HOXD9-Induced MiR-451a/TIA1 Is Implicated In The Regulation Of Human Anaplastic Thyroid Carcinoma Proliferation And Metastasis

Background:The anaplastic thyroid carcinoma(ATC),is an extremely uncommon form of thyroid cancer that only accounts for 1-2% of all thyroid cancers.It is mainly characterised by a rapidly growing and enlarging anterior cervical mass causing pressure on the trachea.Although ATC accounts for only a small percentage of thyroid cancers,the prognosis is extremely poor.Metastases are the primary cause of death in ATC,and approximately 20-50% of patients develop distant metastases.The median survival time for patients with ATC is between 3 and 6 months.Surgery for ATC is frequently ineffectual,despite the availability of surgical treatment,radiation,and chemotherapy;hence,understanding the molecular pathways that underlie its development is essential to the search for a treatment that is effective.ATC is made up of undifferentiated cancer cells and a tumour microenvironment.Undifferentiated cancer cells in an ATC exhibit both a high rate of proliferation and a high capability for metastasis.One of the fundamental changes in ATC cells is the avoidance of cell death,while tumour metastasis is a key step in the development of ATC,predicting a more advanced stage and a worse prognosis.A number of different cellular processes are involved in the progression of ATC.These processes include the breakdown of the extracellular matrix,epithelial-mesenchymal transition(EMT),tumor angiogenesis,the formation of an inflammatory tumour microenvironment,and dysfunction of programmed cell death mechanisms.Apoptosis is the most common form of programmed cell death.Apoptosis helps prevent ATC metastasis by destroying ATC cells.However,evasion of apoptosis can lead to ATC proliferation,development,and treatment resistance.EMT is a process that enables tumor cells to lose some of their epithelial features and adopt some mesenchymal properties,allowing them to become more invasive.As a result,the investigation of the molecular mechanisms that underlie ATC resistance to apoptosis and the induction of EMT lays the groundwork for the creation of molecularly targeted treatments.Homozygous cassette genes(HOX)are a superfamily of genes that encode transcription factors that play critical roles.Transcription factors produced by HOX genes are key regulators of development and affect a wide range of cell functions,including proliferation,differentiation and death.Dysregulation of these proteins,then,is certainly a factor in the emergence and spread of cancer.HOXD9,a member of the HOX family,has been linked to the emergence and spread of cancer.Excessive expression of HOXD9 in colorectal tumor cells has been demonstrated to induce aggressive metastasis in liver cancer,and HOXD9 expression is closely linked with metastatic invasion.Overexpression of HOXD9 in gastric cancer cells,on the other hand,has the potential to greatly boost cell proliferation,migration,and invasion.As a result,it is not apparent what role or method HOXD9 plays in regulating ATC’s development.Methods:1.Expression of HOXD9 in human undifferentiated thyroid cancer and its clinical significance.(1)RT-PCR analysis of mRNA levels of HOXD9,miR-451 a,Ki-67,PCNA,CCNA2 and CCNB1 in thyroid cancer tissues and adjacent normal tissues of thyroid cancer patients;(2)Western blot examined the expression of HOXD9 in the human ATC cell lines8505 C,FRO and HTH7 and the thyroid follicular cell line Nthy-ori 3-1 as well as the expression of HOXD9 and TIA1 in cancer tissues and their paired adjacent normal tissues from three different thyroid cancer patients;(3)Detection of miR-451 a expression in thyroid cancer tissues and adjacent normal tissues using in situ hybridization staining;(4)Statistical analysis of the correlation between mRNA levels of HOXD9 and mRNA levels of Ki67,PCNA,CCNA2 and CCNB1 in thyroid cancer tissues.2.HOXD9 regulates proliferation and apoptosis in human undifferentiated thyroid cancer cells.(1)HOXD9 expression in 8505 c cells was altered using overexpression or knockdown techniques;(2)RT-PCR analysis of HOXD9 mRNA levels in 8505 c cells;(3)Expression levels of HOXD9 and apoptosis-related markers in 8505 c cells were detected by western blot;(4)The effect of HOXD9 on the proliferation of 8505 c cells was examined using CCK-8 and cell clone formation;(5)Transwell migration and stromal gel invasion assays to investigate the effect of HOXD9 on the migration and invasion of 8505 c cells;(6)Detection of the effect of HOXD9 on 8505 c cell apoptosis using flow cytometry assay.3.HOXD9 promotes EMT and metastasis in human undifferentiated thyroid cancer cells by regulating miR-451 a.(1)The effect of disrupting or overexpressing HOXD9 and miR-451 a on the expression levels of EMT-related proteins and apoptosis-related markers was examined using western blot;(2)Relationship between EMT-related protein E-cadherin and N-cadherin expression levels following interference or overexpression of HOXD9 and miR-451 a by immunofluorescence assay;(3)Altered expression of HOXD9 and miR-451 a by transfection technique;(4)Detection of the effect of HOXD9 and miR-451 a expression levels on ATC cell proliferation using cell clone formation;(5)Transwell migration and stromal gel invasion assays to investigate the effect of HOXD9 and miR-451 a expression levels on the migration and invasion of ATC cells;(6)Flow cytometry assay to detect the effect of HOXD9 and miR-451 a expression levels on the apoptosis of ATC cells.4.Regulation of the HOXD9/miR-451a/TIA1 axis in human undifferentiated thyroid cancer.(1)Alteration of HOXD9 and TIA1 expression in 8505 c cells using overexpression or knockdown techniques;(2)Western Blot analysis was used to detect the protrin expression of HOXD9 and TIA1;(3)Immunoprecipitation(Co-IP)analysis of the direct interaction between HOXD9 and TIA1;(4)Luciferase reporter gene assay for the interaction between miR-451 a and HOXD9,TIA1 and miR-451a;(5)RNA immunoprecipitation(ChIP)assay for direct binding of miR-451 a to HOXD9;(6)Statistical analysis of the correlation between mRNA levels of HOXD9 and miR-451 a in thyroid cancer tissues.5.Silencing HOXD9 attenuates tumour growth in a mouse thyroid cancer xenograft tumour model.(1)The effect of silencing HOXD9 on thyroid cancer growth in vivo was assessed using a xenograft tumour model;(2)In vivo imaging(IVIS)on tumour-forming models to track tumour growth;(3)Western blot analysis was used to detect the expression levels of HOXD9 and TIA1 in mouse tumour tissues;(4)Fluorescent TUNEL assay was used to determine apoptosis in mouse tumour tissues;(5)Immunohistochemical analysis of HOXD9,Ki67 and CD31 expression in mouse tumour tissues.6.HOXD9 promotes tumourigenesis and metastasis in vivo.(1)A thyroid cancer lung metastasis model was used to analyse the effect of HOXD9 on ATC metastasis in vivo,and IVIS tracked the growth of lung tumours;(2)Use of a fluorescent TUNEL assay to determine apoptosis in mouse lung tumour tissue;(3)Immunohistochemical analysis of HOXD9,E-Cadherin and N-Cadherin expression in mouse lung tumour tissues.Results:1.Expression of HOXD9 in human undifferentiated thyroid cancer and clinical significance.The expression of HOXD9 was shown by immunohistochemistry and Western Blot to play a key role in the development and progression of ATC.The correlation between HOXD9 and the proliferation markers PCNA,Ki67,CCNA2 and CCNB1 revealed that HOXD9 may promote the proliferation of ATC.Meanwhile,we showed that HOXD9 is a valuable prognostic biomarker for ATC by comparing the effect of HOXD9 on the overall survival of ATC patients.Meanwhile,we found that miR-451 a and TIA1 could be downstream regulators of HOXD9.2.HOXD9 regulates migration,invasion,proliferation and apoptosis of human undifferentiated thyroid cancer cells.In order to determine the effects of HOXD9 on the migration,invasion,proliferation,and apoptosis of ATC cells,we altered the expression of HOXD9 by either overexpressing or knocking it down using specific techniques.According to the findings,disruption of HOXD9 effectively promoted apoptosis in 8505 c cells while significantly inhibiting migration,invasion,and proliferation of 8505 c cells.When HOXD9 was overexpressed,the opposite of the expected result was observed.According to the findings,HOXD9 caused 8505 c cells to undergo apoptosis,which in turn blocked the cells’ ability to migrate,invade,and proliferate.3.HOXD9 promotes EMT and metastasis of human undifferentiated thyroid cancer cells by regulating miR-451 a.We explored the GEO database to locate the dataset(GSE104006),which led to the discovery of nine miRNAs that have the potential to play a role in the regulatory ATC process’ differential expression.The combination of the UCSC and JASPAR databases allowed for the discovery of HOXD9 as an upstream regulator of the miR-451 a gene.Altering the expression of HOXD9 and miR-451 a was accomplished by the use of techniques such as HOXD9 knockdown,miR-451 a inhibitor,HOXD9 overexpression,and miR-451 a mimics.Interfering with HOXD9 was observed to induce apoptosis and block the EMT process,as well as prevent ATC cells from growing,migrating,and invading new territories,respectively.These effects on ATC cells were partially mitigated by the addition of miR-451 a inhibitors,which were used in conjunction with HOXD9 knockdown.Overexpression of miR-451 a in conjunction with HOXD9 had the dual effect of inhibiting the enhancement of cell growth,migration,and invasion by HOXD9,while at the same time decreasing the rise in apoptosis and increasing the EMT process.4.Regulation of the HOXD9/miR-451a/TIA1 axis in human undifferentiated thyroid cancer.The expression of HOXD9 and TIA1 in 8505 c cells was altered using overexpression or knockdown techniques,and HOXD9 expression in ATC cells was also increased or decreased when by overexpression or si-TIA1.Also,we used Co-IP analysis to show a direct interaction between HODX9 and TIA1.The luciferase reporter gene assay showed that the promoter region of miR-451 a binds directly to HOXD9 and the ChIP assay showed that miR-451 a binds directly to HOXD9.Statistical analysis showed that miR-451 a expression in human ATC tissues was negatively correlated with HOXD9 expression.When we inhibited HOXD9 expression in 8505 c cells,it increased the expression level of miR-451a;while overexpression of HOXD9 decreased the expression level of miR-451 a.5.Silencing HOXD9 attenuates tumour growth in a mouse thyroid cancer xenograft tumour model.In our study,we investigated the effect of HOXD9 on thyroid cancer growth in vivo by establishing a thyroid cancer xenograft tumour model and measuring tumour growth progression using IVIS.Our results show that silencing HOXD9 alleviates thyroid cancer growth and progression,as evidenced by a significant reduction in tumour weight and volume.When HOXD9 was inhibited,protein levels of HOXD9 and TIA1 were lower and levels of miR-451 a were higher.When HOXD9 was inhibited,the rate of apoptosis was significantly increased,cell proliferation and microvessel density were reduced,and EMT was inhibited.6.HOXD9 promotes tumourigenesis and metastasis in vivo.To assess the effect of HOXD9 on thyroid cancer metastasis in vivo.We established a thyroid cancer lung metastasis model and combined it with IVIS to explore the effect of silent HOXD9 on metastasis levels in a thyroid cancer lung metastasis model.The results showed that the lung bioluminescence signal intensity was significantly reduced in nude mice inoculated with si-HOXD9 cells compared to the normal inoculated 8505 C cell line.In addition,when HOXD9 was inhibited,the number of metastatic nodules and lung weight in the lung were significantly reduced and the overall physiology of the lung was improved;when HOXD9 was knocked down,apoptosis in the lung tissue was increased.Immunohistochemical results showed a decrease in N-Cadherin expression and an increase in E-Cadherin expression when HOXD9 was inhibited.ConclusionsOur study shows that HOXD9 is closely related to the development and progression of ATC.aberrantly high HOXD9 expression leads to ATC disease progression,and the possible regulatory mechanism is that HOXD9 induces miR-451 a to directly target TIA1 to inhibit ATC apoptosis and promote EMT thereby enhancing its proliferation and invasive ability.In vivo experiments showed that downregulation of HOXD9 in ATC cells prevented tumor growth in a mouse xenograft model and ATC cell metastasis in a lung metastasis model.Our study suggests that targeting HOXD9 can influence ATC proliferation and metastasis,and therefore HOXD9 could be a potential target for ATC diagnosis and management.