Samples. We obtained 25 CRC specimens (12 from males and 13 from females) with intact adjacent tissues (> 4-cm margins) from Shenzhen Nanshan Hospital (affiliated with Guangdong Medical College) between June of 2014 and December of 2015. The patients of origin had a mean (± standard deviation) age of 59.2 ± 13.1 years (range, 32–84 years). All patients were informed of the sample donation and this study was approved by the ethical review committee of the hospital. None of the patients had received radiotherapy or chemotherapy before the tumor resection surgery. The specimens were preserved in liquid nitrogen or neutral formaldehyde.
Reagents. TRIzol reagent, Lipofectamine® 2000, RNA reverse transcription and amplification kits, and human endothelial SFM medium were purchased from Life Technologies (Carlsbad, CA). High-glucose DMEM was purchased from HyClone Laboratories (Logan, UT). Matrigel matrix glue was purchased from BD (Franklin Lakes, NJ). All primers were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd (Guangzhou, China).
Monoclonal rabbit anti-human PTEN (ab154814, clone EPR7495; Abcam) and GAPDH (10494-1-AP; Proteintech Co.) primary antibodies and goat anti-rabbit secondary antibody (111-035-003; Jackson Laboratory, Bar Harbor, ME) were used for immunoblotting. Mouse anti-human CD31 (ZM-0044, clone 1A10; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China) direct antibody was used with a Vectastain Elite ABC system kit (PK-6102; Vector Laboratories, Burlingame, CA) for IHC.
Cell lines and culture. HCT116, SW620, HT29, and SW480 CRC cells (donated by the Chinese University of Hong Kong) were cultured in high-glucose DMEM complete medium. Human umbilical vein endothelial cells (HUVECs; donated by Sun Yatsen University) were cultured in complete medium at 37 °C under 5% CO2 and passaged until stable (2 ~ 3 generations). Cells were undergoing logarithmic growth when subjected to experiments.
qRT-PCR detection of miRNA-92a expression.
Total RNA was extracted from CRC tissues and cells with TRIzol reagent and 1-µg RNA samples were subjected to reverse transcription at 42 °C for 60 min in accordance with the RNA reverse transcription kit instructions. Subsequently, 1-µl cDNA aliquots were subjected to PCR amplification for 40 cycles under the following conditions: 50 °C for 2 min, 95 °C for 10 min, denaturation at 95 °C for 15 s, and extension at 60 °C for 1 min.
Expression of miRNA-92a in tissues was determined by the absolute quantitative method. A miRNA standard was subjected to 10-fold gradient dilution, and the standard and sample were detected by PCR. A standard curve and regression equation were established to calculate miRNA-92a concentrations in samples; miRNA quantification was standardized according to total RNA quantities in samples, and the results are shown as miRNA content (in fmol) per µg of total RNA. RNA extracted from cells was quantified relative to U6 as an internal reference, and the relative expression of miRNA-92a in each group was calculated by the 2−△△Ct method. Each experiment was performed in triplicate and mean of the three values obtained was determined.
Detection of angiogenesis by IHC.
CRC tissue specimens were prepared by conventional dehydration and paraffin embedding. The paraffin-embedded specimens were sliced into 4-µm-thick sections with a microtome. High-pressure/heat epitope retrieval was performed with the sections in citrate buffer. Expression of CD31 in vascular endothelial cells was detected by IHC with mouse anti-human CD31 antibody (1:100 in phosphate buffered saline) performed according to the Vectastain Elite ABC system kit instructions (antibody omitted in negative control; known positive sample used as a positive control).
Angiogenesis was analyzed in terms of microvascular density (MVD) of the CD31-immunopositive signal (brown staining). High-vessel-density areas were selected under a low power microscope (100×), and then five fields of view were selected randomly for imaging under higher power microscopy (400×) and averaged.
HCT116 and SW620 cells in a logarithmic growth stage were subjected to transient transfection. The cells were seeded in 6-well plates, and then cultured in 1 × DMEM medium containing 10% fetal bovine serum with 5 µl of transfection reagent and an experimentally indicated miRNA molecule (50 nM miRNA-92a mimic or 100 nM miRNA-92a inhibitor) until ~ 60% fusion was observed. Serum-free 1 × Opti-MEM medium was added to each well to a final volume of 2 ml, and transfection was allowed to proceed for 6 h at 37 °C under 5% CO2, and then the medium was exchanged for new medium. Cells were collected 48 h after transfection for RNA extraction, and cell proteins and serum-free culture supernatant (conditioned medium) were collected 72 h after transfection for use in subsequent experiments.
Tube formation assay. HUVECs (1 × 106/well) were cultured for 24 h in Matrigel® matrix (50 µl/well) in 96-well plates. Conditioned medium (100 µl/well; from the above transfections) containing HCT116 or SW620 cells transfected with miRNA-92a mimic or inhibitor, respectively, was added to each well. HUVECs were allowed to grow into closed tubule-like structures for 6 h at 37 °C under 5% CO2, and the number of tubules formed in each well was counted. Triplicate wells were set up for each group, and three views were imaged for each well. Averages of the three views were calculated for each well.
Immunoblot detection of PTEN.
Protein concentrations in the 72-h transfections of CRC cells described above were determined by Bradford assay (Sangon Biotech Co., Ltd). The proteins were extracted by cell lysate RIPA, and the total protein concentrations of the extracted protein samples were determined by the bicinchoninic acid assay method. Protein samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (30 µg/lane) and the resultant bands were electrotransferred to polyvinylidene difluoride membranes. Subsequently, the membranes were blocked in 5% skim milk for 1 h, incubated with rabbit anti-human PTEN antibody (1:2000) at 4 °C overnight, and then rinsed in Tris buffered saline with 0.1% Tween20 three times (10 min per rinse). The rinsed membranes were incubated with goat anti-rabbit antibody (1:4000) for 1 h at 37° C, and then ECL developer was applied to the membrane. The processed membranes were imaged with a Bio-Rad ChemiDoc XRS+ system for densitometry analysis of PTEN protein levels.
Statistical analyses were conducted in SPSS19.0 software (IBM). Following confirmation of a normal distribution with Kolmogorov-SmiRNAnov normality tests, t tests were used for inter-group comparisons. Correlations between miRNA-92a expression and angiogenesis MVD in CRC tissues were assessed by Pearson analysis. P values < .05 were considered significant.