MiR-146b-5p In Papillary Thyroid Cancer In TPC-1

BackgroundMicro RNAs is a kind of total length of about 21 to 25 nucleotides of non-coding small single-stranded RNA molecules, these Micro RNAs can identify specific target m RNA, regulate the expression of genes in the transcription level, so as to participate in the regulation of cell growth, proliferation, differentiation, apoptosis, and other life activities. Thyroid cancer is the most common malignant tumor of human endocrine system,the global incidence of thyroid cancer is rising by 4% year on year, thyroid cancer has become the top of the head and neck malignant tumors. Reports from different countries in recent years revealed the incidence of thyroid cancer growth is given priority to PTC. Because the early PTC has no obvious symptoms, often resulting in patients had lymph node metastasis or have to invade surrounding tissue and organs. According to reports, the five-year survival rate of PTC patient after invasion and distant metastasis is only 35%. Therefore, exploring the effective therapeutic target, looking for safer and more effective therapeutic approach of PTC is always a difficult problem to improve the PTC survival rate and quality of life to be settled urgently.Preliminary study of applicants, found the Micro RNA microarray highthroughput screening of differentially expressed PTC in different stage and normal tissues of Micro RNA, mi R-146b-5p were significantly high expression in early stage and late stage in PTC organization, at the same time, along with the increase of staging of tumor, the expression level is increasing, is is revealed that mi R-146b-5p plays an important role in the occurrence, development and metastasis of PTC.Mi R-146 b is located in chromosome 10, with a high degree of sequence homology with mi R-146 a. He and Sheu method founded that mi R-146 b is overexpression in PTC tissues through the study of gene chip technology and RT-PCR. Kim, Xing and Chou found that the expression of mi R-146 b in BRAF gene mutation tissue increased significantly, the mutation of BRAF gene is associated with the invasion of PTC, which may be a molecular mechanism of overexpression of mi R-146 b in PTC. But now there has been no relevant reports on mi RNA-146b-5p in PTC cell biology effect and related mechanism, through the study of this subject, evidence is expected to acquired of mi R-146b-5p on biological behaviors of PTC effect, in order to establish mi R-146b-5p as the PTC diagnosis and prognostic monitoring biological indicators and targets for therapy and lay the theoretical foundation.This experiment through the TPC-1 cell papillary thyroid carcinoma line stably transfected with the overexpression of mi R-146b-5p, later use CCK-8, scratch test, with the changes of Transwell cell infection test and flow cytometry technology contrast before and after transfection of TPC-1 cell migration, proliferation and apoptosis and infection capacity. To explore the relationship between overexpression of mi R-146b-5p and the development of thyroid papillary carcinoma and mi R-146b-5p mechanism in papillary thyroid carcinoma. ObjectiveThis experiment was constructed by lentiviral vectors and lentiviral packaging and through slow virus infection test, allows the hsa-mir-146b-5p to obtain stable high expression in the TPC-1 cells lines of papillary thyroid carcinoma, the test for the next further research on the effect of overexpression of mi R-146b-5p on biological characteristics of papillary thyroid carcinoma TPC-1 cells proliferation, apoptosis, migration, infection etc. lay a solid foundation. Methods1.The construction of lentivirus plasmid:(1) Construction of overexpression of mi R-146b-5p lentivirus plasmid(2) The overexpression of mi R-146b-5p of lentivirus plasmid was transfected into TPC-1 cells of thyroid papillary carcinoma, to acquired stable overexpression of mi R-146b-5p cell monoclonal.2. mi R-146b-5p in papillary thyroid cancer in TPC-1(1) To observe the effect of overexpression of mi R-146b-5p on proliferation of TPC-1 cells: Through detection of CCK-8 method,it is show that, overexpressing group, empty vector group and negative control group all also can obviously promote the proliferation of TPC-1 cells. Three groups of cells all increased in number, but express group cell proliferation faster(P<0.05).(2) Analysis the effects of overexpression of mi R-146b-5p on cell cycle and cell apoptosis of TPC-1 cells: through flow cytometry analysis technology, we can see the apoptosis rate of transfected mi R-146b-5p with the control group is significant difference, the apoptosis rate was obviously decreased.(3) Study the effect of overexpression of mi R-146b-5 effect on cell invasion and migration of TPC-1 cells: through scratch test and Transwell cell infection test,it is observed that transfected mi R-146b-5p group, cell migration and colonization ability significantly enhanced. ResultsBy the lentiviral packaging transfected mi R-146b-5p into thyroid papillary carcinoma TPC-1 cells,the number of cells all were increase, but the proliferation of TPC-1 cells of transfection group of were faster.(P<0.05).Transfection of mi R-146b-5p gene was examined by CCK-8 and flow cytometry, all three guoup can obviously promote the thyroid papillary carcinoma TPC-1cells proliferation, but the proliferation of TPC-1 cells transfection group was more obvious.(P<0.05).Flow cytometry to detect cell apoptosis rate: the apoptosis rate between transfected with mi R-146b-5p and the control group and empty vector group of TPC-1 cells have significant difference, the apoptosis rate was obviously decreased.(P<0.05).By scratch test,it is observed that after transfection of mi R-146b-5p, migrating cells number of24 h, 48 h, 72 h were higher than empty vector group and control group, the migration ability of TPC-1 cells of transfected with mi R-146b-5p was increased obviously(P<0.05).Transwell cell infection test infection ability of transfection group, empty vector group and control group, by crystal violet staining, counting the amount of cells, transfection group was significantly higher than that of empty vector group and control group, the infection ability of transfected group of TPC-1 cells increased significantly(P<0.05). ConclusionAfter a lentiviral transfection with mi R-146b-5p, the transfected cells use CCK-8, scratch test, Transwell cell infection test and flow cytometry detection: the proliferation of TPC-1 cells was facilitated, reduced apoptosis, migration and colonization ability significantly enhanced. It is showed that in mi R-146b-5p play an important role in the development of thyroid papillary carcinoma TPC-1 cells, overexpression of mi R-146b-5p can promote the abnormal proliferation of TPC-1 cells, this effect may be achieved through mi R-146b-5p control a suppressor gene of the target protein, inhibit the gene remains to be further analysis and research.