H827 H827-7 CCK-8. Gefitinib. A549 H358 H1299 H1650 H1975 mir-7. the half growth inhibition concentration IC H827

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1 Chin J Surg OncoApril Vol. 7 No. 2 mirna BK siwenliu1989@ 126. com fjif@ vip. sina. com mir-7 Gefitinib H827 7 /GR 7 CCK-8 RT-PCR H827 7 /GR A549 H358 H1299 H1650 H1975 mir-7 H827 /GR the half growth inhibition concentrationic 50 H827 H827 /GR A549 H358 H1299 H1650 H1975 mir-7 P < mir-7 mir-7 microrna-7 doi /j. issn Difference of mirna-7 expression in the non-small cell lung cancer with different sensitivity to gefitinib and their clinical significance LIU Siwen CAO Haixia WU Jianzhong MA Rong JING Changwen WANG Zuo FENG Jifeng. Department of Medical OncologyJiangsu Cancer HospitalNanjing China Corresponding author FENG Jifeng fjif@ vip. sina. com Abstract Objective To detect the difference of mirna-7 expression in the non-small cell lung cancer with different sensitivity to gefitinib and explore their clinical significance. Methods The gefitinib resistant lung cancer cell line 7 /GR was established from H827 by a method of repeated treatment with high dose of gefitinib for a short period. The 7 / GR cells were monocloned through the method of limiting dilution. CCK- 8 was used to test the sensitivity of H8277 /GR and the monoclonal cells to gefitinib. RT-PCR was used to detect the difference of mirna-7 expression in H8277 /GR and the monoclonal cellsand other lung cancer cell lines with different sensitivity to gefitinibsuch as A549H358H1299H1650 and H1975. Results The drug resistance index of H827 / GR was larger than 100. The half growth inhibition concentrations IC 50 of the six resistant monoclonal cell lines were different. The mirna-7 expression of H827 /GR resistant monoclonal cell linesand gefitinib resistant cell lines such as A549H358H1299H1650 and H1975 were all lower than that of the gefitinib sensitive cell line H827 P < Conclusions The mirna-7 expression

2 Chin J Surg OncoApril Vol. 7 No of gefitinib resistant cell lines is lower than that of gefitinib sensitive cell lines in the non small cell lung cancer. This suggests that the drug resistance of the non small cell lung cancer may be associated with the low expression of mirna-7 and it may be a potential molecular marker of the sensitivity of lung cancer to the drugs. Keywords gefitinib sensitivity mirna-7 non-small cell lung cancer % H827 28% 1 non-small cell lung cancernsclc 80% NSCLC - epidermal growth factor RPMI % receptor tyrosine kinase inhibitoregfr-tki um NSCLC 5 NSCLC EGFR 10-7 um 7 /GR 1 EGFR-TKI 2 RPMI-1640 RNAs micrornasmirnas 20 ~ RNA mrna HCC827-7 /GR 2 DNA 7-n n mirna CCK8 PCR NSCLC H H827 /GR EGFR- / 96 TKI A549 H358 H μm 10 μm 5 μm 2. 5 μm μm H1650 H1975 mir h 1 CCK-8 the half growth inhibition concentration IC RI = IC 50 / IC 50 3 Gibco Real time-pcr mir-7 CCK ml / NanoDropND h Trizol NanoDrop7300Real-time PCR System Applied Bi- RNARNA osystems 9700 Thermocycler Recover All Total Nucleic Acid Isolation Kit Applied Biosystems has-mirna-7-5p RNU6B RiboBio Real-time RR037ATakaRa SYBR Select Master Mix Applied Biosystems 10% Gibco 100U /ml 100U /ml ~ 0. 2 cdna RNU6B cdna SYBR-Green PCR SYBR Green Realtime PCR Master Mix 10 μl 5 μm 0. 8 μl 5 μm 0. 8 μlcdna 2 μldepc 20 μl 7300 Realtime PCR Applied Biosystem

3 Chin J Surg OncoApril Vol. 7 No min s60 1 min Ct MiRNA Ct 4 Ct = Ct - Avg. Ct = CtmiRNA-7 - CtU6 - Avg. CtmiRNA-7 - CtU6 SPSS16. 0 x ± s t P < GR 6 7GR CCK8 H h IC ± μm A549 H358 H1299 H1650 H μm 2. 5 μm 5 μm 10 μm 20 μm 7 48h 7 /GR RI 100 IC ± μm 10 μm mir-7 qrt-pcr mir-7 H827 1 A549 H358 H1299 H1650 H1975 mir-7 H827 A549 H358 H1650 vs H827 P < H1299 H1975 vs H827 P < mir-7 H827 vs H827 P < /GR IC h IC 50 μm 7 /GR > > > ± > > 10 1 MiR-7 vsh827 * P < ** P < MiR-7 vsh827 * P < ** P < 0. 01

4 Chin J Surg OncoApril Vol. 7 No ERK1 /2 EGFR EGFR mir-7 mir-7 6 EGFR NSCLC A549 H358 H1299 EGFR H827 EGFR E746-A750 H1650 EGFR E746-A750 H1975 EGFR L858R T790M 5 7 /GR RT-PCR mir-7 7 /GR CCK mirnas H1975 mir-7 RNA H827 P < Argonaute RNA Ⅱ RNA Ⅲ Drosha Dicer pri-mirna Drosha / DGCR8 7- A mir-7 70 mirna EGFR pre-mirna 7 Dicer /TRBP mir-7 PZA Drosha pre-mirna 22nt mirna mirnamirna RNA RNA-induced silencing complex RISC mrna 8 mirna let-7a mir-21 mir-31 mir-34 mir mir-7 IRS1 IRS2 EGFR PAK1 p21 /CDC42 /RAC mir-7 EGFR 3 -UTR 3 12 mir-7 EGFR mrna mir-7 B protein kinase B PKB 1 /2 extracellular regulated protein kinases1 / A549 H358 H1299 H1650 H827 mir-7 P < NSCLC 1Siegel RMa JZou Zet al. Cancer statistics 2014 J. CA a cancer journal for clinicians Jeong YXie YLee Wet al. Research resource Diagnostic and therapeutic potential of nuclear receptor expression in lung cancer J. Mol Endocrinal EGFR J Bartel DP. MicroRNAs genomicsbiogenesismechanismand function J. Cell Di Leva GGarofalo MCroce CM. MicroRNAs in cancer J. Annu Rev Pathol Rho JKChoi YJLee JKet al. Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549a non-small cell lung cancer cell line J. Lung Cancer Schmittgen TDLivak KJ. Analyzing real-time PCR data by the comparative CT method J. Nat Protoc

5 Chin J Surg OncoApril Vol. 7 No. 2 p53 EGFR ZR2012HL wangxinzhao@ 163. com drzhiyongyu@ aliyun. com p53 EGFR p53 EGFR P > /6 CK5 /6 p53 EGFR P < Luminal T1 non-luminal P < p53 EGFR r = P < TNBC p53 EGFR r = P < p53 EGFR P > p53 EGFR TNBC TNBC TNBC p53 EGFR doi /j. issn Correlation analysis between p53 and EGFR expression in distinct molecular subtypes of invasive breast cancer WANG Xinzhao 1 SUN Jujie 2 LIU Qi 3 ZUO Wenshu 1 ZHENG Meizhu 1 MA Sugang 4 YU Zhiyong Department of Breast Surgery Shandong Cancer Hospital Jinan China 2. Department of Pathology Shandong Cancer Hospital 3. Department of Thyroid Gland and Breast Surgery Weifang Traditional Chinese 檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪 8Zhang ZWang JHe Jet al. Genetic variants in MUC4 gene are associated with lung cancer risk in a Chinese population J. PloS One e Lee YAhn CHan Jet al. The nuclear RNase III Drosha initiates microrna processing J. Nature O Day ELal A. MicroRNAs and their target gene networks in breast cancer J. Breast Cancer Res Negrini MFerracin MSabbioni Set al. MicroRNAs in human cancer from research to therapy J. J Cell Sci Pt Jiang LLiu XChen Zet al. MicroRNA-7 targets IGF1R insulin-like growth factor 1 receptor in tongue squamous cell carcinoma cells J. Biochem J Kefas BGodlewski JComeau Let al. MicroRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma J. Cancer Res Webster RJGiles KMPrice KJet al. Regulation of epidermal growth factor receptor signaling in human cancer cells by micror- NA-7 J. J Biol Chem EGFR J