br Total RNA from the cells was
Total RNA from the cells was extracted by TRIzol (Invitrogen, Carls-bad, CA, USA). After determination of RNA concentration using NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA), 1 mg total RNA was reversely transcribed into cDNA using the PrimeScript RT reagent kit with gDNA Eraser Kit (Takara Bio, Otsu, Shiga, Japan). Following the addition of 5 g DNA Eraser Buffer and gDNA Eraser, DNA removal was conducted at 42 C for 2 min. Next, RNA was reversely transcribed into cDNA. qRT-PCR was performed using a ABI7500 quantitative PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA) with the SYBR PremixExTaq (TliRNaseHPlus) kits (Takara Bio, Otsu, Shiga, Japan). U6 was regarded as the internal reference for miR-126-3p, whereas glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was consid-ered the internal reference for the others. The 2 DDCt method was applied to express the ratio of expression of the target gene in the experimental group and the reference group. The formula was as fol-lows: DDCT (comparative threshold cycle) = Ctthe experimental group Ctthe NC group, DCT = Ctgene Ctinternal control.49 The cycle threshold
(Ct) was considered to be reflective of the number of amplification cycles when the real-time fluorescence intensity reached the set threshold, and the amplification was logarithmic. The primers used in the reaction are displayed in Table 2. The primers used were provided by Gemma Pharmaceutical Technology (Shanghai, China).
Western Blot Analysis
The total protein was extracted and the protein concentration was as-sayed by bicinchoninic BAY-598 (BCA) kit (Thermo Fisher Scientific, Waltham, MA, USA). After polyacrylamide gel electrophoresis for 35 min at constant voltage 80 V and 45 min at 120 V, total protein (30 mg) was transferred to polyvinylidene fluoride membrane (Amer-sham Biosciences, Boston, MA, USA), sealed for 1 h with 5% skimmed milk powder, followed by the removal of the sealing liquid. After that, the membrane was incubated with rabbit monoclonal anti-body to CD63 (1:1,000, ab134045), rabbit polyclonal antibodies to Hsp70 (1:1,000, ab79852), Ki67 (1:5,000, ab15580), VEGF (1:1,000, ab2349), MMP-14 (1:5,000, ab38971), COX-2 (1:1,000, ab15191), and GAPDH rabbit antibody (1:5,000, ab181602) overnight at 4 C. All the aforementioned antibodies were purchased from Abcam (Cambridge, UK). The membrane was washed three times with PBS-Tween 20 (PBST) (containing 0.1% Tween-20 PBS buffer), 10 min for each. After the addition of horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), the mem-brane was incubated for 1 h, scanned, and developed under an optical luminescence instrument (General Electric Company, Boston, MA, USA). ImageProPlus 6.0 (MediaCybernetics, MD, USA) software was used for protein band scanning and analyzing the relative protein expression. Three parallel experiments were repeated.
Xenograft in Nude Mice
Male BALB/c nude mice (4–6 weeks, 20–25 g) were obtained from the experimental animal center (Chinese Academy of Sciences, Shanghai, China) were fed at a constant temperature (25 C–27 C) and constant humidity (45%–50%). The nude mice were randomly classified into two groups with 12 mice placed in each group. After anesthesia, the mice were inoculated with cells. The pancreatic cancer cells exhibiting logarithmic growth were resuspended in 50% Matrigel (BD Biosci-ences, Bedford, MA, USA) with the cell concentration adjusted to 2 106 cells/mL, and 0.2-mL single-cell suspension (4 105 cells) was subcutaneously injected into left subaxillary skin of each nude mouse, respectively. After the tumor had grown to approximately
100 mm3, the mice were injected with lentivirus-packaged BMSCs. BMSCs were transiently transfected with miR-NC or miR-126-3p.
After the BALB/c nude mice were injected with lentivirus-packaged BMSCs, the tumor volume (mm3) was calculated using the formula: (L W2)/2, in which L was the tumor length unit and W was the width unit of the tumor. After seven rounds of treatment, the nude mice were euthanized by means of anesthesia. The tumors were collected and weighed, with certain tumor tissues reserved for qRT-PCR and western blot analysis.
Statistical analysis was conducted using SPSS 21.0 (IBM, Armonk, NY, USA) software. Normal distribution test and homogeneity of variance were conducted. Data displaying normal distribution were expressed as mean ± SD. The data of patients with pancreatic cancer and pancreatitis were compared using an independent sample t test. Data failing to conform to homogeneity of variance were corrected by Welch’s t test. One-way ANOVA was used for comparison among multiple groups, while data between two groups were compared by Tukey for backing test. All the data with skew distribution were tested by nonparametric tests. Data at different times points were analyzed by repeated-measurement ANOVA. p < 0.05 was considered to be indicative of statistical significance.