| BackgroundAs a kind of endocrine system malignant tumor,thyroid cancer has more and more attention.Most countries in the world thyroid cancer incidence on the rise every year,and also increase gradually increased,in many developing countries,thyroid carcinoma has been incorporated into one of the most common malignant tumor.The world health organization(WHO)announced the latest official figures,and the American cancer society(ACS),epidemiology,according to a report released in recent decades,thyroid cancer in most countries and regions all over the world have high incidence and presents the trend of increased year by year.In the United States,thyroid cancer in persons over the age of 65 has high average annual growth rate,can amount to 8.8%,is the fastest growing cancer of all malignant tumor.Epidemiological statistics show that China thyroid cancer incidence in a straight line after the founding of the rising trend,and with the people’s living standards improved,these diseases are growing more quickly.Classify according to the pathological types of thyroid carcinoma,papillary thyroid carcinoma(PTC),respectively,thyroid follicular carcinoma(FTC),anaplastic thyroid carcinoma(ATC)and medullary thyroid carcinoma(MTC)four types.Among them,the most common type of thyroid cancer pathology for PTC,more than 80%of all thyroid cancer.Clinical statistics show that China since reform and opening up,the incidence of thyroid papillary carcinoma increased 5.7 times in nearly 26 years,it has brought the huge economic pressure to the society.Thyroid papillary carcinoma longer occur in women,especially women aged 30 to 50 thyroid papillary carcinoma has high incidence,but much lower incidence in men than women.Studies have reported finding,using 131 I treatment of postoperative patients with thyroid papillary carcinoma,better prognosis results,application of 131 I and jointly TSH suppression therapy of postoperative patients with thyroid papillary carcinoma has more curative effect,nearly 95%of the patients with thyroid papillary carcinoma after surgery and 1311 treatment,postoperative survival time can be up to 25 years,but there are 10%15%of thyroid papillary carcinoma patients after surgery and 1311 treatment effect is not ideal,such as tumor recurrence and distant metastasis,and this kind of tumor recurrence and metastasis often leads to poor prognosis in patients with,greatly limits the curative effect of thyroid papillary carcinoma is expected.Looking for has a higher accuracy of diagnosis and treatment,therefore,the way and method of thyroid papillary carcinoma,for the occurrence of thyroid papillary carcinoma has a important role in prevention.With the continuous development of biological medicine,molecular pathogenesis of thyroid papillary carcinoma received more in-depth research,the application of the biological target therapy in thyroid papillary carcinoma and received more and more attention to by the medical concept of the new cancer precision medicine,it is through the cancer genome sequencing and large data analysis,so as to make accurate diagnosis,a new mode of prevention,treatment,and makes the cancer early diagnosis and treatment level has improved.Precision medicine for the treatment of thyroid papillary carcinoma mainly through from gene level to study the molecular mechanism of action,in the occurrence and development of thyroid papillary carcinoma key gene in the process of change,to find a more effective new targets for the treatment of thyroid papillary carcinoma,and individual differences,formulate the corresponding treatments of thyroid papillary carcinoma can be early discovery,early diagnosis and early treatment,to achieve the purpose of prevention and cure of thyroid papillary carcinoma.ObjectiveTo explore expression profile of the long noncoding RNA LINC00538 in papillary thyroid cancer.This part of study is focused on experiment of expression difference of the long noncoding RNA LINC00538 expression profile in clinical specimens of papillary thyroid cancer.Based on clinical evidence in part I,we explored regulatory mechanism of the long noncoding RNA LINC00538 on papillary thyroid cancer in vitro.Target inhibition of the long noncoding RNA LINC0053 8 was performed in mice with siRNA.Effect of of the long noncoding RNA LINC00538 on papillary thyroid cancer in vivo was assessed,including proliferation of tumor,migration of tumor and tumor colony.At present,in the study of a wide variety of tumor diseases,long chain noncoding RNA as accurate medical star molecule has been more and more attention.In the early 21st century,the researchers through the mouse genome was found in a cDNA library on a large scale of long chain non-coding RNA,such RNA encoded protein,its transcription,the length of more than 200 nucleotides can carried out on the protein expression before and after the transcription regulation.Long chain non-coding RNA in tumor cells involves complicated regulatory mechanism,and observed in clinical trials of a long chain noncoding RNA and the correlation of tumor often confined to the phenomenon.Some scholars found in the study of non coding RNAs,highly translation potential area in 1/2 noncoding RNAs have found that in the non-coding RNAs can come short peptide by tumor cytoplasm,whereas most noncoding RNA can be targeted regulatory protein mRNA expression.Therefore,finding potential regulatory proteins can further understand the non-coding RNA LINC00538 in thyroid papillary carcinoma play a regulatory role in the process of the occurrence and development as well as the mechanism.Information released according to the existing biological database,we use the non-coding RNA sequence pairs LINC00538 has found the tumor cell signaling pathways of protein mRNA sequence,and found that the epithelial calcium sticky-cadherin(E),nerve calcium sticky element(N-cadherin)and proteins(vimentin)waveform LINC00538 and non-coding RNA sequences are highly pairing,suggesting noncoding RNA LINC00538 is likely to be targeted by combining these protein mRNA and protein expression.It should be pointed out that,long chain caused by the combination of mRNA noncoding RNA transcription regulation role mostly suppressed,minority is activated,the need for specific experiments to explore.Therefore,we will explore noncoding RNA LINC00538 for thyroid papillary carcinoma regulation function of the tumor,in order to provide new targets for the clinical treatment.Methods(1)The PTC specimens and normoal adjacent tissues were collected from 88 patients at Affiliated hospital of Inner Mongolia medical university、Inner Mongolia autonomous region people’s hospital and The first hospital of Hohhot.All samples were collected between May 2011 and September 2014.All patients with papillary thyroid cancer were enrolled randomly by statistical software.All enrolled patients were informed with details of present clinical trial,and signed freely informed consent.Present clinical trial was reviewed and approved by ethics committee of Inner Mongolia Medical University.Diagnosis of all enrolled patients was confirmed by pathological examination.All enrolled patients are initial episodes of papillary thyroid cancer,and had no surgery history.Moreover,all enrolled patients did not receive any chemotherapy,radiotherapy or papillary thyroid cancer related treatment.All clinical specimens of papillary thyroid cancer or thyroid normal tissue were removed and promptly storage in liquid nitrogen container(-80℃).All clinical specimens were stored in laboratory of Guangzhou liyin biotechnology co.LTD.Primers of RNA LINC00538 were designed,and prepared for subsequent PCR experiment.Data of PCR were analyzed with 2-△△ct method.T test were performed to analyze inter-group difference.Long term follow-up was performed to collect prognosis data of patients after surgery.Main outcome measures include year survival rate,clinical stages,tumor metastasis and preoperative tumor volume.Mann-Whitney test was performed to analyze difference of separate samples.Kaplan meier survival curve was established to assess association between prognosis of papillary thyroid cancer and the long noncoding RNA LINC00538 expression.Fisher exact test was performed to assess association between clinicopathologic feature of papillary thyroid cancer and the long noncoding RNA LINC00538 expression.(2)All cell lines were purchased from shanghai institute of cell biology.Papillary thyroid cancer cell lines include TPC1、B-CPAP、IHH-4 and CG3.Normal thyroid cell line was Nthy-ori 3-1.All cell lines were routinely cultured(DMEM medium containing 5%fetal bovine serum,5%C02 and 37℃).PCR experiment was performed to assess expression of the long noncoding RNA LINC00538 in all cell lines,and specific cell lines with high expression of the long noncoding RNA LINC00538 were enrolled for further experiment.Synthesis of siRNA was performed for the long noncoding RNA LINC00538,and siRNA of the long noncoding RNA LINC00538 was treated to specific cell lines.CCK-8 experiment was performed to examine proliferation of tumor cells.Transwell migration experiment was performed to assess migration of tumor cells.Colony formation test was performed to examine tumor colony.Cellular proteins were extracted for further experiment.Western blot was performed to examine expression of E-cadherin,N-cadherin and vimentin.T test were performed to analyze inter-group difference,and P<0.05 was considered as statistical difference.(3)All mice in this part of study were purchase from Shanghai Jiesijie laboratory animal technology company.Papillary thyroid cancer TT cell line was purchased from shanghai institute of cell biology.20 wild type BALB/c male mice(8-week,average weight 22g)were injected with TT cell suspension(2×106).Tumor body formed at 4-week after injection.Mice were randomly assigned for control siRNA treatment or siRNA of the long noncoding RNA LINC00538.Tail vein injection was performed for siRNA treatment.Anti-tumor rate was assessed at 2-week after siRNA treatment.Real-time PCR was performed to assess expression of the long noncoding RNA LINC00538.Proteins in tumor body were extracted for further experiment.Western blot was performed to examine expression of E-cadherin,N-cadherin and vimentin.T test were performed to analyze inter-group difference,and P<0.05 was considered as statistical difference.Results(1)① PCR results showed that,compared with thyroid normal tissue,the long noncoding RNA LINC00538 expression in papillary thyroid cancer increased significantly(P<0.05).②Kaplan meier survival curve showed that,compared with thyroid normal tissue,high expression of the long noncoding RNA LINC00538 in papillary thyroid cancer could cause poor prognosis,and the main manifestation was gradual decrease of year survival rate(P<0.05).5-year survival rate of patients with high expression of the long noncoding RNA LINC00538 was less than 40%,while 5-year survival rate of patients with normal of low expression of the long noncoding RNA LINC00538 was more than 60%,suggesting the long noncoding RNA LINC00538 was a predictive index of poor prognosis for papillary thyroid cancer.③Fisher exact test showed that,the long noncoding RNA LINC00538 expression was associated with clinicopathologic feature of papillary thyroid cancer.Patients with high expression of the long noncoding RNA LINC00538 were more susceptible for tumor metastasis,volume increase of tumor and poor clinical stage.(2)①Comapred with normal thyroid cell line Nthy-ori 3-1,TPC1 and B-CPAP cell lines had the most significant increase of the long noncoding RNA LINC00538(P<0.05).② CCK-8 experiment showed that,compared with TPC1 and B-CPAP cell lines with control siRNA,proliferation of TPC1 and B-CPAP cell lines with siRNA of the long noncoding RNA LINC00538 was significantly inhibited(P<0.05).③Transwell migration experiment showed that,compared with TPC1 and B-CPAP cell lines with control siRNA,migration of TPC1 and B-CPAP cell lines with siRNA of the long noncoding RNA LINC00538 was significantly inhibited(P<0.05).④Colony formation test showed that,compared with TPC1 and B-CPAP cell lines with control siRNA,colony ability of TPC1 and B-CPAP cell lines with siRNA of the long noncoding RNA LINC00538 was significantly inhibited(P<0.05).⑤ Western blot showed that,compared with TPC1 and B-CPAP cell lines with control siRNA,N-cadherin and vimentin of TPC1 and B-CPAP cell lines with siRNA of the long noncoding RNA LINC00538 significantly decreased,while expression of E-cadherin was increased(P<0.05 =.(3)① Compared with mice under control siRNA treatment,siRNA of the long noncoding RNA LINC00538 significantly inhibited growth of papillary thyroid cancer.Anti-tumor rate was 47.0%.②Western blot showed that,compared with mice under control siRNA treatment,N-cadherin and vimentin of mice with siRNA of the long noncoding RNA LINC00538 significantly decreased,while expression of E-cadherin was increased(P<0.05).Conclusions(1)Activation of N-cadherin and vimentin related pathways and suppression of E-cadherin related pathways was associated with tumorpromoting mechanism of the long noncoding RNA LINC00538 on papillary thyroid cancer.Target inhibition of the long noncoding RNA LINC00538 could attenuate multiple clinicopathologic features of papillary thyroid cancer,including proliferation of tumor,migration of tumor and tumor colony.(2)Activation of N-cadherin and vimentin related pathways and suppression of E-cadherin related pathways was associated with tumorpromoting mechanism of the long noncoding RNA LINC00538 on papillary thyroid cancer.Target inhibition of the long noncoding RNA LINC00538 could attenuate multiple clinicopathologic features of papillary thyroid cancer,including proliferation of tumor,migration of tumor and tumor colony.(3)Target inhibition of the long noncoding RNA LINC00538 could decrease expression s of N-cadherin and vimentin,and increase expression of E-cadherin.Target inhibition of the long noncoding RNA LINC00538 could maintain promising tumor inhibition effect on papillary thyroid cancer in vivo. |
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