel-miR-13894,and novel-miR-21009 can be stably expressed in esophageal cancer cells and esophageal epithelial cells,while novel-miR-7121 is only expressed in esophageal cancer cells.The amplification curves were well paralleled,and the melting peaks were specific.The results of TA cloning and sequencing of the fluorescent quantitative PCR products showed that the waveforms of the sequencing peaks were specific and had no heterozygous peaks.The fluorescence quantitative PCR products of the four candidate novel miRNAs were identical to those predicted by bioinformatics.126 patients with esophageal cancer diagnosed by pathology or endoscopy from 2009 to 2012 in Huai'an First People's Hospital were recruited.Fluorescent quantitative PCR was used to detect the expression of candidate novel miRNAs in esophageal cancer and adjacent tissues.The results showed that novel-miR-4885,novel-miR-7121 and novel-miR-21009 were significantly overexpressed in esophageal cancer tissue(P<0.05),which was 1.77,1.38,1.91 times that of the control group,respectively.While there was no significant difference in the expression of novel-miR-13894 between esophageal cancer tissues and adjacent tissues(P>0.05).The data were subjected to conditional logistic regression analysis.The results showed that the increased expression of novel-miR-4885,novel-miR-7121,and novel-miR-21009 significantly increased the risk of esophageal cancer(P<0.05),while the increase in the expression of novel-miR-13894 was not associated with the risk of esophageal cancer(P>0.05).(2)Biological function of candidate novel miRNAsFlow cytometry was used to detect the cell cycle distribution of EC109 cells after miRNA mimic transfection for 48 h.The results showed that the proportions of G1,S,and G2 phase in the novelmiR-4885 transfection group and the control group were(52.33±0.83)% vs(50.43±0.81)%,(28.80±0.50)% vs(25.98±0.34)%,(18.87±1.26)% vs(23.59±0.93)%;the proportions of G1,S,and G2 phase in the novel-miR-7121 transfection group and the control group were(49.80±1.69)% vs(44.76±1.11)%,(29.77±0.49)% vs(32.77±0.32)%,(20.43±1.50)% vs(22.48±1.05)%;the proportions of G1,S,and G2 phase in the novel-miR-13894 transfection group and the control group were(49.49±1.48)% vs(49.70±0.92)%,(31.35±1.57)% vs(30.78±0.78)%,(19.36±0.52)% vs(19.51±0.92)%;the proportions of G1,S,and G2 phase in the novel-miR-21009 transfection group and the control group were(56.55±1.70)% vs(50.43±0.81)%,(29.16±1.02)% vs(25.98±0.34)%,(14.29±0.68)% vs(23.59±0.93)%.The ratio of G1 and S phase in the novel-miR-4885 and novelmiR-21009 transfection group was significantly higher than that of control group(P<0.05);the ratio of G1 phase in the novel-miR-7121 transfection group was significantly higher than that in the control group(P<0.05),while the ratio of the S phase was significantly lower than that in the control group(P<0.05);the cycle distribution of novel-miR-13894 transfection group was not statistically different from that of the control group(P>0.05).Flow cytometry was used to detect the apoptosis of EC109 cells after miRNA mimic transfection for 48 h.The early apoptosis rates of novel-miR-4885 transfection group and control group were(10.30±1.01)% vs(5.57±1.17)%;the early apoptotic rates of novel-miR-7121 transfected group and the control group were(7.35±0.35)% vs(7.90±2.69)%;the early apoptotic rates of novel-miR-13894 transfected group and control group were(5.90±1.04)% vs(5.83±1.91)%;the early apoptotic rates of novel-miR-21009 transfected group and control group were(8.70±0.78)% vs(5.57±1.17)%.The early apoptosis rate of cells transfected with novel-miR-4885 and novel-miR-21009 were significantly higher than that of the control group(P<0.05),while there was no significant difference in the early apoptosis rate between the novel-miR-7121,novel-miR-13894 transfected groups and the control groups(P>0.05).The proliferation of EC109 cells after miRNA mimic transfection was detected by EdU staining.The proliferation rates of novel-miR-4885,novel-miR-7121,novel-miR-13894,and novel-miR-21009 were(37.25±4.09)%,(31.32±3.53)%,(31.25±1.21)%,(30.71±4.25)%,respectively.Compared with the control group(31.36±2.98)%,the cell proliferation rate of the novel-miR-4885 transfection group was significantly increased(P<0.05),while there was no significant difference in cell proliferation rate among novel-miR-7121,novel-miR-13894,novel-miR-21009 transfection group and the control group(P>0.05).Transwell chamber was used to detect the migratory ability of miRNA mimic transfected EC109 cells.The number of cells passing through the membrane in the novle-miR-4885,novel-miR-7121,novel-miR-13894 and novel-miR-21009 transfection group was 31.60±3.89,35.60±5.02,37.40±3.92,36.80±5.37,respectively.Compared with the control group 19.90±4.72,the number of cells passing through the membrane in all four miRNA mimic transfection groups was significantly increased(P<0.05).Transwell chamber was used to detect the invasive ability of miRNA mimic transfected EC109 cells.The number of cells passing through the membrane in the novel-miR-4885,novel-miR-7121,novel-miR-13894 and novel-miR-21009 transfection group was 45.60±6.36,29.87±7.40,25.00±3.20,34.30±7.36,respectively.Compared with the control group 19.50±5.36,the number of cells passing through the membrane in all four miRNA mimic transfection groups was significantly increased(P<0.05).(3)Preliminary exploration of the mechanism of candidate novel miRNAs promoting cell migration and invasionAfter transfecting EC109 cells with novel-miR-4885 mimic,an over-expression cell model of novel-miR-4885 was obtained.The expression level of mRNA and protein of novel-miR-4885 candidate target gene and the binding of novel-miR-4885 and 3'UTR of candidate target gene mRNA were detected.Subsequently,the expression of EMT-associated protein and the changes of cell adhesion were detected.The expression level of migration and invasion associated candidate target mRNA before and after RIP in the novel-miR-4885 transfection group was detected by fluorescence quantitative PCR.The results showed that the relative expression of ?-catenin mRNA was significantly decreased by 89.8% in novel-miR-4885 transfection group(P<0.05)compared with control group and the expression level of ?-catenin protein in the novel-miR-4885 transfection group was significantly down-regulated by 57.9%(P<0.05),which was consistent with the change of mRNA expression.RIP analysis displayed the expression of ?-catenin mRNA was 2.87 times that of the control group(P<0.05).The ?-catenin 3'UTR vector was constructed,and the binding of novel-miR-4885 to ?-catenin 3'UTR was verified by a dual luciferase reporter assay.The results showed that the relative luciferase activity in the pmirGLO-WT+novel-miR-4885 mimic group was significantly decreased,which was 27.5% lower than that in the pmirGLO-WT+mimic NC group(P<0.05).There was no significant difference in the relative luciferase activity between the pmirGLO-MUT+novel-miR-4885 mimic group and the pmirGLO-MUT+mimic NC group after the binding site of novel-miR-4885 and ?-catenin 3'UTR was mutated(P>0.05).The expression levels of EMT-associated proteins ZO-1,N-cadherin,E-cadherin,and b-catenin in novel-miR-4885 transfection group were detected by Western Blot.The results showed that the relative expression levels of ZO-1,N-cadherin,E-cadherin,and b-catenin proteins in the novel-miR-4885 and control groups were(0.45±0.12 vs 0.92±0.11),(0.82±0.06 vs 0.51±0.05),(0.59±0.03 vs 0.93±0.03),(0.82±0.04 vs 0.54±0.07).Compared with the control group,ZO-1 and E-cadherin proteins were significantly down-regulated in novel-miR-4885 transfection group by 51.6% and 37.2%(P<0.05),respectively;while N-cadherin and b-catenin proteins were significantly upregulated in novel-miR-4885 transfection group by 66.7% and 51.9%(P<0.05),respectively.Cell adhesion assay was performed to evaluate the adhesion of novel-miR-4885 transfected cells and control cells.The results showed that the absorbances of novel-miR-4885 transfected group and control group were(1.12±0.09)and(1.78±0.02),respectively,the absorbance of novell-miR-4885 transfection group was decreased by 37.1%(P<0.05).?.Conclusions:(1)Real-time RT-PCR and TA cloning experiments preliminarily confirmed that novel-miR-4885,novel-miR-7121,and novel-miR-21009 are novel miRNAs related to esophageal cancer,and novel-miR-13894 is novel miRNA.(2)Increased expression of novel-miR-4885,novel-miR-7121,and novel-miR-21009 can significantly increase the risk of esophageal cancer,which are risk factors for esophageal cancer;while the increased expression of novel-miR-13894 can significantly reduce the risk of esophageal cancer,which is a protective factor for esophageal cancer.(3)novel-miR-4885,novel-miR-7121,and novel-miR-21009 can affect the cycle distribution of esophageal cancer cells;novel-miR-4885 and novel-miR-21009 can induce early apoptosis of esophageal cancer cells;novel-miR-4885 can promote proliferation of esophageal cancer cells;novelmiR-4885,novel-miR-7121,novel-miR-13894,and novel-miR-21009 can promote the migration and invasion of esophageal cancer cells.(4)?-catenin is a functional target gene of novel-miR-4885;novel-miR-4885 can attenuate the adhesion of esophageal cancer cells by targeting ?-catenin,which in turn promotes EMT of esophageal cancer cells to a certain extent,leading to enhanced migration and invasion of esophageal cancer cells. |
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