| MicroRNAs (miRNAs) are short, non-protein-coding RNAs and transcripts that are 18-24 nt in length. MiRNAs regulate gene expression at the post-transcriptional level by binding to complementary sites in the 3'untranslated regions (3' UTRs) of their target mRNAs. And miRNAs regulated downstream target genes though the post transcriptional. Recently, a large number of studies have reported that miRNAs are dysregulated in carcinomas via a series of biological processes. This influences the development of tumors by affecting processes including cell proliferation, apoptosis, the cell cycle, and invasion and migration. MiRNAs play a vital role in breast cancer because they are involved in tumor progression, and especially in blocking apoptosis and promoting uncontrolled cell disruption. MiR-204 was reported to downregulate endometrial carcinoma, gastric cancer, though binfing with target genes and participating in various cellular pathways to affect the formation of tumor. The level of miR-204 was lower in breast cancer tissues than in adjacent normal breast tissues. And levels of expression of miR-204 were associated with TNM stage and distant metastasis. But the specific regulatory mechanism and biological function is still less.FOXA1 is a member of the FOX family of transcription factors and is also known as hepatocyte nuclear factor 3a (HNF3?). Initially, FOXA1 was identified in the liver and reported to be a transcriptional regulator of transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT). High levels of FOXA1 have been reported in types of tumors, in addition to breast cancer. Whereas FOXA1 is usually associated with hundreds of gene to regulate cell signaling pathway, and further participate in the tumor of the process.MicroRNA database shows that there may be binding sites between miR-204 and FOXA1 3 'UTR region, whether miR-204 can regulate breast cancer by binding FOXA1, the conjecture need further experimental verification.Part 1. the expression of miR-204 and FOXA1 in breast cancer tissues and cellsObjective: To detect the expression of miR-204 and FOXA1 in breast cancer tissues and breast cancer cell lines MCF-7, and to analyze the relationship with clinicopathological features, and to preliminarily determine the relationship between the expression of miR-204 and FOXA1 in breast cancer.Methods: 1105 cases of breast cancer tissues and normal breast tissues were from TCGA database to detect the expression of miR-204 and FOXA1 mRNA.And analyze the relationship between genes and the clinicopathologic features.The correlation analysis of miR-204 and FOXA1 was detected. The expression of miR-204 and FOXA1 was detected by qRT-PCR on breast cancer cell line MCF-7. Meta analysis was used to evaluate the prognosis of miR-204.Results: The expression of miR-204 was significantly lower in BC than that of in corresponding normal breast tissues (P < 0.001), and the level related wuth lymph node metastasis (P = 0.026), distant metastasis (P < 0.001), PR (P = 0.044)and Her2(P = 0.014). Whereas FOXA1 showed a high level in cancerous tissues compared with the corresponding normal breast tissues (P < 0.001), and expression associated with lymph node metastasis (P = 0.022), age (P < 0.001),ER (P < 0.001)and PR (P < 0.001). The expression of miR-204 and FOXA1 was negatively correlated in breast cancer (r = - 0.082, P = 0.014). In BC cell line MCF-7, the expression of miR-204 was significantly lower than normal mammary epithelial cell line HBL-100, whereas FOXA1 in MCF-7 was higher than that in HBL-100. The results of meta analysis showed that low miR-204 expression had a higher risk of death.Conclusion: miR-204 and FOXA1 are involved in the formation of breast cancer,and there may be some relationship between the two. MiR-204 can be used as a protective factor.Part 2. Effect of miR-204 on the biological behavior ofbreast cancer cell line MCF-7Objective: To detect the effect of miR-204 on the biological function of breast cancer cell line MCF-7.Methods: Lentiviral transfection was performed to construct miE-204 stably overexpressing cell lines; the effect on the proliferation of miR-204 was using MTT assay; flow cytometry detected effects of miR-204 on apoptosis and cell cycle; Effects of miR-204 on migration and invasion was tested by transwell.Results: Proliferation was detected by MTT on 24h, 48h, 72h and 96h, 72h began proliferation began to decline (P < 0.01). A inhibitory effect being observed in 72h. The rate of apoptosis in miRNA-204 overexpression group reached (34.7 ± 1.9) %,was significantly higher than that in the negative control group and blank control group (P < 0.001), early apoptosis effect is particularly obvious. Cells block in G2 / M phase. The number of cells that were transfected with the miR-204 overexpression group (75 ± 9) penetrated the membrane at a strikingly lower rate than the Control (254 ± 17) and NC (242 ±22) groups (P < 0.05). Transwell migration assays performed without Matrigel revealed that the number of cells that penetrated the membrane (102 ± 8) was clearly lower in the miR-204 overexpression group than in the Control (298 ± 18)and NC (277 ± 15) groups (P < 0.001).Conclusion: miR-204 could inhibit the proliferation, promote apoptosis and make the cell block in the G2/M phase, and inhibit cell migration and metastasis.Part 3. Relationship between miR-204 and FOXA1 in breast cancer and biological information analysisObjective: To study the molecular mechanism of miR-204 and FOXA1.Methods: Predicted complementary binding sites between miR-204 and FOXA1 in microRNA databases (http://www.microma.org/microma/home.do). Dual luciferase reporter assay was used to detected the combination between miR-204 and FOXA1, mutation test was to observe activity. Western blot determined change of FOXA1 protein level. The neetwork of FOXA1 was analyzed in STRING databases (http://string-db.org/).Results: miR-204 regulation FOXA1 3UTR, inhibit the expression of the fluorescent enzyme (P < 0.001), regulatory disappeared in the combination with the mutation site. WB detected changes in miR-204 and the FOXA1 protein levels had the opposite change. The interaction protein network of FOXA1 mainly has 10 genes, They are AR ,ESR1, NKX2-1, INS, SCGB1A1, SHH,FOXA2, TFF1, NKX3-1 and TTR.Conclusion: mR-204 targeted FOXAlin breast cancer MCF-7 cell line, '' which provides clues and theoretical basis for the further exploration of the mechanism of miR-204. |
PDF Full Text Request