| BackgroudCRC (colorectal cancer) is one of the common malignant tumors. In china, along with the development of economy, the life style changing, especially diet, the incidence of CRC is increasing. The morbidity and mortality is third and fifth in malignant. As is known to us that the morbidity of colorectal cancer is a multi-factor, multi-step complex process. In the early time of colorectal cancer, up to90%of patients can be cured by surgical if the disease is detected at the early stage, but unfortunately most patients were diagnosed at an advanced stage and the prognosis is poor. Thus, strengthen the basic research of the mechanism in the development of colorectal cancer, found and identified with high specificity and sensitivity of colorectal cancer metastasis molecular marker, providing the new target for the development, prognosis and intervene of colorectal cancer, it’s significantly for improving the cure rate of colorectal cancer.,The discovery of tiny RNA(microRNA)is a groundbreaking scientific discovery in the21st century, a large number of researchs have shown that the miRNA plays an important role in the development and progression, metastasis of tumor. MiRNAs are an abundant class of17-25nucleotides small noncoding RNAs, aslo a set of short RNA which are no-encoding protein sequences, miRNA widely exist in eukaryotes. Mature miRNA binds to its complementary site of an mRNA by base-pairing to regulate the expression of gene. To play a role by inducing target mRNA degradation or translational inhibition. MiRNA participate in regulating cell differentiation, growth, development, proliferation, metabolism, apoptosis and so on, the imbalance expression of miRNA will lead to serious diseases such as cancer.MiRNA is closely associated with tumorigenesis, a large number of reports revealed that miRNAs are abnormal expression in a variety of cancers. MiRNA play as a oncogene or anti-oncogene in the development and progression of cancer. MiRNA regulate the progress of tumorigenesis by regulating cell proliferation, migration and invasion, EMT(epithelial-mesenchymal transition), angiogenesis and apoptosis. In recent year, more and more researchs revealed that along with the imbalance expression of miRNA in the development process of the colon cancer. Some miRNA play a role by regulating oncogenes or tumor suppressor genes in colon cancer, other oncogenes or tumor suppressor genes play a role by regulating miRNA.A good deal of researchs show that miRNA is also involved in regulating Wnt signaling pathway, we know that WNT/β-catenin pathway plays an important role in the tumorigenesis and metastasis of colorectal cancer. Research shows that microRNA-145can play a role in colorectal cancer cells through targeting catenin delta-1to regulate WNT/β-catenin signaling pathway. In addition, research revealed that miRNA can regulate target genes to regulated WNT/β-catenin pathway is also involved in the tumorigenesis and metastasis in oral squamous cell carcinoma, lung cancer, pancreatic cancer, prostate cancer and esophageal cancer.In previous study, our lab found that the miR450b-5p was differentially expression in microarray analysis of colorectal cancer tissues. Base on the previous study, the purpose of this study is to examine the expression of miR450b-5p in CRC tissues and cells. Analysis the role of miR450b-5p in prognosis, investigate the effection on biological characteristics in CRC cells and explore the molecular mechanisms of miR450b-5p in CRC cells, we hope that this study will improve the better understangding of CRC and find new targets of molecular targeted therapy for CRC.Materials and Methods1. Clinical specimens 90cases of colorectal cancer tissues,30cases of primary CRC biopsy specimens and their matched adjacent tissues were obtained from Nanfang Hospital (Southern Medical University,Guangzhou,china). All specimens were diagnosed colon cancer in pathology, the patients have not received chemotherapy treatment. The research protocols were approved by Ethics Committee of Nanfang Hospital.2. Cell cultureThe human CRC cell lines SW480and HCT116were cultured in PRMI-1640with10%FBS, HEK-293FT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with5%FBS, all cells were cultured in a humidified atmosphere of95%air and5%CO2at37℃.3. Real-time PCRTotal RNA was extracted from cells or tissues. Reverse transcription into cDNA by All-in-OneTMFirst-Strand cDNA Synthesis Kit or Reverse Transcriptio nSystem, then detected by All-in-OneTMqPCR Mix or SYBR Green Realtime P CR Master, all samples were normalized to internal controls and fold changes were calculated through relative quantification (2-△△Ct)4. Western blotTotal Protein was extracted from cells and quantitated using BCA assay, the protein lysates were separated by10%-13%SDS-PAGE, and electrophoretically transferred to PVDF membrane, then the membrane was incubated with antibodies and detected by chemiluminescence.5. Cell biology experimentsColony formation assay, Soft argar assay, MTT assay and detect cell cycle by Flow cytometry.6. Luciferase report assayAccording to the instructions of Dual-Luciferase(?)Reporter Assay System, at48h post-transfection with Luciferase Reporter genonic vector and mimics/inhibitor, collected and detected the cell lysis buffer, relative luciferase activity was measured by the activity of firefly luciferase and renilla luciferase.7. Immunofluorescence staining Using immuno-fluorescence staining method to detect the expression of βcatenin in colorectal cancer cells after overexpressing or disturbing miR450b-5p.8. Cytoplasmic and nuclear protein extraction assayAccording to the instructions of ProteoJETTMCytoplasmic and Nuclear Protein Extraction Kit, collected and lysed cells, cytoplasmic protein and nuclear protein were extracted respectively, western blot detected the the expression of interest protein in nucleus protein and cytoplasmic protein, respectively, and cytoplasmic protein used a-tubulin and nuclear proteins used LaminB1as an internal control.9. Vector construction(1) Plvthm/miR450b-5p and plvthm/miR450b-5p mutant:Use human colon cancer cell genomic DNA as template.A486bp fragment of miR450b-5p was amplified by PCR and cloned into Plvthm. The vector was named plvthm/miR450b-5p. Then use plvthm/miR450b-5p as template, change the acting site of miR450b-5p, construction the plvthm/miR450b-5p mut.(2) PGL3-SFRP23’-UTR-WT and PGL3-SIAH13’-UTR-WT:Human colon cancer cell genomic DNA as template, SFRP2-3’-UTR (492bp) and SIAH1-3’-UTR (429bp) were amplified by PCR and cloned into PGL3-Basic vector. The vector were named PGL3-SFRP23’-UTR-WT and PGL3-SIAH13’-UTR-WT.10. Production and purification of lentivirusThe transfer and the packaging plasmids were co-transfected into293FT c ells using CaPO4precipitation. The supernatant was harvest48-72h post-transfe ction. Then, the viral particles were concentrated by ultracentrifugation, and the viral pellets were resuspended. The titration of lentiviral suspension was calcu lated by making a tenfold serial dilution.11. Cell transfection(1) Transient transfection:The transfection was performed using LipofectamineTM2000reagent.(2) Lentivirus infection:The packaged lentivirus were used to infect CRC cell lines. Following fluorescence-activated cell sorting, the GFP+cells were isolated, and the cell lines were established, which could stably overexpress miR450b-5p.(3) miRNA and plasmid co-transfection:According to the instructions of LipofectamineTM2000reagent. A working concentration of miRNA was50nM,the recombinant plasmid PGL3-SIAH1-3’-UTR or PGL3-SFRP2-3’-UTR was0.5μg, and the concentration of pRL-SV40was O.O5μg in a24-well plate.12. Animal experimentsSW480/miR450b-5p and control cells SW480/plvthm randomly were inoculated in12nude mices subcutaneously, respectively, each group included six mices. Measure the length and diameter of tumorevery every six day with a vernier caliper, when the tumor volume exceeded900mm3, the experimental animals were sacrificed, removed tissue to Formalin-fixed, and then proceed to next step.13. ImmunohistochemicalThe nude mice tumor tissue made into paraffin sections, then by immunohistochemistry, Ki67staining, Determinate the result by the way of semi-quantitative.14. Statistical analysisSPSS13.0software was used for statistical analysis. The correlation between the expression of miR450b-5p and clinical pathological features in colorectal cancer were determined by Chi-square test and Spearman rank correlation analysis; Survival curves were plotted by Kaplan-Meier method, and using the Log-Rank test to compare the statistically difference in survival curve; Levels of miR450b-5p in primary colorectal tumors compared with their adjacent normal tissues were determined by the" Wilcoxon matched pair test; MTT assay were analyzed by factorial design analysis of variance. Realtime PCR, colony formation assay, softargar assay and Luciferase report assay were determined by two independent t-test. P<0.05was considered statistically significant.Results1. Identificate the expression of miR450b-5p in colorectal cancer and analysis the role of miR450b-5p in clinical prognosis.Real-time quantitative PCR was used to detect the expression of miR450b-5p in 30cases of paired colorectal cancer tissues, the result revealed that compaired with paraneoplastic normal tissue, the expression of miR450b-5p in colorectal tissue was higher significantly. Wilcoxon matched pair test revealed that the difference between two group was considered statistically significant (P<0.05). In addition, RT-qPCR was used to detected the expression of miR450b-5p in90cases of colorectal cancer tissues. The result revealed that the high expression of miR450b-5p was associated with pathological grade, clinical stage. Follow-up analysis in colorectal cancer revealed that the long-term survival in colorectal cancer patients with high expression of miR450b-5p below the patients with low expression of miR450b-5p (Log-Rank test, P=0.007).2. The impact of miR450b-5p on the biological characteristics in CRC cellsDetected the expression of miR450b-5p in SW480, HCT116, HT29, KM12, SW620, DLD1, LS174t, M5, HCT115, it revealed that the expression in HCT116was higher than SW480.After HCT116and SW480were transfected with miR450b-5p mimics or inhibitor, respectively. The results showed that the expression of miR450b-5p was up-regulated (P<0.05); but transfected with miR450b-5inhibitor, the expression of miR450b-5p was reduced (P=0.001). All these datas demonstrated that the transient transfection could effectively upregulate or downregulate the expression of miR450b-5p.The result of MTT assay displayed that miR450b-5p overexpression can promoted cell growth in SW480and HCT116cells at5day post-transfection (P<0.001); and disturbed miR450b-5p can inhibited cell growth (P<0.001).As demonstrated in colony formation assay, the colony number was15±2.645in SW480/plvthm and44±13.527in SW480/miR450b-5p (P=0.022), and the colony number was113±6.111in HCT116/Plvthm and606±8.000in HCT116/miR450b-5p (P<0.001). On the other hand, SW480and HCT116were transfected with miR450b-5p inhibitor, compared with control group, the colony number in SW480and HCT116were reduced (P<0.05). Softagar assay showed that the colony number was12.3±2.5in SW480/plvthm, and23.6±4.1in SW480/miR450b-5p; the colony number was14±3.0in HCT116/plvthm and37.0±4.0in HCT116/miR450b-5p, compared with control group, the groups of miR450b-5p overexpression formed more and larger colonies (P<0.05). On the contrary, SW480and HCT116were transfected with miR450b-5p inhibitor, compared with control group, the colony number in SW480inhibitor and HCT116inhibitor were small and the number was reduced (P<0.05)Flow Cytometry detected the cell cycle distribution revealed that SW480and HCT116cells transfected with miR450b-5p mimics displayed an decreased percentage of cells in G1-phase and more cells in S, G2/M phase (P<0.05), SW480and HCT116cells transfected with miR450b-5p inhibitor revealed opposite result, the percentage of cells in G1-phase were inceased and fewer cells in S, G2/M phase (P<0.05). These results suggested that miR450b-5p can promoted cell proliferation by regulating cell cycle.3. The effection of miR450b-5p on colorectal cancer cells in vivo tumor ability The growth of subcutaneous tumors in nude mice in miR450b-5p group was markedly increased compared to blank group (P<0.001).4. miR450b-5p inhibited the expression of SIAH1and SFRP2via binding to their3’UTRCotransfected PGL3-SIAH1/PGL3-SFRP2, miR450b-5p mimics, miR450b-5p inhibitor and miR450b-5p-mut into SW480and HCT116, the result of dual luciferase reporter assay showed a significant decrease of luciferase activity when miR450b-5P mimics and PGL3-SIAH1-3’UTR or PGL3-SFRP2-3’UTR was co-transfected in SW480and HCT116cells, On the contrary, a significant increase of luciferase activity was examined when miR450b-5P inhibitor and PGL3-SIAH1-3’UTR or PGL3-SFRP2-3’UTR was co-transfected in SW480and HCT116cells (P<0.05).The results of Western blot and qRT-PCR showed that compaied with control group, the protein and mRNA expression of SIAH1and SFRP2were decreased in miR450b-5p groups. When SW480and HCT116cells transfected with miR450b-5p inhibitor, the protein and mRNA expression of SIAH1and SFRP2were increased. These results suggested that SIAH1and SFRP2were targets gene of miR450b-5p.5. miR450b-5p can activate WNT/β-catenin pathway and promote the tumorigenesis of colorectal cancer.The result of TOP/FOP Dual-luciferase reporter assay showed that SW480and HCT116cells were transfected with miR450b-5p mimics have a higher Wnt/β-Catenin transcriptional activity than negative control cells (P<0.05). While the activity of Wnt/β-Catenin was decreased in SW480and HCT116Cells which were transfected with miR450b-5p inhibitor (P<0.05).The result of western blot after cytoplasmic and nuclear protein extraction assay revealed that the expression of β-catenin in the nuclei was up-regulated in the SW480/mimics and HCT116/mimics cells as compared with the control cells. On the contrary, the expression of β-Catenin was down-regulated in the SW480/miR450b-5p inhibitor or HCT116/miR450b-5p inhibitor cells as compared with control cells. At the same time, immunofluorescent staining showed that β-Catenin was mainly expressed on the cell membrane in SW480/NC or HCT116/NC cells, Less expression in the cytoplasm and nucleus.but when miR450b-5p overexpression in SW480and HCT116, β-Catenin was significantly enhanced in the cytoplasm and nucleus, when transfected with miR450b-5p inhibitor, the expression of P-Catenin was decreased in the cytoplasm and nucleus. These dates showed that miR450b-5p likely to make β-Catenin cytoplasmic accumulation and transport into the nucleus, activating Wnt signal pathway.Conclusions1. miR450b-5p at low expression in colorectal cancer, and is associated with clinical stage, pathological grade and long-term prognosis.2. miR450b-5p can promote the proliferation of colorectal cancer cells, participate in the development of colorectal cancer, affect CRC cell proliferation in vivo and vitro, which functions as a potential Oncogene in CRC.3. SIAH1and SFRP2are targets of miR450b-5p in CRC cells. 4. miR450b-5p possibly increase non-phosphorylated levels of β-Catenin, and make β-Catenin accumulation in the cytoplasm and transport into the nucleus, thereby activating the Wnt signal pathway to promote the proliferation of CRC cells. |
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