1、Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer,Li Jianfang 2010-10-27,microRNA定义,1993年，Ambros首先在Cell杂志上撰文发现了一段非编码的单链小分子RNA，具有调节发育时相的作用，这是对microRNA的首次报道。近年来， Cell/Nature/Scicence连续把RNA的研究进展列为“十大科技突破之一”，其中最引人注目的是microRNA。,microRNA定义,microRNA：一类18-24个核苷酸的非编码的小R
2、NA分子，由发夹结构单链RNA前体经过Dicer酶加工而成。不同于siRNA（双链）但是和siRNA密切相关。据推测，这些非编码小分子RNA（miRNAs）参与调控基因表达，但其机制区别于siRNA介导的mRNA降解。 miRNAs的表达方式各不相同，具有分化的位相性和时序性 ：在不同组织、不同发育阶段中miRNA的水平有显著差异。,Development of microRNA,miRNA的功能,对一部分miRNAs的研究分析提示：miRNAs参与生命过程中一系列的重要进程，包括早期发育(Reinhart 2000)，细胞增殖，细胞凋亡，细胞死亡(Brennecke 2003），脂肪代谢（X
3、u 2003)和细胞分化(Kawasaki 2003)。此外，一个研究表明，2个miRNAs水平的下降和慢性淋巴细胞白血病之间的显著相关，提示miRNAs和癌症之间可能有潜在的关系(Calin 2002)。 然而，大多数miRNAs的功能仍然是个谜。,miRNA的作用方式,最早被发现的两个miRNAslin-4 and let-7被认为是通过不完全互补结合到目标靶mRNA 3非编码区端，以一种未知方式诱发蛋白质翻译抑制，进而抑制蛋白质合成，阻断mRNA的翻译。多个果蝇miRNAs也被发现和他们的目标靶mRNAs的3非编码区有部分同源。由于miRNAs和其潜在的目标靶之间并非完全互补，这使得通过
4、信息学的方法鉴定miRNA的目标靶位点变得困难。因而也无法确定miRNAs的作用方式是什么，以何种机制影响mRNA的翻译，以何种方式调控基因表达。miRNAs的作用目标靶和活性机制一直是各地的研究人员的关注热点。,研究miRNA的新工具,小分子RNA的分离 ： 现行的RNA纯化方法包括有机溶剂抽提+乙醇沉淀，或者是采用硅胶膜离心柱的方法来纯化RNA。小分子RNA往往被淘汰掉，因而不适用于小分子RNA。miRNA Isolation Kit主要是采用玻璃纤维滤膜离心柱(glass fiber filter，GFF)方法分离。小分子RNA探针的制备 ：要准备目的基因的一小段寡核苷酸序列，3端另外增
5、加8个和T7启动子互补的碱基，将这段寡核苷酸和T7启动子引物退火，用Klenow大片断补齐得到双链的转录模版，然后用T7 RNA聚合酶、rNTP和标记物混合，体外转录得到标记的小分子RNA探针。,研究miRNA的新工具,小分子RNA的检测： 由于小分子RNA是一类很小的分子，部分小分子RNA表达水平可能很低，因而需要极为灵敏而定量的分析工具，由于其分子很小，用RT-PCR的方法来定量研究非常困难。 目前多数采用Northern Blots一种技术复杂而费力的方法。改进的新方法：将同位素标记好的小分子RNA探针和待检测样品混合杂交，未杂交的RNA和多余的探针用单链核酸酶消化，然后使核酸酶失活，并
6、纯化杂交的RNA分子，最后通过变性胶电泳放射自显影检测结果。,Introduction,Papillary thyroid carcinoma (PTC) is the most prevalent endocrine malignancy in humans 12. 70% of PTCs harbor genetic alterations in RET, RAS, or BRAF ，and linear signaling along the RET-RAS-BRAF-ERK pathway is key to their development 13,14. In particular
7、, RET gene rearrangements occur in up to 43% of PTCs 15.,Introduction,In this study： the potential involvement of let-7 miRNA in PTC development is investigated. Because let-7 was demonstrated to inhibit RAS expression, it might provide a tumor suppressor function in thyroid cancers with MAPK activa
8、tion. Our data suggest that let-7 miRNA is an essential regulator of thyroid carcinogenesis.,Materials,PTC3-5 19 and PCCL-BRAF cells obtain doxycycline(DOX) inducible expression of RET/ PTC3 and BRAF V600E oncogenes from PCCL3 rat thyroid cells.PCCL3 Cells of rat thyroid cultivated in the absence of
9、 DOX were used as controls.,Materials,TPC-1, a human PTC cell line spontaneously harboring the RET/ PTC1 rearrangement 20 .TPC-1 cells transfected with pH1-RNApuro-let-7f plasmids using Lipofectamine 2000 (Invitrogen Life Technologies). TPC-1 cells transfected with pH1-RNA puro-control plasmids.,Met
10、hods,PCR ,Western Blot ,MTTStatistical Analysis:The results are presented as the mean SD and submitted to analysis of variance using the Bonferroni t-test. Differences were considered significant at P 0.05.,Results,expression of let-7f miRNA in PCCL3 Rat Thyroid Cells,RT-PCR was used to confirm the
11、efficient induction of RET/PTC3 expression of the PTC3-5 cells with DOX for 72 hours (Figure 1A). The induced expression of the RET/PTC3 oncogene in PTC3-5 cells led to a strong reduction (76%) in let-7f miRNA expression (Figure 1B).,expression of let-7f miRNA in PCCL3 Rat Thyroid Cells,However,( PC
12、CL-BRAF cells ) induction of the BRAF oncogene in PCCL3 cells treated with DOX for 72 hours (Figure 2A) did not modulate let-7f mature miRNA expression (Figure 2B).,Results 1Doxycycline-Inducible Expression of RET/PTC3 in PCCL3 Rat Thyroid Cells,Effects of let-7f on MAPK Signaling Pathway in TPC-1 C
13、ells,The observation that let-7f expression decreases in response to RET/PTC activation led us to explore the possibility that let-7f- reduced expression might contribute to papillary thyroid cancer development. To test this idea, let-7f was introduced into the TPC-1 cells, which spontaneously harbo
14、r the RET/PTC1 rearrangement.,Effects of let-7f on MAPK Signaling Pathway in TPC-1 Cells,Real-time PCR was used to evaluate the expression of let-7f mature miRNA in TPC-1 control (CTR) and TPC-1 let-7 stable clones. let-7f was observed to have a basal expression level in TPC-1 cells, which was subst
15、antially increased after introducing the let-7f plasmid (Figure 3A).,Effects of let-7f on MAPK Signaling Pathway in TPC-1 Cells,ERK Signaling Pathway is classical one of three MAPK Signaling Pathway. the potential influence of let-7f over expression on ERK activation was evaluated. As expected, resu
16、lts of the Western blot analysis demonstrated that let-7f induction resulted in a decrease in phosphorylation of the ERK protein in TPC-1 cells (Figure 3B), even without altering H-RAS and K-RAS protein levels.,Figure 3. Stable transfection of let-7 miRNA in TPC-1 cells (B) Total cellular proteins w
17、ere pre- pared and subjected to 10% SDS-PAGE electrophoresis.Immunoblot analyses were performed using specific antibodies against K-RAS, H-RAS, phosphorylated ERK (p-ERK), ERK1/2, and -tubulin. *P = .017 versus TPC-1 CTR.,Growth Inhibitory Effect of let-7f miRNA on TPC-1 Cells,The MTT assay was used
18、 to examine whether let-7f expression is associated with PTC cell growth. let-7f overexpression markedly inhibited proliferation of TPC-1 cells by 55% (Figure 4).,Growth Inhibitory Effect of let-7f miRNA on TPC-1 Cells,The transcriptional expression of genes in the cell cycle, such as MYC, CCND1 (cy
19、clin D1), and CDKN1A (p21), was examined by quantitative RT-PCR. let-7f inhibited the mRNA expression of cell cycle stimulators such as MYC (17%) and CCND1 (14%). However, this small RNA increased the transcriptional expression of CDKN1A (18%), a gene that acts as a cell cycle inhibitor (Figure 5).,
20、Results 3Growth Inhibitory Effect of let-7f Mature miRNA on TPC-1 Cells,Effects of let-7f on Expression of Thyroid Differentiation Genes,Transcription of specific molecular markers of thyroid differentiation such as thyroid transcription factor 1 (TITF1), thyroglobulin (TG), and sodium iodide sympor
21、ter (NIS), was analyzed. let-7f induces a three-fold increase in TITF1 mRNA expression and also a slightly in- crease in TG transcription when compared with control cells (Figure 5). However, NIS mRNA expression was not detected in TPC-1 cells even with let-7f overexpression (data not shown).,Result
22、s 3Growth Inhibitory Effect of let-7f Mature miRNA on TPC-1 Cells,Discussion,let-7f down-regulation,Despite all the information concerning changes in the expression of miRNA in thyroid cancer, the functions of these molecules in thyroid tumorigenesis are poorly understood. In this study, we have ide
23、ntified a functional association of let-7f miRNA with thyroid cancer for the first time.,let-7f down-regulation,The let-7 family of miRNA includes 14 isomers; each isomer is typically located on a different chromosome 8. This study focused on let-7f, because it has been previously shown to inhibit p
24、roliferation of lung cancer cells 29. We observed a reduced expression of let-7f mature miRNA in PCCL3 cells with activation of RET/ PTC3. This suggests that let-7f down-regulation is an important step in the biologic effects mediated by the RET/ PTC oncogene.,reduce cell growth,a previous study fro
25、m patients with papillary carcinoma and normal thyroid tissues showed that let-7f is downregulated in this cancer 28. To clarify the function of let-7f in the thyroid cancer, exogenous let-7f was overexpressed in TPC-1 papillary cancer cells that harbor the RET/PTC1 rearrangement.,reduce cell growth
26、,Although transfection of let-7f in TPC-1 cells repressed ERK phosphorylation, significant modulation of K-RAS and H-RAS proteins was not observed after the induction of let-7f. Nevertheless, the modulation of genes related to cell cycle may explain, at least in part, the reduced cell growth of TPC-
27、1 cells transfected with let-7f.,enhance expression of differentiation markers,During malignant progression, thyroid cancer cells undergo dedifferentiation, becoming more aggressive and refractory to treatment. We demonstrate that let-7f is able to enhance the expression of some of differentiation m
28、arkers in TPC-1 cells, including TITF1.mice lacking this transcription factor are unable to develop a thyroid gland 33.,enhance expression of differentiation markers,let-7 may also play an important role in thyroid development through the regulation of TITF1 expression. Therefore, it is evident that
29、 let-7f is critical for proper regulation of thyroid cell growth and differentiation. It is known that let-7 has other targets in addition to RAS with respect to cancer, including the HMGA2 oncogene 34.,inhibitory effects on the MAPK pathway,However, there is compelling evidence in the literature sh
30、owing that activation of the MAPK signaling pathway is required for the development of RET/PTC-positive PTCs. Thus, the effects of let-7f in this specific cell (TPC-1) are likely due to its inhibitory effects on the MAPK pathway. Whether let-7f truly interacts with other mRNA in TPC-1 cells is a que
31、stion that remains to be answered.,inhibitory effects on the MAPK pathway,A few studies used miRNA expression profiling to identify four miRNA (miR-146, miR-221, miR-222, and miR-181b) with high levels of expression in cancerous tissues 28,35. In addition, it has been shown that the BRAF mutation an
32、d RET/PTC rearrangements, have global effects on the miRNA expression profile 26,27. our work demonstrates an association between this new class of small RNA and the development of thyroid carcinogenesis.,summary,In summary, we show that reduced expression of let-7f is associated with RET/PTC malignant transformation. Furthermore, restoration of let-7f expression attenuates RET/PTC-mediated oncogenesis by preventing MAPK Signaling Pathway. Our data support a suppressor function of let-7f miRNA in thyroid cancer, revealing this molecule as a potential therapeutic target in patients.,
