Bioinformatic Analysis
CancerMIRNome (http://bioinfo.jialab-ucr.org/CancerMIRNome/) is a comprehensive database for facilitating the use of publicly available cancer miRNome data to assist in miRNA research in various cancers[16]. We focused the different expression between tumor and normal samples, ROC analysis, and KM survival analysis for miR-128-1-5p in COAD and READ by this database. Expression of miR-128-1-5p in serum in both healthy and different cancer types also explored in CancerMIRNome.
Human tissue samples
All human CRC and their adjacent normal tissues were obtained from patients who received surgery in the Department of Colorectal Surgery between January 2006 and October 2012. All patients did not receive anticancer treatments before operation and written informed consent was obtained from all patients enrolled in this study. The study protocol was reviewed and approved by the Institutional Human Experiment and Ethic Committee of Fudan University Cancer Hospital. After collection, the fresh tissues were immediately frozen in liquid nitrogen for RNA isolation.
Cell lines and cell culture
The human CRC cell lines (HT29, RKO, SW480, SW620 and LOVO) and the normal human intestinal epithelial cell line (HIEC) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured as described previously [17].
RNA extraction and Real-time PCR analysis
Total RNA was extracted using Trizol reagent (Invitrogen, Thermo Fisher Scientifc, Inc., Carlsbad, CA) according to the manufacturer's instructions. qRT-PCR was carried outas described previously[17]. The primers were obtained from Sangon (Shanghai, China), the primers sequences were as follows: miR-128-1-5p: forward, 5ʹ-CCACAGAGAGGATTCCAGAA-3ʹ, reverse, 5ʹ-TCCAGGCTCTGTATAGAAGCT−3ʹ; U6 RNA: forward, 5ʹ-CTCGCTTCGGCAGCACA-3ʹ, reverse, 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ; PRKCQ:forward, 5ʹ-ATGTCGCCATTTCTTCGGATT-3ʹ, reverse, 5ʹ-ACATACTCTTTGACGAGCACAG−3ʹ; GAPDH: forward, 5ʹ-AGCCTTCTCCATGGTGGTGAA-3ʹ, reverse, 5ʹ-ATCACCATCTTCCAGGAGCGA−3ʹ.
Lentivirus production and transduction
Lentivirus-medicated miR-128-1-5p overexpression vector was purchased from GenePharma (Shanghai, China). Lentivirus was produced by transfecting 293T cell linewith Lenti-Pac HIV Expression Packaging Mix and the lentiviral vector using Lipofectamine 2000 (Life Technologies Corporation, Carlsbad, CA, USA). Stably transfected cell lines were established by infecting SW620 and LOVO cells with lentiviruses containing 2 μg/mL puromycin. The full-length sequence of PRKCQ was cloned into the pcDNA3.1 vector and empty vector was used as negative control simultaneously.
Cell proliferation and colony formation assay
The CCK-8 assay was performed as our previous study [17]. In brief, 2×103 cells were cultured in a 96-well plate and cultured for 24-48-72-96 h. Then 10 μL CCK-8 solution was added to each well and incubated for 2 h at 37˚C. Finally, the absorbance was measured at 450 nm. For colony formation assay, 8×102 cells were seeded in 6-well plates and cultured for 2 weeks at 37°C. Cell colonies were fixed with methanol and stained with 0.5% crystal violet. Colonies containing more than 50 cells were counted and photographed.
Flow cytometric analysis
Cells were dissociated with trypsin without EDTA, subsequently incubated with Annexin V- DY-634 PI Apoptosis Staining / Detection Kit (Abcam, Cambridge, United Kingdom). The ratio of apoptotic cells was detected with a flow cytometry (FACScan, BD Biosciences).
Vector constructs and luciferase reporter assay
The full-length of wild type 3’UTR of PRKCQ was cloned into the psi-CHECK2 vector (Promega, Madison, WI, USA) to form the reporter vector PRKCQ-wild-type (PRKCQ-WT). The corresponding mutant was created by mutating the miR-128-1-5p seed region binding site, which was named PRKCQ-MUT. All clones were verified by DNA sequencing. MiR-128-1-5p or vector was co-transfected with luciferase reporter vectors into 293T cells using LipofectamineTM 2000.Cells were harvested 36 h post-transfection and luciferase activities were analyzed by the dual-luciferase reporter assay system (Promega) in a luminescence reporter gene assay system (PerkinElmer, Norwalk, CT, USA) according to the manufacturer’s instructions.
Protein isolation and western blotting
Total protein was extracted using RIPA lysis buffer from Cell Signaling Technology (CST, Beverly, MA, USA) according to the manufacturer’s protocol. Western blotting was performed using standard protocols as described previously[17]. The primary antibodies used in this study included: PRKCQ (Proteintech Group, Inc, IL, USA), Caspase-3 (CST), Bax (CST), Bcl-2 (CST), and GAPDH (Proteintech Group, Inc).
Xenograft model
4–6-week-old female immune-deficient nude mice (BALB/c null) were purchased from Slaccas (Slaccas Laboratory Animal, Shanghai, China). All animal procedures were performed in accordance with guidelines of the care and use of laboratory animals of Fudan University and approved by the Animal Ethics Committee of Zhongshan hospital. Stable miR-128-1-5p-overexpressing LOVO and control cells (1 × 107 cells) in 100 ul serum free medium were subcutaneously injected into the flank region of the mice (8 for one group). Tumor size was measured with a slide caliper every week. Mice were sacrificed 4 weeks later and the tumors were removed for further investigation.
Immunohistochemistry Staining
Immunohistochemistry (IHC) staining was carried out as our previously described (23). Briefly, formalin-fixed tissues were embedded in paraffin and cut into 4-μm-thick sections. Subsequently, the specimens were dewaxed, hydrated, antigen retrieved by heat, blocked, and then incubated overnight at 4°C with primary antibody Ki67 (CST). Following, sections were incubated with HRP conjugated secondary antibodies. Finally, slides were treated with 3, 3’-diaminobenzidine (DAB) substrate and visualized under a Nikon Eclipse Ti inverted microscope (Nikon, Amstelveen, The Netherlands).
Statistical analysis
The results were presented as the mean ± standard error of the mean (SEM). Student’s t-test was used to assess differences between two groups. One-way analysis of variance (ANOVA) was used to compare differences among three groups or more. Association between miR-128-1-5p and PRKCQ expression was assessed using Spearman rank correlation test. The χ2 test was performed to analyze the relationship between miR-128-1-5p expression and the clinicopathological characteristics. Statistical analysis used SPSS 18.0 (SPSS, Chicago, USA) and a P < 0.05 was considered statistically significant.