| Background:The incidence of thyroid cancer has been increasing recently.It has been the most common malignant tumor of the endocrine system and the most common malignant tumor in women under the age of thirty worldwide.Thyroid cancer can be divided into four pathological subtypes: thyroid papillary carcinoma(papillary thyroid cancer,PTC),thyroid follicular carcinoma(folicular thyroid cancer,FTC),)and medullary thyroid carcinoma(medullary thyroid cancer,).MTC and(anaplastic thyroid cancer,ATC).Papillary thyroid cancer(PTC)is the most common pathological subtype of thyroid cancer,accounting for 80% ~ 90% of the total thyroid cancer.Most PTC patients had a good prognosis after complete surgical resection,with a 5-year survival rate of more than 95%.However,the survival rate of patients with tumor recurrence and local and extensive lymph node metastasis was reduced to 40%.Histone methylation abnormal is a common cause of cancer initiation and progression.KDM1 A can interact with a variety of protein complexes(Corest,NuRD and RCOR2),receptors(estrogen,androgen,and TLX),noncoding RNA)(HOTAIR,SRA and Terras),small RNA(miR-137 and miR-329),Non histone(p53 E2F1 and DNMT 1)and transcription factors(TLA and snail).These compounds have complex functions which regulated a variety of physiological and pathological processes,including the cancer development.It has been reported that KDM1 A was overexpressed in prostate cancer,breast cancer,oral cancer,colorectal cancer,neuroblastoma,glioblastoma,acute myeloid leukemia,T cell acute lymphoblastic leukemia and so on.However,the expression and function of KDM1 A in PTC is unclear.Tissue inhibitor of metalloproteinases(TIMPs)is a family of endogenous inhibitor of matrix metalloproteinase family(MMPs).These endogenous secreted proteins inhibit all matrix metalloproteinases,and each TIMP is targeted at multiple MMPs.Each MMP has a wide range of protein substrates,so the inhibition mediated by TIMP can form a strong linkage system in regulating homeostasis in vivo.Many studies have reported that TIMP1 is abnormally expressed in many kinds of cancers such as lung cancer,brain cancer,prostate cancer,breast cancer,colon cancer and many other malignancies.However,the role of TIMP1 in thyroid neoplasms is rarely reported.The aim of this study was to investigate the expression of KDM1 A in PTC tissues and its effect on the function of PTC cells,and determined whether KDM1 A involved in promoting PTC development by acting on TIMP1.Further research explained the mechanism of TIMP1 regulation by KDM1 A.Methods: 1.We collected 60 pairs of PTC tissues and adjacent non-cancerous from September 2014 to February 2017 from thyroid surgery,the first affiliated Hospital of China Medical University,and the clinicopathological features were statistically analyzed.The expression of KDM1 A in 60 pairs of PTC tissues and corresponding paracancerous tissues was determined by qRT-PCR method.?2 test was used to analyze the relationship between the expression of KDM1 A and clinicopathological data in PTC tissues.2.RNAi was used to down-regulate the expression of KDM1 A,qRT-PCR was used to detect the interference efficiency.Transwell and wound healing assay were used to detect the down-regulation of KDM1 A on the migration and invasion of PTC cell line.pulmonary metastasis assay in nude mice was performed to detect whether knockdown KDM1 A could inhibit PTC metastasis ablility in vivo.3.QRT-PCR and Western blot were used to detect the effect of KDM1 A on invasion related proteins.The activity of metalloproteinases was detected by gelatin zymography.QRT-PCR and Western blot were used to detect the expression of TIMP1 after transfection with si-KDM1 A.The expression of downstream invasion-associated proteins was detected by Western blot and the activity of metalloproteinases was detected by gelatin zymogram.The effects of co-transfection of si-KDM1 A and si-TIMP1 on the migration and invasion of PTC cells were detected by Transwell and wound healing assay.The expression of H3K4me2 and H3K9me2 after down-regulation of KDM1 Awere detected by Western blot.Chromatin immunoprecipitation(ChIP)assay was used to detect the coprecipitation between KDM1 A and TIMP1 gene transcription initiation site.The binding of H3K4 me 2 and H3K9me2 to TIMP1 after transfection of si-KDM1 A was detected by ChIP assay.Results: 1.The expression of KDMs was detected in 16 pairs of PTC and corresponding paracancerous tissues.KDM1 A,KDM5A and KDM7 A were found to be overexpressed in PTC tissues.KDM1 A was selected as the follow-up study.The results showed that the expression of KDM1 A in 60 patients was significantly higher than that in the corresponding adjacent tissues,and the expression of KDMA in PTC was positively correlated with the age of less than 55 years and lymph node metastasis.2.Knockdown KDM1 A inhibited the migration and invasion ability of the PTC cells.To further investigate the effect of KDM1 A on tumor metastasis,we performed pulmonary metastasis assay in nude mice by injecting sh-KDM1 A and sh-NC transfected PTC cells through tail vein.The result showed that the number of visible nodules in the sh-KDM1 A group were signifcantly increased when compared to those in the sh-NC group.3.Knockdown KDM1 A weaken the expression and activity of the invasive-associated protein MMP9 at the protein level.Moreover,the expression of TIMP1 increased significantly after knockout of KDM1 A at both mRNA and protein levels.Rescue experiments showed that the expression and activity of MMP9 in PTC cells co-transfected with si-KDM1 A and si-TIMP1 were significantly higher than those in PTC cells transfected with si-KDM1 A alone.The inhibition of PTC migration and invasion induced by KDM1 A knockout was recovered to a certain extent.After transfection of si-KDM1 A,the total expression of H3K4me2 and H3K9me2 protein was decreased.ChIP assay showed that H3K4me2 and H3K9me2 directly binds to the region of transcription initiation site of TIMP1 gene,and compared with the cells transfected with si-NC.si-KDM1 A transfected cells significantly increased the H3K4me2 expression,but H3K9me2 did not change significantly,suggesting that KDM1 A plays a role by regulating the demethylation of H3K4me2.Conclusion: KDM1 A overexpression in PTC tissues and cell lines,and high expression of KDM1 A positively associated with tumor metastasis.KDM1 A promote the cell migration and invasion through inhibit TIMP1 transcription,decrease the expression of TIMP1 and increase the expression and activity of MMP9 by binding to the region of TIMP1 transcription initiation site and demethylate H3K4me2. |
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