| Lung cancer is the most common cause of cancer-related mortality worldwide. Although new chemotherapy agents and radiotherapy have improved survival and quality of life of patients, the overall five-year survival rate for such tumor is lower than 15%. In vitro data have shown that only 1 in 1,000 to 5,000 lung cancer cells forms colonies in soft agar assay, indicating that not every lung cancer cell was capable of tumor initiation. Evidence is accumulating that a subset of cancer cells within some tumors, contain a minor population of"cancer stem cell"(CSC). CSC have been isolated and expanded from leukemia, and several solid tumors, including melanoma, breast, brain, prostate, pancreatic and colon carcinomas. These cells display unlimited proliferation potential, ability to self-renew and capacity to generate a progeny of differentiated cells. Therefore, focusing research efforts on the CSC may drive important advances in our understanding of cancer biology and developing potential cures for these devastating diseases.MiRNAs are small (22–25 nucleotides in length) noncoding RNAs that can effectively reduce the translation of target mRNAs by binding to their 3-untranslated region (UTR). let-7 is originally identified in Caenorhabditis elegans as a regulator of developmental timing and cellular proliferation. The expression level of let-7 is reduced in human lung cancer, and it is show to be the anticancer miRNA that represses RAS and/or HMGA2 expression at the translational level. A recent study suggests that let-7 suppresses tumorigenesis via alteration of self-renewal and differentiation of breast cancer stem cells. Therefore, analyzing the relationship between lung cancer stem cell and let-7 may insight into let-7-mediated tumor suppression and also establish unique treatment for lung cancer.ObjectiveTo isolate and characterize of tumorigenic, stem-like Sca-1 positive cells in lewis lung carcinoma( LLC) cell line, and research the expression levels of let-7 to assess the function of let-7 in Sca-1+LLC cells.Methods 1. Immunofluorescence staining technology was used to detect the expression of the putative stem cell marker Sca-1 in lewis lung carcinoma. Then Sca-1+LLC cells and Sca-1-LLC cells were sorted by magnetic activated cell sorting from lewis lung carcinoma cell line, and the purity was analyzed by flow cytometry. After isolation, Sca-1+LLC cells and Sca-1-LLC cells were cultured in 10% FBS RPMI-1640 culture medium or serum-free medium supplemented with bFGF, EGF, and ITS. To test the hypothesis that Sca-1+LLC cells were enriched for tumor-initiating cells, the ability of self-renewal, differentiation and resistant to drugs were evaluated in vitro. In vivo, Sca-1+LLC and Sca-1-LLC cells were inoculated into C57BL/6 mice at different injection dose to assess the tumorigenic distinction.2. Total RNA was extracted respectively from Sca-1+LLC cells and Sca-1-LLC cells using Trizol reagent. Then the TaqMan?MicroRNA Assays were used to detect and accurately quantify mature let-7 family, including of let-7a, let-7c, let-7d, let-7f, and let-7g. The relative amount of gene transcripts was normalized to snoRNA202.3. In order to generate the recombinant lentiviruses which carrying pre-let-7a gene, the pre-let-7a cDNA was amplified by PCR technique and subcloned into the lentiviral vector, pGCSIL-GFP, to generate the lentiviral expression vector, pGCSIL-let-7a. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pGCSIL-let-7a with packaging plasmids pHelper 1.0 and pHelper 2.0. The viral supernatant was collected 48 h after transfection, and viral titers determined by transducing 293T cells at serial dilutions and analyzing GFP expression by real-time PCR.4. The LLC cells were infected by lentiviral expression vector and the control vector at the MOI of 2, 20, or 200, and the infectivity was determined by counting the cells that showed fluorescence under microscopic examination. Then the Sca-1+LLC cells were sorted by magnetic activated cell sorting and respectively named Lenti-let-7/Sca-1+LLC cells and Lentivector/Sca-1+LLC cells. The difference between Lenti-let-7/Sca-1+LLC cells and Lentivector/Sca-1+LLC cells were evaluated by self-renewal assays, proliferation assays, chemoresistance assays and tumorigenicity assays.5. To assess the effectiveness of in vivo gene therapy, some groups received let-7a lentiviral vector or control vector intratumoral injection when the volume of lewis lung carcinoma reached to 100～200mm3, and other groups received let-7a lentiviral vector in combination with paclitaxel. Tumor size was followed in the case of treatment with lentiviral vector, and the mice were sacrificed after 18 d to assess tumor formation rate, weight, lung metastasis, and the expression of K-Ras protein.Results1. Under the fluorescence microscope, a small proportion LLC cells had sparse light of red fluorescence, and flow cytometry shown that (10.23±1.65) % of LLC cells expressed the membrane antigen Sca-1. After isolation by Magnetic Cell Sorting, the Sca-1 positive fraction was raised to (93.92±1.07) %. Under the serum-free medium, Sca-1+LLC cells could grow as nonadherent, multicellular spheres, and had clonogenic frequencies of 10%. Whereas, the clonogenic frequencies was only 1% in Sca-1 - LLC cells. Under serum-cultured conditions, Sca-1+LLC cells could differentiate into Sca-1-LLC cells but Sca-1-LLC cells could not differentiate into Sca-1+LLC cells. Sca-1+LLC cells exhibited higher resistance to chemotherapeutic drugs than Sca-1-LLC cells.The drug-resistance machanism may be relate to the over expression of ABCG2 mRNA. In vivo, the tumor formation ability of Sca-1+LLC cells were 25 times higher than Sca-1-LLC cells.2. Real-time PCR analysis showed that let-7a, let-7d and let-7g were markedly reduced by 1.32-fold, 1.27-fold, and 1.45-fold relative to control, respectively. The changes in expression levels of let-7c and let-7f were not significantly different from controls.3. The sequence of cloned DNA was in conformity with the sequence deposited in GeneBank (MI0000557) by PCR, enzyme digestion and sequencing. The recombinant lentiviruses which carrying pre-let-7a gene could be product by co-transfection pGCSIL-let-7a to 293T cells, and the titer of virus reached to 2×109TU/ml.4. The infectivity of the lentiviral vector was approximately 70% when infected at the MOI of 20. After infection, the percent of Sca-1+LLC cells in LLC cell line was decreased to 5.47%. Comparing with the lentivector/Sca-1+LLC cells, the lenti-let-7/Sca-1+LLC cells had lower clone formation efficiency, and more sensitiveness to chemotherapeutic drugs. lentivector/Sca-1+LLC cells generated a tumor with 2×104cells compared with at least 5×10~5 needed for lenti-let-7/Sca-1+LLC cells.5. A single intratumoral injection of the let-7a lentiviral vector can significantly reduce the volume of primary tumor and the number of lung metastasis. Moreover, let-7a lentiviral vector can completely regressed tumors in combination with paclitaxel in 33% of animals.These inhibition was correlated with a decrease in K-Ras protein.Conclusion1. Sca-1 is one of the markers for cancer stem cells in Lewis lung carcinoma cell line, and Sca-1+LLC cells exhibit characteristics of a tumor-initiating, cancer stem cell phenotype.2. let-7 expression is markedly reduced in Sca-1+LLC cells compared with Sca-1-LLC cells, and maintenance of an undifferentiated state of Sca-1+LLC cells require reduced let-7 levels.3. The let-7a lentiviral vector offers a powerful new strategy for lung cancer gene therapy. Combination of let-7 and chemotherapy may be an effective way to cure lung cancer.
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