Introduction
PDAC is a fatal type of cancer worldwide; it has a high mortality rate and a 5-year survival rate of less than 5% [
1]. Among malignant tumors, PDAC is the fourth leading cause of death among malignant tumors in the United States [
2]. In recent years, the incidence and mortality rates have shown an increasing trend in China [
3]. Some studies predict that the global fatality rate will be the second highest in the world by 2030 [
4,
5]. Although the understanding of PDAC is gradually deepening, the pathogenesis remains unclear. The serum level of CA19-9 is the most commonly used diagnostic marker for PDAC, but the current screening methods cannot achieve effective early diagnosis [
6]. In addition, patients are still mostly treated with surgeries and diagnosis relies heavily on imaging technology, which is the main constraint on their prognosis. Therefore, exploring the novel mechanisms of PDAC is an effective way to seek new drug targets and therapeutic strategies.
MiRNAs are a crucial cluster of ncRNA which are 21–25 nucleotides long [
7]. They have the potential to be therapeutical for disease genes by complementing the 3’UTR of mRNAs and forming an RNA-induced silencing complex (RISC) to block the activity of gene transcription [
8‐
10]. Furthermore, miRNAs can regulate various biological processes such as proliferation, metastasis, apoptosis, and DNA damage by regulating the expression of target genes [
11‐
14]. Recently, some studies have shown promise in suppressing tumor progression [
15‐
17]. For instance, it is well known that mutations in KRAS contribute to PDAC tumorigenesis, but there are no effective therapeutic ways of targeting KRAS until now. Hence, the emergence of miRNAs compensates for the lack of drugs by targeting mutant KRAS such as miR-96, miR-126, and miR-143 which showed an inhibitory effect by targeting KRAS to regulate RAS family proteins [
18]. However, the mechanisms of miRNAs during PDAC progression are still elusive.
The suppressor of cytokine signaling 2 (SOCS2) is a member of the suppressor of cytokine signaling (SOCS) family. SOCS2 plays a tumor suppressor role in multiple tumors. Previous studies have shown that inhibiting SOCS2 can promote the proliferation and metastasis of colon cancer [
19,
20]. SOCS2 is also a target of several miRNAs such as miR-196b, miR-301a-3p, and miR-3648, which regulate the proliferation and metastasis of malignant tumors [
21‐
23]. However, the molecular mechanisms of SOCS2 in PDAC tumorigenesis are still obscure.
CircRNAs are another type of functional ncRNA generated by back-splicing and covalent linking with the 3’ splice site and the 5’ splice site [
24,
25]. In recent years, many studies have shown that circRNAs are aberrantly expressed in various cancers [
26‐
29]. CircRNAs can combine with miRNAs through miRNA recognition elements (MREs) [
30]. Therefore, circRNA sponges may be an effective mechanism to play an important role in tumors relying on ceRNAs surrounding circRNAs, miRNAs, and targets [
24,
25,
31]. Moreover, circRNAs function as miRNA sponges in many facets of PDAC such as proliferation, metastasis, angiogenesis, and apoptosis [
32].
In the present study, our results indicated that miR-194-5p was significantly upregulated in PDAC tissue compared to tumor-adjacent tissue, and clarified the underlying mechanism by which circPVRL3 competitively absorbs miR-194-5p as miRNA sponge to regulate the expression of the target gene SOCS2 and activate the PI3K/AKT signaling pathway to promote the progression of PDAC.
Materials and methods
Cell culture
All PDAC cell lines (PANC-1, BxPC-3, AsPC-1, CFPAC-1 and SW1990), and HPDE-C7 were purchased from American Type Culture Collection (ATCC). BxPC-3 cells were cultured in RPMI-1640 medium (Gibco, USA), HPDE-C7 cells were cultured in minimum essential medium (MEM) (HyClone, USA) and SW1990 cells were cultured in Leibovitz's L-15 medium with 10% fetal bovine serum (FBS) (HyClone, USA) and 1% penicillin–streptomycin solution (Solarbio, China). The remaining cell lines were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) medium with 10% FBS and 1% penicillin–streptomycin solution. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Transfection and cell treatment
Plasmids and small interfering RNA (siRNA) transfections were performed using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Hsa-miR-194-5p mimic or mimic NC miRNA (RiboBio, China) was transfected into cells at a final concentration of 50 nM; hsa-miR-194-5p inhibitor or inhibitor NC miRNA (RiboBio, China) was transfected into cells at a final concentration of 200 nM. The plasmids, pcDNA3.1-SOCS2 (GenePharma, China) and pLC5-ciR-circPVRL3 (GENESEED, China) were transfected (2.5 µg) into cells. si-SOCS2 and si-circPVRL3 (RiboBio, China) were transfected into cells at a final concentration of 100 nM. The sequences of the siRNAs were listed in Additional file
1: Table S3. For lentivirus transduction, 10
6 PDAC cells were incubated in a 6-well plate with 1 ml of medium containing 10 µl (10
8 U) of lentivirus particles and 5 µg/ml polybrene for 24 h.
CCK-8 assay
Cells were seeded at 5 × 103 cells per well in the 96-well plates. All groups were examined at 1 to 5 days by using a CCK-8 (Bioground, China) assay, and the absorbance was measured at 450 nm with a microplate reader. All assays were repeated 3 times.
EdU proliferation assay
To visualize cell proliferation, cells transfected with miR-194-5p mimic or inhibitor were cultured on 24-well plates, and cells were incubated with the thymidine analog EdU at a final concentration of 100 µM for 2 h. Then, the cells were fixed with 4% paraformaldehyde and an EdU proliferation assay was performed using the EdU Cell Proliferation Kit (BeyoClick™ EdU-594 #C0078L, Beyotime, China) following the manufacturer’s protocol. Cells were observed by immunofluorescence microscopy (Olympus, Tokyo, Japan).
Flow cytometry for cell cycle analysis
Transfected cells in 6-well plates were digested with trypsin and fixed with 70% ethyl alcohol at 4 °C overnight. The next day, the cells were washed once with PBS and resuspended in 1 ml PBS, and then stained using the propidium iodide method following the manufacturer’s protocol (Beyotime, China). Cell cycle analysis was performed by using a FACSCalibur FACS scanner (Becton Dickinson).
The transfected cells were seeded in the 6-well plates at 103/well. After 10–14 days of cell culture, the cells were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, China). The number of colonies was analyzed by the ImageJ software (National Institutes of Health, Bethesda, MD).
Wound-healing and Transwell migration assays
For the wound healing assay, cells were seeded into 6-well plates after transfection, and the cell monolayer was scratched with a 10 µl sterile micropipette tip to create artificial wounds. Representative images were captured at 0 and 24 h. For Transwell migration assays, were performed using cell culture inserts with 8 μm pore size transparent PET membranes (Corning, MA, USA). A total of 5 × 104 cells were resuspended in serum-free medium and placed into the upper chamber, and medium supplemented with 10% FBS was added to the lower chamber. After 10 h or 12 h, the migrated cells were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, China), and the migrated cells were counted by ImageJ software.
Real-time quantitative PCR assays and RNase R treatment
Total RNA extracted with a TRIzol Reagent (Bioground, China) was reverse‐transcribed according to the procedures of a PrimeScript™ RT reagent Kit with gDNA Eraser Kit (Takara, Kusatsu, Japan). For miRNA reverse‐transcribed reactions, the procedures followed the TaqMan™ MicroRNA Reverse Transcription Kit (#4366596, Thermo Fisher Scientific, USA). The reverse‐transcribed reactions were performed on a Thermal Cycler Dice instrument (Bio-Rad Laboratories Inc., Hercules, CA, USA). mRNA expression was quantified with an SYBR Premix Ex Taq II (Takara, Kusatsu, Japan) and miRNA expression was quantified with a TaqMan Universal Mastermix II, no UNG kit (#4440040, Thermo Fisher Scientific, USA). The real‐time quantitative PCR(RT‒qPCR) system reaction conditions were as follows: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 34 s with 40 cycles for mRNA; 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min for miRNA. Primers for hsa-miR-194-5p and U6 reverse‐transcribed and amplified were purchased from Thermo Fisher (Thermo Fisher Scientific, USA). The U6 or GAPDH was used for normalization, and all of the reactions were performed in triplicate. The relative levels of gene expression were calculated using the 2
−ΔΔCt method. Divergent primers and convergent primers were used to validate circRNA. Primer sequences are listed in Additional file
1: Table S2. For RNase R treatment, 5 U RNase R was added to the digest 1 μg of RNA for 30 min at 37 °C. The relative RNA levels were examined by agarose gel electrophoresis.
Western blotting
Total protein was extracted from transfected cells that were lysed with RIPA buffer (Solarbio, China) containing the protease inhibitor PMSF. Proteins were separated by SDS‒PAGE and blotted onto PVDF membranes. Primary antibodies against P21, E-cadherin, N-cadherin, CDK2, CDK4, Cyclin D1, Cyclin E1, p-AKT, AKT, p-PI3K, PI3K, GAPDH (1:1000; Cell Signaling Technology) and SOCS2(1:1000; ABclonal) were incubated overnight at 4 °C. Following incubation with secondary HRP-conjugated antibodies (1:5000; Cell Signaling Technology), the membranes were incubated for 1 h at room temperature. The chemiluminescence signal was detected using an ECL chemiluminescence system (Bioground, China).
Luciferase reporter assay
The wild-type (WT) sequence and mutant-type (Mut) of the circPVRL3 as well as the WT and Mut 3ʹUTR sequences of the SOCS2 that contained the predicted binding site of the hsa-miR-194-5p were subcloned them into the GP-miRGLO reporter vectors (constructed by GenePharma, China). A Dual-Luciferase Reporter System (Promega, USA) was applied to examine relative luciferase activities.
RNAscope assay
The expression of hsa-miR-194-5p in the tissue microarray was tested by using the RNAScope 2.5 HD-RED assay (324500, Advanced Cell Diagnostics, Newark, CA) following the manufacturer’s protocol. The probes used included the positive control probe U6 (727871-S1) (positive control, 313901) and the negative control probe scramble (727881-S1) (negative control, 310043) (Advanced Cell Diagnostics, Newark, CA).
Fluorescence in situ hybridization (FISH)
Cells were seeded in the confocal dishes at 1 × 104 cells per dish. Cells were fixed with 4% paraformaldehyde. For the FISH assay, Cy3 labeled cirPVRL3 (hsa_circ_0004639) and FAM labeled hsa-miR-194-5p were synthesized by GenePharma. The probe signals were observed by the RNA FISH Kit (GenePharma, China) following the manufacturer’s instructions. Images were captured by a Leica confocal microscope. The probe sequence of circPVRL3 was: 5′-GCTAAGGCACCTGCTCCACACAGAT-3′, and that of miR-194-5p was:5′-TCCACATGGAGTTGCTGTTACA-3′.
mRNA-sequencing (mRNA-seq) and bioinformatics analysis
RIP assay
RIP assays were performed by a Magna RIP™ Kit (Millipore, Burlington MA, USA) according to the manufacturer's protocol. Cells were lysed in RIP lysis buffer, and RIP lysate was incubated with the magnetic beads conjugated with AGO2 (Abcam ab186733) or IgG antibody and rotated overnight at 4 °C. The immunoprecipitation products were pulled down by the bead-antibody complex and washed with wash buffer. Immunoprecipitated RNAs were analyzed by RT‒qPCR and normalized to the input control.
Animal xenograft model
Animal experiments were approved by the Ethics Committee of Chongqing General Hospital. Four-week-old nude mice were used in the study. Approximately 5 × 106 PANC-1 or CFPAC-1 cells were injected into the armpits of nude mice subcutaneously (5 mice per group). When the tumor grew up to 5 mm × 5 mm (approximately 2 weeks), it was injected with 1 nmol miR-194-5p agomir or 5 nmol antagomir with a multipoint injection of the tumor. For the rescue animal models, nude mice were subcutaneously injected with 5 × 106 with Lv-circPVRL3 or Lv-NC (Cyagen, China) with CFPAC-1 cells into the armpits of nude mice subcutaneously and treated with/without miR-194-5p agomir/antagomir following the methods above. The tumor size was measured every 7 days, and the mice were sacrificed at 1 month after injection. The tumors were fixed in 4% paraformaldehyde and embedded in paraffin.
Immunohistochemistry
Paraffin sections of tumor tissues were cut at a thickness of 5 μm. Sections were incubated with the primary antibody anti-Ki-67 (CST, #9949, 1:200); P21 (CST, #2947, 1:50); E-cadherin (Proteintech, #20874-1-AP, 1:2000); N-cadherin (Proteintech, #22018-1-AP, 1:1000) at 4 °C overnight. Sections were incubated with HRP‐polymer‐conjugated secondary antibodies after washing with phosphate‐buffered saline, and then they were immunostained using a DAB plus kit (ZSGB-Bio, Beijing, China).
Statistical analysis
All data are presented as the mean ± standard deviation (SD). Evaluation of clinical characteristics was performed by the chi-square test and differences between the two groups were analyzed using Student’s t-test. The paired samples were subjected to a paired-samples t-test. All statistical analyses were performed with the SPSS 20.0 software (IBM Corp., Armonk, NY, USA), and two-tailed P values of < 0.05 were defined as statistically significant. The degree of significance is expressed as: *p < 0.05, **p < 0.01, or ***p < 0.001.
Discussion
PDAC is a systemic disease prone to local progression and characteristic of metastasis. However, the pathogenesis mechanism of PDAC remains unclear. The commonly mutated genes in PDAC include KRAS, TP53, SMAD4, and CDKN2A, but related specific drugs are currently unavailable. The lack of specific targets and the toxic side effects of chemotherapy are obstacles to the treatment of PDAC [
41,
42]. ncRNA is a core component of transcription, and miRNAs are posttranscriptional regulators that regulate nearly 50% of protein-coding genes [
9]. In addition, miRNAs have shown promise and have provided an approach for the diagnosis and treatment of PDAC. miRNAs downregulate the expression of target genes by the sponge effect. Meanwhile, miRNAs are also subjected to the regulation of long non-coding RNAs (lncRNAs) or circRNAs simultaneously [
25]. This ceRNA mechanism not only alters the temporal and spatial transcriptome but also plays an important role in tumor progression. In this study, we proposed that miR-194-5p expression at a high level, which is regulated by circPVRL3, promotes PDAC cell proliferation and migration by targeting SOCS2 and activating the PI3K/AKT signaling pathway.
Previous studies have reported that miR-194-5p is upregulated in PDAC and is related to PDAC tumor growth, which our findings are consistent with previous research. Studies revealed that miR-194-5p acts as a tumor promotor by targeting DACH1 [
43]. Another study showed that lncRNA H19 could absorb miR-194-5p and upregulate PFTK1 through the WNT signaling pathway to promote the proliferation and migration of PDAC based on the lncRNA‒miRNA-mRNA regulatory network [
44]. However, previous studies lacked verification with a large number of samples to some extent. In the present study, we used a tissue microarray containing 58 PDAC patient tissues, and six cell lines were used to examine the expression of miR-194-5p. We found that the expression of miR-194-5p was upregulated in PDAC tissue and two PDAC cell lines by RNAscope and RT‒qPCR. Next, we examined the function of miR-194-5p using gain-of-function and loss-of-function methods in vitro and in vivo. These results revealed that overexpressed miR-194-5p could accelerate the process of the cell cycle, and promote DNA replication and migration. Another study also reached a conclusion that was consistent with this study [
45]. In the animal model, we found that overexpression of miR-194-5p also accelerated tumor growth and increased the percentage of Ki-67-positive cells verified by IHC. Therefore, miR-194-5p served as a tumor promotor in PDAC progression.
Subsequently, we further sought to identify the target gene of miR-194-5p and identified SOCS2 as the target gene that had been screened. Moreover, miR-194-5p combined with the 3’UTR of SOCS2 mRNA directly according to bioinformatics analysis and the results of mRNA-seq. The relationship between miR-194-5p and SOCS2 was confirmed by dual-luciferase reporter assay and RIP assay. Our results verified that overexpressed miR-194-5p sharply downregulated the level of SOCS2. Consequently, we intensively explored the regulatory mechanism of SOCS2. SOCS2 is one of the SOCS family members. Previous studies indicated that a decreased expression of SOCS2 was associated with the progression and poor prognosis of hepatocellular carcinoma (HCC) [
46]. Similarly, SOCS2 was identified as a target of miR-196a and miR-196b that modulated the JAK/STAT signaling pathway affecting the progression of HCC [
47]. Our results indicated that SOCS2 acted as a tumor suppressor gene in PDAC by inhibiting cell proliferation and migration by functional assays. Meanwhile, combined with SOCS2 is a negative regulator of the JAK/STAT signaling pathway which the PI3K/AKT signaling pathway is downstream of it. We predicted that SOCS2 affected biological function by regulating the PI3K/AKT signaling pathway. The PI3K/AKT signaling pathway is one of the essential pathways for regulating cell proliferation and the cell cycle and is critical in tumor progression [
48,
49]. Our results showed that the phosphorylation levels of PI3K and AKT were enhanced by overexpressing miR-194-5p, which was opposite to the effect of knockdown miR-194-5p. Moreover, we observed that SOCS2 could partially reverse the expression of p-AKT and p-PI3K. Therefore, our results indicated that the activity of the PI3K/AKT signaling pathway was stimulated by miR-194-5p.
CircRNAs are one of the ncRNAs that are indispensable for tumorigenesis and tumor progression. Generally, circRNAs exert effects by absorbing target miRNAs, which is an important way to modulate tumor progression. The importance of circRNAs in the PI3K/AKT signaling pathway has been demonstrated in many studies. CircRNA-miRNA‒mRNA is a core regulatory network in different types of cancer. A previous study showed that circRNAs targeting different miRNAs regulated cancer progression via the PI3K/AKT signaling pathway mostly through this regulatory axis [
50]. In colorectal cancer, circIL4R targeting miR-761 promoted proliferation and metastasis by activating the PI3K/AKT signaling pathway [
51]. In osteosarcoma, circ_001422 targeted miR-195-5p and promoted the progression and metastasis through the miR-195-5p/FGF2/PI3K/Akt axis [
52]. To explore the underlying molecular mechanism of miR-194-5p, we used previous circRNA profiling data and bioinformatics methods to identify the target of miR-194-5p. Finally, we obtained circPVRL3 (hsa_circ_0004639), which was derived from the exons 2,3 and 4 of the parental gene PVRL3. Our results demonstrated that circPVRL3 could inhibit tumor proliferation and migration in vitro and in vivo. Similarly, the downregulation of circRNA_0066779, which is derived from the exons 4, 5, 6, and 7 of the PVRL3 gene, promoted the progression of gastric cancer cells by targeting several miRNAs [
53]. The dual-luciferase and RIP assays showed that circPVRL3 could directly combine with miR-194-5p. The co-localization of circPVRL3 and miR-194-5p by FISH validated the existence of the circRNA-miRNA‒mRNA network. Rescue experiments revealed that circPVRL3 absorbs miR-194-5p directly and regulates SOCS2 to manipulate the phosphorylation level of the PI3K/AKT signaling pathway.
The subsequent metastasis function of miR-194-5p through the circPVRL3/miR-194-5p/SOCS2 axis requires to perform further validation in vivo in the future. Because miRNAs could be used as biomarkers [
54], another deficiency is the significance of the clinical diagnosis of miR-194-5p among PDAC patients need to be verified. Meanwhile, one study indicated that exosomes derived from dying tumor cells in PDAC after radiotherapy delivered miR-194-5p and further regulated E2F3 to enhance the survival of residual tumor repopulating cells (TRCs) [
55]. Another study predicted circulating MIR1307 as a potential biomarker of patients who may benefit from FOLFIRINOX [
56]. Therefore, miR-194-5p may be a potential diagnostic or prognostic biomarker in serum or other body fluid samples.
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