Experimental Studies On Therapy For Endometriosis With Short Hairpin RNA Targeting Survivin Mediated By Lentivirus

BackgroudEndometriosis is one of the most common gynecologic diseases with an increasing prevalence. Endometriosis presents in approximately 10 to 15 percent of reproductive age women. It often causes infertility, dysmenorrhea, dyspareunia and chronic pelvic pain. Although endometriosis is a kind of benign disease, it has many similar characters to malignant disease, such as cellular proliferation, infiltration and recidivation. Endometriosis is hard to be cured because of its recrudescence and has heavy impacts on women's body and mind health.The treatments for endometriosis include surgery and medication. Operation is good for cleaning the lesion, restoration of pelvic anatomy, releasing symptom, and increasing the chances of conceiving. But its recurrence rate is still up to nearly 50% except radical surgery which would result in the loss of reproductive and endocrine abilty. Currently available medical therapies are designed to inhibit ectopic lesions by depressing the ovarial function to redcuse the level of estrogen directly or indirectly. However, none of these drugs can eradicate the disease and the substantial side effects. So far, recurrence is the key point to treatment of endometriosis because the true cause of the disease is ambiguous. All of the therapies are lack of the exact target. It is very urgent to find out the pathogenesis of endometriosis and get effective, safe, long-lasting treatment measures for endometriosis. The transplantation theory that Sampson proposed in 1927 is still recognized widely. However, the incidence of reflux of shed endometrial fragments is up to 76%～90% which is not to accord with the incidence of endometriosis. It is suggested that the refluent endometrial fragments is only the inducement. Recent studies indicated that the biological behavior of cell adhesion, invasion, planting, growth and transfer is the key to the pathogenesis of endometriosis. There are more and more researches indicated:Spontaneous apoptosis is decreased and proliferation is inereased in women with endomertiosis. This showed that apoptosis is the key to maintain stability of endometrial structure and function. The change of apoptosis characteristics is important for ectopic endometrial cells to have the ability to implant and grow. At the same time, many studies indicated that angiogenesis play a important role in formation and development of endometriosis, the formation and growth of ectopic lesions is depend on the new blood vessels. Therefore, promoting apoptosis and blocking angiogenesis are effective strategy for prevention and control endometriosis.Survivin is the strongest member of inhibitor of apoptosis proteins family and play an important role in the regulation of apoptosis. It expresses selectively in human embryonic tissues and tumor cells but almost not in normal differeniiated human tissues except for thymus, testes and secretory phase endometrium. survivin not only inhibits cell apoptosis, but also involves in angiogenesis. Therefore, survivin has been paid significant attention as a new target for anti-tumor and anti-vessel therapy.Recent studies have shown that survivin expression in ectopic endometrium was much higher than that in normal endometrium and eutopic endometrium. Those results indicate that survivin gene inhibits cell apoptosis in ectopic endometrium, destroies the balance between proliferative and apoptosis, favor abnormal proliferative, so it may contribute to the formation and progression of endometriosis. As above, If survivin expression have been suppressed, the occurrence and development of endometriosis would be blocked result from promotion of ectopic endometrial cells apoptosis and inhibition of angiogenesis. Therefore, survivin gene may be an ideal target for treatment of endometriosis.The important aim of gene therapy is the continually and steadily expression of obtained genetic material in transferred cell. Because the inhibition effect caused by RNA interference was fast, efficient and high stable, RNA interference is the most effective method of the sequence-specific post-transcriptional gene silencing in biology. It has become a powerful tool in exploring gene function and gene therapy for tumor and infectious diseases. Nevertheless, the key to the success of gene therapy is selecting suitable vector. Lentiviral vector become the focus of current attention widely due to its unique advantage. Lentiviral vector system have many advantages such as infection of non-dividing cells and divided cells, small immune response, reusable applications, huge gene fragments carried, long-term stable expression and high biological safety.ObjectiveThe feasibility and effectiveness of lentivirus mediated short hairpin RNA (shRNA) to repress the expression of survivin and promote apoptosis in ectopic endometrium cells were investigated. Furthermore, the effectiveness and security of lentivirus mediated shRNA targeting survivin gene to inhibit the establishment and growth of ectopic lesion in nude mice were studied. The main object is to seek a novel treatment strategy for endometriosis.Methods1. Construct a lentiviral expression vector mediated shRNA targeting human survivin gene and package lentivirus particles. Human survivin gene shRNA sequence was designed using software available on-line. After synthesis and annealing, the shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin-shRNA, which was subsequently confirmed by PCR and DNA sequencing analysis. The recombinant plasmid, packaging plasmid pHelper 1.0 and pHelper 2.0 were co-transfected into human embryonic kidney epithelial cell line (293T cells) and high titer replication defective lentivirus particles were collected and determined. 2. Study the effect of lentiviral vector-mediated shRNA on the expression of survivin mRNA and protein and its influence on the proliferation and apoptosis in human ectopic endometrial cells. The ectopic endometrial cells from patients with endometriosis were isolated and purified by trypsin digestion and pasted wall purification. The cellular morphology was observed by optical microscope. Endometrial cells were identified by immunocytochemistry. Lentiviral vector mediated shRNA targeting survivin gene (LV-survivin-shRNA) was transfected into human ectopic endometrial cells, the empty vector and culture media as control, respectively. The survivin mRNA and protein expression were detected by RT-PCR and Western-blot. The proliferation and apoptotic rate of cells after transfection were assayed by methy-thiazoly-tetrazolium and flow cytometry.3. Investigate the effect of LV-survivin-shRNA on angiogenesis and growth of endometriosis like lesions in the chick embryo chorioallantocic membrane (CAM). Eutopic endometria from women with endometriosis were transplanted onto the non-vascular region of CAM. After successful established, the human endometriosis CAM angiogenesis models were randomly divided into 4 groups (n=30). It include inoculated alone group, LV-survivin-shRNA group, LV-NC-GFP group and empty vector group. Another 7 days later, CAMs were photographed under the anatomical microscope after entirely exposure. Graph software automatically counted vascular area and the area of CAM. Transplant specimens were analyzed by histology. Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method was used to detect cells apoptosis of endometriotic lesions.4. Animal endometriosis models of nude mouse were established by inoculating human endometrial fragments. Forty five female nude endometriosis models were randomized into three groups,15 mice each group, including LV-survivin-shRNA group, LV-NC-GFP control group and blank control group when ectopic lesion diameter reached 5mm-10mm. Endometriosis models of nude mouse were administrated with LV-survivin-shRNA, LV-NC-GFP and PBS into the transplants. The activity and appetite of those mice were monitored each day, and the body weight of each mouse was weighing on the first day and the last day. The ectopic lesion sizes were measured every three days. All of these mice were killed at 15th day after treatment. The mRNA and protein expression of survivin and caspase-3 in ectopic lesion after transfer was detected by RT-PCR and immunohistochemistry method, respectively. TUNEL method was used to detect endometriosis like lesions cells apoptosis. Meanwhile we detected the level of steroid hormone and histological change in mice uterus, ovary, liver and kidney.Results1. PCR analysis and DNA sequencing confirmed that the shRNA sequence was successfully inserted into the lentiviral vector. The titer of concentrated virus was 8×108TU/ml.2. The cell culture of ectopic endometrium was successfully established. Both endometrial epithelial cell (EEC) and endometrial stromal cell (ESC) were existed in the culture and stained positively by cytokeratin and vimentin monoclonal antibody respectively. The life span of EEC and ESC can last 5 weeks and 14 weeks, respectively. We could see the expression of green fluorescent protein after lentivirus infecting primary ectopic endometrial cells for 24h, the strongest expression of EGFP could be seen after 72h. Lentivirus of Multiplicity of Infection (MOI) less than 10 had low transfection efficiency, but when viral MOI was equal to 50, the transfection efficiency is high, up to 80%, and cell morphology and growth of cells are the same as the proliferation of normal cells. When MOI is equal to 100, the cells became obviously pathological cells, such as round and floating cells with normal proliferation affected. After transfected with LV-survivin-shRNA for 72 h, the expression of survivin mRNA and protein in human ectopic endometrial cells was inhibited by 64.20% and 58.79% respectively. After transfected with LV-survivin-shRNA for 3 weeks, the expression of survivin mRNA in human ectopic endometrial cells was still inhibited by 70.93%. The growth speed of the human ectopic endometrial cells transfected with LV-survivin-shRNA was significantly slowed than other two groups. There was a significant difference between them (P<0.001) except at the first day after being transfected. There was no difference between the growth speed of non-transfected cells and that of empty vector transfected cells (P>0.05). The apoptosis rate of survivin-shRNA group reached (41.61±3.64)%, which was significantly higher than those of non-transfected group (7.07±1.16)% and empty vector transfected group (9.14±1.88)% (P<0.001). There was no difference between the apoptosis rate of non-transfected cells and that of empty vector transfected cells (P=0.087).3. The human endometriosis chick chorioallantoic membrane angiogenesis models(hEMCAM) were established successfully. Transplantation of endometrium onto the CAM led to a strong angiogenic response in the chicken tissue. The vessels grew radially around focus and the density of newborn blood vessels increased significantly in the hEMCAM. The angiogenesis and endometriosis-like lesions formation were significantly suppressed after treatment with LV-survivin-shRNA in comparison with the control groups. The transplantation achievement ratio of LV-survivin-shRNA group was significantly lower than those of other groups (P<0.001). Quantitative analysis shows that the ratio of vascular area (VA)/CAM of LV-survivin-shRNA group is significantly reduced (P<0.001). There is no significant difference between the other three groups (P>0.05). The apoptosis rate of endometriosis-like lesions after treatment with LV-survivin-shRNA was significantly higher than those of other three groups(P<0.001), and accomponied by various degree of necrosis in the endometriosis-like lesions under microscopic observation.4.50 nude mice were modeled by subcutaneous implantation by injection with the endometria from endometriosis patients, there are 45 animal endometriosis models were established successfully. After endometrial fragment being inoculated for 10 days, lesion nodes (5mm-10mm in diameter) were found. There were no significant difference in volume of the endometrial lesion and weight of the nude mice before treatment among blank control group, negative control group and LV-survivin-shRNA group. At the 15th days after treatment, compared with the other two control groups, the volume and weight of the lesion nodes were decreased obviously (P<0.001). Microscopic examination showed that the glandular epithelium of LV-survivin-shRNA group had partially degenerated, and necrotic debris was present in the endometrial stroma. The difference was not statistically significant between the two control groups. The results of RT-PCR and immunohistochemisty staining showed that the expression of survivin mRNA and protein in ectopic lesions of the LV-survivin-shRNA group were significantly less than the two control groups (P<0.001). The expression of survivin mRNA and protein in ectopic lesions were inhibited by 70.88% and 66.38%, respectively. Nevertheless, the mRNA and protein expression of caspase-3 in ectopic lesions of the LV-survivin-shRNA group were significantly higher than the two control groups (P<0.001). The apoptosis rate of LV-survivin-shRNA group was significantly higher than that of blank control group and negative control group (P<0.001). There was no difference between the apoptosis rate of blank control group and that of negative control group (P=0.952). There were no obvious changes in the nude mouse liver, kidney, uterus and ovary were observed through routine pathological check after the LV-survivin-shRNA treatment. At the 15th days after treatment, there was no significant difference in concentration of E2 and FSH among three groups. During administrating, side effects were not found in the LV-survivin-shRNA group.Conclusions1. The recombinant lentiviral vector mediated shRNA targeting survivin gene have been constructed successfully. The recombinant lentiviral vector is effective, safe and convenient, which is convenient to observed because of GFP and can be packaged high-titer, high-purity lentivirus.2. The primary culture method for ectopic endometrial cells by trypsin digestion and pasted wall purification was convenient and of high performance. It can be applied to culture EEC and ESC incorporately.3. LV-survivin-shRNA can infect ectopic endometrial cells effectively in vitro and can inhibit the expression of survivin gene, which resulted in suppressing the proliferation and promoting the apoptosis in human ectopic endometrial cells.4. The CAM endometriosis models have been constructed successfully. LV-survivin-shRNA can effectively inhibit angiogenesis induced by eutopic endometrium onto the CAM, obviously promote the apoptosis in ectopic endometrial cells and remarkably suppress endometriosis-like lesions formation. 5. The endometriosis mice models of subcutaneous implantation were established successfully. The model of subcutaneous implantation is more convenient to observe. The modeling method is simple and accomponied by high achievement ratio.6. Lentiviral vector-mediated shRNA can be transferred into mice ectopic lesion and can effectively inhibit the expression of survivin gene and activate the expression of caspase-3, which leaded to obviously promote the apoptosis in ectopic endometrial cells and remarkably suppress the growth of endometriosis like lesions in nude mice.7. There were no significant side effects were observed. The therapy of endometriosis with lentiviral vector-mediated shRNA was effective and safe. It lays the foundation for the experimental study and future clinical application.