1. MicroRNAs’ Transfection Affect Biological Behavior Of Lung Cancer Cell Lines And The Relationship Between Micrornas’ Expression And Prognosis In NSCLC2. Mutation Analysis Of EGFR, K-Ras And B-Raf By X-TAG Multiplexed Microsphere-based Flow Assay An

Background MicroRNAs is a kind of no-coding RNA. MicroRNAs repress geneexpression by inhibiting mRNA translation or by promoting mRNA degradation. Theyregulate multiple important cellular processes ranging from cell proliferation to apoptosis.Aberrantly expressed microRNAs are a hallmark of cancer, and microRNA expressionprofiling is associated with tumor progression and response to chemotherapy, suggestingtheir potential application as prognostic and predictive biomarkers. But, the role ofmicroRNAs in lung cancer remains elusive.Methods We have examined expression of miR-888，miR-892b，miR-550a，miR-34ain14cell lines, including9lung cancer cell lines and5HBE cell lines. We transfectmiR-888，miR-892b，miR-550a，miR-34a in4lung cancer cell lines, A549, H1299,H460, H1299, respectively. Then get the growth curve of the cancer cell lines whichtransfected Lentiviral-Hsa-miRs. We have therefore retrospectively examined expressionof miR-888，miR-892b，miR-550a，miR-34a，miR-30b, miR-30c, miR-221, miR-222,miR-103and miR-203in41Formalin-Fixed Paraffin-Embedded tissue samples fromNon-Small Cell Lung Cancer (NSCLC) patients when tyrosine kinase inhibitors (TKIs)were used as first line therapy. The short-term response after TKIs therapy, progressionfree survival, overall survival and other clinical characteristics were compared betweenmiRNAs expression high level and low level cohorts. Results We found significant miR892b expression difference between lung cancer celllines and HBE cell lines, except H1299cell line. The expression of miR-550a in lungcancer cell lines is higher than the expression in HBE cell lines. The expression of miR-34a in lung cancer cell lines is lower than the expression in HBE cell lines. Lentiviralcoated hsa-miRs has a high transfect rate. GFP expression stability, after more than10generations pass, the cell still stable express GFP. The expression of miRNAs weresignificantly increased. Parts of the cells transfected Hsa-miRNAs grow faster or slower,significantly. In the lung cancer patients’ FFPE tissue, We found significant correlationbetween expression of miR-203and the patients gender. Expression of miR-888，miR-892b，miR-550a，miR-34a，miR-30b, miR-30c, miR-221, miR-222and miR-103werenot significantly correlated with patients’ age, gender, histology, stage or smoking status.Furthermore, miR-30b and miR-30c expression correlated with short-term response anddisease control rate when TKIs used as first line treatment. Kaplan-Meier analysis furtherrevealed that the expression of miR-30b, miR-30c and miR-103a predicted overallsurvival rate(Log rank P=0.013，0.006and0.038, respectively) in the examined cohort.Expression level of miR-203was significantly correlated with progression free survivalwhen TKIs used as first line therapy (Log rank P=0.005).Conclusions Taken together, our study identified miRNAs expression specific in cellsand tissues. Cells transfection with miRNAs can affect the biological characteristics. TheFormalin-Fixed Paraffin-Embedded tissue samples can be used to detect the expressionlevel of miRNAs. Our study identified miR-30b, miR-30c and miR-103a as usefulprognostic predictors in NSCLC patients who underwent first line treatment with TKIs.The miR-30b and miR-30c expression level also could be used to forecast the reponse ofthe NSCLC patients when TKIs used as first line treatment. Objective: To investigate gene mutation information of EGFR, K-Ras and B-Raf inChinese patients with NSCLC，analyze the relationship between gene mutation and theclinicopathological feature, EGFR-TKIs efficiency and the value of prognosis.Methods: Using x-TAG multiplexed microsphere-based flow assay to detect EGFR/K-Ras/B-Raf mutation of126NSCLC patients in Beijing Chest Hospital from March2006to May2011，and analyzing the relationship between gene mutation information and theclinicopathological character, gene mutation’s possibility of being a prognosis andpredict maker.Methods: Using x-TAG multiplexed microsphere-based flow assay to detect EGFR/K-Ras/B-Raf mutation of126NSCLC patients in Beijing Chest Hospital from March2006to May2011，and analyzing the relationship between gene mutation information and theclinicopathological character, gene mutation’s possibility of being a prognosis andpredict maker.Result：49patients are detected EGFR mutation in126patients with NSCLC（mutation rate is38.9%）. All the mutation are single point mutation, and no multiple points mutation are detected. We didn’t find the EGFR exon20mutation in these patients.EGFR mutation rate of adenocarcinoma and bronchioloalveolar are higher than that ofnon-adenocarcinoma (P=0.038). EGFR mutation rate is higher in female patients orpatients without smoking history than male patients or patients with smoking history(P=0.044, P=0.042, respectively) in NSCLC patients. The overall survival of the patientswith EGFR sensitive mutation is longer than those patients without EGFR sensitivemutation, but didn’t find sensitive difference (P=0.169). The response of the patients withEGFR sensitive mutation be given TKIs as first line therapy is better than those patientswithout EGFR sensitive mutation, didn’t find sensitive difference (P=0.159).10patients are detected K-Ras mutation in100patients with NSCLC （mutation rateis10.0%）. We found6kinds of K-Ras mutatoin totally. They are K-Ras codon12G12Rmutatoin；K-Ras codon12G12V mutatoin；K-Ras codon12G12C mutation；K-Rascodon13G13Dnutation； K-Ras codon61Q61H muttoin； K-Ras codon61Q61Lmutation.4cases have both EGFR sensitive mutation and K-Ras mutatoin.All themutation are single point mutation, and no multiple points mutation are detected. Wedidn’t find the relationship between K-Ras mutation and clinicopathological feature, suchas gender, smoking statues, pathology and age. But the overall survival of the patientswith K-Ras mutation is shorter than those ptients without K-Ras mutation (P=0.758).10patients are detected B-Raf mutation in110patients with NSCLC （mutation rateis9.1%）. We found1kind of B-Raf mutatoin, B-Raf Exon15codon600V600Emutation. We didn’t find the relationship between B-Raf mutation andclinicopathological feature, such as gender, smoking statues, pathology and age.Conclusion: In this study, the rate of EGFR sensitive mutation is similar as the otherreports in Asian people, is higher than Caucasian. EGFR mutation rate is relative with theclinicopathological, such as gender, pathology, smoking statue and race. The patients with EGFR sensitive mutation have better therapy response and longer overall survival.The patients with K-Ras mutation have lower response to the therapy and shorter overallsurvival than those patients without K-Ras mutation. The rate of B-Raf is lower than10%, the mutation statures doesn’t have relationship with the clinocopathologicalfeature.In this study, we use the x-TAG multiplexed microsphere-based flow assay todetect the gene mutaion. It can be used as clinical detection.