| Background: Breast cancer is one of the most lethal cancers in humans. The majority of patients died of local recurrence despite aggressive medical intervention including surgery, radiotherapy, chemotherapy. In recent years, gene therapy of tumors has become the most noticeable research field. It's the fifth model followed surgical therapy, radiotherapy, chemotherapy and immunotherapy. Osteopontin (OPN) is a secreted arginine- glycine- (RGD) -containing phosphoprotein with cell adhesive and aspartate chemotactic properties in vitro and in vivo. It is closely associated with infiltrating macrophage in tumors and it can directly stimulate macrophage migration, which has made it a key target as a molecule likely to be important in mediation tumor metastasis. It has been shown that osteopontin is up-regulated in many kinds of cancer tissues, including hepatocellular carcinoma, breast cancer, prostate cancer, ovarian cancer, brain cancer and lung cancer. Elevated osteopontin (OPN) transcription often correlates with increased metastatic potential of cancers. The aim of study is to research the effect of ANOPN gene on proliferation and metastasis of the breast cancer.Methods:1. Construction of the mammalian expression recombinant pcDNA3.1- ANOPN A OPN gene recombinant expression vector plasmid was constructed by RT-PCR from human umbilical vein endothelial cell gene and cloned into a mammalian expression vector pcDNA3.1(+) at the sites of Xho I and Hindâ…¢,and named pcDNA3.1-ANOPN. The recombinant was cleaved with appropriate endoneuclear and sequenced. The results showed that the inverse direction of the insert was found to be correct, while no rearrangement was found.2. Integratuion and expression of ANOPN gene in breast cancer cell line (MDA-MB-231) and effects to breast cancer cells in virtoThe recombinant pcDNA3.1-ANOPN was introduced MDA-MB-231 by LipofectinTM Reagent. After the cells resistant to G418, we obtained positive cell clones (MDA-ANOPN), and vector-transfected cells MDA-vect and blank cell MDA. OPN gene mRNA RT-PCR assay, immunocytochemistry assay showed that ANOPN gene has been integrated into MDA- ANOPN cells and stably expressed. The characteristics of the ANOPN expressing MDA-ANOPN cells were studied. Compared with the vector-transfected cells (MDA-vect cells) and MDA cells,the MDA-ANOPN cells showed that: (1) the growth rate of MDA-ANOPN cells was diminished and their doubling-time increased; (2) The ability of MDA-ANOPN cell's adhere, invasion and migration diminished; (3) Immunocytochemistry analysis also showed that the products of OPN gene have decreased in MDA-ANOPN cells.4. Effects of ANOPN to breast cancer cells in vivoTo study the effects of OPN gene on the growth of transplantation tumor of nude mice of breast cancer cell line (MDA-MB-231). MDA, MDA-vect and MDA-ANOPN cells were transplanted into nude mice, then the time of tumor formation, growth rates and weight of tumor were observed. The tumor volume was calculated, and growth curve was plotted. Representative histological sections were taken from mice bearing transplantation tumor either treated or control groups, and expression of OPN was detected. In MDA-ANOPN nude mice, the expression of ANOPN gene significantly suppressed the growth rates and elongated the time of tumor formation. ANOPN is able to significantly inhibit the growth rate of transplantation tumor of nude mice of MDA-MB-231 cell line, and dramatically depress microvessel density and the quantity of pulmonary metastasis in vivo. Above of all functions of ANOPN gene may be related to that it suppressed the expression of MDA-MB-231 cell line OPN gene.Conclusion: With these, we conclude that ANOPN gene is able to significantly suppress the malignant phenotye of breast cancer and. The above results showed that ANOPN gene possesses important effects on gene therapy of breast cancer. Our study provided data for gene therapy of tumor and possible of using ANOPN gene to improve tumor treatment.
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