| Breast cancer is a common gynecological malignancy negatively affecting the health of women. With developments in early diagnosis strategies and standardized multidisciplinary treatment technology of breast cancer, the mortality rate of patients has gradually decreased, but the morbidity rate is increasing annually, resulting in breast cancer as the most common tumor in women. Hence, intensively studying the mechanisms underlying breast cancer incidence and development, to provide theoretical guidance for early clinical diagnosis and prognosis efficacy assessment, has been a focus in fundamental and clinical tumor exploration.Human epididymis protein 4(HE4) is a secreted glycoprotein rich in cysteine. Since 1991, when it was discovered by Kirchhoff et al., high expression of HE4 has been observed in malignant tumors such as ovarian, lung and breast cancers. As confirmed by many clinical studies, HE4 has high sensitivity and specificity in tumor serological diagnosis, and can assist clinical diagnosis and prognosis estimation. Currently, the clinical research of HE4 mostly focused on the serological detection of ovarian tumor. Preliminarily study of the mechanisms revealed that HE4 is closely associated with the biological characteristics of ovarian cancer cell such as proliferation, apoptosis and invasion. Recent studies have found a high correlation between HE4 expression level and breast cancer. HE4 expression is associated with lymph node metastases in breast cancer and may be a predictor of breast cancer recurrence. Combined detection of CA153 and HE4 can significantly improve the diagnostic accuracy and sensitivity of breast cancer patients. However, relevant biological functions and the mechanisms of HE4 in breast cancer development remain unclear.The prevention and treatment of breast cancer requires the development of early diagnostic technology and the clarification of molecular mechanisms. As a mature tumor diagnosis marker, HE4 test project has been carried out in a certain range of our country, and the main detection methods are enzyme linked immunoassay and chemiluminescence method. Lack of rapid detection technology is a serious impediment to the application of HE4 in clinical disease screening and diagnosis. Therefore, it is urgent to develop HE4 diagnostic reagent with convenient operation. As a new type of rapid quantitative detection technology, the fluorescence immunity chromatography detection technology has the characteristics of high sensitivity, wide detection range, fast results, low price and other characteristics. Its performance index is much higher than other rapid detection technology, and has gradually become a hot spot in the development of diagnostic reagents.To clarify the biological functions and relevant mechanisms of HE4 in breast cancer development, and to provide effective support for clinical diagnosis, HE4 was studied in the following two ways:(1) HE4 expression was observed in human breast cancer tissues and cell lines, and an HE4 RNAi lentiviral vector was constructed, to clarify the influences and relevant molecular mechanisms of HE4 gene silencing on cell proliferation, apoptosis and migration, and to observe its influence on the growth of transplanted tumors in nude mouse models, thus providing insight into the molecular treatment of breast cancer;(2) The HE4 recombinant protein was obtained through genetic engineering and protein purification technology, and a fluorescence immunochromatographic assay for HE4 was preliminarily established, to provide new insights for the research and development of HE4 diagnostic reagents in China.Part I HE4 expression differences between breast cancer tissues and cells, and establishment of an HE4-silenced breast cancer cell lineObjectiveTo detect expression of HE4 m RNA and protein in breast cancer tissues and cells, and obtain breast cancer cell lines with low HE4 expression through the construction of a lentiviral vector. To lay the foundation for further research on the role and mechanisms of HE4 in breast cancer cells.Methods1. Thirty samples of breast cancer and paracancerous tissues were collected for paraffin block preparation, and expression changes of HE4 protein were determined by immunohistochemical assay(IHC).2. Real-time fluorescence quantitative PCR(Real-time PCR) and Western blot were performed to detect HE4 expression in the normal human breast cell line Hs 578 Bst and breast cancer cell lines MDA-MB-231 and MCF7.3. An HE4 RNAi lentiviral vector was constructed to infect an MDA-MB-231 breast cancer cell line. Real-time PCR and Western blot were performed to detect HE4 gene and protein expressions in infected cells, and breast cancer cell line with low HE4 expression were obtained.Results1. IHC showed HE4 was expressed in both breast cancer and paracancerous tissues, whereas HE4 expression was significantly elevated in breast cancer(P<0.05) compared to that in paracancerous tissues.2. Real-time PCR and Western blot showed that HE4 expression levels were higher in the breast cancer cell lines MDA-MB-231 and MCF7 than that in the normal human breast cell line Hs 578Bst(P<0.05), and the expression in MDA-MB-231 was higher than that in MCF7. Therefore, the MDA-MB-231 cell line was employed in subsequent studies.3. The HE4 RNAi lentiviral vector was successfully constructed and verified by real-time PCR and Western blot to have an about 75% infection rate in MDA-MB-231 cell line.ConclusionsHE4 expression was significantly elevated in both breast cancer tissues and cells, indicating that it could play an important role in breast cancer development. HE4 low-expression breast cancer cell line were also successfully constructed.Part II Influences and related mechanisms of HE4 on breast cancer cellsObjectiveTo explore the influences and related mechanisms of HE4 on proliferation, apoptosis, and migration of breast cancer cells utilizing HE4-silenced MDA-MB-231 breast cancer cells and the nude mouse model.Methods1. An MTT assay was performed to investigate the influence of HE4 gene silencing on cell proliferation ability.2. Cell scratch and Transwell assays were performed to determine the influence of HE4 gene silencing on cell migration ability.3. TUNEL staining and flow cytometry were performed to determine the influence of HE4 gene silencing on cell apoptosis ability.4. Western blot was performed to investigate the influences of HE4 gene silencing on protein expression of ERK, p-ERK, p-c-Jun, c-Fos, p-Akt, Akt, cleaved caspase-3, caspase-3, MMP-2 and MMP-9.5. A nude mouse model with transplanted tumor was established to observe the influence of HE4 gene silencing on the tumorigenic ability of breast cancer cells.6. HE staining was performed to observe the morphological changes of tumorigenic cells, and an IHC assay was performed to observe expression changes of HE4, MMP-2 and MMP-9 proteins.Results1. The MTT assay showed HE4 gene silencing caused significantly reduced cell proliferation(P<0.05) compared with that in the control group.2. The cell scratch assay showed that HE4 gene silencing caused significantly weakened cell healing ability(P<0.05) and the transwell assay showed that the number of migrated cells significantly decreased after HE4 gene silencing(P<0.05), indicating that HE4 gene silencing reduced cell migration ability compared with that in the control group.3. Flow cytometry and TUNEL staining showed that the number of apoptotic cells significantly decreased after HE4 gene silencing(P<0.05) compared with the control group.4. Western blot showed HE4 gene silencing caused reduced expression levels of relevant proteins including p-ERK, p-c-Jun, c-Fos, p-Akt, MMP-9 and MMP-2(P<0.05), and increased expression level of cleaved caspase-3(P<0.05).5. The nude mouse model with transplanted tumor showed that tumors in the HE4 gene-silencing group grew more slowly, and clear differences in tumor size were observable 5 days after cell transplantation(P<0.05), compared with that in the control group.6. IHC showed that the expression of HE4, MMP-2 and MMP-9 were significantly reduced in the HE4 gene-silencing group compared with that in the control group(P<0.05).ConclusionsThe HE4 gene may affect proliferation, apoptosis and migration abilities of breast cancer cells through ERK-Jun-Fos and Akt signaling pathways. HE4 gene silencing had obvious inhibitory effects on tumor growth in the nude mouse model with transplanted tumors.Part Ш Establishment and preliminary assessment of HE4 fluorescence immunochromatographic assayObjectiveTo obtain recombinant HE4 protein through genetic engineering and purification, for preparation of a suitable reference; and to preliminarily establish an HE4 fluorescence immunochromatographic assay.Methods1. A PET32a-HE4 plasmid was constructed and transformed into Escherichia coli BL21(DE3), the induced expression of the recombinant HE4 protein was optimized, and its immunogenicity was tested.2. Nickel ion exchange chromatography was performed to purify recombinant HE4 protein and determine its purity.3. A commercially available kit was used to calibrate the recombinant HE4 protein and prepare a series of references for laboratory use; the stability of the prepared references was also verified.4. The development and preliminary assessment of an HE4 fluorescence immunochromatographic assay were performed.By optimizing the amount of EDC, labeled and coating antibodies, the coupling microsphere dilution ratio and nitrocellulose membrane, and related techniques of an HE4 fluorescence immunochromatographic assay were determined, and the linear range, precision, stability and recovery rate of the assay were assessed. Forty serum samples with a fixed HE4 level were used in a comparative assay to preliminarily assess the detection method.Results1. A prokaryotic expression system of the HE4 protein, BL21-PET32a-HE4, was obtained, and the optimal induced expression conditions of the protein were determined: IPTG concentration: 0.2 mmol/L; temperature: 37°C and duration: 3 h.The soluble expression of the HE4 protein was achieved. SDS-PAGE showed that purity of purified HE4 recombinant protein was over 90%.2. A series of linear references(15 pmol/L、150 pmol/L、300 pmol/L、750 pmol/L、1500 pmol/L), a series of linear range references(5 pmol/L、10 pmol/L、15pmol/L、50 pmol/L、150 pmol/L、300 pmol/L、750 pmol/L、1000 pmol/L、1200 pmol/L、1500 pmol/L、1800 pmol/L、2000 pmol/L), and a precision reference(750 pmol/L) were prepared. The stability test showed that the references maintained good stability after being stored at 4°C for 7 days or at-80°C for 6 months.3. Determination of related techniques of the HE4 fluorescence immunochromatographic assay: nitrocellulose membrane: No.01; amount of EDC: 1 μL fluorescent microspheres with a solid content of 1.05% corresponding to 8 μg EDC; amount of labeled antibody: 1 μL fluorescent microspheres with a solid content of 1.05% corresponding to 5 μg labeled antibody; coating antibody of line T: 1.5 mg/m L; coupling microsphere dilution ratio: 300 times. The linear range: 15-1500 pmol/L; Intra-assay coefficients of variation(CV): 7.9 %; inter-assay CV: 9.8 %; stability: stably stored at 37°C for 7 days; recovery rates of high-, medium- and low-value references were 99.75 %、99.8 %、96 %, respectively.4. The assay and Roche detection reagents were both used to test the 40 HE4 serum samples, and the results showed a good correlation between the two, the regression equation is: Y=0.9286x+26.737,R2=0.9462.ConclusionsA recombinant HE4 protein was successfully obtained through genetic engineering and protein purification technology, and was utilized to calibrate a series of references for laboratory use. An HE4 fluorescence immunochromatographic assay was preliminarily established, which will provide technical support for the subsequent final establishment of an HE4 fluorescence immunochromatographic assay. |
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