Construction of Galectin-3siRNA and pIRES-IL-24-TRAIL Vector and Its Influences on the Proliferation and Apoptosis of Breast Cancer Cells
|School||Southern Medical University,|
|Keywords||Galectin-3 Survivin Ki-67 siRNA Interleukin-24 Tumor necrosisfactor-related apoptosis inducing ligand Triple-negative breast cancer Breast CancerStem Cells Proliferation Apoptosis|
Background and objectiveBreast cancer is one of the most common malignant tumor in women. In our country it is accounted for7%～10%of whole body various malignant tumors. During recent years, in our country, especially in some big cities and coastal economic rapid development areas, the incidence is rising rapidly. It becomes the main disease of harming to women health. Since the past two decades, great progress has been made in the diagnosis and treatment of breast cancer, whose development has been achieved good results especially in the surgical treatment. At the same time, the further understanding of the biological behavior of breast cancer has been made, and the occurrence and development of tumor are further described by modern molecular biology techniques. According to the prognosis of breast cancer, especially the patient with advanced invasion and metastasis who has poor prognosis, invasion and metastasis mechanism has also become the researching hot pot today. It is well known that the invasion and metastasis of malignant tumor has a multi-factor, multi-step process, but the specific mechanism remains unclear.About1.2million women developed breast cancer in the world each year, accounting for18%of all female cancers, in which more than170000are triple-negative breast cancer (TNBC), accounting for10%-23.8%of the patients with breast cancer. TNBC has aggressive, early recurrence, progress fast, short survival time, special clinical biological behavior, and unsensitive for most endocrine therapy and targeted therapy of breast cancer (for its Her-2expression and female progesterone receptor positive expression), worse prognosis than other types of breast cancer. Traditional mode of cancer therapy (surgery, radiotherapy, chemotherapy) still is the principal means of cancer treatment. However, as for further research in the molecular mechanism of tumor cell growth, proliferation, and apoptosis, with the revolutionary progress of molecular biology, treatment of tumor has been into the era of human biological treatment, make human targeted treatment of tumor to be realization. Targeted therapy is a relative or absolute targeted therapy, by using certain genes or gene expression product who can express in tumor cells, but not in normal cells to kill tumor cells maximally and damage to the normal cells minimally. Molecular targeted therapy of breast cancer is a pointer to the breast cancer occurrence and development of cancer gene expression and its related product for treatment.The treatment of breast cancer molecular targeted drugs by blocking tumor cells or related cell signal transduction, to control the change of cell gene expression, and inhibit or kill tumor cells. Because TNBC have poor histologic classification of breast cancer, no expression of ER, PR and its HER-2, and associated with p53mutations, the expression of EGFR, at present the common molecular targeted drugs are useless for this subtype, although the subtypes of neoadjuvant chemotherapy with higher total response rate and pathological response rate, overall is still the worst prognosis.There is currently no TNBC treatment guidelines, and its treatment generally carried out according to the conventional treatment standards. In recent years, many researchers have launched multiple international, multicenter clinical study, think that triple negative breast cancer mainly choose Paclitaxel and cisplatin,, anthracycline-based and molecular targeted therapy drugs. Currently looking for TNBC new therapeutic targets is a difficulty and focus of research in the world.In recent years, several studies have shown that inhibition of Galectin-3expression can influence the proliferation and apoptosis of tumor cells, but few studies for breast cancer, especially triple-negative breast cancer. Galectin-3(Gal-3) is a member of the carbohydrate-binding protein family known as lectins. It plays a role in cell-cell and cell-matrix interactions, cell growth, cell-cycle regulation, apoptosis, cell damage and repair processes, neoplastic transformation, and metastasis. In recent years, several studies have documented overexpression of Gal-3in different human tumors, including large-cell lymphoma, colorectal carcinoma, breast carcinoma, hepatocellular carcinoma, brain tumors, melanoma, and thyroid carcinoma.Survivin is an inhibitor-of-apoptosis gene,described as capable of regulating both cell proliferation and apoptotic cell death. Mapped to chromosome17q25, survivin consists of four exons and three introns, encoding1417kb message. Studies of common human cancers have shown survivin protein expression in all cancer types, ranging from approximately35%survivin-positive patients in gastric to approximately100%in malignant melanomas. In the majority of cases, survivin protein has been associated with poorer prognosis. In comparison to fetal and cancer cells, relatively little expression of survivin has been detected in many normal adult tissues. Ki-67is expressed in all phases of the cell cycle except GO, and it can be used to evaluate the biologic behavior and proliferation activity of cells. The overexpression of Ki-67has been observed in many malignant tumors. Since its discovery in the early1980s, there has been interest in the role of Ki-67as a proliferation marker in cancer, particularly lymphomas, breast, endocrine and brain cancers. It is commonly used as a complement to grading systems that include mitotic counting as a sign of proliferation. Ki-67is one of the five genes of proliferation that contributes an importance weight to the Oncotype score, out of16cancer-associated genes. First, this topic study the invasion and metastasis of breast cancer mechanism by Survivin, Galectin-3and Ki-67, to guide the clinical diagnosis, classification, prognosis, and treatment. This experiment explores the relationship between SiRNAGalectin-3and TNBC in proliferation and apoptosis by RNA interference, and understands the role of Galectin-3playing in biology behavior of TNBC, which supports it becoming the new target for gene diagnosis and treatment of TNBC.In addition,5-year survival rate of breast cancer is about50%-60%, in which nearly50%patients relapse after treatment. Average survival time of the patients with advanced breast cancer is only18-30months. Although there are many alternative treatment of breast cancer, poor prognosis, side effects, and easily invading outside the mammary gland make it become one of the most difficult treating tumors. Traditional treatment only can kill tumor cells in splitting phase, once treatment stops, cancer stem cell will continue to proliferate and differentiate, which leads to tumor recurrence or metastasis. This is the "dandelion" phenomenon.In2003, Al-Hajj, etc in the primary lesion of patients with breast cancer molecular markers, found with CD44+/CD24-/Lineage-phenotype of breast cancer cells in the immune deficiency animal model has significant tumorigenic ability, the transplanted tumor cells and primary focal tumor are the same, after multiple generation cell culture, but also has strong tumorigenicity, indicating that this type is similar to mammary gland stem cell self-renewal and differentiation of the essence, it is called breast cancer stem cells. Breast cancer stem cells (breast cancer stem cells, BCSCs), BCSCs is considered is the basis of tumor occurrence, development and maintenance, and tumor recurrence and metastasis, and resistance to therapy. Current research suggests only a handful of BCSCs enter the cell cycle, and most of BCSCs in static state, so the BCSCsRg conventional chemotherapy drugs is not sensitive. Existing physical, chemical and drug treatment for the majority of tumor cells, the tumor in a handful of BCSCs can’t achieve the most effective to kill effect, can prevent malignant tumor recurrence and metastasis, and tumor recurrence and metastasis is the leading cause of death in patients. Existing breast cancer treatments designed to kill or remove all the tumor cells as much as possible, but for the few stem cells exist in the tumors in the cells, but to little effect, after treatment residual cancer stem cell proliferation, enough to make tumor recurrence and metastasis. Breast cancer stem cell surface markers CD44expression, high protein, MDR, ABC ABCG2, etc., but has yet to find specific targeted therapy of markers.At present, breast cancer stem cell targeted therapy research mainly has:(1) the DNA damage repair blockers, such as ADP ribose polymerase1(PARPI) blockers;(2) the cell surface receptors, such as EGFR, C-Kit;(3) vascular epidermal growth factor receptor vascular endothelial growth factor (VEGF) receptor inhibitors;(4) the Src kinase inhibitors;(5) mammals rapamycin target protein in mammalian target of rapamycin (roTOR);(6) interleukins IL;(7), tumor necrosis factor related apoptosis inducing ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), etc. IL-24(Melanoma diffrentiation-associated gene-7/interleukin-24, MDA-7/IL-24) is a new, cell factor characteristics of the tumor suppressor genes, is IL-10new members of the family of cytokines. Its code is secreted protein product(secreted MDA-7/IL-24protein, sMDA-7/IL-24), same as interleukin function of the immune system, and can inhibit tumor growth and specific ability to induce tumor cell apoptosis. The present experimental study showed that IL-24in inhibition and differentiation of tumor cells, promote tumor cell apoptosis; Inhibition of tumor angiogenesis around at the same time; Enhance the sensitivity of tumor cells to radiotherapy and chemotherapy; Inhibition of tumor cell invasion and metastasis plays an important role. IL-24has pleiotropic, selective, overlapping, collaborative, antagonistic, and network duality function characteristics. But in the study found that IL-24is not entirely eliminate cancer grown in nude mice. To solve this problem there are two ways of:(1) and other cytokines joint;(2) make cytokines can efficiently express stable role in the tumor site. Reports to IL-24can induce and enhance the Tumor Necrosis Factor related apoptosis inducing ligand (Tumor Necrosis Factor-related related apoptosis inducing ligand, TRAIL) activity. Tumor necrosis factor Related Apoptosis Inducing Ligand (Tumor necrosis factor-Related Apoptosis Inducing Ligand, TRAIL) element2Ligand or Apoptosis (Apo-Ligand2, Apo2L) is a myocardial cDNA library by Wiley, etc from the cloned, because of its amino acid sequence with TNF superfamily structure characteristics and can induce Jurkat cells and EB virus transformation of human lymphocytes Apoptosis and its name, because of their different sources can be induced by Tumor cells and virus into cells Apoptosis, and has no toxicity to the cells of normal tissue, won’t cause normal cells Apoptosis, and coordination with other antitumor drugs combined application can kill Tumor cells, and so become a research hotspot in the field of Tumor treatment, is a new type of antitumor drugs. Current research think TRAIL through the main death receptor DR4and DR5in cell membrane by its extracellular region combined with TRAIL after activation, the function of intracellular death domain DD signal conduction, apoptosis, and activate the intracellular FADD-caspase pathway, activation of independence in mitochondria and mitochondrial dependent two pathways mediated apoptosis.This experiment intends to restructure an IL-24-TRAIL gene vecter, and try to solve the shortcomings of cytokine treatment for breast cancer stem cells such as poor curative effect, great side effect and unguarantee safety and so on, which provides new experimental and theoretical basis for breast cancer stem cell gene therapy. Make breast cancer patients especially triple negative breast cancer benefit from this project. This study has potential huge social benefit and economic benefit.Methods and materials1. The Expression of Galectin-3in Breast Cancer and the correlation of Galectin-3, Survivin and Ki-67.1.1The expression of Galectin-3. Survivin and Ki-67in breast cancer (120cases) and paracacinoma normal tissues(120cases) were evaluated by SP immunohistochemistry.1.2The relationship between the expression level of Galectin-3, Survivin and Ki-67, and the clinical and pathological features of breast cancer.1.3The correlation of Galectin-3and Survivin expression in breast cancer was analysis Spearman rank test.1.4The correlation of Galectin-3and Ki-67expression in breast cancer was analysis Spearman rank test.1.5The correlation of Survivin and Ki-67expression in breast cancer was analysis Spearman rank test.2. Influences of Inhibiting Galectin-3expression by RNA Interference Technique on the Proliferation, Apoptosis and Invasion of Triple-negative Breast Cancer Cells MDA-MB-2312.1Galectin-3small interfering RNA (siRNA) was constructed by chemistry synthesis. Effective siRNA was transfected into breast cancer cell MDA-MB-231(as Galectin-3siRNA group).2.2Non-transfected cells were used as blank control and negative RNA as negative control. Inverted fluorescence microscope and RT-PCR was used to verify the interference result.2.3Cellproliferation (absorbance value) was observed by XTT after interference for24,48,72and96hours. Cell apoptosis was measured with flow cytomter.2.4Wound healing test and transwell test were performed for cell motility and invasion assay.3. Construction of IL-24-TRAIL vector and its influences on the motility and apoptosis of Breast Cancer Stem Cells3.1cDNA fragment encoding human TRAIL gene were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with the total mRNA isolated from human placenta tissue as template. The PCR amplified fragment of TRAIL gene was cloned into pIRES vector.3.2cDNA fragment encoding human IL-24gene were amplified. The PCR amplified fragment of IL-24gene was subcloned into the recombinant eukaryotic expression vector pIRES-TRAIL to construct pIRES-IL-24-TRAIL plasmid after sequencing.3.3Plasmid DNA of pIRES-IL-24-TRAIL was transfected into Breast Cancer Stem Cells MCF-7and MDA-MB-231with the help of Lipofectamine2000transfection reagent.3.4Cell apoptosis was measured with flow cytometer.3.5Wound healing test was performed for cell motility assay.4. statistical analysisAdopt SPSS13.0statistical software analysis the experimental results. analysis the relationship betewwn Galectin-3, Survivin, Ki-67protein expression and clinical pathological data (including the patient’s age, sex, tumor grade and TNM stage) by the Chi-square test. Using Pearson column connection number (C) and Chi-square test for paired data, analysis further the correlation of Galectin-3, Survivin, Ki-67protein. Experimental data are list as x±s, using one-way ANOVA comparing between groups: homogeneity test for variance first, if the variance is homogeneity, choose LSD method, if the variance is not homogeneity, Brown-Forsythe approximate variance analysis method is used for calibration, using factorial ANOVA analysis whether there is interaction between teams and time points, with P <0.05has significant difference, two tailed test.Results:1.The Expression of Galectin-3in Breast Cancer and the correlation of Galectin-3, Survivin and Ki-67.1.1The expression of Galectin-3and Ki-67in breast cancer was also significantly higher than that in paracacinoma normal tissues (75.00%VS8.33%, χ2=109.70, P<0.001;62.50%VS6.67%, χ2=82.68, P<0.001). The expression of Galectin-3and Ki-67was correlated with the pathological grade and node metastasis (χ2=8.571, P=0.014;χ2=4.444, P=0.035;χ2=6.834, P=0.033;χ2=18.809, P<0.001). There were no correlations among their expressions and the age or tumor size (χ2=0.012, P=0.913;χ2=0.212, P=0.645;χ2=0.192, P=0.661;χ2=0.042, P=0.837)1.2The positive rates of Survivin were70.83%in breast cancer,5.00%in nomal tissues. The positive rate of Survivin expression was significantly higher in breast cancer than that in normal tissues (χ2=110.50, P<0.001), and was correlated with node metastasis (χ1=9.076, P<0.001). There were no correlations between the expression o f Survivin and the age, histological grade or tumor size (χ2=0.012, P=0.913;χ2=0.212, P=0.645;χ2=0.192, P=0.661;χ2=0.042, P=0.837) 1.3The expression of Galectin-3was positively correlated with Survivin and Ki-67(χ2=24.27, C=0.41, P<0.001; χ2=27.23, C=0.43, P<0.001; χ2=26.18, C=0.42, P<0.001)2. Influences of Inhibiting Galectin-3expression by RNA Interference Technique on the Proliferation, Apoptosis and Invasion of Triple-negative Breast Cancer Cells MDA-MB-2312.1After transfected24hours, the transfection efficiency rate by inverted fluorescence microscope was80.60±5.62%. RT-PCR showed that the expression of Galectin-3was markedly decreased in Galectin-3siRNA group after transfected (0.077±0.003VS0.072±0.004VS0.016±0.001, F=479.7, P<0.001)2.2Cells proliferated still in the blank group and negative contro group after24,48,72and96hours, but cells grew inhibited and declined gradually in Galectin-3siRNAgroup(1.61±0.05、1.86±0.04、2.04±0.26、2.80±0.07VS1.60±0.07、1.77±0.10、2.01±0.37、2.49±0.15VS1.18±0.04、0.97±0.02、0.68±0.15、0.46±0.12).There was the interaction effect between the interference time and the experiment group (F=39.18, P<0.001)2.3The apoptosis rate by flow cytomter was slight higher in the Galectin-3siRNA group than the normal and blank controlgroup (36.99%±0.27%VS2.22%±0.03%VS3.84%±0.05%, F=44058.82, P<0.001)2.4The results of Transwell experiment indicated that the number of cells in down-pore of micro-membrane in the transfected group was less than that in the control group(154.70±24.01VS465.00±31.76VS322.30±23.18, F=102.30, P<0.001). Wound-healing assay shows that cell motility could be effectively suppressed by Galectin-3siRNA (Healingrate:48.39%±1.63%VS92.62%±2.88%VS88.49±2.25%, F=308.80, P<0.001) 3. Construction of IL-24-TRAIL vector and its influences on the motility and apoptosis of Breast Cancer Stem Cells3.1cDNA sequence encoding human TRAIL and IL-24gene were successfully cloned, and recombinant eukaryotic expression vector pIRES-IL-24-TRAIL was constructed.3.2The apoptosis rate of MDA-MB-231by flow cytomter was higher in the transfected group than the controlgroup (27.91%±1.05%,68.81%±5.53%VS10.54%±1.13%,9.52%±0.95%, F=272.90, P<0.001). The apoptosis rate of MCF-7by flow cytomter was higher in the transfected group than the controlgroup(13.69%±1.09%,58.71%±3.03%VS3.31%±0.22%,4.32%±0.47%, F=774.60, P<0.001)3.3Wound-healing assay shows that cell motility could be more effectively suppressed in the transfected group than the controlgroup (MDA-MB-231:7.47%±1.53%,30.21%±1.29%VS91.77%±2.25%,87.54%±2.93%, F=716.40, P<0.001; MCF-7:39.8%±1.22%,31.85%±1.33%VS93.34%±2.33%,89.54%±2.15%, F=848.10,P<0.001)Conclusion1. The Expression of Galectin-3in Breast Cancer and the correlation of Galectin-3, Survivin and Ki-67.Galectin-3, Survivin and Ki-67may play an important role in the carcinogenesis and progress of breast cancer through the inhibition of apoptosis. They may play synergetic roles in the process of carcinogenesis of breast cancer.2. Influences of Inhibiting Galectin-3expression by RNA Interference Technique on the Proliferation, Apoptosis and Invasion of Triple-negative Breast Cancer Cells MDA-MB-231 The interference of RNA inhibits the expression of the Galectin-3and the proliferation of MDA-MB-231cells significantly, while it promotes the apoptosis of tumor cells and impede tumour cell invasion.3. Construction of IL-24-TRAIL vector and its influences on the motility and apoptosis of Breast Cancer Stem CellspIRES-IL-24-TRAIL was constructed successfully, and it can promotes the apoptosis of tumor cells and impede tumor cell motility.Innovation1. Breast cancer development involving multiple genes and various regulatory factors interaction, is a complex process. Survivin, Galectin-3and Ki-67have obvious relevance the prognosis of breast cancer, which can be used as prognostic indicators of breast cancer. Further research in the specific inner mechanism of them is needed,which provides a new target therapy for breast cancer.2. The invasion and metastasis of breast cancer is a multi-factor and multi-step process. The project researchs in mechanism of invasion and metastasis in breast cancer by clinical and molecular biology aspects, using RNA interference technique, immunohistochemistry, RT-PCR, apoptosis and analysis technology to research, which is of great depth and breadth. There are many research about the Galectin-3SiRNA in the treatment of respiratory and digestive tract tumor at home and abroad, but there are few studies on triple-negative breast cancer. This reseach has important application value and theoretical significance.3. Although international has started using recombinant IL-24or TRAIL to treat some malignant tumors in experimental studies, but there is no joint used in breast cancer. This research project is proposed pIRES as a carrier of the IL-24and TRAIL gene, building pIRES-IL-24-TRAIL, which can express has activity-biology IL-24and TRAIL in the tumour for a long time, to improve the effect of tumor therapy, and because it is humanized, small molecular weight, strong penetrating power, weak immunogenicity and the characteristics of easy to large-scale preparation, it can achieve the ideal security while exerting biological missile power. Through literature search, there is no similar report. The drug listed successfully will brought good news the patients of breast cancer, which has great economic and social benefits.