| Anaplastic thyroid caner(ATC)is one of the most malignant and very poor prognostic pathological types of all thyroid tumors,which is often diagnosed clinically at an advanced stage.Due to its lack of differentiation and inability to accumulate iodine,it cannot be treated with radioiodine.Doxorubicin(DOX)is currently the first-line chemotherapeutic agent for the treatment of ATC,but resistance of ATC to doxorubicin during the late treatment period is the main cause of final chemotherapy failure.However,the current research progress on the mechanism of doxorubicin resistance in ATC is slow,and the development of resistance reversal agents is even lagging behind.The mechanism of resistance to doxorubicin chemotherapy is not well understood,and recent studies have shown that abnormally expressed multiple microRNAs(miRNAs)are involved in the formation of multidrug resistance in tumors.MiRNAs are a class of highly conserved endogenous,non-coding,single-stranded RNA molecules that are involved in the development of a variety of tumors.We identified microRNA-27b-3p(miR-27b-3p)and peroxisome proliferator-activated receptor gamma(PPAR-y)as further research targets through database searches such as GEO and TCGA,bioinformatics analysis,and a large review of the literature.Previous studies have found that the abnormal expression of miR-27b as well as PPAR-y is associated with drug resistance in various tumors such as gastric cancer,liver cancer,skin cancer,and breast cancer,but the expression of both in ATC and their effects on doxorubicin treatment sensitivity have not been reported.In order to elucidate whether miR-27b-3p and PPAR-y are involved in the formation of drug resistance in ATC cells and how to reverse the resistance of ATC cells to doxorubicin,we investigated the targeted regulation relationship between miR-27b-3p and PPAR-y in ATC cells,the effect of dynamic expression of miR-27b-3p and PPAR-γ on doxorubicin resistance in ATC cells,and the upstream and downstream genes of the miR-27b-3p/PPAR-γ axis that may be involved in the regulation at the same time,providing a theoretical basis for reversing doxorubicin resistance in ATC.Part Ⅰ Construction of doxorubicin-resistant human anaplastic thyroid carcinoma cell lineObjective:To establish doxorubicin-resistant human anaplastic thyroid carcinoma cells and describe their biological characteristics,and to detect the expression difference of P-gp in resistant and sensitive strains at mRNA and protein levels to verify the successful establishment of resistant strains and provide a scientific research model for the next step in the study of the mechanism of tumor drug resistance.Methods:1.Human thyroid anaplastic carcinoma cell lines SW1736 and 8305C were used as parental cells,and doxorubicin was used as a screening drug to construct SW1736/DOX and 8305C/DOX-resistant cell line models by stepwise increasing doxorubicin concentrations and intermittent induction in vitro.2.CCK-8 assay was used to detect the inhibition of resistant strain cells and their parental cells after treatment with different concentrations of doxorubicin in order to select the most appropriate intervention concentration and treatment time.3.Differential expression of the resistance-associated protein P-gp was detected by real-time PCR and western blotting in cells of resistant and sensitive strains.4.Using adenoviral transfection technique,the stably expressing fluorescent LUC cell lines SW1736-LUC/DOX and 8305C-LUC/DOX were constructed for later animal experiments.Results:1.The resistant cell lines,named SW1736/DOX and 8305C/DOX,were obtained by continuous induction with low concentration of doxorubicin,gradual increase of doxorubicin concentration in the culture medium,and intermittent induction screening in vitro for 3 months.Through experiments,we found that the increase in the inhibition ratio of resistant SW1736/DOX and 8305C/DOX cells was significantly lower than that of the parental cell line under gradually increasing doxorubicin concentrations.2.The IC50 of SW1736 cells to doxorubicin was 0.82 μM,the IC50 of SW1736/DOX cells to doxorubicin was 8.83 μM,and their resistance coefficient RI was 10.73 as measured by CCK-8 assay;the IC50 of 8305C cells to doxorubicin was 1.77μM,the IC50 of 8305C/DOX cells to doxorubicin was 9.22μM,and their resistance coefficient RI was 5.41.According to the conventional grading of RI,SW1736/DOX and 8305C/DOX cells were moderately resistant to doxorubicin and had significant resistance compared with parental SW1736 and 8305C cells.The optimal doxorubicin concentration of 1 μM and the action time of 48 hours were also selected.3.qRT-PCR results showed that P-gp mRNA expression was significantly up-regulated in the resistant cell lines SW1736/DOX and 8305C/DOX compared with their parental cell lines SW1736 and 8305C,with significant differences(p<0.01);Western blot results showed that P-gp protein expression was significantly increased in the resistant cell lines SW1736/DOX and 8305C/DOX,with significant differences,suggesting that the ATC-resistant strain was successfully constructed.4.Using adenoviral(pHBLV-CMV-MCS-EF1-fLUC-T2A-PURO)transfection technology,we obtained cell lines SW1736-LUC/DOX and 8305C-LUC/DOX stably expressing the LUC gene for later animal experiments.Conclusion:The process of drug resistance acquisition by the two ATC cells was gradually changed in a significant dose-dependent manner,and the resistant strain cells were more resistant to doxorubicin drugs than their parental cells,and the high mRNA and protein expression of the resistance-associated protein P-gp in the resistant strain also verified the successful construction of the resistant cell line.Using the SW1736/DOX and 8305C/DOX models,we could study the alteration of their drug resistance at the subsequent level of microRNAs regulation,and further carry out studies to reverse drug resistance based on this.Part Ⅱ Relationship between the expression level of miR-27b-3p and the sensitivity of human anaplastic thyroid carcinoma cells to doxorubicinObjective:To identify differentially expressed miRNAs that may be associated with adriamycin resistance in ATC by bioinformatics analysis combined with literature review,and to further select miRNAs aberrantly expressed in ATC adriamycin-resistant strain cells,and to detect the relationship between their expression levels and adriamycin drug sensitivity in ATC cells by in vitro and in vivo experiments.Methods:1.Differentially expressed miRNAs were analyzed by searching and downloading datasets related to thyroid cancer chemoresistance through the GEO database and TCGA database on the NCBI website.Differential miRNAs that may be associated with adriamycin chemoresistance in anaplastic thyroid carcinoma were selected as the main study subjects.2.Among a variety of related miRNAs,qRT-PCR was used to further select miRNAs that were significantly differentially expressed in ATC-resistant strain cells and parental cells.3.In vitro experiments:By controlling miRNA expression(miRNAinhibitor),the changes in the sensitivity of resistant cell lines to adriamycin in the transfected and non-transfected groups were observed.4.In vivo experiment:ATC-resistant cell line xenograft models in vivo were constructed.The growth curve of xenograft tumors in experimental group and control tumor-bearing mice was observed by knocking down the target miRNA(miRNAantagomir),and the expression changes of target miRNA in the xenograft tumors were analyzed by qRT-PCR.Results:1.We used the TCGAbiolinksR package to download the TCGA-THCAmiRNA-seqreadcount data with the clinical sample data and the ggplot2R package to plot the volcano plot.A total of 394 differential miRNAs were found,including 264 up-regulated miRNAs and 30 down-regulated miRNA1s.By consulting a large number of literature reviews,we selected miRNAs closely related to anaplastic thyroid carcinoma and involved in the regulation of chemoresistance in many different malignancies:miR-200 family(including miR-200a,miR-200b,miR-200c,miR-141),miR-205 and miR-27b as target miRNAs for next research.2.The expression levels of the above miRNAs in ATC cell lines SW1736 and 8305C and their resistant strains SW1736/DOX and 8305C/DOX were screened and detected by RT-PCR experiments,and the results showed that the expression levels of miR-27b-3p in the resistant strain cells were significantly increased,with a significant difference compared with their parental cells(p<0.01).Therefore,we selected miR-27b-3p as the target miRNA for studying the mechanism of adriamycin resistance in ATC.3.In order to determine whether the expression level of miR-27b-3p has an effect on the drug resistance of ATC cells,we transfected miR-27-3pinhibitor and scramblemiRNA in resistant cell lines and detected the changes in the sensitivity of transfected control cells to the chemotherapeutic drug doxorubicin in resistant cell lines using the CCK-8 assay.The results showed that compared with the control group,the cell inhibition ratio gradually increased and the IC50 value was significantly lower in the group transfected with miR-27-3p inhibitor with increasing drug concentration,and the difference was statistically significant(p<0.01),suggesting that inhibiting miR-27b-3p expression could significantly improve the sensitivity of ATC cells to adriamycin.4.To further validate the regulation of miR-27b-3p on adriamycin resistance in ATC in vivo,we constructed an in vivo subcutaneous xenograft model.In the experimental group,after intratumoral injection of miR-27b antagomir,the growth of transplanted tumors was significantly slowed down;the tumor weight change was consistent with the volume change after removal of the tumor.Comparison of the gross photographs of the transplanted tumors in the two groups of tumor-bearing mice further showed that injection of miR-27b antagomir enhanced the antitumor effect of doxorubicin.Consistent with the results of in vitro cell experiments,the expression of miR-27b-3p was significantly reduced in the transplanted tumor tissues after miR-27b antagomir interference.The above in vivo experimental results confirmed that knockdown of miR-27b-3p could enhance the chemosensitivity of ATC-resistant cells to adriamycin drugs.Conclusion:miR-27b-3p is significantly highly expressed in adriamycin-resistant cells of anaplastic thyroid carcinoma,and inhibition of miR-27b-3p expression can enhance the sensitivity of anaplastic thyroid carcinoma cells to adriamycin chemotherapy.Part Ⅲ miR-27b-3p regulates doxorubicin resistance in human anaplastic thyroid carcinoma cells by targeting PPAR-γObjective:To investigate the regulation of adriamycin resistance in ATC cells by miR-27b-3p by targeting PPAR-y and its molecular mechanism,and to preliminarily expand the upstream and downstream signaling molecules of miR-27b-3p/PPAR-γ regulatory axis.Methods:1.The target genes downstream of miR-27b-3p were predicted by multiple databases and subjected to Venn analysis,GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the target gene set using the clusterProfilerR package,while combining the TCGA-THCAmRNA-seqreadcount data with the clinical sample data,the target genes related to thyroid cancer were selected as the next study subjects.2.The binding sites of miR-27b-3p to target genes were clarified by bioinformatics analysis.3.Whether this gene is a direct target of miR-27b-3p was verified by dual-luciferase reporter assay.4.miR-27b-3p mimics were transfected in SW1736 and 8305C cells,and changes in target gene mRNA as well as protein levels were observed by qRT-PCR and WB experiments to clarify the correlation between the two expressions.5.The differential expression of this target gene in mRNA and protein between resistant cell lines and parental cell lines was identified by qRT-PCR and WB experiments.6.The overexpression plasmid of this gene was constructed and transfected to observe the changes in the sensitivity of resistant cell lines to adriamycin.7.SW1736 and 8305C cells were transfected with miR-27b-3p mimics,the mRNA expression levels of downstream transcription factors in the transfected non-transfected group were detected by qRT-PCR,the significantly differentially expressed downstream genes were selected,their expression in the resistant and sensitive strains was detected by ELISA experiments,and whether they were involved in the regulation of adriamycin resistance in ATC cells by miR-27b-3p was verified by rescue experiments.8.Western blot was used to detect changes in the content of phosphorylated NF-κB p65 in the cytoplasm as well as nucleus of resistant cells,respectively;BAY11-7082,an inhibitor of NF-κB p65,was transfected in resistant cell lines SW1736/Dox and 8305C/Dox,and the mRNA expression changes of miR-27b-3p and its target genes were detected by qRT-PCR to verify whether NF-κB p65 affected adriamycin resistance in ATC cells by affecting the regulation of its target genes by miR-27b-3pResults:1.The target genes of miR-27b-3p were predicted by TargetScan database,miRDB database and miRTarBase database for Venn analysis,while a total of 17 target mRNAs could be predicted in the three databases.Functional enrichment analysis was performed for the mRNA genes screened for miR-27b-3p,and GO analysis revealed that the significantly differentially expressed genes were mainly involved in reproductive system development,stress response to immobilization,regeneration of animal organs,occurrence of embryonic epithelial morphology,and placental development in biological processes;they were mainly concentrated in exogenous components of the cytoplasmic membrane and membrane,ubiquitin ligand complexes,asymmetric synapses,and ER lumen in cellular components;and they mainly affected protein C-terminal binding and VEGF receptor binding in molecular functions.KEGG enrichment analysis revealed that differentially expressed genes were mainly focused on miRNA regulation in malignant tumors,MAPK signaling pathway,p53-related pathway,and thyroid cancer regulation.From the KEGG signaling pathway analysis,we can see that PPAR-y is closely related to thyroid cancer,and we searched the selected differential genes by Gepia online tool and found that PPAR-y gene was significantly underexpressed in thyroid cancer compared with the cancer-adjacent group.Previous studies have suggested that high expression of PPAR-y gene can improve the efficacy of radioiodine therapy in thyroid cancer.We used TCGAbiolinksR package to download TCGA-THCAmRNA-seqreadcount data and clinical sample data.After analysis,it was found that the expression of PPAR-y in thyroid cancer radiotherapy group was significantly down-regulated compared with non-radiotherapy group.In addition,we found that PPAR-γ is also involved in the formation of chemoresistance in breast,ovarian,and lung cancers by consulting the literature.We therefore hypothesized that PPAR-γ is an important target of miR-27b-3p in regulating ATC chemoresistance,so miR-27b-3p/PPAR-y was selected as the object of our next study.2.TarScan4.0 software was used to analyze the targeting effect of miR-27b on PPAR-y.The results showed that the possible PCT value of miR-27b targeting PPAR-γreached 73,and the percentage probability of contextscore reached 67.We also used the miRanda database to analyze the target of miR-27b,and the results showed that the5’seed region of miR-27b-3p had seven nucleotides that were completely complementary to the 3’-UTR of PPAR-y and located between bases 82-883.miR-27b-3p mimics were transfected in ATC cell lines,and the results of qRT-PCR and WB experiments showed that the PPAR-y mRNA and protein levels of the transfected group were significantly reduced compared with the control group,and the difference was statistically significant(p<0.01),indicating that overexpression of miR-27b-3p effectively regulated the expression of PPAR-y mRNA and protein in anaplastic thyroid cancer cell lines,which were significantly negatively correlated4.In the two groups of cells transfected with wild-type pmirGLO-PPARG-3’UTR-wt reporter plasmid,the luciferase activity of the group co-transfected with miR-27b-3pmimics was 45.2%of that of the blank control group,with a significant statistical difference(p<0.01);however,in the two groups of cells transfected with mutant pmirGLO-PPARG-3’UTR-mut plasmid,there was no significant difference in the luciferase activity of the control group co-transfected with miR-27b-3pmimics,suggesting that miR-27b-3p could significantly inhibit the expression of luciferase reporter gene containing the 3 ’UTR of PPAR-y,and this inhibition could be reversed by a mutation of five nucleotides at the binding site.Thus,there is a targeted regulation of PPAR-y by miR-27b-3p,indicating that PPAR-y is indeed a direct target gene of miR-27b-3p5.The results of RT-PCR and WB experiments suggested that the mRNA and protein levels of PPAR-y in resistant strains were significantly lower than those in parental cell lines,and the difference was statistically significant(p<0.01)6.PPAR-y plasmid was constructed and transfected into resistant cell lines SW1736/DOX and 8305C/DOX,and the results showed that the IC50 of resistant strains to adriamycin was significantly decreased,suggesting that overexpression of PPAR-y could significantly improve the sensitivity of ATC cells to adriamycin.7.After transfection of miR-27b-3p mimics in ATC cell line,the mRNA expression of downstream gene bFGF was significantly decreased,while the expression of c-Myc,Bc12,and VEGF was not significantly different,and the concentration of bFGF in the culture medium of the transfected group was determined by ELISA,and the same results were obtained.The bFGF expression of the resistant cell line was significantly lower than that of the parental cell line by ELISA.These results suggest that upregulation of miR-27b-3p levels has an inhibitory effect on the expression of bFGF,and rescue experiments confirmed that overexpression of PPAR-y can reverse this inhibitory effect.The above results suggest that bFGF is positively regulated by PPAR-y and may be involved in the regulation of adriamycin sensitivity in ATC cells by the miR-27b-3p/PPAR-y axis.8.The total protein and nuclear protein expression of p65 were significantly increased in the resistant cell lines SW1736/DOX and 8305C/DOX,suggesting that NF-κB p65 presented an activated state in the resistant cell lines.After NF-κB p65 was inhibited,the expression of miR-27b-3p was also significantly down-regulated,while the expression of PPAR-γ was significantly up-regulated compared with the control group.The above results suggest that miR-27b-3p level up-regulation in ATC-resistant cells is associated with NF-κB p65 activation,and NF-κB p65 may have an effect on ATC resistance as an upstream regulatory gene of the miR-27b-3p/PPAR-y regulatory axis.Conclusion:Overexpression of PPAR-γ can increase the sensitivity of ATC cells to adriamycin;PPAR-y is a direct target gene of miR-27b-3p,and miR-27b-3p is involved in regulating adriamycin resistance in ATC cells by targeting PPAR-γ;NF-κB p65 and bFGF,as upstream and downstream signaling molecules of the miR-27b-3p/PPAR-γaxis,respectively,are involved in regulating adriamycin resistance in ATC cells. |
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