| BACKGROUND AND OBJECTBreast cancer has become the first common malignance among developed countries and cities in China. The incidence of breast cancer worldwide is growing at0.2to8%annual rate. Every year about1.4million women are diagnosed as breast cancer, and about0.5million people died. Epidemiological data indicates that the incidence and mortality are both increasing in recent years in China. And the patients are becoming much and much younger. The Survival rate is closely related to the clinical stage and phenotype of breast cancer. Distant metastasis is often the lethal factor for breast cancer. Macia and other studies have shown that the5-year survival rate of breast cancer was83.3percent, which amounted to97.1%in patients with stage I, but for patients of stage IV, the rate was only24.5%. Among them, about15-20percent is triple negative phenotype. The disease progression is rapid in these patients, and the recurrence and metastasis rate are both higher than other phenotypes. The five-year survival rate is only about15percent of these patients.Currently the treatment methods for breast cancer include surgery, radiotherapy, chemotherapy, endocrine therapy and targeted therapy. Surgery is the fundamental means for breast cancer. It gets rapid progress in recent years. But the aim of progression is mainly focused on reducing operation trauma and artistic, the rate of recurrence and metastasis is still high after operation. Postoperative radiotherapy, especially for breast-conserving surgery, can reduce the recurrence rate, but it is unprofitable for metastasis. Clinical doctors and researchers put into much vigor and financial resources in past years, and obtained considerable progress. The efficiency of chemotherapy is much higher than most solid malignances, and it can exactly decrease the recurrence and metastasis rate for breast cancer. Moreover, chemotherapy can increase the long-term survival rate. But because of the intolerable side effects and secondary resistance, the effect of chemotherapy for advanced and metastatic breast cancer patients is slight. Endocrine therapy for breast cancer is representative. It is reported that5-10years of tamoxifen can significantly improve the long-term survival rate for hormone receptor positive breast cancer patients. The side effects of endocrine therapy are well tolerable. So it can be long-term used. But endocrine therapy is only applied to ER and/or PR-positive patients without any effect on triple negative patients. Targeted therapy of breast cancer emerged almost the earliest in solid malignances and succeeded hugely. The targeted drug named as Trastuzumab was written in NCCN guidelines. It was reported that Trastuzumab was used alone or combined with chemotherapy drugs could significantly improve the efficacy of chemotherapy and prolong the long-term survival of breast cancer patients with Her-2/new gene over expression. As the same question with endocrine therapy, Trastuzumab is only valid for Her-2positive patients, also useless for triple negative breast cancer patients. For triple negative breast cancer patients, operation is also the primary method, and radiotherapy should be given to most parts after operation. Chemotherapy for these patients is also adopted common regimens in breast cancer patients without endocrine and trastuzumab indications. And as mentioned before, the prognosis of patients with triple negative phenotype is much worse than other types of patients. Therefore, it is urgent to find a new and more effective with less toxic treatment method, to improve the long-term survival rate of triple negative breast cancer.Recent researches support the view that an important reason for the occurrence and development of malignancy is the lost of normal apoptosis among those already transformed cells. It is known that there are many factors involved in the induction and inhibition of apoptosis. In these factors, the interaction between the receptors and the ligands on the cell surface plays a dominant role. TRAIL, named as tumor necrosis factor-related apoptosis-inducing ligand, is one of the tumor necrosis factor families. It has become the focus of current researches. The five kinds of specific binding of TRAIL receptors, namely TRAIL R1, TRAIL R2, TRAIL R3, TRAIL R4and osteoprotegerin (OPG), determine the biological effects of TRAIL, which plays an important role in the process of inducing functional apoptosis. TRAIL can selectively induce apoptosis in a variety of malignancies, without killing effect on normal cells. This feature makes a distinction from targeted therapy from chemotherapy. So the side effect of TRAIL is minimal, and it can be well tolerated. When TRAIL is used alone or combined with chemotherapy drugs, it can block the progression of multiple malignances. In addition, TRAIL can reverse the resistance to chemotherapy drugs, and improve the efficacy of chemotherapy. Therefore it has broad clinical application. Many researches have confirmed that TRAIL is a high efficient anti-tumor drug targeting with low toxicity in pre-clinical and clinical trials. Besides this, there are studies have shown that TRAIL is also effective for triple negative breast cancer, especially for those with mesenchymal phenotype, in which the role of the base-like cells are more prominent. But recent researches suggest that nearly half of the breast cancer cell lines are resistance to TRAIL, including all of the ER-positive cell lines and most of the Her-2-positive cell lines. But most of the triple negative breast cancer cell lines are sensitive to TRAIL. But with the time of application prolonging, it is easy to appear secondary resistance to TRAIL, and the data showed that once the tumor cells were resistant to TRAIL, the drug-resistant tumor cells could present enhanced ability of invasion. So once resistance emerged, the disease will harvest rapid clinical progress. The mechanism of resistance to TRAIL and enhanced invasiveness reason is unclear. So it is important to explore possible ways for TRAIL resistance mechanisms. Thus we can provide meaningful guidance for the clinical treatment of breast cancer, and to provide ways to reverse TRAIL resistance.METHODS AND RESULTSTo reveal the mechanisms of TRAIL resistance, we first produced recombinant human TRAIL (rhTRAIL) according to methods reported in the literature, and confirmed its effectiveness by MTT analysis. To build TRAIL resistance cell line, we select MDA-MB-231breast cancer cell line. Because MDA-MB-231cells are triple negative breast cancer cells, consistent with our research purpose, and ER-positive breast cancer cells, for example MCF7breast cancer cell line and other most cell lines as described before are naturally resistant to TRAIL. Of course, we also conducted MTT assay on MCF7and reached the same conclusion. After culture the cells routinely, we used the method of increasing the concentration of rhTRAIL gradually to build TRAIL resistant cell line. First, the MTT assay showed that when reaching IC50of MDA-MB-231cells to rhTRAIL, the concentration of rhTRAIL was12ng/ml, so we selected the eighth concentration, i.e.1.5ng/ml of rhTRAIL as the initial one. Then we cultured the cells conventionally. After passage at least3times, we gradually increased the concentration of rhTRAIL. About six months later, the concentration of rhTRAIL reached4mg/ml as the final concentration. The cells survived well. MTT cell viability detection displayed that in the concentration of rhTRAIL10ng/ml and above, the relative cell viability of rhTRAIL resistant MDA-MB-231(231T) cells was significantly different from MDA-MB-231(231) cells, p<0.01, indicated that the difference was statistically significant. Thus we confirmed that rhTRAIL resistant cell line was successfully constructed. Then we observed the form of231T cells under a microscope and found that drug-resistant cell lines aggregated into wheat-like structure, morphological differences compared with231cells. What we considered that TRAIL-resistance cells occurred the process of EMT, so then we applied western blot to detect the expression levels of EMT-related proteins, including N-cadherin, fibronectin and vimentin, found that the expression of these three proteins in231T cells was significantly higher than that in231cells, respectively2.79,2.16and2.13fold, and the p value was less than0.01. And Snail, which plays a central role in the EMT process, was also raised1.97-fold in231T cells than in231cells, p<0.05, meaning the difference was statistically significant. Because Snail was a transcriptional factor, then we applied real-time quantitative PCR (RT-PCR) to detect the mRNA levels of multiple transcriptional factors associated with EMT, including Snail, Twistl, Zeb2, Foxql, FoxC2, found that only Snail and Twistl in231T cells raised, indicating that EMT occured via Snail/Twist pathway. Since studies have found that EMT can enhance the invasive ability of cells, so we conducted Transwell assay on TRAIL-resistant MDA-MB-231cells to check their abilities of migration and invasion, and the results showed that they were both significantly enhanced.To further investigate the mechanism of TRAIL-resistance and the occurrence of EMT, we used western blot assay to test the expression level of PTEN protein in MDA-MB-231cells and TRAIL-resistance cells, and found that compared with231cells, PTEN expression in231T cells was significantly reduced, as69%of231cells. The P value was less than0.05. To clarify that PTEN was the reason for inducing TRAIL resistance and EMT, we used small interfering technology to knockdown the expression of PTEN in231cells and confirmed that the expression of PTEN was down-regulated dramatically in MDA-MB-231cells which were transfected by siRNA of PTEN (231-siPTEN) by western blot. The control group cells were named as231-n. Subsequently, we performed MTT assay, western blot and transwell testing on231-siPTEN and231-n cells. MTT assay showed that after interfering PTEN expression,231-siPTEN cells were resistant to rhTRAIL. P values were less than0.05at different concentrations of rhTRAIL when compared with the231-n cells, which confirming that PTEN was involved in TRAIL resistance process of MDA-MB-231cells. Western blot results showed that the reduction of PTEN expression induced EMT, thus verified that PTEN also participated in the occurrence of EMT. Next we performed Transwell experiments. The results further validated that after interference the expression of PTEN, the abilities of migration and invasion of231-siPTEN cells were both enhanced, so our results showed that PTEN down-regulation induced TRAIL resistance, EMT and enhancement of cell migration and invasion.In order to find an upstream regulator of PTEN, to further explain the molecular mechanisms of resistance to TRAIL and EMT. we used target scan software to predict the miRNAs that regulate PTEN. According to the results, we used RT-PCR method to detect miR-21, miR-221, miR-222, miR-23b-3p and miR-214expression level in231and231T cells We found that compared with MDA-MB-231cells, miR-21, miR-221and miR-222expression levels were significantly up-regulated in TRAIL resistance cells. The up-regulation of miR-21was most significant, about140times than231cells, followed by miR-221, about90times. Then we used transfection technique to inhibit the expression of miR-21in231T cells, named as231T-si21cells and231T-n as the control group. We also conducted MTT assay to detect the sensitivity to rhTRAIL and western blot to detect the PTEN expression. The results showed that down-regulation of miR-21expression could not reverse the resistance to rhTRAIL of231T cells and the level of PTEN protein did not change. So we considered that miR-21was not the upstream regulator of PTEN in231T cells and it did not participate in TRAIL resistance. Then according to the results of RT-PCR, we analyzed miR-221that ranked No.2. As those done to miR-21, we still use the transient transfection to inhibit the expression of miR-221in231T cells, and verified its transfection efficiency by RT-PCR. As before, the cells that were transfected by simiR-221were called231T-si221, and the control group cells were named as231T-n. Then we also detected the expression of PTEN by RT-PCR and western blot. The results showed that both on the mRNA and protein levels, PTEN were significantly up-regulated. Therefore, we concluded that PTEN gene is the target of miR-221in231T cells.In order to clarify the role of miR-221in TRAIL-resistance process, we analyzed drug sensitivities to rhTRAIL of231T-si221and231T-n cells by MTT assay. Data showed that miR-221knockout could partially restore drug sensitivity of cells to rhTRAIL in231T cells. Then we tested EMT-related proteins that were tested before, including N-cadherin, fibronectin, vimentin, and transcription factor Snail by western blot, and found that they were all down-regulated in231T-si221cells compared with231T-n cells. EMT process was reversed. While the subsequent transwell test also confirmed that knockdown the expression level of miR-221, the the capacities of migration and invasion were both decreased. P values were all less than0.01, the difference was statistically significant. All these results indicated that miR-221induced TRAIL-resistance, EMT and enhancement abilities of cell migration and invasion by targeting PTEN.CONCLUSION1. TRAIL-resistance MDA-MB-231cell line was successfully constructed, and its morphology was different from that of MDA-MB-231cells.2. TRAIL resistant cells appeared EMT, enhancement of cell migration and invasion capacities. 3. MiR-221partly induced TRAIL resistance, EMT and enhancing the abilities of migration and invasion in231T cells by targeting PTEN.SIGNIFICANCE1. Reveal one of the mechanisms of TRAIL-resistance, EMT, and the reason of the enhancement of migration and invasion.2. Confirm that in TRAIL-resistance breast cancer cells, PTEN is a target gene of miR-221.3. Provide a theoretical basis for TRAIL clinical application and avoidance of drug resistance. |
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