ABSTRACT
Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.
ABSTRACT
Objective To compare different diagnosis values of age-specific procalcitionin (PCT) and C-reactive protein (CRP) on early-onset neonatal sepsis (EONS) during the first 72h of life.Methods From September 2008 to December 2015,96 neonates (including 2 confirmed sepsis and 94 clinical sepsis) without severe complications were chosen as the EONS group and 170 non-infectious newborns as the control group.A total of 605 blood samples were collected from all 266 newborns.Serum concentration of PCT and CRP were measured in both the EONS group and the control group at each age over the first 72 h of life.The diagnostic value of PCT and CRP within 1 ～ 12 h and 13 ～ 24 h and 25 ～ 48 h and 49 ～ 72 h of life was evaluated by calculating the cut-off values,sensitivity,specificity,and the area under the receiver operating characteristic curve (ROC).Results PCT and CRP levels of neonates within each age in the EONS group were significantly higher than those in the control group during the first 72h of life.(all P ＜ 0.05).Within 1 ～ 12 h,13 ～ 24 h,25 ～ 48 h and 49 ～ 72 h of life,the cutoff value of PCT was 0.45 μg/L (sensitivity 84.2％,specificity 74.4％),1.885 μg/L (sensitivity 73.5％,specificity 75％),0.995 μg/L (sensitivity 82.4％,specificity 74.1％) and 0.51 μg/L (sensitivity 83.3％,specificity 79.2％) respectively;that of CRP3 185 mg/L (sensitivity 68.4％,specificity 82.1％),6.29 mg/L (sensitivity 58.8％,specificity 89.7％),8.615 mg/L (sensitivity 54.3％,specificity 93.9％) and 10.27 mg/L (sensitivity 59.1％,specificity 100％) respectively,and the area under ROC of PCT for the diagnosis of EONS was 0.767,0.754 and 0.755,and 0.8 respectively;that of CRP 0.773,0.8,0.815 and 0.789 respectively.Conclusions There are age-specific cut-off values of PCT and CRP in the diagnosis of EONS without severe complications during the first 72 h of life.Both PCT and CRP are moderately accurate for the diagnosis of EONS.PCT may be more helpful for the early diagnosis of EONS for its higher sensitivity but CRP presents higher specificity.