| With the early prevention, early diagnosis and development of targeted treatment, although the incidence of breast cancer has decreased and the survival rate increased gradually, but the breast cancer is still a major worldwide health problem, is one of the most common malignancies in women. There is still many issues about mechanism, recurrence and resistance to treatment remains to be further elucidated.RNA interference(RNAi) is a sequence-specific post-transcriptional, genesilencing mechanism that is induced by double-stranded RNA. RNAi has become a valuable research tool for specific genes of interest and large-scale gene screening, but it also used as a potential tool in biotherapy and medicine. Small interfering RNAs(si RNAs) is a type of centre to RNA interference,20-25 base pairs in length, roling a notable tool for gene silencing pathway. Lentiviral-mediated si RNA is able to infect non-dividing cells,and long-term and efficiently silence the target gene by delivering a significant amount of RNA into the host cell.Interferon-induced transmembrane proteins(IFITM) are a family of transmembrane proteins that contain two alpha helical domains. To date, there are five IFITMs(IFITM1, IFITM2, IFITM3, IFITM5, and IFITM10) that are all clustered on chromosome 11(11p15.5) with less than 2-kb next to each fragment. All five genes encode 125-133 amino acids and have two transmembrane domains. IFITM1,-2 and-3 attributed to the antiviral proteins but little is known of the roles of IFITM5 and IFITM10 in humans. They can inhibit invasion of pathogenic viruses, such as HIV virus, influenza virus, vesicular stomatitis virus, West Nile virus and dengue fever virus. At the same time, certain species of IFITMs protein may be involved in multiple cellular processes, including the regulation of immune cells, somitogenesis, germ cells homing and mature, cell proliferation and cardiomyocytes development. However, recent studies have found that it plays an important role in human cancer development, and IFITM levels can be upregulated or downregulated depending on the cancer types. IFITMs are significantly upregulated in colorectal cancer, regulating migration and invasivenessof tumor cells, whilegastric cancer cellsalso has been associated with high levels of IFITM1 protein expression. In the screening of gene expression in human melanoma cell lines, IFITM expression was decreased. Wang et al identified that the expression of IFITM2 protein was significantly increased in HER2-overexpressing mouse mammary tumors, and Abba et al recently reported that expression of IFITM3 m RNA was significantly upregulated in invasive breast cancer tissues compared to DCIS. However, up to date, only a few studies have reported the role of IFITM3 in breast cancer.In this study, we firstly detected the relation between the expression of IFITM3 protein with in adjacent normal tissue, premalignant and invasive breast tissues, and then investigated the effects of knockdown IFITM3 with lentiviral sh RNA on the regulation of breast cancer cell proliferation,migration and invasion.Method:1. The expression of IFITM3 in breast cancer: a total of 81 patients of breast cancer and the matched normal tissue specimens were obtained for immunohistochemical analysis of IFITM3 expression and its clinical pathological significance;2. The expression of IFITM3 gene in breast cancer cell line was detected and screened by PCR(real-time quantitative PCR) method. The expression of endogenous IFITM3 was inhibited by lentivirus-mediated sh RNA, and the silence efficiency were detected by Real-time PCR and Western Blot in breast cancer cell lines MDA-MB-231 and MCF-7;3. The effect of downregulation IFITM3 on the proliferation of breast cancer cells was detected by MTT assay, Brdu assay, flow cytometry, and plate clone formation assay. Western Blot analysis was used to detect the change of cyclin D1,CDK4 and P21 protein in breast cancer cells;4. The effect of downregulation IFITM3 on the migration and invasion of breast cancer cells were detected by Wound-healing assay and Transwell invasion assay. Western Blot was used to detect the change of MMP-9 protein in breast cancer cells;5. After estrogen dependent breast cancer cell line MCF-7 was treated with 0.1% DMSO,1n M 17β-estradiol, 1μM 4-hydroxytamoxifen and 1μM Fulvestrant for 24 h,the change of IFITM3 m RNA were detected by Real-time PCR analysis.Result:1. 81 cases of breast carcinoma specimens were divided into 32 cases of ductal carcinoma in situ(DCIS) group and 49 cases of invasive ductal carcinoma(IDC) group. In DCIS group,immunohistochemical staining showed that 9(28.12%) cases were strong positive for IFITM3 expression,13(40.63%)cases were negative in tumor tissue, and 3 cases(9.38%) were positive, 21(65.62%)cases were negative in the matched normal tissue. In the 49 IDC group, 29(59.18%) cancer tissues had strong IFITM3 expression, and 5(10.20%) patients had negative expression, and 4 cases(8.16%) were strong positive, and 28 cases(57.14%) were negative in the matched normal tissue. In IDC group,IFITM3 strong expression was significantly correlated with expression of ERα, and was correlated with PR expression, but was not correlated with HER-2 et al expression; while in DCIS group,ERα, PR and HER-2 had no correlation with IFITM3 strong expression. Clinical features of breast cancer, including age, tumor size, lymph node metastasis and pathological grade was compared in DCIS group and IDC group, and IFITM3 strong expression did not show a correlation with these clinical features.2. Real-time PCR method to detect the expression of IFITM3 m RNA in 5 breast cancer cells, the results showed that breast cancer cell line MCF-7, MDA-MB-231 cells have a relatively higher expression of IFITM3 m RNA. The lentivirus-carried IFITM3 sh RNA vector were successful packaged, and the packaged virus infected human breast cancer cells MDA-MB-231 and MCF-7. The expression of IFITM3 in breast cancer cell line MCF-7 and MDA-MB-231 cells was effectively silenced by sh RNA IFITM3;3. MTT assay showed that the cells proliferation in si-IFITM3 group became slower contrast to control group from the second day to the fifth day; Brd U assay showed that the DNA synthesis ability will be suppressed in si-IFITM3 group contrast to the control group. Compared with the control group, the flow cytometry found that IFITM3 knocking down induced cell cycle arrest at G0/G1 phase in MCF-7 and MDA-MB-231 cell in si-IFITM3. Cloning formation assay showed that the colony formation ability decreased in two si-IFITM3 group contrast to control. Knockdown of IFITM3 in breast cancer cells supressed the expression of Cyclin D1 and CDK4,and increased P21 expression;4. At the same time, knockdown of IFITM3 in breast cancer cell line MCF-7 and MDA-MB-231 supressed the cell migration and invasion, and also decreased MMP-9 expression;5. 17β-estradiol and 4-hydroxytamoxifen upregulated the express of IFITM3 m RNA in breast cancer MCF-7 cells contrast to control group; and Fulvestrant treatment of breast cancer MCF-7 cells supressed IFITM3 m RNA expression contrast to control group.Conclusion:1. The expression of IFITM3 in human breast cancer tissues was higher than that in the adjacent tissues. The strong expression of IFITM3 in breast invasive ductal carcinoma was correlated with the expression of ERα and PR.2. In vitro,silencing IFITM3 significantly supressed cell proliferation ability in breast cancer cell line, and reduced the expression of Cyclin D1, CDK4, and upregulated the expression of P21.3. After silencing IFITM3 in vitro, the cell migration and invasion ability of breast cancer cell lines were significantly decreased, and MMP-9 were down regulated.4. Estrogen and estrogen receptor antagonists changed the expression of IFITM3 in ERα positive breast cancer cell MCF-7.These results suggest that IFITM3 plays an important regulatory function in the proliferation and invasion of breast cancer cells. IFITM3 expression is closely related to the occurrence and development of breast cancer. |
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