MiR-199a Suppresses The Metastasis Of Mouse Breast Cancer Cell Line 4T-1 By Targeting DDR1

Breast cancer, secondly only to lung cancer, is one of the most prevalent female tumors, which seriously threaten women’s health. The pathogenesis of breast cancer is complex, but the essential cause is known as genetic changes, including the inhibition of the tumor suppressor genes and the activation of oncogenes. All the stages of breast cancer are regulated by the changing in genetic information, like initiation, growth, invasion and metastasis. The leading cause of breast cancer death is distant metastasis, like migrating to brain, lung, liver and bone. Accordingly, it is very important to explore the molecular mechanisms for the therapy of breast cancer. Currently, the mainstay clinical treatments of breast cancer are surgery, chemotherapy, hormonal therapy and molecule-target therapy. Although the survival rate of breast cancer has been improved by chemotherapy, drug resistance has frequently appeared which makes the treatment difficult. Consequently, as a new part of oncotherapy, molecule-target therapy has been paid more and more attention. It’s significant to explore the molecular mechanisms of breast cancer and find the new target of molecule-target therapy.miR-199 family is formed of miR-199a-1, miR-199a-2 and miR-199 b. Two mature microRNA products of miR-199 a, miR-199a-5p and mi R-199a-3p, are expressed from the miR-199 precursors that map to chromosome 19(miR-199a-1) and chromosome 1(miR-199a-2) in humans. The studies indicated that miR-199 a was overexpressed or decreased in different tumors, which suggested that miR-199 a regulated tumor suppressor genes or oncogenes to control the development or progression of tumors.DDR(discoidin domain receptor) family is formed of DDR1 and DDR2. DDR2 has only one isoform, while DDR1 has 5 different isoforms(DDR1a-DDR1e) and DDR1 c is the longest one. DDR1 a, DDR1 b and DDR1 c encode functional receptor tyrosine kinases, while DDR1 d and DDR1 e lack kinase activity. DDR1 mRNA is found in many tissues in human and mice, especially in brain, lung, kidney and spleen. The studies showed that DDR1 could regulate the interaction between tumor cells and collagen to play an important role in the development of many tumors, such as lung cancer, hepatocellular carcinoma, and breast cancer. Expression dysregulation and dysfunction of DDR1 are closely related to the development of tumor and the prognosis of the patients.In this study, we will use the following experiments to explore whether miR-199 a and DDR1 play a crucial role in the metastasis of breast cancer and whether miR-199 a regulates the expression of DDR1 in breast cancer.Part I Overexpression of miR-199 a suppresses the metastasis of mouse breast cancer cell line 4T-1Objectives: To investigate the effect of miR-199 a up-regulation on metastasis of 4T-1 cell lines.Methods: 4T-1-Luc cells were transfected with the constructed recombinant plasmid pcDNA3.1-miR-199 a to establish the cell line which stably expressed miR-199 a. The expression level of miR-199 a in 4T-1-Luc cell was determined by QRT-PCR. TranswellTM assay was used to detect the effect of mi R-199 a on metastasis of 4T-1 cell lines with higher level of miR-199 a in vitro. 4T-1-Luc cells that stably expressed miR-199 a were injected into BALB/c mice via tail vain injection and lung colonization was quantified by bioluminescence imaging(BLI) per week after inoculation to detect the effect of miR-199 a on metastasis of 4T-1 cell lines in vivo. Furthermore, we used H&E(hematoxylin and eosin) staining and immunohistochemistry of mouse lung tissue to proof the previous results.Results: Recombinant plasmid pcDNA3.1-mi R-199 a was successfully constructed; over-expression of miR-199 a suppressed magration capacity of 4T-1 cell lines in vitro; over-expression of miR-199 a suppressed lung metastasis of 4T-1 cell lines in vivo.Conclusions: miR-199 a could inhibit lung metastasis of 4T-1 breast cancer cell lines.Part II DDR1 is a bona fide target gene of miR-199aObjectives: To confirm the relationship between miR-199 a and DDR1.Methods: Using the methods of bioinformatics, we predicted that 3’untranslated region of DDR1 gene had the miR-199 a binding sites in human and mouse. The human and mouse DDR1 3’UTR wild type or mutants that each lacks one miR-199 a binding site were cloned by PCR. Dual luciferase reporter gene assay was used to verify that DDR1 was directly regulated by miR-199 a and confirm its targeted binding sites.Results: miR-199 a down-regulated the expression of heterozygous wild DDR1 and the miR-199 a binding sites were confirmed.Conclusions: DDR1 was a target gene of miR-199 a.Part III The role of DDR1 on metastasis of breast cancerObjectives: To examine the effect of silencing DDR1 on lung metastasis of breast cancer.Methods: Defective interfering particle of DDR1(shDDR1) was successfully constructed, which was transfected to 293 T cells for packaging lentivirus particles. 4T-1-Luc cells were infected with the lentivirus particles(sh DDR1) to establish the cell line that stably expressed shDDR1. QRT-PCR and Western blot assays were performed to detect the expression of DDR1. TranswellTM assay was used to detect the effect of DDR1 on metastasis of 4T-1 cell lines with lower level of DDR1 in vitro. 4T-1-Luc cells that stably expressed shDDR1 were injected into BALB/c mice via tail vain injection and lung colonization was quantified by bioluminescence imaging(BLI) per week after inoculation to detect the effect of DDR1 on metastasis of 4T-1 cell lines in vivo. Furthermore, we used H&E staining and immunohistochemistry of mouse lung tissue to proof the previous results.Results: Defective interfering particle of DDR1(shDDR1) was successfully constructed. Silencing the expression of DDR1 suppressed magration of 4T-1 cell lines in vitro. Silencing the expression of DDR1 suppressed lung metastasis of 4T-1 cell lines in vivo.Conclusions: Silencing the expression of DDR1 could suppress lung metastasis of 4T-1 breast cancer cell lines.Part IV Clinical significance of miR-199Objectives: To explore the clinical significance of miR-199 in breast cancer.Methods: The TCGA database was used to study the relationship between mi R-199 and the prognosis of patients with breast cancer by statistical analysis.Results: The patients with higher expression of miR-199 will survive longer.Conclusions: miR-199 could increase the survival rate of patients with breast cancer.