| Breast cancer is the most common malignant tumor of women all over the world, which is a serious threat to women’s health. With the changes in living envirbnment, lifestyle and diet, the incidence of breast cancer around the world increases significantly and exhibits a youthful trend. Morbidity and mortality of female breast cancer in China rising steadily as their number one killer has become a threat to women’s lives and health. In order to facilitate the classification, diagnosis and treatment of breast cancer and assessment and prediction of patient’s clinical outcomes, it used to classify the disease according to morphology of breast cancer for easy distinction traditionally. As the depth and maturity of modern techniques for breast cancer research, types of breast cancer classification become much more than before, with classification standards being more specifications, which help scientific research, diagnosis, and treatment of breast cancer a lot. Breast cancer has of with a high degree of heterogeneity features, and it can be divided into different subtypes according to different biomarkers. At present, the most studied classification of breast cancer is the method based on the gene chip technology, which is also called molecular typing method. The molecular typing can divide breast cancer into5subtypes, that is luminal subtype A, Luminal subtype B, HER2over-expression subtype, basal-like subtype and normal breast-like subtype. Besides the molecular typing, there is also another typing method which can classify breast cancer according to the expression of ER, PR and Her-2.Triple-negative breast cancer, which can be called TNBC for short, is a special subtype of breast cancer that is characterized by failing to express ER, PR and Her-2. Because of the high degree of heterogeneity of breast cancer, biological behaviors and clinical manifestations of TNBC are quite different from other types of breast cancer. TNBC account for9%-16%of all types of breast cancer, which is easy found in young female patients and brac1gene mutation carriers. Compared with other subtypes of breast cancer, TNBC is more easily to early recurrence and distant metastasis with shorter disease-free survival and overall survival rate. The prognosis of TNBC is poorer than other types. TNBC and basal-like subtype are two different kind of breast cancer, coinciding with each other to a large extent. According to a recent study,86.1%percent of TNBC belongs to basal-like subtype and67.7%of basal-like subtype breast cancer conforms to TNBC. Therefore, TNBC has some behaviors of basal-like subtype breast cancer, such as highly invasive, higher Nuclear splitting phase, more mitotic count. At present, treatments of TNBC include surgery, chemotherapy and radiotherapy; unfortunately none is effective. As the only systemic therapy for TNBC, chemotherapy is the cornerstone of TNBC treatment. In addition, triple-negative feature makes TNBC insensitive to hormone therapy. And no targeted therapy is specified to TNBC. Targeted therapies for other molecules (such as EGFR) are in the process of preclinical studies or clinical trials, making widely clinical application in the short term impossible. Therefore, longking for effective treatment to reduce early recurrence and distant metastasis of TNBC and enhance chemosensitivity is particularly important.beclin1gene is the first autophagy-related gene found in mammalian cells by scholars Liang et al in1998, located on chromosome17q21and structurally similar to the yeast Atg6protein. Autophagy is a physiological process, in which cellular contents and organelles are enclosed by double-membrane structure, which is called autophagic vesicles, and delivered to the lysosomes for degradation and recycle. The most important function of Beclin1is to interact with Vps34proteins to form the Ⅲ-PI3K complex. Through interaction, Beclin1can function at the start of autophagy process. In addition, Beclin1contains the BH3domain in its structure, by which Beclin1can interact with Bcl-2protein family members Bcl-2and Bcl-XL, the so-called anti-apoptotic protein family, thus involved in the apoptosis process. Remarkably, conjunction between Beclin1and Bcl-2or Bcl-XL protein may not affect the apoptosis process but the autophagy process.Currently, beclin1is considered a tumor suppressor gene, loss of heterozygosity of which may lead cells to malignant transformation. In a study conducted by Yue et al, beclin1(-/-) mice often died at early stage of embryo and beclin1(+/-) mice can survive with a significant increase of spontaneous tumors incidence. Liang et al found that MCF-7cells with stable expression of Beclin1increased autophagic effect and reduced cells proliferation in vitro. These cells also have reduced tumor-forming ability in vivo. In addition to the role in autophagy process, Beclin1can also inhibit cell proliferation, induce apoptosis and cell cycle arrest, prevent inflammation and decrease mutations of genome. And Beclin1is closely related to some tumor-associated signaling pathways, such as mTOR signaling pathway, Rb signaling pathway and PTEN signaling pathway. Studies have found that expression of Beclin1is lower in various maligant tumor tissues than normal tissue. Currently, the role and status of Beclin1in malignant tumors remains controversial. Therefore, researchers generally categorize beclin1as a candidate tumor suppressor gene, and related researches require further to be carried on.Epithelial-mesenchymal transformation (EMT) refers to a specific physiological or pathological process, during which epithelial phenotype cells lose polarity and transform into mesenchymal phenotype. EMT process can reduce adhesion capacity of cells and enhance migration and invasion ability of cells. EMT can also make cells resistance to apoptosis. Judging from the cellular level, change of cells showed cells phenotype from epithelial to mesenchymal during EMT process. But at the molecular level, EMT process contains extremely complex molecular transformation. Generally, conversion of EMT markers include reduced expression of epithelial markers, such as E-cadherin, and increased expression of mesenchymal markers, such as N-cadherin, Vimentin, Snail, etc. Apart from its physiological role, EMT can function in several pathological processes such as cancer. EMT can enhance invasion and migration capabilities of tumor cells by transform the phenotypes of cells to another, thus leading to increased rate of tumor recurrence and distant metastasis.Distant metastasis is one of the most important biological characteristics of malignant tumor. Clinically, most cancer patients died of metastatic tumors, not of primary tumors. The number of cells that undergo EMT process in tumor tissue is related with tumor invasion, metastasis and recurrence. Breast cancer also goes though EMT phenomenon frequently. More and more studies have confirmed that EMT plays an important role in the invasion and metastasis process of TNBC. Studies have found that about70%of primary breast tumors have a decreased or even absent expression of E-cadherin. Aditya Bardia et al found that breast cancer circulating tumor cells mainly rendered as a mesenchymal phenotype. EMT is positive related with higher histological grade of TNBC. Significantly reduced expression of E-cadherin and increased expression of N-cadherin, Vimentin and Snail demonstrated that EMT phenomenon is relatively common in TNBC.EMT and autophagy are two very common and important processes, both of which play an important role in both physiological and pathological cases. Because EMT and autophagy can also play important role in development and progression of tumor, more and more attention has been put into the study of relationship and interactions between them. When Lv Q et al study the effect of DEDD on breast cancer, they found that DEDD can reverse the EMT process and activation of autophagy may participate in this process. Further research found that DEDD can interact with the PI3K III/Beclin1autophagic complex to maintain its stability, in tum to activate autophagy-lysosomal degradation system and increase the degradation of Snail that is a kind of EMT-related transcription factor. By increasing the degradation of Snail, DEDD can inhibit and reverse the process of EMT. Peng Y et al have found that inhibition of autophagy could reduce resistance to anoikis and significantly inhibit pulmonary colonization of hepatocellular carcinoma. Further research has shown that inhibition of autophagy cannot influence invasion, metastasis and EMT process of hepatocellular carcinoma. Thus, autophagy is likely involved in tumor metastasis through enhancing resistance to anoikis of tumor cells and promoting the colonization process. Li j et al have found that autophagy activated by starvation can induce EMT process of hepatocellular carcinoma to regulate and control cell invasiveness, in which process TGF-beta/Smad3signaling pathway may has a very important role. Akalay et al research and found that EMT can make cancer cell resistant to cytotoxic T lymphocyte-induced lysis, and autophagy may also participate in this process; inhibition of autophagy can restore sensitivity of cancer cells to cytotoxic T lymphocytes induced Lysis. Therefore, the relationship between EMT and autophagy and their effect on tumor invasion and metastasis are still disputed, and so are in different kinds of tumors, which suggests the need for further study on them.In addition their role in tumor invasion and metastasis, EMT and autophagy can also affect other aspect of tumor, such as drug resistance. Mani et al found for the first time that EMT can have effect not only on breast cancer cell phenotypic changes but also on the proportion change of breast cancer stem cells. And this phenomenon has been verified by multiple studies and researches. It is generally recognized that cells of basal-like subtype breast cancer that have undergone EMT process and express markers of tumor stem cells are easily to metastasize distantly. EMT can contribute to increase in proportion of tumor stem cells in TNBC, which is critical to drug resistance, relapse and metastasis of breast cancer. In addition, autophagy may participate in maintaince of breast cancer stem cells. Therefore, EMT and autophagy may be involved in phenotype formation process of cancer stem cells and drug resistance in breast cancer.A large number of studies have confirmed that Beclin1plays an important role in drug resistance and regulation of chemosensivity of breast cancer. During the process in which Beclin1regulates and controls chemosensivity of breast cancer, autophagy-dependent and autophagy-dependent mechanisms both are important. Beclin1can significantly improve the sensitivity of tumor cells,such as thyroid carcinoma, ovarian cancer, cervical cancer and gastric carcinoma, et al, to chemotherapeutic drugs by enhancing the apoptosis or of autophagic cell death induced by chemotherapeutic agents to kill tumor cells. But there were also findings are inconsistent with this. In glioblastoma, knockout of beclin1can improve the sensitivity of tumor cells to TRAIL-induced apoptosis; in hepatocellular carcinomas, Sorafenib can downregulate the expression of Mcll, leading to break-down of the interactions between Beclin1and Mcll. Elevated levels of Beclin1in tumor cells lead to increased activation of autophagy, thus increasing the resistance of tumor cells to Sorafenib. Therefore, effect of Beclin1on chemosensitivity is still inconclusive, suggesting that chemotherapy drugs, tumor type, and stimulus intensity and many other respects can affect the function of Beclin1.The aim of our study is to study effect of beclin1gene on EMT process and chemosensivity of TNBC cells by overexpressing Beclin1in the TNBC BT-549cell line, further exploring the possibility to reduce recurrence and metastasis of TNBC as well as to enhance chemosensitivity. Our study is divided into two parts:Part I aims to construct a TNBC BT-549cell line with stable expression of Beclin1, then to observe the influence of Beclin1on EMT process of BT-549cells and on invasion and migration capability of BT-549cells; Part II aims to observe the effect of Beclin1on sensitivity to chemotherapeutic agents paclitaxel and5-FU of BT-549cells and further to explore related mechanisms.Part I Effect of beclin1gene on EMT process of BT-549cellsObjective The aim of this part is to construct a TNBC BT-549cell line with stable expression of Beclin1, then to observe the influence of Beclin1on EMT process of BT-549cells and on invasion and migration capability of BT-549cells.Method The pLenO-GTP-BECN1eukaryotic expression vector was transfected into BT-549cells through lentiviral transfection method to construct a cell line with stable expression of Beclin1. Fluorescence microscope, real-time PCR and western blot method were used to identify whether the cell line is needed. Cells in this study can be divided into3groups:the pLenO-GTP-BECN1group, the pLenO-GTP group and the blank control group. Transwell matrigel invasion assay and transwell migration assay are used to test the ability of BT-549cells to invade and migrate. Expressions of EMT-related markers (such as E-cadherin, N-cadherin, Vimentin and Snail) are detected via real-time PCR and western blot method.Result After lentiviral transfection and puromycin selection, expression of GFP in pLenO-GTP-BECN1group of BT-549cells was significantly higher than the pLenO-GTP group and the blank negative group. Results of real-time PCR and western blot showed that mRNA and protein expression of Beclin1in pLenO-GTP-BECN1group was significantly higher than the other two groups (p<0.01), suggesting that successful construction of BT-549cells with stable expression of Beclin1was completed. The transwell matrigel invasion assay showed that invasion ability of pLenO-GTP-BECN1group cells was significantly lower than the other groups (p<0.01). The transwell migration assay showed that migration ability of pLenO-GTP-BECN1group cells was significantly lower than the other groups (p<0.05). The expression tested in our study showed that expression of E-cadherin in pLenO-GTP-BECN1group was significantly higher than the other two groups (p<0.05), as N-cadherin, Vimentin and Snail were significantly lower in contrast (p<0.05).Conclusion Beclin1could reverse EMT process of BT-549cells and reduce the ability of BT-549cells to invade and migrate. Part II Influence of beclin1on chemosensivity of BT-549cellsObjective The aim is to observe the effect of Beclin1on sensitivity of BT-549cells to paclitaxel and5-FU and further to explore the related mechanisms.Method Different concentrations of5-FU and paclitaxel were used to treat cells of each group for a period of time respectively. Then MTT assay was conducted to detect proliferation inhibition rate. Annexin V-APC/7-AAD method was used to detect apoptosis rate of BT-549cells treated by50mg/ml5-FU and200μg/L paclitaxel for48h respectively. Hoechst33342dying method was used to detect the nuclear changes and AO dying method to detect formation of autophagic acid vesicles. Western blot was conducted to detect the expression of apoptosis related protein (such as Caspase-3, Caspase-8) and autophagy related protein (LC3B).Result MTT assay showed that proliferation of BT-549cells in pLenO-GTP-BECN1group had been significantly inhibited compared with the other two groups (p<0.05). Annexin V-APC/7-AAD method and Hoechst33342dying method results showed that after treated by5-FU and paclitaxel, apoptosis rate of pLenO-GTP-BECN1group of BT-549cells was more highly elevated than the other two groups (p<0.05). The AO dying method showed that after treated by5-FU and paclitaxel, autophagy was more significantly activated in pLenO-GTP-BECN1group of BT-549cells than the other two groups (p<0.05). The results of western blot showed that after treated by5-FU and paclitaxel, expression of apoptosis and autophagy related protein of pLenO-GTP-BECN1group of BT-549cells was significantly higher than the other two groups (p<0.05).Conclusion Beclin1could significantly enhance chemosensibility of BT-549cells. The mechanisms underlying include that Beclin1could enhance sensitivity of BT-549cells to anti-tumor drugs induced apoptosis and that Beclin1causes overactivation of autophagy in BT-549cells when treated by chemotherapeutics. |
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