miR-132调节非小细胞肺癌hedgehog信号通路受体基因PTCH1及细胞增殖、迁移和侵袭.pdfVIP

杨秀方 ，等．miR一132调节非小细胞肺癌 hedgehog信号通路受体基 因PTCH1及细胞增殖、迁移和侵袭 293 Here，wereporttheidentification ofm iR一132 ScriptRT reagentKit (TaKaRa，Tokyo，Japan) in NSCLC cel1． W e further proved m iR一132 according to the manua1．The specific reverse negatively modulates PTCH1 expression in transcription (RT) primer of miR一132 was 5一 NSCIC celldirectly，indicating thatmiR一132may GTCG—TATCCAGTGCAGGGTCCGAGGTATT— havean importantroleinHedgehogpathway．Our CGCACTGGATACGACCGACCA一3． Real—time investigations willbe helpful to illuminate the quantitativePCR (RT-qPCR)wasrunonaLight-Cycler functions of miRNA and their impact on cell Roche480 (RocheAppliedScience，Shanghai，China) proliferation，migrationandinvasion． withreactionsconsistedofahotstart(10minat95℃)， 40cyclesof 15 secat95 ℃ and 60 secat6()℃． M aterialsandM ethods Transcriptlevelsforeachtargetgenewerenormalizedto 18srRNA levelsby using comparative threshold cycle CeJlculture Human NSCLC cellline H 1299 (Ct) method， in which fold difference = (American Type CultureCollection，Manassas，VA， 2一‘ ”：or by comparative CT USA)andbul selectedH1299 cellsweregrown in methods．in which fold difference = 2 ．Each RPM I1640 medium ，supplemented with 10 FBS， samplewas run in triplicate to ensure accuracy． 2．383 mg／mL HEPES and 0．11mg／mL sodium Relativeexpressionlevelsof JR-132。P丁CH 1and pyruvateat37 ℃ in ahumidifiedatmosphereof5 18srRNA were detected．Theprimersofm iR一132 C02．Allcellculturemediaandreagents[theRPMI were 5 CGGCGG ACAGTCTACAGCCA 一3 and 1640 medium，fetalb