Study Of Mir-150's Expression And Mechanism Of Action In Papillary Thyroid Carcinoma

BackgroundThyroid cancer(TC)is the most common malignant tumor in thyroid-related diseases.It originates from cells of different origins located in the thyroid gland at the base of the neck.Thyroid carcinomas have now been classified in several forms and high variability has been observed in molecular,cellular and clinical features.The most common form has a better prognosis,but some very aggressive thyroid carcinomas are characterized by poorly differentiated cells and no effective treatment is currently available.Although the cause of thyroid carcinoma is not fully understood,it is generally considered to be the relationship between the innate and postnatal environment,and many genetic factors involved in its pathogenesis have been found.In recent years,microRNA(miRNA)have attracted a great deal of attention as novel small molecule markers.MiRNA regulate many aspects of tumor progression including cell proliferation,migration,invasion,angiogenesis and anti-apoptosis.The process by which miRNA are formed generally occurs in the nucleus by RNA polymerase II to form primary miRNA(pri-miRNA)>1000 kb followed by a nuclear microbiome consisting of RNase III enzyme Drosha and its cofactor Dgcr-8 Cutting.This activity produces pre-miRNA of 65 nucleotides in length.Cytoplasmic Transporting by EXP5 and Cytoplasmic RNAse III Endonucleases dicer catalyzes the formation of inosine through adenosine deaminase(ADAR).As a single-stranded,highly conserved non-coding small RNA,miRNA is closely related to many human diseases including tumors.Initially,miRNA were identified as a negative regulator that plays an important role in gene expression by exerting their effect in a sequence complementary manner by binding with the 3'UTR of the messenger RNA(mRNA)encoded by their target protein.However,recent data suggest that miRNA regulation requires a more sophisticated post-transcriptional control system.There are detailed literatures on how miRNA inhibit and activate gene expression by interacting with complementary regions in the 3'UTR of promoters,and coding regions of the mRNA targets.Abnormalities of miRNA in vivo may be associated with the occurrence and development of many cancers.Oncogene activity of miRNA(oncomiR)or tumor suppressor gene activity of miRNA is a cancer-associated microRNA(miRNA).The expression of onco-miRNA and tumor suppressor of miRNA is a state of dynamic equilibrium under physiological conditions.However,the imbalance can cause malignant transformation of the body cells,thus promoting tumorigenesis.It is well known that,miRNA can specifically target the 3'UTRs of certain messenger RNA(mRNA)to prevent them from encoding specific proteins or directly degrading their mRNA.Disorders of certain microRNA are closely linked to the development and progression of specific cancers.Some studies have reported that the 'abnormal expression of miRNA in vivo and the occurrence and development of thyroid carcinoma are closely related.The occurrence and development of miRNA and thyroid carcinoma are closely related to the clinical diagnosis and treatment of thyroid carcinoma miRNA.Since RNA are very unstable in the blood,they are rapidly degraded by the body's RNases.Initially,scholars thought that miRNA would not be retained in serum.And then,miRNA in cells were often used to analyze the expression level of miRNA.However,subsequent studies showed that miRNA can be found in the blood's extra nuclear bodies and some of the particles.They also found that these miRNA are capable of intracellular signaling and can be secreted into the placenta and breast milk.MiR-150 is a family of microRNA precursors found in mammals,including humans.Mature miRNA sequences(approximately 22 nucleotides)are excised from the hairpin of a precursor digested by the enzyme dicer,and then linked to RNA-induced silencing complex(RISC)complex that affects RNA interference.Studies showed that miR-150 is already associated with many cancers.For example,it is thought to promote the proliferation of cancer cells in gastric cancer and is also found to be expressed more than 50-fold in osteosarcoma.In addition,the expression of miR-150 also affects the levels of GAB1 and FOXP1 proteins in malignant and normal B cells and regulates their BCR signaling pathway.Increasing evidence showed that miRNA are involved in the development of human malignancies,including thyroid tumors.Recent reports suggest that these small noncoding RNA can be used to develop and improve the diagnostic characteristics of thyroid carcinomas.MiR-150 may play a role of suppressor gene in many cancers.Although miR-150 has been reported in many cancers,the role and underlying functional mechanisms in thyroid carcinomas have not been well studied.ObjectiveThe occurrence of tumors is related to multiple reactions and mutations during the process of tumorigenesis.Dysregulation expression of miRNA play an important role in tumorigenesis.Furthermore,the abnormal expression of miRNA has a close relationship with the occurrence and development of cancer.Recently,the role of miR-150 in melanoma,lung cancer and hepatocellular carcinoma has been reported.However,the role of miR-150 in thyroid carcinoma cells remains unclearn The purpose of this study was to determine the role of miR-150 in thyroid carcinoma cells and to further explore the mechanism of miR-150 in papillary thyroid cancer,which may provide an early diagnosis,mid-term treatment and clinical prognosis for papillary thyroid cancer as a new molecular marker.This study will be carried out from the following aspects:Detection of miR-150 in papillary thyroid cancer tissues and the paired normal paracancerous tissue.Construction the overexpression and knockdown plasmids of miR-150 and validation the efficiency of these plasmids;whether overexpression or knockdown of miR-150 can regulate cell viability;cell proliferation,colony formation,cell cycle process and cell apoptosis and other biological effects in thyroid carcinomas;prediction the target genes of miR-150,and verification the relationship of miR-150 and target genes using EGFP fluorescence reporter system,RT-qPCR and Western blot and other experimental techniques;The functional role of target gene on cell proliferation,colony formation,cell cycle process and cell apoptosis and other biological characteristics in thyroid carcinomas.Methods1.The expression level of miR-150 was detected by real-time fluorescence quantitative PCR(RT-qPCR)in papillary thyroid carcinoma tissues and normal thyroid tissues.2.MTT and colony formation assays showed the proliferation of thyroid carcinoma cells.3.Flow cytometry cell cycle and apoptosis assays were used to detect the effects of miR-150 on the cell cycle and apoptosis in papillary thyroid cancer cells.4.Bioinformatics software predicted the target genes of miR-150.5.Western blot and immunofluorescence assays were used to verify the activation of miR-150 and RAB11A-mediated WNT/?-catenin pathway.6.RT-qPCR assay detected the expression level of miR-150 and RAB11A in TPC-1 transplanted tumor tissues.And the RAB11A protein levels in TPC-1 transplanted tumor tissues were examined by Western blot.Results1.Real-time quantitative PCR was used to detect the expression level of miR-150 in 10 paired papillary thyroid carcinoma tissues and their adjacent normal tissues:The expression level of miR-150 in papillary thyroid carcinoma was significantly lower than that in adjacent normal tissues.2.The cell viability of Klcells and TPC-lcells was measured by MTT assay.After transfection of pri-miR-150,pcDNA3,ASO-miR-150 and ASO-NC inKl cells and TCP-1cells,the changes of groups were different.The viability of K1 and TCP-1 cells transfected with pri-miR-150 were weaker than that in the pcDNA3 treated group.Colony formation assay showed that colony formation ability was significantly increased in K1 and TCP-1 cells transfected with ASO-miR-150,while the clonogenicity was decreased in cells transfected with pri-miR-150.Flow cytometry cell cycle assay showed that over-expression of miR-150 inhibited the transition of G1 phase to S phase in K1 cells and TPC-1 cells,while blocking miR-150 promotedthe cell cycle progress.Flow cytometry cell apoptosis assay showed that miR-150 overexpression promoted the apoptosis of K1 cells and TPC-1 cells.;However,blocking miR-150 inhibited the apoptosis of K1 and TPC-1 cells.3.The target gene of miR-150 was predicted by TargetScan,miRBase and miRanda.RAB11A was the alternative target gene of miR-150,RAB11A mRNA levels were reduced by 45%and 57%in miR-150-overexpressing K1 and TPC-1 cells;while RAB11A mRNA levels were increased by 3.8-fold and 4.7-fold in ASO-miR-150 transfected thyroid carcinoma cells.Furthermore,overexpression of RAB11A promotes the malignant phenotype of thyroid carcinoma cells.4.EGFP reporter assayshowed that the intensity of EGFP fluorescence expression of RAB11A 3'UTR decreased by about 50%and 52%respectively in miR-150-overexpressing cells compared with the control group.However,the EGFP fluorescence intensity of RAB11A 3'UTR increased by about 43%and 45%in ASO-miR-150-treated cells,respectively.However,no significant change in the EGFP fluorescence intensity of the mutated RAB11A 3'UTR was observedin pri-miR-150 or ASO-miR-150 transfected cells.5.RT-qPCR assay was used to detect the expression levels of RAB11A in 10 paired papillary thyroid cancer tissues and adjacent normal tissues,p-actin was used as an endogenous control in the experiment.Compared with the adjacentcontrol group,the expression of RAB11A mRNA levels were significantly increased about 8.3 fold.Compared with the control group,the number of colony-forming cells increased about 1.5 fold and 1.8 fold after RAB11A overexpression,respectively.Knockidown of RAB11A decreased the clone number of K1 cells and TPC-1 cells by 70%and 50%,respectively.Overexpression of RAB11A,accelerated K1 cells and TPC-1 cells from G1 to S phase conversion,and knockdown of RAB11A in K1 cells and TPC-1 cells inhibitedthe cell cycle progress.Overexpression of RAB11A inhibited the apoptosis of K1 and TPC-1 cells and decreased the relative apoptotic index.However,knockdown of RA311A promoted the apoptosis of K1 and TPC-1 cells,and the relative apoptosis index increased.In addition,miR-150 inhibits RAB11 A-mediated activation of WNT/?-catenin in thyroid carcinoma cells.6.In nude mice experiment,the expression level of miR-150 and RAB11A were detected by RT-qPCR assay in two groups of nude mices.Compared with the control group,the expression level of miR-150 was significantly increased and the expression level of RAB11A was significantly reduced in pri-miR-150 treated mices;The protein expression level of RAB11A in TPC-1-transplanted tumors was detected by Western blot assay.The results showed that the expression level of RAB11A protein was lower in miR-150 transfection group.Conclusion1.The expression level of miR-150 in papillary thyroid carcinoma was lower.2.Overexpression miR-150 will weaken cells' reproduction ability and inhibit the cell cycle from G1 to S,as a result,this procedure speed cells' apoptosis.3.RAB11A is miR-150's direct target gene.miR-150 inhibit the expression of RAB11A in transcription level?It is possible that miR-150 develop its carcinostasis by target RAB11A.4.RAB11A can enhance papillary thyroid cancer cell's proliferation ability,speed cell cycle and inhibit cells' apoptosis.miR-150 inhibit WNT's signal path by regulating RAB 11 A.5.miR-150 can inhibit growth of transplantation tumor of nude mouse in internal body.The overexpression of miR-150 can decrease RAB11A's level in tumor.