MiR-223 Mediated Regulation Of The Malignant Phenotype Of Lewis Lung Cancer Cell Line

Lung cancer is the leading cause of cancer deaths worldwide because of its high incidence and mortality, the most common forms is the so-called non-SCLC (NSCLC) including adenocarcinoma (AC), squamouscell carcinoma (SCC) and large cell carcinoma (LCC). Despite continuous efforts to improve the therapeutic response,the overall five-year survival rate for such tumors is lower than 15%. Recently, the cancer stem cells (CSCs) theory has been proposed to explain the tumor heterogeneity and the carcinogenesis process. These cells can be expanded in vitro as tumor spheres, while reproducing the original tumor when transplanted in immunodeficient mice. Differences of Genetic expression and surface marker between non-CSCs and CSCs may provide novel therapeutic targets.MicroRNAs (miRNAs) are endogenous small noncoding RNAs (18– 25 nucleotides) that negatively regulate the gene expressions at the posttranscriptional level by base pairing tothe 3' untranslated region of target messenger RNAs. It is revealed that miRNAs regulate various physiological and pathological pathways such as cell differentiation, cell proliferation, and tumoriogenesis. microRNA expression patterns as potential biomarkers for diagnosis, prognosis, personalized therapy,and disease management is just starting to emerge.Growing evidences suggest regulatory roles of miRNAs in malignant phenotype of cancer stem cells. The expression pattern of miRNA is often developmentally regulated and/or tissue specific, although some miRs are steadilyexp ressed in the whole organism. Our research would afford new avenues for exploring the functional impact of miR-223 on proliferation and differentiation such as malignant phenotype of cancer metastatic stem cells.objective1. To find out and identify the metastatic tumor stem-like cells in lewis lung cancer cell line.2. To analyze the differential expressions of miR-146a, miR-206, miR-223 and let-7c-1, such as cell differentiation-related miRNAs,in CXCR4-positive and CXCR4-negative subsets of LLC,and then find the common bioinformatics features of miR-223.3. To explore the functional impact of miR-223 on proliferation and differentiation such as malignant phenotype, and illuminate the molecular mechanism of the regulation of miR-223 on the IGF1R signaling pathway.Methods1. The expression of Sca-1 and CXCR4 in LLC were measured by flow cytometry, and dual-color immunofluorescent staining cells were observed by laser scanning confocalmicroscope (LSCM). After the CXCR4-positive cells were isolated from LLC by magnetic cell sorting, its properties were evaluated by their tumorigenic and metastatic potentials.2. Total cellular RNA were extracted by Trizol, expression of four miRNAs were detected by real-time fluorescence quantitative PCR (TaqMan probe), and the most differentially significant expression of miRNA was predict potential target genes. Immunohistochemistry was carried out to confirm differential expression of the key molecule of certain research value within CXCR4-positive and CXCR4-negative subsets growing tumor tissue, and a BLAST search based on orthologs was performed to identify homologies of its 3'-UTR.3. A recombinant eukaryotie expression vector , pGenesil-mmu-mir-223 , was constructed and expressed in LLC. After 48 hours, the effects of miR-223 on adhesion and motility potential of LLC were investigated with wound assay and transwell assay. The cell cycle of LLC treated with miR-223 were analyzed by flowcytometry. The production of VEGF and MMP9 were detected by ELISA . Realtime-RT-PCR and western Blot were used to investigate the effects of miR-223 on the expression of predicted target gene.Results1. Sca-1and CXCR4 positive cells were counted for 0.1% of the total number of LLC,and CXCR4+ cells for 0.18% by flow cytometry. Dual-color immunofluorescent staining cells were identified by LSCM. Trypan Blue staining analysis shown the viability of LLC was (95.58±0.87) %. After isolation, the viability of the CXCR4-positive LLC was(94.96±0.76) % ( P > 0. 05). CXCR4-positive LLC suspension cultured in a serum-free medium, cell spheres were formed. CXCR4- positive LLC generated a tumor with 7×103 cells compared with at least 2×105 needed for CXCR4-negative.2. Most of CXCR4-positive LLC were close to vascular endothelial cells,aberrant vasculature around it was forming. The expression of VEGF mRNA in CXCR4-positive LLC(0.440±0.006) was higher than that in CXCR4-negative LLC (0.290±0.003) (P<0.01), MVD of CXCR4 -positive subsets growing(56.87±4.83) was higher than of CXCR4-negative subsets growing tumor tissue(43.52±4.91) (P<0.01).3. Transmembrane cells of CXCR4-positive LLC (109.67±20.03) was more than of CXCR4-negative LLC (53.67±13.01) (P<0.05), the expression of MMP9 mRNA in CXCR4-positive LLC(0.622±0.014) was higher than that in CXCR4-negative LLC (0.579±0.017) (P<0.05), and the metastases rate in CXCR4-positive group (2×104 inoculated) was clearly higher than that in CXCR4-negative group (5×105 inoculated).4. The chemotherapy sensitivity to cisplatin of CXCR4-positive LLC were lower than of CXCR4-negative LLC (P<0.05) .The expression of ABCG2 mRNA in CXCR4-positive LLC(0.524±0.008) was higher than that in CXCR4-negative LLC (0.387±0.007) (P<0.01).5. Compared to CXCR4-negative subsets , the expression of four miRNAs were lower in CXCR4-positive subsets ,and expression of miR-223 has the most significant difference (Fold Change = 8.26). Through softwares forecasting, miR-223 have potential target sites of IGF1R, IGFBP5, Pik3cb , ELK-1and E2F1 mRNA, such as key molecular of IGF1R signaling pathway. The expression of IGF1R mRNA in CXCR4-positive LLC(0.421±0.007)was higher than that in CXCR4-negative LLC(0.185±0.007) (P<0.01), and the expression of IGF1R of CXCR4-positive subsets growing tumor tissue was significantly higher than of CXCR4-negative subsets. miR-223 was lowly expressed in CXCR4 positive cell from Lewis lung carcinoma cell lines. Position 238～244nt and 688～695nt in target sequences of 3'-UTR of IGF1R was high homologous by screening.6. After 48h, the relative moving distance on matrigel of miR-223 expressed group(218.67±39.80μm)was shorter than of normal control group(341.67±35.92μm) (P<0.05), the counting Transmembrane cell of miR-223 expressed group (60.67±12.66) was lower than of normal control group(100.33±14.01) (P<0.05), the concentration of MMP9 in the supernatant of miR-223 expressed group (214.16±28.18 pg/mL) was Less than normal control group(1139.14±50.13pg/mL) (P<0.01), and the proportion of G0/G1 phase cell in miR-223 expressed group was reduced from 53.8% to 45.3%, while the proportion of G2 phase cell in miR-223 was increased from 4.29% to 13.2%.7. After 48h, the expression of IGF1R, CXCR4, CDK2, and MMP9 mRNA in miR-223 expressed group were reduced respectively to 17,12,25 and 15 fold of normal control group,and the expression of IGF1R, CXCR4, CDK2, and MMP9 protein were decrease. in additional,AKT and ERK signaling were significantly inhibited.Conclusion1. CXCR4-positive LLC had some properties of cancer metastic stem cells, such as the ability to self-renew and metastases.2. miR-223 in the CXCR4-positive LLC cells was significantly lower expression,and it may be related to regulation of the IGF1R signaling pathways.3. As miR-223 overexpressed, the expression of predicted target genes such as IGF1R, CXCR4 and CDK2 were decreased, Akt and Erk pathway as the downstream of IGF1R were significantly inhibited, The secretion of VEGF and MMP9 was reduced, the invasion and migration of LLC was obviously supressed, and the proportion of G0/G1 cells was declined. Therefore, we conclude that miR-223 was extensively involved in the regulation of IGF1R signaling pathway in LLC.