ABSTRACT
Objective To investigate the efficiency of Trichostatin A (TSA) in inducing cell apoptosis and altering the Notch pathway genes expression in PANC-1 cells line.Methods The survival rate and apoptosis of PANC-1 cells were measured by MTT assay and Hoechst 33258 staining,respectively.mRNA expression levels of the genes,numb,gcn512,dll3,hes6,eaf2,cytohesins,in PANC-1 cells were assessed by real-time quantitive PCR.Western blot was used to measure the expression of bcl-2,bax,actived caspase-3 and NICD protein which was the biologically active form of Notch-1.Results After culturing with 0.1,0.2,and 0.4 μmol/L TSA for 24 hours,the cellular survival rate of PANC-1 cells significantly decreased to 72％,58％ and 39％,respectively.The survival rate of PANC-1 was negatively correlated to time length of culture with TSA.Increased apoptosis of PANC-1 cells after 12,24 and 36 h culture with TSA was detected by Hoechst 33258 staining.Western blotting showed that the expression of bax,actived caspase-3 and NICD protein increased while the bcl-2 protein decreased after culture with TSA.In real time quantitive PCR assessment,the mRNA expression of numb and hes6 in PANC-1 cells were upregulated by TSA (P ＜ 0.05),while the mRNA expression of gcn512 and dll3 were down-regulated by TSA (P ＜ 0.05).While mRNA expressions of eaf2 and cytohesin1,2,3,4 were not affected by TSA.Conclusions TSA induces apoptosis of pancreatic cancer cell line PANC-1.The Notch signal pathway may be involved in inducing cellular apoptosis of PANC-1 when cultured with TSA.
ABSTRACT
Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.