| Epithelial ovarian cancer has the highest death rate in all gynecologic cancer, so it has been considered as a "silent killer". More than 70% of patients have advanced stages at diagnose, and tumors have metastasis, 5-years survival rate just about 30%.Gynecologist and gynecologic oncology researchers have focused on the methods of early detection and early treatment of patients with ovarian cancer for many years.So far,the standard treatment of ovarian cancer is surgical treatment in combination with platinum-based chemotherapy, however, chemotherapy-resistant has been an insurmountable problem. As such, there is an unmet need for finding a new treatment method to increase therapeutic efficacy of ovarian cancer patients.As a new methord for treating cancer, gene therapy brought hope to mankind.Along with the research on the molecular mechanisms associated with the occurrence and development of human cancers, gene therapy and cell transplantation immunotherapy has become a new field of cancer therapy. More and more researchers have used different approaches for target treatment, and some researches have made considerable progress in animal experiments, and some cancer gene therapy and cell transplantation immunotherapy strategies have been used to clinical application stage.However, gene therapy and cell transplantation immunotherapy for cancer treatment as a new means of cancer treatment method is necessary for further study and exploration.Mesenchymal stem cells (MSCs) are primitive cells which have the effect of self-renewal and multi-differentiation. Many studies have shown that MSCs have the tumor tissue tropism that can be used as a carrier for targeted cancer therapy. Human umbilicalcord mesenchymal stem cells (hUCMSCs) have no possible to promote solid tumor growth and invasion in cancer therapy; on the contrary, hUCMSCs can inhibit the classical Wnt/?-catenin signaling pathway, which can inhibit the proliferation of tumor cells.IL-21 is an immune regulatory factor that has a variety of biological functions,and it can mediate innate immunity to adaptive immunity, and involve in humoral and cellular immune responses. IL-21 may promote B and T cell proliferation, promote cord blood progenitor cell differentiation to NK cells, and increase it's toxicity to target cells.More and more studies have shown that IL-21 cytokine has strong potential in the anti-tumor therapy. Therefore, we transfered IL-21 into lentivirus carrier pHAGE-CMV-MCS-IZsGreen, and used the recombinant lentivirus pHAGE-IL-21 in tumor therapeutic experiments. Genetically modified cells has been concerned in gene therapy, and gradually become an important means for overcoming many diseases.MicroRNA (miRNA) is a class of small non-coding RNA molecules recently discovered, and it regulats the gene expression in post-transcriptional level MiR-200 family (miR-200a, miR-141, miR-200b, miR-200c, miR-429) is a newly discovered tumor metastasis-associated miRNA,and influences tumor metastasis by regulation of epithelial-mesenchymal transition (EMT). A series of studies showed that there was abnormal expression of miR-200c in ovarian cancer, and that downregulation of miR-200c may be associated with the progression of ovarian cancer, and may be one of adverse prognostic factors of ovarian cancer.There is a part of gene therapy for ovarian cancer that has been used to clinic, but the majority still remains to be in the animal experimental stage, and there are still many problems to be solved in clinical application. For examples,how to choose a more effective target gene; how to make the recombinant vectors with the therapeutic effect transfer efficiently and express the gene products stably; how to make the gene therapy in combination with other treatments effectively. Based on consideration of the above problems, we designed this study. We used the lentivirus which carry interleukin-21 (IL-21) gene to transfecte the hUCMSCs, and combined with microRNA-200c (miR-200c)to treat ovarian cancer.1. ObjectiveThis study was designed to combine gene therapy with cell transplantation therapy,use the lentivirus carrying IL-21 gene, and infect the hUCMSCs, resulting in the cytokine IL-21 being carrid to the tumor site by hUCMSCs, which will play anti-ovarian cancer effects depend on the synthetic action of IL-21, hUCMSCs, and miR-200c. We also investigated and analyzed the possible mechanisms of this efficacy,hoping to provide some experimental evidences for anti ovarian cancer in clinical trials by miR-200c combined with gene therapy based on hUCMSCs carrying IL-21.2. Methods2.1 The fragment of IL-21 gene was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen to construct recombinant plasmid pHAGE-IL-21 and was identified, and then the recombinant was used in lentivirus packaged, and was also identified.2.2 hUCMSCs was primary cultured, and identified by its surface molecular markers and osteoblastic and adipogenic differentiation assays.2.3 hUCMSCs was infected by recombinant lentivirus-IL-21, and the expression and activity of IL-21 in hUCMSCs was detected.2.4 Experiment in vitro: 1) miR-200c mimic was transfected into SKOV3 cells and identified; 2) Cell co-cultured: cells were co-culatured in the "Trans-well" system, and divided into seven experimental groups: medium control group, hUCMSCs group,hUCMSCs-LV-Vec group, hUCMSCs-LV-IL-21 group, miR-200c group, miR-200c combined with hUCMSCs-LV-IL-21 group, cisplatin (DDP)group; 3) Cell proliferation was measured by MTT assay; 4) Scratch test was used to detect cell migration ability; 5) Cell invasion ability were detected by transwell cell matrigel invasion assay.2.5 The model of human ovarian cancer cell line SKOV3 xenografts was established in nude mice injected with SKOV3 cells, and the tumor bearing nude mice were divided into seven groups for subsequent experiments: physiological saline control group,hUCMSCs group, hUCMSCs-LV-Vec group, hUCMSCs-LV-IL-21 group, miR-200c group, hUCMSCs-LV-IL-21 combined with miR-200c group, cisplatin group. The tumor size, tumor pathology and organ metastasis was observed. The peripheral blood of nude mice were collected to do routine blood test, and the function of liver and kidney was preliminary tested in order to evaluate the safety of hUCMSCs, IL-21, lentivirus and miR-200c used in the the treatment of transplanted tumor in nude mice.The levels of IFN-?, IL-21 and TNF-a was respectively tested in peripheral blood. The expression of NKG2D and MIC A in tumor tissue were detected by real-time quantitative PCR assay. Immunohistochemistry and Western blotting was used to detect the expression of?-catenin, cyclin-D1,E-cadherin, ZEB1,Glil and Gli2 in tumor tissues.3. Results3.1 The recombinant plasmid pHAGE-CMV-MCS-IzsGreen-IL-21 was established, and the recombinant was verified by the analysis of endonuclease digestion and sequencing, suggesting the recombinant was successfully constructed, and we named it for pHAGE-IL-21. psPAX2, pMD2. G,and pHAGE-IL-21 plasmids were co-transfected into HEK 293T cells, green fluorescent protein could be observed after 24 h,and more green fluorescent protein could be seen after 48 hours. The fluorescence of HEK 293T cells infected with lentivirus was observed in 48h, and the lentivirus titer is around 1.0×108TU/ml, which confirmed the lentivirus was packaged successfully. The restructured lentivirus and empty lentivirus were labeled 'LV-IL-21' and 'LV-Vec' respectively.3.2 hUCMSCs was cultured successfully, and the third generation of hUCMSCs was identified by flow cytometry (FCM). The results indicated CD34 (-), CD45 (-), CD29(+),CD90 (+),which was agreed with hUCMSCs cell phenotypes. Osteogenic and adipogenic differentiation assays were carried for 21 days in the culture media respectively, and the results were presented as both positive reactions.3.3 hUCMSCs was infected by recombinant lentiviral (LV-IL-21) and empty lentiviral vector (LV-Vec) respectively,and selected by puromycin. hUCMSCs-LV-IL-21 and hUCMSCs-LV-Vec were acquired from the screening assay. The biological activity of IL-21 in hUCMSCs-LV-IL-21 was confirmed by spleen cell proliferation experiment.3.4 The results of experiment in vitro: (1) Mimic miR-200c was transferred into SKOV3 cells confirmed by RT-PCR. (2) The proliferation rate of hUCMSCs-LV-IL-21 combined with miR-200c group was significantly decreased in 4,6,8 days compared with the other 6 groups detected by MTT assay, and the difference was statistically significant (p < 0.05). The comparison results of cell proliferation rate in all 6 groups are as follows: DDP group <miR-200c group <hUCMSCs-LV-IL-21 group =hUCMSCs group= hUCMSCs-LV-Vec group < control group ("<" means the result was statistically significant difference (p<0.05),"=" means the results showed no significant difference (p>0.05)). (3) Scratch test showed that the fracture closure of the cells was significantly decreased in hUCMSCs-LV-IL-21 combined with miR-200c group after inoculation 24 and 72 hours, and the difference was statistically significant (p<0.05) compared with the other six groups. The cells fissure closure were decreased in hUCMSCs group, hUCMSCs-LV-Vec group and hUCMSCs-LV-IL-21 group compared with normal control group, which was statistically significant (p<0.05), but there were no significant difference among these three groups (p>0.05). The fissure closure degree in miR-200c group was lower than that of hUCMSCs-LV-Vec, hUCMSCs and hUCMSCs-LV-IL-21 groups (p<0.05),but higher than that of DDP group (p<0.05). (4) Transwell assay showed that the invasion ability of SKOV3 cells was significantly decreased in the hUCMSCs-LV-IL-21 group combined with the miR-200c group, and there was a significant difference between the hUCMSCs-LV-IL-21 group and other 6 groups (p<0.05). The invasion ability of hUCMSCs, hUCMSCs-LV-Vec and hUCMSCs-LV-IL-21 groups was lower than that of normal control group, which was statistically significant (p<0.05);however, there was no significant difference among these three groups (p>0.05). The invasion ability of SKOV3 cells in miR-200c group was lower than that in hUCMSCs,hUCMSCs-LV-Vec and hUCMSCs-LV-IL-21 groups (p<0.05),but higher than that in DDP group (p<0.05).3.5 The results of animal experiments showed that (1) the inhibition effects of seven treatment groups on ovarian cancer growth in nude mice respectively was: hUCMSCs-LV-IL-21 combined with miR-200c group>cisplatin group> miR-200c group ?hUCMSCs- LV-IL-21 group> hUCMSCs group = hUCMSCs-LV-Vec group> control group. ( 2 ) The blood routine index including red blood cell count (RBC),hemoglobin (HGB), hematocrit (HCT), platelet count (PLT) and white blood cell count(WBC), percentage of neutrophils (N%), and blood biochemical indexes including serum total protein (TP), albumin (ALB), globulin (GLB), alanine aminotransferase(ALT) and aspartate aminotransferase (AST), glucose (GLU), urea nitrogen (BUN),creatinine (CR), glycerol (TG), cool three of total cholesterol (CHOL), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in nude mice had no obvious difference (p>0.05). (3) After four weeks of treatment, the serum levels of IFN-y, IL-21 and TNF-a in nude mice were significantly increased in hUCMSCs-LV-IL-21 combined with miR-200c group compared with other six groups, and the differences were statistically significant. (p<0.05 or p <0.01). (4) The killing activity of mouse spleen cells to target cells YAC-1 and SKOV3 were significantly enhanced in hUCMSCs-LV-IL-21 group compared with control group and hUCMSCs-LV-IL-Vec group (p<0.01). (5) The growth of tumor cells were active with obvious karyokinesis in control group; the infiltration of mononuclear cells and necrosis were observed in hUCMSCs group and hUCMSCs-LV-Vec group; both the degree of infiltration of mononuclear cells and necrosis was heavier in hUCMSCs-LV-IL-21 group than those of miR-200c group, cisplatin group and aforementioned groups, in which the necrosis of tumor tissue was very severe in hUCMSCs-LV-IL-21 combined with miR-200c group,accompanied with a large number of mononuclear cell infiltration. (6) Routine HE staining showed there was no signs of ovarian cancer metastasis or tumor generation in mice lung, liver, stomach and spleen in sacrificed mice. (7) The results of immunohistochemical assay and WB test showed that the expression of ?-catenin and cyclin-D1 was decreased in six groups except the control group, which was significantly decreased in hUCMSCs-LV-IL-21 combined with miR-200c group compared with theother groups, and the differences were statistically significant(P<0.05); the expression of ZEB1 was down regulation but the expression of E-cadherin was upregulation, which can be respectively observed in HUCMSCs group, hUCMSCs-LV-Vec group,hUCMSCs-LV-IL-21 group, miR-200c groups, hUCMSCs-LV-IL-21 combined with miR-200c groups, in which these changes was obviously found in hUCMSCs-LV-IL-21 combined with miR-200c group, and there is a statistically significant difference brtween hUCMSCs-LV-IL-21 combined with miR-200c group and other groups (P <0.05) . The reduced expression of Gli1 and Gli2 in hUCMSCs, hUCMSCs-LV-Vec, hUCMSCs-LV-IL-21, miR-200c, miR-200c combined with hUCMSCs-LV-IL-21 groups was observed,of which the hUCMSCs-LV-IL-21 combined with miR-200c group has the obvious change compared with the other groups,and there were statistically significant differences (P <0.05).(8) Fluorescence quantitative PCR showed that the expression of MIC A and NKG2D was no significant change in the control group, hUCMSCs group,hUCMSCs-LV-Vec group, and miR-200c group, however, they were increased in cisplatin group, and the difference were statistically significant between cisplatin group and other four groups (MIC Ap <0.01,NKG2D p <0.05). The expression of MIC A and NKG2D were significantly increased in the hUCMSCs-LV-IL-21 group and the hUCMSCs-LV-IL-21 combined with miR-200c group versus the cisplatin group(statistical significance p < 0.05 (MIC A), p <0.01 (NKG2D)). However, there was no statistically difference (P> 0.05) in the expression of MIC A and NKG2D between hUCMSCs-LV-IL-21 group and hUCMSCs-LV-IL-21 combined with miR-200c group.4. Conclusions(1) hUCMSCs could be successfully isolated and cultured from the umbilical cord, and hUCMSCs could be successfully infected by LV-IL-21.(2) hUCMSCs-LV-IL-21 could increase the secretion of IFN-y and TNF-a to promote the numbers and activity of NK cells,playing a good role of anti-ovarian cancer in nude mice. The results lay a foundation for the clinical use of hUCMSCs for gene therapy of ovarian cancer.(3) hUCMSCs-LV-IL-21 combined with miR-200c has a good efficacy against ovarian cancer and the mechanisms may be that 1) IL-21 actives the NK cells, up regulates the expression of NKG2D and MIC A in tumor tissues, and increases the levels of IFN-y and TNF-a in the serum of nude mice, and these comprehensive effects play a role of killing ovarian cancer; 2 ) hUCMSCs and miR-200c down regulated the expression of ?-catenin,cyclin-D1,ZEB1,Glil and Gli2, up regulated the expression of E-cadherin in tumor tissue, and the cooperativity suppressed the growth and metastasis of ovarian cancer. The findings provides a new strategy and method for the treatment of ovarian cancer. |
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