MicroRNA-29b-2-5p inhibits cell proliferation by directly targeting Cbl-b in pancreatic ductal adenocarcinoma

Freshly isolated human PDAC tissues from 120 patients and adjacent pancreatic tissues were obtained with informed consent from the Department of Pathology, the affiliated Shengjing Hospital, China Medical University, between January 2009 to Feburary 2011. The clinic-pathologic characteristics and prognosis were available for 120 patients. The patients had not received chemotherapy or radiation therapy prior to surgery.

Each case diagnosis and histological grade, there are two pathologists confirmed based on the American joint committee on pathological diagnosis. Patient information included age, gender, location of tumor, Maximum tumor diameter, differentiation, surgical margins, pT category, pN category, vessel invasion, vascular tumor thrombus, adjacent organs invasion, pTNM category and Overall survival(OS). The maximal tumor size was defined as the maximum diameter on pathologic analysis. The tumor was staged according to the American Cancer Association (TNM’s AJCC staging system) 2010. The final survival data were collected in 31 December 2014. During the 120 cases, 20 cases were analyzed with miRNA microarray. Because they were similar in clinic-pathologic features and treatment but were different in outcomes. The medium OS used as cut off value reference to previous studies [27, 28]. Half of the patients died within the first year of diagnosis were classified as “poor prognosis” with median OS of 6.3 months. Patients who survived more than 21 months had a median OS of 48.0 months, which classified as the “good prognosis” group. The background of the clinic-pathologic characteristics of the 20 patients has been published on our previous study [25]. This study was approved by the Human Ethics Review Committee of China Medical University (protocol #: 2015PS63K); informed consent was obtained from all patients in accordance.

The human pancreatic adenocarcinoma cell lines SW1990(#TCHu201), Capan-2(#SUER0449) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and Suer Biological Technology(Shanghai, China) respectively. Before the experiments, the two cell lines were authenticated on cell micrograph compared to the cell lines on ATCC. The cell lines were maintained in RPMI 1640 medium that contained 10% heat-inactivated foetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml) under 5% CO2 at 37 °C.

MiR-29b-2-5p mimic and the negative control were obtained from RiboBio (Guangzhou, China). p3XFLAG—CMV9(NC) and p3XFLAG—CMV9 Cbl-b (OE Cbl-b) were obtained from Sigma(USA). The small interfering RNA sequences (Genepharma, Shanghai, China) for Cbl-b was 5′-CCUGAUGGGAGGAGUUAUAtt-3′ (sense), 5′-UAUAACUCCUCCCAUCAGGtt − 3′ (antisense).MiRNAs and siRNAs transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

The levels of total human microRNAs’ expression were quantified using a GenoSensor’s GenoExplorerTM microRNA microarray (Tempe, AZ, USA). The hybridized miRNA chips were scanned and analyzed using an Axon GenePix 4000B scanner and GenePix Pro software (Molecular Devices, CA, USA).

Total RNA extracted as described above [25]. For miRNA detection, reverse transcription was performed using One Step PrimeScript® miRNA cDNA Synthesis kit (Takara, Japan), and real-time polymerase chain reaction (PCR) was carried out using SYBR® premix Ex Taq™ II (TaKaRa, Japan) with the ABI 7500 Sequence Detection System (Applied Biosystems, Foster, CA). The sequences (TaKaRa, Japan) for miR-29b-2-5p was 5′-CCTTCGACATGGTGGCTTAGAAA-3′, and U6 was 5′-GCTTCGGCAGCACATATACTAAAAT-3′(sense) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′(anti-sense). The PCR conditions were 30 s at 95 °C, followed by 45 cycles at 95 °C for 5 s, and 58 °C for 25 s. Data were analyzed using the Applied Biosystems 7500 software program (version 2.3) with the automatic Ct setting for adapting baseline and threshold for Ct determination. The threshold cycle and 2-^ΔΔCt method were used for calculating the relative amount of the target RNA.

For mRNA detection, reverse transcription was performed using the M-MLV Reverse Transcriptase System (Promega, USA). RT-PCR was performed with the following primer pairs for Cbl-b: forward (5′-CGCTTGACATCACTGAAGGA-3′); and reverse (5′-CTTGCCACACTCTGTGCATT-3′). GAPDH was used as a control: forward (5′-GTGGGGCGCCCCAGGCACCA-3′); and reverse (5′-CTCCTTAATGTCACGCACGATTTC-3′). PCR conditions for Cbl-b were 95 °C for 5 min, 30 cycles at 95 °C for 30 s, 59 °C for 30 s, 72 °C for 30 s, and 1 cycle at 72 °C for 10 min. GAPDH were 95 °C for 5 min, 33 cycles at 95 °C for 30 s, 56 °C for 45 s, 72 °C for 45 s, and 1 cycle at 72 °C for 10 min. The amplified products were separated on 1% agarose gels, and stained with ethidium bromide and visualized under UV illumination.

To evaluate the effects of miR-29b-2-5p on cell growth, SW1990 and Capan-2 PDAC cells were incubated in the 6-well plates (3 × 10⁵ cells per hole) in triplicate. The next day, the cells were transfected with miR-29b-2-5p mimics or negative control mimics (NC; Ribobio, China) or OE Cbl-b/NC(1.5 μg) using Lipofectamine 2000 (Invitrogen). The final concentration was kept constant (50 nmol/L). Measure the culture of cell proliferation, cell in 2 ml medium, counted manually after 24, 48, 72, and 96 h use the hemacytometer (Hawksley, West Sussex, UK) and bright field microscope. It combined with Trypan blue staining method to determine growth state of dispersed cells.

Western blotting was performed as our previously described [29]. The primary antibodies, anti-Cbl-b, anti-b-actin, anti-p53, anti-Bax-2, anti-Bcl-1, anti-GAPDH, anti-UB were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-IgG was from Cell Signaling Technology (Beverly, MA). Enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate; Pierce, USA) were used to analysis proteins. The final result was analyzed by NIH Image J software.

Cells were fixed with 70% ice-cold ethanol overnight. Fixed cells were resuspended in PBS containing 10 μg/ml propidium iodide (PI, KeyGEN, China), 0.1% Triton, and 20 μg/ml RNase A (KeyGEN) and were incubated for 30 min in the dark. Finally, the samples were evaluated by flow cytometry and the data were analyzed with Flow Cytometry (BD Accuri C6; BD Biosciences, San Jose, CA, USA) and analyzed with WinMDI version 2.9 software (The Scripps Research Institute, La Jolla, CA, USA).

Transfected cells were cultured in six-well plates. Samples were subsequently stained using an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (cat no. BMS500FI-100; Invitrogen; Thermo Fisher Scientific, Inc.) and the number of apoptotic cells was determined by FACS Calibur flow cytometry (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s protocol. Finally, the results were analyzed with WinMDI v.2.9 software (The Scripps Research Institute, La Jolla, CA, USA).

All in vivo studies were approved by the Institutional Review Board of China Medical University. These animals were cared of in accordance with institutional ethical guidelines of animal care. Female SPF BALB/c nude mice were bought from Vitalriver (Beijing, China). Mice were sacrificed in gas chamber and by cervical dislocation to confirm death according to the protocol filed with the Guidance of Institutional Animal Care and Use Committee of China Medical University. SW1990 cells (1 × 10⁶) with 0.15 ml PBS subcutaneous injected into mice’s right shoulder area. A week after the cells injected, randomly divided into two groups, each group of three mice, and mir-29-2b* agomir or mir-NC agomir (40 ul saline 5 nmol/L, Ribobio technology, Guangzhou, China) treatment by subcutaneous injection every 2 days. Every 2 days with a caliper measuring the volume of tumor, the calculation of tumor volume, use the following formula: V = 1/2 (width×length×height).Body weights were also recorded. With the protocol to the Animal Care and Use Ethnic Committee the China Medical University under the protocol number 16080 M, the tumor-bearing mice were sacrificed by cervical dislocation when the mice became moribund or on day 15.

SW1990 cells were seeded at 3 × 10⁵ per well in six-well plates and incubated overnight; Cells were transfected with NC (1.5 μg), OE Cbl-b (1.5 μg) 24 h every six wells. The next day, the cells with OE Cbl-b treated with or without proteasome inhibitor PS341 (5 nM) for 24 h. After removal of the medium, cells were transferred to 1.5 ml EP tube for transient centrifugalization. Cell pellets were washed by ice-cold PBS for two times. For immunoprecipitation, cells were collected with denaturation buffer to separate protein complexes. Cell lysates were incubated with p53 antibody or immunoglobulin-G (1–4 μg, Cell Signaling Technology, MA) at 4 °C overnight followed by the addition of 20 μl of protein G-Sepharose beads (Santa Cruz Biotechnology) for an additional 2 h at 4 °C. The immunoprecipitated proteins with 3 × sampling buffer were eluted by heat treatment at 100 °C for 5 min.

Pancreatic cancer cells grew on Lab-Tek chamber slides (Nunc S/A, Polylabo, France). The following day, miR-29b-2-5p or NC (50 nmol/L) treated into cells for 48 h, 3.3% paraformaldehyde fixed for 15 min, 0.2% Triton X-100 permeabilized for 5 min, 5% bovine serum albumin (BSA) blocked for 1 h. And the cells incubated with anti-Cbl-b and anti-p53 antibody (Santa Cruz, CA) at a dilution of 1:200 overnight at 4 °C. Blocking solution for 1 h at room temperature with Alexa Fluor 546-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) in the dark. Nuclei was stained by 4′-6-diamidino-2-phenylindole for 5 min. The cells were visualized by fluorescence microscopy (BX53, Olympus, Japan).

One hundred of formalin-fixed, paraffin-embedded PDAC tissues were used for IHC. All sections were performed using the following antibodies: anti-Cbl-b (Santa Cruz Biotechnology) using S-P immunohistochemical kit (Fuzhou Maixin Biological Technology Ltd., Fujian, China) as described previously [30]. The scanning the entire tissue specimen evaluated the staining under low magnification (× 10) and confirmed under high magnification (× 20 and × 40). Visualized and classified the protein expression was based on the percentage of positive cells and the intensity of staining. Tumors with < 10% Cbl-b expression were regarded as negative or weak (0),10–70% were regarded as moderate (1) and ≥ 70% were considered positive (2). The cut off of weak-medium-strong is 10 and 70% respectively. Final scores were assigned by two independent pathologists.

Statistical analysis was performed using the GraphPad Prism software (La Jolla, CA, USA). Overall survival (OS) was defined as the time from the date of the surgery to the date of death or the last contact, i.e., the date of the last follow-up visit. Kaplan-Meier estimate was used to analyze the survival data and the statistical significance was evaluated by the log rank test. ROC curve from the point to cut off value is based on the previously study [31]. Multivariate analysis was performed using the multivariate Cox proportional hazards model (forward), which was fitted using all of the clinic-pathologic variables. Chi-square test was used to evaluated the correlation between miR-29b-2-5p expression levels and the clinical characteristics. The differences between groups were assessed by Student’s t-test or Mann-Whitney U test. For correlation analysis, the non-parametric Spearman r tests were applied. All means were calculated from at least three independent experiments. Two-sided P values < 0.05 were considered to be statistically significant. SPSS software (version 13.0; SPSS, Inc. Chicago, IL, USA) was used for statistical analysis.

The flowchart of patient selection and schematic design were shown in Fig. 1a. We performed a comprehensive microarray analysis to compare miRNA expression profiles in pancreatic tissues from two groups of participants. Our previous study showed that patients with good prognosis, median OS was 48.0 months, compared to 6.3 months in those with poor prognosis. There was no statistically significant differences in the remaining clinical and pathological features between the two groups, corroborating previous findings [25]. The good prognosis group had 22 miRNAs significantly upregulated (miR-29b-2-5p, etc.) as demonstrated by miRNA microarray analysis [25]. Among these candidate miRNAs, 4 miRNAs are Dead miRNA Entry through miRbase which we cannot get the sequences. We used real-time PCR to test the result of miRNA array. In the rest of 18 candidate miRNAs, 2 miRNAs were opposite from the miRNA array, 16 were coherent with the miRNA array (see Additional file 1: Figure S2.A.B online). We tried to find targets which can be regulated by the miRNAs, and found 7 miRNAs had targets with softwares miRwalk and starBase. Among these candidate 7 miRNAs, miR-29b-5p, miR-891b and miR-490-5p could inhibit proliferation in cell lines, and miR-29b-2-5p was most stable in inhibiting PDAC tumor cell proliferation as well as the result of microarray (see Additional file 1: Figure S2.C online, Fig. 2a). Real-time PCR confirmed that miR-29b-2-5p was associated with better prognosis. MiR-29b-2-5p expression gradually increased from the poor to good prognosis groups (Fig. 1b), and from cancer to adjacent pancreatic tissues (Fig. 1c). Furthermore, high miR-29b-2-5p expression was associated with a median OS of 35.2 months versus 6.4 months for the low expression group (log rank x² = 21.837, p = 0.02; Fig. 1d). A strong correlation between miR-29b-2-5p expression status and OS was demonstrated, confirming that miR-29b-2-5p was a prognostic factor in PDAC.

To verify the prognostic role of miR-29b-2-5p, the expression levels of this miRNA were assessed by qRT-PCR in 100 independent PDAC samples. This validation cohort contained stage I, II and III tumors. Other clinical pathologic features were not significantly different from those of the initial patient cohort (see Additional file 2: Table S1). We also evaluated the correlation between miR-29b-2-5p expression levels and the clinical characteristics using chi-square test (Table 1), found that Gender (p = 0.028), Maximum tumor diameter (cm) (p = 0.11), Differentiation (p < 0.001), Surgical margins (p < 0.001), pT category (p = 0.002), pN category (p < 0.001), Vascular tumor thrombus (p < 0.001), Adjacent organs invasion (p < 0.001), CA19–9((p < 0.001) had correlation with miR-29b-2-5p. MiR-29b-2-5p was detected in all patients. Patients with high miR-29b-2-5p expression had median OS of 18.8 months (95% CI 10.4–27.3 months) versus 12.9 months (95% CI 10.6–15.1 months) for the low expression group (log rank χ² = 9.296, p = 0.002; Fig. 1e). And scatter plot showed that the good prognosis group levels of miR-29b-2-5p in these 100 validation cohort is higher than poor prognosis group (p < 0.001, Fig. 1f). We also use ROC analyses based on clear cut-off values on which expression levels miRNA-29b-2-5p is prognostic relevant. The result is the same as Medium method. (see Additional file 3: Figure S1 online).

Multivariate Cox proportional hazard model (forward) was used to fit all 15 clinical pathological variables. MiR-29b-2-5p was included in the multivariate Cox proportional hazards model (forward) analysis of 100 patients along with prognostic clinic-pathologic factors. High miR-29b-2-5p expression (HR, 0.492; 95% CI, 0.300–0.807; P = 0.005), pT4 category (HR, 1.286; 95% CI 1.004–1.646; P = 0.046), serum CA19–9 level ≥ 37 U/ mL (HR, 3.47; 95% CI, 1.484–8.112; P = 0.004), and poorly differentiated tumor (HR, 1.472; 95% CI 1.016–2.133; P = 0.041) were significant independent prognostic factors associated with OS (Table 2). These data suggested that miR-29b-2-5p represented a tumor suppressor in PDAC.

To assess whether miR-29b-2-5p plays a tumor suppressive role in PDAC development, we first evaluated the effect of miR-29b-2-5p on cell proliferation using the Trypan blue staining method in Capan-2 and SW1990 cells. MiR-29b-2-5p-treated Capan-2 and SW1990 cells exhibited significantly lower growth rates compared with control cells (Fig. 2a, b). Increased miR-29b-2-5p expression upon treatment of the two PDAC cell lines was confirmed by qRT-PCR (see Additional file 4: Figure S3 online). These results provided strong evidence that miR-29b-2-5p was a negative regulator of pancreatic cancer development and progression. To determine whether miR-29b-2-5p could have a potential therapeutic value in vivo, nude mice bearing subcutaneous SW1990 xenografts were treated with miR-29b-2-5p every other day for 14 days. After euthanasia, the tumors were removed from the animals for analysis (Fig. 2c–e). The results suggested that miR-29b-2-5p might have a therapeutic potential for the treatment of PDAC.

To further evaluate whether the miR-29b-2-5p-reduced cell proliferation was due to cell cycle arrest and/or apoptotic death, we first examined the effect of miR-29b-2-5p on cell cycle of SW1990 and Capan-2 cells. Compared with NC, the miR-29b-2-5p mimic significantly enhanced the G0/G1 subpopulation in SW1990 and Capan-2 cells (Fig. 3a). As shown in Fig. 3b, miR-29b-2-5p significantly promoted apoptosis in PDAC cells. In agreement, miR-29b-2-5p significantly reduced the levels of Bcl-2 and cyclinD1, and enhanced Bax2 amounts (Fig. 3c). These data suggested that miR-29b-2-5p up-regulation may promote cell cycle progression and inhibit cell apoptosis in PDAC cells.

We used predicted softwares to screen the target gene of miR-29b-5p. In the top three candidate genes, Cbl-b changed most significantly. Our previous study reported that Cbl-b plays an important role in PDAC. Silencing of Cbl-b expression inhibited proliferation in PDAC cells [25]. In this work, the relationship between miR-29b-2-5p and Cbl-b was assessed. As shown in Fig. 4a, the miRNA/mRNA comparative analysis showed that the 3′UTR of Cbl-b had the binding site for miR-29b-2-5p, at 611–617 nt. To assess whether Cbl-b is regulated by miR-29b-2-5p through direct binding to its 3′UTR, we structured plasmids containing WT or mutant 3′UTR of human Cbl-b fused downstream of the firefly luciferase gene. WT and mutant plasmids were co-transfected into Capan-2 or SW1990 cells, respectively, with miR-29b-2-5p mimic or miR-NC. As shown in Fig. 4b, luciferase activity upon miR-29b-2-5p transfection was significantly reduced. Mutations of the Cbl-b 3′-UTR abrogated the suppressive effect of miR-29b-2-5p. RT-PCR showed that Cbl-b mRNA levels had no changes after miR-29b-2-5p treatment of both Capan-2 and SW1990 cells; miR-29b-2-5p repressed Cbl-b expression through post-transcriptional inhibition in human PDAC cells (Fig. 4c). These results suggested that Cbl-b serves as an actual target of miR-29b-2-5p.

To evaluate the effect of Cbl-b in PDAC cells, the overexpression plasmid targeting Cbl-b p3xFLAG-CMV9-cbl-b (OE Cbl-b) and control plasmid (NC) were transfected into SW1990 and Capan-2 cells. Cells with more than 50% of endogenous Cbl-b expression were used in subsequent experiments (Fig. 4d). The effect of Cbl-b on cell proliferation was assessed by the Trypan blue staining method. The results showed that Cbl-b could promote the proliferation of PDAC cells (Fig. 4e). To determine the impact of miR-29b-2-5p expression on PDAC biology, the levels of this miRNA in SW1990 cells were assessed after transfection with NC and miR-29b expression -2-5p, NC plus Cbl-b, or miR-29b-2-5p plus OE Cbl-b. The results showed that miR-29b-2-5p could effectively reverse the effect of Cbl-b on the proliferation of PDAC cells. (Fig. 4f).

It is well known that the tumor suppressor p53 induces G1 arrest in response to stress. The major downstream effectors of p53 include cyclin D1, Bcl-2 and Bax. Therefore, we further assessed the p53 response after miR-29b-2-5p treatment. As shown in Fig. 5a, miR-29b-2-5p significantly enhanced p53 and p-p53 expression after Cbl-b silencing. Multiple studies showed that p53 ubiquitination and degradation are largely controlled by Mdm2, an E3 ligase. Cbl-b, which is similar to Mdm2, is also an E3 ligase. However, the relationship between Cbl-b and p53 remains undefined. As shown in Fig. 5b, p53 was associated to Cbl-b, with which it could interact (immunoprecipitation, IP) (Fig. 5c). To valuate whether the ubiquitin-proteasome mediated p53 downregulation, the proteasome inhibitor PS341 (5 nM) was incubated for 24 h with SW1990 cells. Interestingly, Cbl-b was associated with p53 in SW1990 cells (Fig. 5d). It is well known that p53 works in the cell nucleus to regulate proliferation. However, it remains unknown p53 is found after Cbl-b inhibition. As expected, miR-29b-2-5p reduced Cbl-b protein expression, while drastically inducing the expression of the nuclear form of p53. Immunofluorescent staining consistently confirmed the induced nuclear p53 expression (Fig. 5e). These findings strongly indicated that miR-29b-2-5p could promote cellular p53 by suppressing Cbl-b, while promoting p53 translocation, from the cytoplasm to the nucleus.

The expression levels of the Cbl-b protein in tissue samples from 100 patients with PDAC were detected by immunohistochemistry. We first assessed the role of Cbl-b in pancreatic cancer; interestingly, Cbl-b amounts showed a significant negative correlation with prognosis in pancreatic cancer. Patients with high Cbl-b expression had a median survival of 13.1 months (95% CI 7.9–18.1 months); those with moderate expression had 22.0 months (95% CI 17.1–26.9 months), and the low expression group 32.4 months (95% CI 24.2–40.7 months; P = 0.001, Fig. 6A). Furthermore, the pancreatic tumor specimens were grouped according to Cbl-b expression levels as negative/weak, moderate, and strong as determined by immunohistochemical staining (Fig. 6B). The expression level of miR-29b-2-5p was negatively correlated with Cbl-b protein amounts in patients with SPSS (Table 3). Collectively, this clinical and experimental study strongly suggested that Cbl-b promotes PDAC growth.