Identification Of Metastasis And Invasion Related Micrornas And Investigate The Function In SCLC

Part Ⅰ Preliminary screening of invasiveness related miRNA of SCLCObjectives:Screening the invasion and metastasis related microRNAs in SCLC patients.Methods:MiRNA micro Array analysis was used to detect the expression difference of miRNAs in untreated patients’ serum between extensive stage and limited stage SCLC patients. Selected the appropriate objects as external reference of serum miRNA and calculated the sample amplification curve to get the best sample adding concentraion. Prism5was used to design miRNA stem-loop primer and RT-PCR primer. The concentration of primer was adjusted to warrant the validity of the results.31SCLC patients’ serums were used to detect the miRNAs expression to further confirm the result of microArray analysis. And21SCLC patients’ in situ cancer tissue were tested by RT-PCR to analysis the expression difference betweeun extended stage and limited stage SCLC pattents and the relationship of miRAN expression between serum and tissue in situ. The patients’ PFS prognosis analyses were made by COX univariate analysis, Kaplan-Meier survival analysis and COX multivariate regression analysis used screened miRANs.Results:MiRNA microArray result showed that there were significant differences between extensive stage and limited stage SCLC patients’serum as blow higher expression:miR-3074-5p(p<0.001), miR-574-5p(p=0.014), miR-874(p=0.038), miR-30e-5p(p=0.040), miR-4746-3p(p<0.001) and miR-4685-3p(p<0.001); lower expression:miR-718(p=0.01), miR-1249(p=0.046), miR-4253(p=0.038), miR-184(p=0.001), miR-4459(p=0.030), miR-4298(p=0.033), miR-4706(p<0.001), miR-4655-5p(p=0.040) and miR-671-5p(p=0.026). Cel-mir-39-3p was chosed as the external reference and25fmol cel-mir-39-3p was determined add into each500ul serum sample. The primers were designed reasonablely as acting stable efficency and specific amplification. RT-PCR results showed that there were significant differences bewteen extended stage and limited stage SCLC patients’ serum as below:higher-expression:miR-3074-5p(p=0.026), miR-574-5p(p=0.010), miR-4685-5p(p=0.026), miR-4746-3p(p=0.019), miR-874(p=0.015); lower-experession:miR-184(p=0.002), miR-4459(p<0.001), miR-4298(p=0.017), miR-4706(p=0.019). The miRANs differneces between extended stage and limited stage SCLC patients’ in situ cancer tissue were including:higer-expression: miR-574-5p(p<0.001), miR-4746-3p(p=0.001); lower-expression:miR-184(p<0.001), miR-4706(p<0.001), miR-4298(p=0.001), miR-4459(p=0.012). A comparison of the miRNA expression relationship between serum and tissues provided the results as miR-184(p<0.001,r=0.978), miR-4298(p<0.001,r=0.966), miR-574-5p(p<0.001,r=0.875), miR-4459(p<0.001,r=0.844), miR-3074-5p(p<0.001,r=0.843) and miR-4706(p<0.001,r=0.725) were all significant associated. COX univariate analysis showed that miR-3074-5p (p=0.039), miR-574-5p (p=0.003) and the metastasis stage (p<0.001) were the risk factors of prognosis and hsa-mir-184was the protection factor of prognosis. Kaplan-Meie survival analysis (Log-Rank analysis) finded that miR-184(p<0.001), miR-574-5p (p=0.001), miR-4459(p=0.04) and metastasis stage (p<0.001) were significantly associated with PFS of patients. COX multivariate regression analysis showed that high expression of miR-574-5p (p<0.001) and metastasis stage (p<0.001) were the independent risk factors for PFS of SCLC patients.Conclusions:①There are miRNAs expression differences between extended stage and limited stage SCLC patients’ serum and in situ tissue;②here are miRNAs significant associate with SCLC patients’ serum and in situ tissue;③miR-574-5p and metastasis stage are the independent risk factors of SCLC patients’ PFS. Part Ⅱ Candidate miRNA affect the biological behavior of SCLC cell lines H446Objectives:Study on the behavior changes of SCLC cell line H446impact of candidate miRNAs. Further determine which miRNAs can affect H446invasion ability.Methods:RT-PCR was used to detect the transfection efficiency of miRNA mimics or inhibitors to H446. MTT assay was used to detect the candidate miRNA impact on H446cell growth, and used colon formaion and growth curve to further verify the function. Wound healing assay and transwell (migration/invasion) assays were used to detect the invasion or migration ability influenced by candidate miRNA.3D cell culture assay was used to find out the ablity change by candidate miRNA of H446to form vascular mimicry. RT-PCR was used to detect the miRNA expression differences between H446and HFL-1cells and media on the purpose of find out whether there existed differences between SCLC cell lines and normal lung-derived cells.Results:Select miR-3074-5p, miR-574-5p, miR-4685-5p, miR-4746-3p, miR-874, miR-184, miR-4459, miR-4298and miR-4706to study their effects on biological behavior changes of H446. The transfection effiency of miRNA mimics or inhibitors were tested by RT-PCR, and reslutes show that the miRNAs expression of H446was increased or reduced in24h,48h and72h (p<0.001). MTT assay indicated that miR-3074-5p mimics can promoted cell growth at48h (p<0.001) and72h (p<0.001), moreover, miR-3074-5p inhibitor showed repression of cell growth at48h (p=0.01)、 72h (p=0.003). RT-PCR resluts pointed that mimics transfection efficiency sustainedly increased to day5(p<0.001), day7(p<0.001), day10(p=0.001) and day15(p=0.002). On the contrary, miRNA inhibitor transfection efficiency recur to normal level since the7th day. All that lead to miRNA mimics can be only used to complete the colony formation and growth curve assay. Colony formation assay showed that transfected miR-3074-5p mimic lead to promoting cell growth of H446(p=0.005). The growth curve showed that miR-3074-5p mimics promote cell growth at day3(p=0.008), day5(p=0.027), day7(p=0.001) and day10(p=0.012). Wound healing assay showed that12h after the wound was made, miR-574-5p mimic (p=0.001) or miR-184inhibitor(p=0.033) promote cell migration, meanwhile, miR-574-5p inhibitor (p=0.046) or miR-184mimic (p=0.042) can inhibite cell migration. This phenomenon lasted to24h and even more obvious.24h after the wound was made miR-574-5p mimic (p=0.003) or miR-184inibitor (p=0.003) still promote cell migration, meanwhile, miR-574-5p inhibitor (p=0.000) or miR-184mimic (p=0.003)can significantly inhibite cell migration. Transwell migration assays confirmed that miR-574-5p mimics promote cell migration (p<0.001), and miR-574-5p inhibitor can inhibit cell migration (p<0.001); miR-184mimic can inhibit cell migration (p<0.001), and miR-184inhibitor can promote cell migration (p<0.001). Further transwell invasion was found that, miR-574-5p mimic promoted cell invasion (p<0.001), and miR-574-5p inhibitor can inhibit cell invasion (p <0.001); miR-184mimic inhibit cell invasion (p=0.001), and miR-184inhibitor can promote cell invasion (p=0.001).3D cell cultures found that miR-184can increase the tube area (p=0.001) and length (p<0.001) at6h, the effect still exits at10h (area:p<0.001, length:p=0.002). RT-PCR results showed that miR-184(p=0.001) and miR-574-5p (p=0.013) in cell culture Medium of H446were more than that in RPMI1640. After transfected miR-574-5p into H44648h, the expression of cell culture medium was increased than before(p=0.002), but after transfected miR-184into H44648h, the expression of cell culture medium was increased without statistical difference than before (p=0.062). The expressin of miR-184(p=0.002) and miR-574-5p (p=0.033) of HFL-1cell culture medium were higher than that in F12K. The expression of miR-184(p=0.01) and miR-574-5p (p=0.013) of H446cell culture medium were higher than that of HFL-1.Conclusions:①miR-3074-5p can promote the growth of H446;②miR-184could inhibit the invasion and migration of H446, miR-574-5p could promote invasion and migration of H446;③miR-184could inhibit the ability of H446vascular mimicry forming;④miR-574-5p expression of SCLC cell line is higer than that of lung fibroblasts compared;⑤miR-574-5p and miR-184were secreted miRNAs, transfected miR-574-5p mimic into H446could increase the secretion. Part Ⅲ Forecasting and identification target genes of hsa-mir-574-5p and hsa-mir-184Objectives:Identification the gene targets of miR-574-5p and miR-184Methods:Bioinfomatic analysis was made to forecast the gene targets of miR-184and miR-574-5p. RT-PCR was used to detecte the impact of miR-184and miR-574-5p on their targets’ mRNA. Western Blot was used to detecte the impact of miR-184and miR-574-5p on their targets’protein expression. Dual luciferase reporter assay was used to confirm the targets of miR-184and miR-574-5p.Results:Bioinfomatic analyses predicted the binding sites for miR-184and miR-574-5p and choose the one involved in invasion and migration was selected as PTPRU and EPAS1. RT-PCR results showed that miR-574-5p mimic can refrain PTPRU mRNA expression (p=0.008), and miR-574-5p inhibitor can promote the expression of PTPRU mRNA (p=0.000). MiR-184mimic can refrain the expression of EPAS1mRNA (p=0.039), and miR-184inhibitor can promote the expression of EPAS1mRNA (p=0.005). Western blot results indicated that miR-574-5p mimic reduce the expression of PTPRU protein in H446, meanwhile miR-574-5p inhibitor increased the expression of PTPRU protein. MiR-184mimic reduced the expression of EPAS1protein in H446, miR-184inhibitor increase the expression of EPAS1 protein in H446. PCR and gene sequencing results showed that dual luciferase reporter plasmid was constructed successfully. Dual luciferase reporter showed that: miR-574-5p acted directly on the3’UTR region of PTPRU (p=0.004.); miR-184act directly on the3’UTR region of EPAS1(p<0.001).Conclusions:①PTPRU is the direct target of miR-574-5p;②EPAS1is the direct target of miR-184. Part Ⅳ Preliminary investigate the mechanism of hsa-mir-574-5p and hsa-mir-184affact invasion and imgrationObjectives:Preliminary investigate the regulation mechanism of hsa-mir-574-5p and hsa-mir-184involved in H446invasion and migration capabilities.Methods:RT-PCR was used to detect the mRNA expression of β-catenin in H446after transfected with miR-574-5p or miR-184mimic or inhibitor. Western blot was used to detect the total protein and tyrosine phosphorylation of β-catenin in H446after transfected with miR-574-5p or miR-184mimic or inhibitor.Results:RT-PCR results showed that miR-184mimic reduce the expression of β-catenin mRNA (p=0.000), and hsa-mir-184inhibitor increase the expression of β-catenin mRNA (p=0.005). But miR-574-5p mimic (p=0.608) or inhibitor (p=0.273) did not change the mRNA expressin of β-catenin. Western Blot results confirmed that miR-184mimic can increase the protein expression of β-catenin while miR-184inibitor decreased the expression. The expression of total β-catenin did not change after transfected with miR-574-5p mimic or inhibitor, but the tyrosine phosphorylation state of β-catenin significant increasd or decreased.Conclusions:①miR-184can inhibit the mRNA and protein expression of β-catenin in H446;②miR-574-5p can inhibit the tyrosine dephosphorylation of β-catenin in H446.