| The incidence of thyroid carcinoma(TC)accounts for about 3% of systemic malignancies.It ranks first among endocrine system malignancies and is also one of the fastest growing malignancies in recent years.The main reason for the growth rate is papillary thyroid carcinomas(PTC),accounting for about 80% of TC.Therefore,it is of great significance to explore the biological markers and prognostic evaluation indicators related to the occurrence and development of PTC.MicroRNA(miRNA)is a type of non-coding single-stranded RNA molecule with a length of 18 to 22 nucleotides.With the in-depth research of precision medicine,exploring the mutual regulation mechanism of miRNA and gene has a good prospect for the diagnosis and treatment of PTC.The previous research of ourgroup showed that there is a high expression of TFF3 in PTC tissues.Toexplore the regulation of TFF3 by miRNAs may provide potential targets for the treatment of PTC.First,the bioinformatics website was used to predict the miRNAs that may regulate TFF3 are miR-7-5p and miR-143-3p.Real-time quantitative PCR(RT-qPCR)was used to analyze the expression levels of miR-7-5p and miR-143-3p in PTC and normal tissues adjacent to carcinoma.Then the correlation was furtheranalysedbetween miR-7-5p and miR-143-3p and the clinicopathological characteristics of PTC patients(such as age,gender,clinical stage,tumor size and lymph node metastasis).Then,TPC-1 cell,the papillary thyroid carcinomas cell line was divided to 3 groups.TPC-1 cells transfected with miR-7-5p and miR-143-3p respectively,to harvest mimics-7-5p or mimics-143-3p over-expressed groups as experimeal group;the negative control group was transfected with the corresponding mimics-NC;TPC-1cell without any treatment as blank control group.Inorder to verify the targeting relationship between two miRNAs and TFF3,the dual-luciferase reporter gene experiment was used and dividided to experimemtal group and negative control group by co-transfected miR-7-5p mimics/miR-143-3p mimics and corresponding mimics-NC with the wild-type and mutant TFF3 luciferase vectors into TPC-1 cells.Westernblot experiments simultaneously verified the expression of TFF3 in the mimics-7-5p group/mimics-143-3p group,the corresponding negative control group and the blank control group.Cell growth curve,scratch test,and Transwell test were used to detect the effects of miR-7-5p and miR-143-3p on the proliferation,migration and invasion of TPC-1 cells.Finally,the effect of overexpression of miR-7-5p and miR-143-3p on the epithelial-mesenchymal transition(EMT)marker(E-cadherin,N-cadherin and Vimentin)of TPC-1 cells by western blot and immunocytochemical staining.Results:(1)The target miRNAs that may regulate TFF3 were selected by bioinformatics technology as miR-7-5p and miR-143-3p.(2)Compared with normal tissues adjacent to carcinoma,the expression of miR-7-5p and miR-143-3p in PTC were significantly lower,and the expression levels were0.6 times and 0.47 times of those in adjacent tissues,respectively.There was adifferences tatistically significant(P<0.01 or P<0.01);(3)The relationship between the expression levels of miR-7-5p or miR-143-3p and the clinicopathological characteristics of PTC patients: The low expression of miR-7-5p and miR-143-3p is correlated with clinical stage and tumor size(P<0.01),but not correlated with the age and gender of patient’s(P>0.05);the decreased expression of miR-7-5p is associated with lymph node metastasis,while miR-143-3p has nothing to do with lymph node metastasis.(4)The expression levels of miR-7-5p and miR-143-3p in PTC cell lines(TPC-1,BCPAP,K1)were significantly lower than the normal thyroid(Nthy-ori 3-1)cells,while TPC-1 cells had the lowest expression levels of miR-7-5p and miR-143-3p(P<0.01).(5)The results of the dual luciferase reporter gene shows that the luciferase activity of the TFF3 in experimental group,carrying the wild-type TFF3 reporter gene plasmid co-transfected with miR-7-5p ormiR-143-3p was respectively reduced(P<0.01 or P<0.01);while the relative fluorescence intensity had no significantly difference compared with negtive groups when the experimental group,carrying with mutant TFF3 reporter gene plasmid co-transfected with miR-7-5p or miR-143-3p.These indicates that miR-7-5p and miR-143-3p can specifically bind to the 3’UTR region of TFF3.(6)The growth curve shows that from the first day,the growth of mimics-7-5p group and mimics-143-3p group cell were significantly lagging behind that of TPC-1 and mimics-NC groups,the growth rate was slowed down,and the proliferation ability was significantly reduced(P<0.05 or P<0.01);while the mimics-NC group and TPC-1 group have no significant difference in cell proliferation.(7)The results of the cell scratch experiment shows that the cell migration rate of mimics-7-5p group and mimics-143-3p group were significantly lower than that of mimics-NC group and TPC-1 group(P<0.05 or P<0.01),while there was no statistical difference in migration ability between negative groups and blank group.(8)The results of the cell invasion experiment shows that compared with negative groups and blank group,the number of invasive cells in the mimics-7-5p group and mimics-143-3p group were significantly reduced(P<0.05 or P<0.01),indicating that mimics-7-5p group and mimics-143-3p group can significantly inhibit the invasion of TPC-1 cells;while the negative groups and blank group have no significant difference in cell invasion ability.(9)Western blot results show that the expression levels of EMT index N-cadherin and Vimentin protein in mimics-7-5p group and mimics-143-3p group decreased,and the expression level of E-cadherin protein increased(P<0.05);the levels of EMT-related proteins between groups of negative groups and blank groupwere no statistical difference.Conclusions:(1).Human PTC tissues have low expression of miR-7-5p and miR-143-3p.(2).Mi R-7-5p and miR-143-3p can inhibit the process of TPC-1 epithelial-mesenchymal transition(EMT)by targeting TFF3,thereby reducing the proliferation,migration and invasion of TPC-1 cells. |
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