| BackgroundMicroRNAs which were found in eukaryotes is a kind of endogenous RNAs,about 21-23 nucleotides.It was caused by regulating the transcription of target mRNA degradation or translational inhibition. It plays an important regulator role in the plant and animal body. So far, in different species we have been found hundreds of unique and mature microRNAs,and more and more new microRNAs were found, and its expression levels are significant differences in different species and tissues.Studies have shown that abnormal expression of microRNAs are closely related with many tumors, including papillary thyroid carcinoma.Abnormal expression of microRNAs play an important role in oncogenes and tumor suppressor genes,and relate with tumorigenesis and poor prognosis. It is highly conserved in evolution, which is a set of encoded protein that does not have a short sequence of RNA open reading frame (ORF). The studies have shown that, although its accounts for only 2% of human genes, regulate gene expression in more than 30% of the human body. MicroRNAs have can identify the specific target mRNA 3’-untranslated region (3’-UTR) and binds, causing the target mRNA degradation or translational repression, and thus the transcription of target gene expression and regulation, and thus regulating many important cellular activities.MicroRNAs play an important role in the tumorigenesis and development process, including cell differentiation, apoptosis, proliferation; invasion, invasion and metastasis of tumor cells.MicroRNA-21 is the earlier one of the small RNA which is also found with different types of tumor-related microRNAs up a brittle region in 17q23.2 chromosome FRA17B, having independent transcription units. Now, studies have shown that the micro RNA-21 in a variety of tumors express as significantly high expression, such as: the malignant glioma, breast cancer, bladder cancer, lung cancer, gastrointestinal and head and neck solid tumors. However, the expression level of microRNA-21 in individual tumors express as low expression, such as in cortisol secretion of pituitary tumor microRNA-21’s expression compared with normal pituitary tissue is 2.4 times lower. The large number of studies based on the above results a hypothesis, high expression of microRNAs through the inhibition of tumor suppressor genes play the role of oncogenes, the low expression of microRNAs. play the role of tumor suppressor genes.They participate post-transcriptional gene regulation.ObjectiveBy constructing microRNA-21 eukaryotic expression vector pEZX-eGFP-microRNA-21 which were transiently transfected with liposome lipofectamine 2000TM recombinant plasmid pEZX-eGFP was-microRNA-21, it is highly expressed in papillary thyroid cancer cells TPC-lfor further study of micro RNA-21 in papillary thyroid cancer cells TPC-1 proliferation, migration, apoptosis of gene regulation mechanisms lay the foundation.Methods(1) After double digestion of colony PCR identification of microRNA-21 eukaryotic expression vector pEZX-eGFP-microRNA-21 were sequenced. (2) Identification of completely correct recombinant plasmid pEZX-eGFP-microRNA-21 by lipid body lipofectamine 2000TM transient transfection of thyroid papillary carcinoma TPC-1 transfection by quantitative real-time PCR identification of recombinant plasmid pEZX-eGFP-microRNA-21, microRNA-21 expression changes in the TPC-1 papillary thyroid cancer cells.(3)By immunohistochemistry and Western blot detection of biological changes in cells transfected with the recombinant plasmid.Results(1) Successfully constructed the eukaryotic expression vector of microRNA-21 identified by sequencing:DNA fragments containing the microRNA-21 were successfully inserted into the plasmid pEZX. (2) The obtained recombinant plasmids were transiently transfected cell lines, compared by real-time quantitative PCR identification of microRNA-21 expression levels and normal thyroid papillary cancer cells TPC-1 was significantly increased (P<0.01). (3) Transfected with the recombinant plasmid, the biological characteristics of papillary thyroid cancer cells a series of changes, immunohistochemistry showed that the cell membrane of the experimental group were brown; Western blot showed that knockdown of PDCD4 expression was decreased (P<0.05)Conclusion(1) Transient transfection pEZX-eGFP-microRNA-21 which were sequenced in papillary thyroid carcinoma TPC-1, microRNA-21 in papillary thyroid cancer cells TPC-1 showed high expression, but PDCD4 expression was significantly lower,prompt in papillary thyroid cancer cells TPC-1, microRNA-21 regulation of PDCD4 protein expression of tumor suppressor genes, tumor suppressor gene PDCD4 protein is an important target genes of microRNA-21.(2) By immunohistochemistry and Western blot experiments, that the biological characteristics of papillary thyroid cancer cells TPC-1, the experimental group a series of changes, suggesting the important role of microRNA-21 in papillary thyroid carcinoma development actor. |
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