Functional And Mechanistic Studies On T Brachyury Transcription Factor And MicroRNA Let7a-2 Negative Feedback Loop In Lung Cancer With Bone Metastasis

BackgroundPrimary lung cancer is the leading cause of morbidity and mortality in China among the malignant tumor. Bone metastasis is one of the major target of hematogenous metastasis. About 30%~40% patients with advanced lung cancer will be suffered bone metastasis. The median survival time in patients with bone metastasis only left 6 to 10 months, and their five-year survival rate is less than 5%.MicroRNA(MiRNAs) are a kind of endogenous about 22 nt RNAs which play important roles in regulating cell differentiation, development, proliferation, apoptosis, et al. The functions of miRNAs are to suppress target gene expression by binding to the specifical sequences of the 3’-untranslated region(3’UTR) of target mRNA. MiRNAs repressing target gene expression via mRNA cleavage and translational repression. MiRNAs can also been controlled at transcriptional or posttranscriptional level. Let7a-2 is a member of let7 family, which are highly conserved in variety species in sequence and functions. Let7 s exist in almost all the normal tissues, while present a low expression in tumor tissues, especially in lung cancer tissue. However, it’s still unclear why the expression of miRNA let7 s are decreased in cancer tissues.T Brachyury transcription factor was firstly found in embryonic notochord. Recently years, researchers determined that Brachyury was abnormally high expressed in tumor tissues, such as lung cancer, breast cancer, colon cancer, prostate cancer, oral squamous cell carcinorma and so on. Brachyury was recognized to be associated with proliferation,differentiation, apotosis and metastasis of cancer cells.ObjectiveResearch the regulation relationships and molecular mechanism between T Brachyury transcription factor and microRNA let7a-2, and find the effect of cell biology behavior in lung cancer, with the aim of laying the foundation of novel antineoplastic therapy in cancer sufferers with Brachyury-positive.Methods1. Test the different expression levels of miRNAs by microarray in brachyury overexpression cell. 2. Use bioinformatics analysis method to find the relationship between Brachyury and let7a-2 in Lung Adenocarcinoma, Lung Squamous Cell Carcinoma, Breast invasive carcinoma, HeadNeck Squamous Cell Carcinoma, Kidney Renal Clear Cell Carcinoma, Urothelial bladder cancer, Ovarian Cancer and Uterine Corpus Endometrioid Carcinoma from Online public database. 3. Choose different cancer cell lines to establish transient Brachyury overexpression or knock-down, and test the expression level of let7a-2 by Real-time PCR. 4. Choose different cancer cell lines to establish transient let7a-2 overexpression or knock-down, and test the expression level of Brachyury by Western Blot. 5. Use Immunohistochemistry and miRNA in situ hybridization histochemistry technology to display the locations of Brachyury and let7a-2 respectively in bone metastasis tissues of lung cancer, and find the relationship. 6. Use luciferase reporter system(pGL4.10) and genetic mutation technology to definite the regulation function and the concrete binding site of let7a-2 by Brachyury. 7. Use luciferase reporter system(pMIR) and genetic mutation technology to definite the regulation function and the concrete binding site of Brachyury by let7a-2. 8. Knock down Brachyury and then inhibit let7a-2 in H460, and overexpress Brachyury and overexpress let7a-2 in H1299, check the expression level of let7a-2 downstream gene. 9. Knock down Brachyury and then inhibit let7a-2 in H460, and overexpress Brachyury and overexpress let7a-2 in H1299, check the expression level of EMT related gene. 10. Knock down Brachyury and then inhibit let7a-2 in H460, check the proliferation, migration and invasion abilities.Result1. Expression level of let7a-2 is down-regulated in Brachyury overexpression cell line H1299. 2. Bioinformatics analysis study show that the relationship of Brachyury and let7a-2 is negatively correlated in Lung Adenocarcinoma and Lung Squamous Cell Carcinoma tissues; the relationship is positive in Breast invasive carcinoma tissues; while there are no correlation in HeadNeck Squamous Cell Carcinoma, Kidney Renal Clear Cell Carcinoma, Urothelial bladder cancer, Ovarian Cancer and Uterine Corpus Endometrioid Carcinoma tissues. 3. Brachyury knock-down were performed in Brachyury-positive cell lines H460, SK-MEL-28 and 7402, the let7a-2 level was increased; Brachyury overexpression were performed in Brachyury-negative cell lines H1299, PC-3 and SW480, the let7a-2 level was decreased. 4. let7a-2 overexpression were performed in Brachyury-positive cell lines H460, SK-MEL-28 and 7402, the Brachyury level was decreased; let7a-2 overexpression were performed in Brachyury-negative cell lines H1299, PC-3 and SW480, the Brachyury level was increased. 5. Negative correlation was found in bone metastasis tissues of lung cancer. 6. Brachyury could directly bind to the promotor region of let7a-2, repressing the expression of let7a-2. 7. let7a-2 could directly bind to the 3’UTR region of Brachyury, repressing the expression of Brachyury. 8. Expression of Cyclin D2 and NRAS were consist with brachyury in H460 and H1299. 9. Expression of E-cadherin were consist with brachyury in H460 and H1299. 10. Brachyury—let7a-2 feedback loop could promote lung cancer cells proliferation, migration and invasion capability.Conclusion1. Negative correlation between T Brachyury Transcription Factor and MicroRNA let7a-2 was existed in variety tumor cells, especially in lung cancer. 2. Negative regulation of each other was existed between T Brachyury Transcription Factor and MicroRNA let7a-2. 3. Brachyury—let7a-2 feedback loop could positively regulate Cyclin D2 and NRAS and negatively regulate E-cadherin. 4. Brachyury—let7a-2 feedback loop could promote lung cancer cells proliferation, migration and invasion capability.