| Thyroid cancer is the most commonly diagnosed type of endocrine tumor,and its incidence has been rapidly increasing in recent years.It is considered to be one of the top 10 forms of malignant tumors.According to 2016 data from the National Cancer Institute,which is located in the United States,the new onset of thyroid cancer in the world increased by an average of more than 5%annually,and papillary thyroid carcinoma(PTC)is the fastest growing malignancy.Although the vast majority of PTC cases have an excellent prognosis,with the 10-year survival rate of more than 90%,approximately 50%of these patients emerge with cervical lymph node metastasis at the initial diagnosis of PTC.Between 10-20%of PTC patients have recurrent and distant metastasis;once the distant metastasis is discovered,the 10-year survival rate of the patients is reduced to about 40%.At present,the main treatment regimen of PTC is surgical resection since chemotherapy is basically ineffective in PTC patients.However,surgical resection has also been controversial among academic researchers in recent years.Some scientists believe that because of the low degree of PTC malignancy and its slow disease progress,patients with<1 cm papillary thyroid microcarcinoma(PTMC)should be temporarily monitored during the follow-up.Other scientists recommended surgical resection and believe that the PTMC was not necessarily equivalent to low-risk cancer;indeed,many patients with microcarcinomas have very rapid disease progression and early emergency of local and distant metastasis.Therefore,in the absence of an effective means of identification,the early diagnosis and treatment of PTC is still necessary.Nevertheless,the detailed molecular mechanisms of PTC onset and progression have not been elucidated.Therefore,the study of high-risk molecular events of PTC and the screening of molecular markers for the characterization of high-risk PTC malignancy and those associated with PTC metastasis can identify the high-risk individuals at an early stage among numerous PTC patients to achieve an effective and early diagnosis and prognosis as well as to conduct stratified treatments in the patients that have important theoretical significance and clinical value.LncRNA is a class of>200 bp non-coding RNA,and more and more evidence has shown that IncRNA plays an important role in gene transcription,modification,and posttranscriptional regulation.Studies have shown that IncRNA plays a crucial part in the progression of various solid tumors,such as gastric cancer,colon cancer,kidney cancer,and pancreatic cancer.LncRNA takes part in tumor suppressor gene or oncogene activity by regulating the chromosomal structure or the expression of the downstream genes.Therefore,IncRNAs are likely to be important molecular markers and potential targets for the diagnosis and treatment of cancer in the future.We used thyroid papillary carcinoma IHH4 cells as the parent strain.Through transwell chamber screening and cell culture techniques,after fifteen generations of screening culture,we successfully got the sub-strain of thyroid papillary carcinoma with high tendency to invasion and metastasis,which is named IHH4-M.A series of in vivo and in vitro experiments were used to examine and confirm the invasive and metastasizing ability of IHH4-M sub-strain.Besides,the differentially expressed IncRNAs and mRNAs were screened by lncRNA-mRNA gene chip,and the differential IncRNA expression profiles and differential mRNA expression profiles associating to metastasis were constructed.The RT-PCR assay were performed in the fresh tumor tissues and normal tissues of 60 patients with papillary thyroid carcinoma.The results showed that IncRNA AP000472.2 was significantly related to capsular invasion and lymph node metastasis of papillary thyroid carcinoma,but was not related to the patient's sex,age,and tumor size.Thus,IncRNA AP000472 was identified as the candidate object of our study.To further confirm the role of IncRNA AP000472.2 in invasion and metastasis of papillary thyroid carcinoma and explore its related molecular mechanisms,we constructed IncRNA AP000472.2 overexpression lentiviruses and RNA interference lentiviruses.The results of overexpression and knocking out IncRNAs AP000472.2 showed that upregulation of IncRNA AP000472.2 expression could promote the process of epithelial-mesenchymal transition(EMT)in papillary thyroid carcinoma IHH4 cells,and significantly increase the ability of proliferation,migration,and invasion of IHH4 cells.On the contrary,knocking out IncRNA AP000472.2 could block the process of EMT in papillary thyroid carcinoma IHH4-M cells,and reduce the proliferation,invasion and metastasis of IHH4 cells.This indicated that:IncRNA AP000472.2 plays a role in promoting the EMT process of IHH4 cells,and increases the proliferation,invasion and metastasis capacity of IHH4 cells,but the relevant molecular mechanism is still not clear.In order to solve this problem,we constructed a IncRNA-mRNA co-expression network based on the IncRNA microarray,and used cis and trans prediction methods to predict the downstream regulated genes of lncRNA AP000472.2.DEAR1 was selected as a candidate downstream regulated gene by RT-PCR,WB and other experiments.To further confirm that whether DEAR1 is a downstream regulated gene of IncRNA AP000472.2,we designed DEAR1 RNA interference experiments and DEAR1 overexpression experiments based on the IncRNA AP000472.2 knockout HH4-M cells and the IncRNA AP000472.2 over-expressed IHH4-C cells.The experimental results showed that after knocking out IncRNA AP000472.2 in IHH4-M cells,the expression of DEAR1 protein was significantly up-regulated,and the expression of SMAD3 protein was down-regulated,resulting the inhibition of TGF-? signal pathway and the decrease of invasion and metastasis ability of IHH4-M cells.The expression of EMT-related markers such as N-cadherin,twist,MMP9 was down-regulated,and when the expression of DEAR1 continued to be knocked down,the expression of SMAD3 protein was significantly up-regulated and TGF-? signaling pathway was activated,and the invasion and metastasis of IHH4-M cells were partially recovered and the expression levels of N-cadherin,twist,MMP9 were partly upregulated.Conversely,when lncRNA AP000472.2 was introduced into IHH4-C cells,the expression of DEAR1 protein was down-regulated,the expression of SMAD3 protein was up-regulated,TGF-? signal pathway was activated,and the invasion and metastasis of IHH4-C cells were significantly enhanced,and the expression of EMT-related markers such as N-cadherin,twist,and MMP9 was up-regulated.However,when the expression of DEAR1 was overexpressed,the expression of SMAD3 protein was significantly down-regulated.TGF-?,the signaling pathway was inhibited,the invasion and metastasis capacity of IHH4-C cells was partially down-regulated,and the expression levels of N-cadherin,twist,and MMP9 were partly downregulated.This suggests that in thyroid papillary carcinoma IHH4-M cells,lncRNA AP000472.2 can down-regulate the expression of DEAR1 protein,and then activate TGF? signaling pathway and EMT process,thereby promoting tumor invasion and metastasis.This study,starting from the construction of high metastatic sub-strains of papillary thyroid carcinoma,screened the lncRNA and mRNA related to metastasis of papillary thyroid carcinoma by microarray and confirmed the role of lncRNA AP000472.2 in the invasion and metastasis of papillary thyroid carcinoma through experiments.The results of the study not only deepen the understanding of the new invasion and metastasis mechanism of thyroid papillary carcinoma,but also are hopeful to provide new early warning biomarkers or drug intervention targets in the early diagnosis and treatment of high-risk PTC patients,and may provide the basis for the further study of the hierarchical diagnosis and precise treatment of thyroid cancer. |
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