Ed with BCA Protein Assay Kit (Pierce, USA). A 20 g sample of the total protein was resolved using 12 SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked in Trisbuffered saline containing Tween 20 (TBST) with 5 nonfat milk at room temperature for 2 h. Primary antibodies to detect PADI4(1:1000, Abcam, USA) and LC-3(1:1000, Novus Biologicals, Inc) were incubated overnight with the membranes at 4 . Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (1:8000, Dako, USA), and proteins were Baicalein 6-methyl etherMedChemExpress Baicalein 6-methyl ether detected by enhanced chemiluminescence (ECL) (Beyotime, USA). GAPDH was used as the internal control to normalize the loading materials.Animal modelAll procedures involving animals were performed in accordance with the institutional animal welfare guidelines of Second Military Medical University. Subcutaneous implantation of HCC cells was performed in armpit areas of nude mice. Mice were examined three times per week. Tumor growth was evaluated by measuring the length and width of the tumor mass. Animals were sacrificed and tumors were removed at the end of theWe collected 5 ml vein blood samples before TACE and after TACE in HCC patients with partial hepatectomy. Chemoresistance were detected by MDR1 mRNA expression in blood samples by using Realtime-PCR. We found that elevated expression of MDR1 after TACE was detected in 72.5 of HCC patients (87/120), and lower expression of MDR1 was detected in 27.5 of HCC patients (33/120) after TACE. Here, MDR1 (+) group means elevated expression of MDR1, and MDR1 (-) group means lower expression of MDR1 after TACE treatment for HCC patients with partial hepatectomy. According to the MDR1 results, all 120 HCC patients were divided into two groups: the chemoresistance group DR1 (+) group, and chemosensitivity group DR1 (-) group (Figure 1A). However, there are no significant difference in clinicopathologic variables between MDR1 (+) group and MDR1 (-) group (Additional file 1: Table S2). Time zero of recurrence-free survival of patients treated with TACE is the date of TACE. All patients underwent ultrasound, computed tomography scan or magnetic resonance imaging of the abdomen once every 2 months after treatment. Kaplan eier analysis indicated that the recurrence-free survival of MDR1 (+) group was shorter than MDR1 (-) group (median: 6 months vs 9 months, respectively; P < 0 ?0001; HR:Fan et al. Cell Bioscience 2014, 4:49 http://www.cellandbioscience.com/content/4/1/Page 4 ofFigure 1 The elevated PADI4 mRNA expression in MDR1(+) HCC patients with TACE. (A) The MDR1 mRNA expression were detected in blood samples before and after TACE. - Ct, -(Ctbefore TACE ?Ctafter TACE); MDR(+):- Ct > =0; MDR(-):- Ct < 0. (B) The progression-free survival (PFS) after transcatheter arterial chemoembolization (TACE) in partial hepatectomy for hepatocellular carcinoma determined by Kaplan-Meier analysis. (C) The PADI4 mRNA expression in HCC tumor tissues associated with chemoresistance. - Ct, -(Ctbefore TACE ?Ctafter TACE); MDR(+):Ct > =0; PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28404814 MDR(-):- Ct < 0.6.339, 95CI : 3.742-10.74, Figure 1B). Interestingly, we found that PADI4 mRNA expression of tumor tissues was higher in MDR1 (+) group (Mean -Ct ?SD = 0.59 ?0.37) than that in MDR1 (-) group (Mean -Ct ?SD = -1.69 ?0.53). PADI4 expression of HCC tissues was significantly difference between chemoresistance patients and chemosensitivity patients (p = 0.0011; Figure 1C).PADI4 enhanced chemoresistance of hepatoc.
