Study Of MicroRNA-146b-3p Promotes Cell Metastasis By Targeting NF2 In Human Papillary Thyroid Cancer

Background:Thyroid cancer(TC)is the most common malignant tumor in endocrine system.The incidence of TC is gradually increasing throughout the world in recent years.Thyroid cancer ranks the fourth or fifth the most common malignancy in women in China or the United States separately.Papillary thyroid cancer(PTC)is the most commonly diagnosed thyroid cancer,accounting for more than 80%of thyroid malignancies.Although PTC is usually indolent,and curable with early diagnosis and surgical thyroidectomy,with an overall 10-year survival rate of more than 90%;a subset of patients who occur local recurrence or distant metastasis may have a poor quality of life and prognosis.Studies shown that the 5 year survival rate of PTC with distant metastasis decreased to 54.1%.In addition,lymph node metastasis(LNM)is common in PTC.Studies have demonstrated that approximately 40%of PTC have LNM at initial presentation.Furthermore,LNM,as one of the aggressive phenotype of PTC,is closely associated with localregional and distant metastasis.Therefore,the study of exploring the molecular mechanism and identification of PTC with LNM,is important for the adjuvant therapy and improving the prognosis.MicroRNAs(miRNAs)are small,noncoding RNAs,21 to 25 single nucleotides in length,involve in the post-transcriptional regulation of eukaryotic organisms.MiRNAs negatively regulate gene expression through binding to the 3’untranslated region(UTR)of target messenger RNAs(mRNAs)and by blocking the translation or degradation of the mRNAs,suggesting that miRNAs may have important roles in physiological and pathological processes,including cell proliferation or apoptosis,development and differentiation and human carcinogenesis.Studies have shown that 50%of miRNA genes are located in tumor-related regions,and dysregulation of miRNA expression has been described in a variety of tumors,including lung,liver,breast,and colorectal cancer.In thyroid cancer,the miRNA expression pattern depends on the cellular origin and the degree of tumor differentiation.Numerous studies have demonstrated an increased aberrant miRNA expression,including miR-146b,miR-221,and miR-222,in PTCs compared to normal thyroid tissues.Recently,further studies have reported that miRNAs such as miR-146b are strongly associated with more aggressive PTC.In humans,miR-146b-5p and miR-146b-3p are products of the same MIR146B gene that is located on chromosome 10 at position q24.32.MiRNAs behave in a dual mode in cancer as either oncogenes or tumor suppressors,depending on tissue type and specific targets.A number of studies have examined the role of miR-146b-5p in cancer,including lung,breast,glioma,and pancreatic cancer.MiR-146b-5p was proven to decrease the migration and invasiveness of cancer cells by targeting MMP16 and EGFR genes and negatively regulating the Nuclear Factor-KB(NF-κB)pathway.Unlike in other cancers,miR-146b-5p has a confirmed oncogenic role in regulating TGF-βsignal transduction by repressing SMAD4 in PTC and promoting metastasis by targeting ZNRF3.While miR-146b-5p is relatively well characterized,such functional studies of miR-146b-3p have rarely been performed.In a human non-small cell lung cancer study,miR-146b-5p and miR-146b-3p had opposite prognostic values,which indicated that although they are encoded by the same gene,miR-146b-3p might have a different role from miR-146b-5p and other important functions in human carcinogenesis.However,the expression of miR-146-3p in PTC and LNM has remained elusive,and the possible roles and mechanisms of miR-146b-3p in human PTC are still not well established.This work focused on miR-146b expression in different PTC tissue samples and cells with different invasion abilities,and explored the relationship between miR-146b-5p or miR-146b-3p and PTC with LNM.Moreover,we confirmed that miR-146b-3p enhanced PTC migration and invasion more obviously than did miR-146b-5p in vivo.Then,we predicted and verified NF2 gene was the direct target gene of miR-146b-3p.Furthermore,we elucidated the mechanism of miR-146b-3p promoting PTC metastasis via in vivo and in vitro.These findings are important for the early diagnosis of PTC patients with the tendency to develop metastases and provided a new target for gene therapy of PTC.Objectives:1.To determine the relationship between miR-146b and papillary thyroid cancer lymph node metastasis by detecting the expression of miR-146b-5p and miR-146b-3p in different aggressive PTC tissue samples and cells.2.To explore the functional role of miR-146b in PTC metastasis by evaluating the migratory and invasive behavior of different PTC cells altered with miR-146b overexpression or inhibition,and further to identify that miR-146b-3p can enhance cell migration and invasion more obviously than did miR-146b-5p.3.To predict and verify the potential target gene NF2 of miR-146b-3p in PTC metastasis.4.To determine that miR-146b-3p promotes PTC metastasis in vivo.5.To elucidate the molecular mechanism of miR-146-3p promoting PTC cells metastasis by directly inhibiting the NF2 gene.Methods:1.Tissue samples:A total of 72 formalin-fixed paraffin-embedded(FFPE)tissue samples from 24 patients were obtained at the Department of Pathology in Shandong Provincial Hospital affiliated to Shandong University.All tissues samples were pathologically confirmed as PTC tissues,matched adjacent non-neoplastic thyroid tissues,normal lymph node tissues,or LNM tissues.2.Cell culture(1)Different PTC cell lines:BHP10-3,BHP10-3SCmice cells and K1 cells were cultured in accordance with the guidance.(2)BHP10-3SCmluc cells:High tumorigenic BHP10-3SCmice cells were stably labeled for bioluminescence and red fluorescent protein and cultured according to the guidance(3)Primary normal thyrocytes(PHT):Human normal thyroid tissue specimens were digested in collagenase I and trypsin for 1 h,then were seeded and cultured in DMEM/F12 medium containing 10%newborn calf serum and bovine thyrotropin.3.MicroRNA extraction,reverse transcription,and quantitative real-time polymerase chain reaction:MiRNA was extracted from FFPE human tissues and human thyroid cell lines.MiR-146b-5p and miR-146b-3p were reverse transcribed and quantified with quantitative reverse transcription polymerase chain reaction(qRT-PCR).RUN6B was used as an endogenous control.4.Recombinant plasmid construction:Recombinant plasmids with 3’UTRs of potential target genes(NF2 and MTSS1)were constructed based on bioinformation software.The oligonucleotides were ligated downstream of the Renilla luciferase open reading frame in the pMIR-REPORTTM miRNA expression reporter vector.5.Cell transfection:All transfections were carried out using Lipofectamine 3000 transfection reagent.For BHP10-3 and K1 cells,microRNA mimics and negative control(miR-NC)were transfected.For BHP10-3SCmice cells,microRNA inhibitor and negative control(anti-NC)were transfected.BHP10-3 and K1 cells were cotransfected with miR-146b-3p mimics and pcDNA3 merlin or pcDNA3(empty vector control),miR-NC(mimics control),or pcDNA3 merlin was respectively transfected into cells as control.6.Dual luciferase activity assay:PTC cells were co-transfected with either miR-146b-3p mimics or inhibitor together with the constructed pMIR-REPORTM miRNA target expression vectors.Luciferase activities were measured using the dual luciferase reporter assay system kit.Values were double normalized to firefly luciferase activity and to cells transfected with empty pMIR-REPORTTM control vectors.7.Cells migration and invasion assays:The effect of miR-146b-5p and miR-146b-3p on PTC cells metastasis was detected using the real-time cell analyzer(RTCA)and TranswellTM well migration and invasion assays.8.Animal studies:(1)For the establishment of the PTC xenograft model,BHP10-3SCmluc cells were inoculated intramuscularly into the left leg of BLAB/c nude mice.After two weeks,mice were imaged for bioluminescence in vivo to exclude any that were not successfully xenografted.(2)MiR-146b-3p agomir was directly injected into the implanted tumor of successfully xenografted nude mice.Tumor and metastasis bioluminescent imaging in vivo was analyzed weekly.At the end of the sixth week after xenografting,bioluminescence imaging in vivo was used to detect the primary tumor and metastasis,red fluorescent imaging ex vivo was used to detect the metastatic tissues.9.Hematoxylin and eosin staining:The tumor tissues,lung,and lymph nodes were embedded in paraffin and stained with hematoxylin and eosin(H&E)to evaluate the histopathological features.10.Protein extraction and Western blotting analysis:Total proteins were extracted from cells or tissue.Western blotting assays were adopted to detect the expression of merlin and GAPDH.11.Statistical analysis:All statistical analyses were performed using SPSS 18.0,and data are expressed as the means±SEMs.Differences between two groups were compared using an unpaired Student’s t-test or Mann-Whitney’s U-test.Analysis of variance was used to compare the means of multiple groups.Differences were regarded as statistically significant at p<0.05.Results:1.MiRNA-146b expression is significantly increased in PTC tissues and further enhanced in metastatic lymph nodes.Median miR-146b-5p and miR-146b-3p expression levels were 42-and 24-fold higher respectively,in PTC than in the non-tumorous thyroid tissues(median expression = 7.59/0.18,12.48/0.51).Intriguingly,median miR-146b-5p and miR-146b-3p expression levels in metastatic lymph nodes were further increased(median expression= 20.94 and 42.58,respectively),Overall,miR-146b was significantly upregulated in metastatic lymph nodes.Moreover,miR-146b-5p and miR-146b-3p expression levels were significantly higher in PTC with LNM than in PTC without metastasis,indicating that expression levels of miR-146b positively correlate with PTC LNM.2.Expression of miRNA-146b is significantly increased in PTC cell lines and positively correlated with cell metastasis abilityBoth miR-146b-5p and miR-146b-3p expression was significantly higher in PTC cell lines(BHP10-3,BHP10-3SCmice and K1 cells)than in the normal human thyrocytes,and miR-146b expression was further increased in BHP10-3SCmice cells with high aggressive abilities compared to BHP10-3 and K1 cells,which indicate that the level of miR-146,especially miR-146b-3p,was positively correlated with the migration and invasion abilities.3.MiR-146b-3p enhances the migratory and invasive abilities more obviously than miR-146b-5p in BHP10-3 cells and K1 cells.Inhibiting miR-146b-3p expression can reduce the metastatic ability of BHP10-3SCmice cells.The migration and invasion capacities of BHP10-3 cells and K1 cells transfected with miR-146b-3p or miR-146b-5p mimics were higher than that of BHP10-3 cells and K1 cells infected with miR-NC.Notably,compared to miR-146b-5p mimics,miR-146b-3p mimics promoted migration and invasion more obviously.The migration and invasion abilities of BHP10-3SCmice cell,which highly expressed miR-146b,decreased significantly after transfection with the miR-146b-3p or miR-146b-5p inhibitor.Consistent with results of the cells transfection with microRNA mimics,inhibition of miR-146b-3p expression reduced PTC migration and invasion more significantly than did inhibition of miR-146b-5p.4.NF2 is a direct downstream target of miR-146b-3pAccording to the bioinformation software,it was found that NF2 and MTSS1 contained two predicted miR-146b-3p binding sites in their 3’UTRs.The study constructed vectors containing wild-type or mutant 3’UTRs of NF2 or MTSS1 into a luciferase receptor vector and co-transfected them with miR-146b-3p mimics or mimics controls into BHP10-3 and·K1 cell lines.Luciferase reporter assays showed that miR-146b-3p obviously inhibits the relative luciferase activities of two wild-type NF2 3’UTRs,while the luciferase activities of the MTSS1 3’ UTRs and two mutated NF23’UTRs were not decreased in PTC cells.Furthermore,the relative luciferase activities of the two wild-type NF2 3’UTRs increased after BHP10-3SC mouse cells transiently co-transfected with 3’UTR of wild-type recombinant NF2 luciferase plasmids with miR-146b-3p inhibitor.These results indicate that miR-146b-3p cold directly bind to the 3’UTR of NF2 but not that of MTSS1.Western blot analysis was performed to observe any changes in merlin protein expression with miR-146b-3p mimics or inhibitor transfection.Endogenous merlin protein expression was significantly inhibited or enhanced after miR-146b-3p mimics or inhibitor transfection.Taken together,these observations indicate that NF2 is a direct downstream target of miR-146b-3p in PTC cells.5.MiR-146b-3p promotes tumor metastasis in vivoPTC xenograft mice were established successfully via intramuscular injection with BHP10-3SCmluc cells,and then intratumorally injected with miR-146b-3p agomir and imaged.Tumor metastasis was detected by bioluminescence imaging in vivo weekly.At the end of the sixth week,not only was tumor metastasis detected by in vivo bioluminescence imaging,but also pulmonary and LNM was observed by ex vivo fluorescence imaging.Compared to total metastasis in the control mice,the total metastasis occurrence number was much higher in the miR-146b-3p agomir-treated mice(1/10 vs.3/5 separately).Moreover,resected tissues from those treated xenograft tumors were analyzed to detect miR-146b-3p and merlin expression.Consistent with the above results,at the endpoint of the experiment,miR-146b-3p agomir treatment significantly increased the expression of miR-146b-3p and decreased the expression of merlin.All these data indicate that high expression of miR-146b-3p cold promote PTC tumor metastasis in vivo.6.Reintroduction of NF2 abrogated the miR-146b-3p-induced metastasis enhancementMiR-146b-3p mimics and pcDNA3merlin were co-transfected into BHP10-3 cells and K1 cells.The expression of merlin recovered after pcDNA3 merlin transfection.TranswellTM assays showed that the migration or invasive capabilities of BHP10-3 cells and K1 cells decreased after cotransfection with miR-146b-3p mimics and pcDNA3 merlin.RTCA assays showed the similar results with TranwellTM assays.Conclusions:1.MiR-146b was significantly upregulated in metastatic lymph nodes.The expression levels of miR-146b-3p and miR-146b-5p were positively associated with PTC with LNM.2.MiR-146b-3p enhanced cell invasion and metastasis more obviously than did miR-146b-5p.3.MiR-146b-3p promotes PTC metastasis by inhibiting target NF2 gene.