| Background and ObjectivePapillary thyroid carcinoma(papillary thyroid carcinoma,PTC)is the most common type of thyroid cancer.In recent years,the incidence of PTC in China has increased significantly,which is a serious threat to human health.Due to the strong invasive ability of PTC and no obvious symptoms in the early stage,patients often have lymph node metastasis or invade surrounding organs and tissues.When patients have lymph node metastasis or invade surrounding organs and tissues their prognosis will deteriorate seriously.In order to improve the survival rate and quality of life of patients with PTC,to explore effective therapeutic targets of PTC and to find a more safe and effective treatment has been the focus of research.MicroRNAs(small molecule RNA,miRNAs)is non-coding single-stranded RNA with a full length of about 21 to 25 nucleotides.MiRNAs can target to recognize and degrade specific mRNA,and participate in a variety of cell biological activities.A large number of studies have found that the abnormal expression of miRNAs may be the inducement of tumorigenesis and metastasis.In recent years,people have also found a variety of abnormal expression of miRNAs in PTC tissues,among which miR-146b-5p,miR-221 and miR-222 have the most obvious changes.Further detection showed that the expression level of miR-146b-5p in PTC tissues was higher than that in normal tissues,and the expression level increased with the increase of tumor stage.Therefore,it is considered that miR-146b-5p may be closely related to the occurrence and development of PTC.Ubiquitin protein-specific protease family(ubiquitin specific protease,USPs)is an important member of the deproteasome superfamily.It contains many subtypes,such as USP22,USP33,USP10,USP2a and so on,and exists widely in cells.USP can selectively degrade or stabilize oncogenes,tumor suppressor genes and various proteins,which plays an important role in the occurrence and development of tumors.According to recent reports,the expression of USP33 is decreased in PTC tissues and it can inhibit the development and metastasis of PTC.Based on previous studies,we assume that miR-146b-5p promotes the proliferation and migration of PTC by regulating USP33.The specific mechanism of the role of miR-146b-5p and USP33 in the occurrence and development of PTC is not clear.Therefore,based on the model of gene overexpression and knockout,we made a preliminary study on the relationship between miR-146b-5p and USP33 in the development of PTC and the influence of miR-146b-5p and USP33 on the development o f PTC.Methods1.The thyroid papillary carcinoma tissues and paracancerous normal tissues of 65 patients who were treated in the Department of thyroid surgery of the first affiliated Hospital of Zhengzhou University from April 2018 to December 2018 were collected and preserved in liquid nitrogen.2.RT-PCR and Western blot were used to detect the expression of microRNA-146b-5p and USP33 in paracancerous normal tissues and PTC tissues,then analyze the correlation between microRNA-146b-5p and USP33 in PTC tissues.3.To construct miR-146b-5p overexpression vector and targeted silencing miR-146b-5p vector,and to establish TPC-1 cell line with stable overexpression and silencing of miR-146b-5p.4.RT-PCR and Western blot were used to detect the expression of miR-146b-5p,USP33 and USP33 mRNA in TPC-1 cells which were stably overexpressed or silenced by miR-146b-5p.5.The cellular biological behavior of TPC-1 cells in each group was detected by CCK-8 and transwell test.6.The effect of overexpression and silencing of miR-146b-5p on the expression of USP33 in vivo was evaluated by tumor formation in nude mice and detection of the expression of USP33 in tumor tissue.7.The data were analyzed by SPSS 25.0 and Prism 8 statistical software,and the statistical methods such as t test,Pearson correlation analysis and Kruskal-Wallis H test were used,the difference was statistically significant when P<0.05.Results1.The expression level of miR-146b-5p in PTC tissues was higher than that in adjacent normal tissues(P<0.05).The expression level of USP33 in PTC tissues was lower than that in normal tissues adjacent to cancer(P<0.05).There is a negative correlation between the expression of miR-146b-5p and USP33 in thyroid papillary carcinoma.2.MiR-146b-5p overexpression vector and targeted silencing miR-146b-5p vector were constructed and transfected successfully,and TPC-1 cell lines with stable overexpression and silencing of miR-146b-5p were established.3.The growth rate and migration ability of TPC-1 cells stably overexpressing miR-146b-5p were significantly higher than those of negative control group(P<0.05).The growth rate and migration ability of TPC-1 cells silenced with miR-146b-5p were significantly lower than those of negative control group(P<0.05).4.In TPC-1 cells with stable overexpression of miR-146b-5p,the expression level of miR-146b-5p was significantly higher than that of negative control TPC-1 cells(P<0.05),while the expression level of USP33 and USP33 mRNA was significantly lower than that of negative control group(P<0.05).The expression level of miR-146b-5p in TPC-1 cells silenced with miR-146b-5p was significantly lower than that in TPC-1 cells with negative control(P<0.05).The expression level of USP33 and USP33 mRNA was significantly higher than that in the negative control group(P<0.05).5.In the tumor tissues of nude mice stably overexpressing miR-146b-5p,the expression level of USP33 was significantly decreased(P<0.05),while in the tumor tissues of nude mice silenced with miR-146b-5p,the expression level of USP33 was significantly increased(P<0.05).Conclusion1.The proliferation and migration ability of TPC-1 cells overexpressing miR-146b-5p was enhanced,while that of TPC-1 cells silenced with miR-146b-5p was weakened.MiR-146b-5p can promote the proliferation and migration of TPC-1 cells and promote the occurrence and development of PTC.2.In PTC tissues,the overexpression of miR-146b-5p suppresses the expression of USP33,the silencing of miR-146b-5p promotes the expression of USP33,and miR-146b-5p has a certain regulatory effect on USP33.The research results connect the role of miR-146b-5p in the development of PTC,which is beneficial to continue to explore the targets and key molecules that inhibit PTC metastasis,to predict the dynamic process and laws of PTC metastasis,and then to find the targets and key molecules of intervention. |
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