Long noncoding RNA LINC00514 accelerates pancreatic cancer progression by acting as a ceRNA of miR-28-5p to upregulate Rap1b expression

PC tissue and adjacent normal tissue were collected from the First Affiliated Hospital of Nanchang University with the informed content of the enrolled patients in this research. Patients received neither chemotherapy nor radiotherapy before surgery. Our study was approved by the Human Research Ethics Committee of Nanchang University.

RNA was extracted by TRIzol reagent (Invitrogen) from tissue samples and cells. Extracted RNA was later reverse transcribed into complementary DNA (cDNA) by PrimeScript RT Reagent (Takara, Japan). A SYBR Green Kit (Takara, Japan) was utilized to perform RT-PCR. GAPDH and β-actin were used as internal controls. Gene expression levels were calculated by the 2^−ΔΔCt method. The primer sequences are shown in Table 1.

The normal pancreatic epithelial cell line (HPDE) and PC cell lines (BxPC-3, SW1990, PANC-1, AsPC-1, Capan-2, and MIAPaCa-2) were purchased from ATCC. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂ in humidified air.

LINC00514 overexpression plasmid and shRNAs against LINC00514 and Rap1b were purchased from Gene Pharma (Shanghai, China) with scramble plasmid and shRNA used as negative controls. MiR-28-5p mimics and miR-28-5p inhibitors were acquired from Gene Pharma as well. All of the above reagents were transfected into cells via TF-3000 Transfection Reagent (Invitrogen) according to the manufacturer’s recommendations.

Cells (1 × 10³ cells per well) were seeded in 6-well plates and then incubated for 10 days. After being washed with PBS three times, colonies were stained with hematoxylin and counted.

To evaluate cell viability, a CCK-8 assay was carried out. Cells (1 × 10⁵ cells per well) were plated in 96-well plates for 0 h, 24 h, 48 h and 72 h. The cell growth rate was analyzed by Cell Counting Kit-8 (Solarbio, China) reagent according to the manufacturer’s instructions. The optical density value was measured by a microplate reader at 450 nm.

A transwell chamber (Corning, Tewksbury, MA) was used to detect cell migration and invasion capacities. Cells (1 × 10⁵) were seeded on the upper chamber covered with Matrigel (Corning, Tewksbury, MA), while DMEM with 10% FBS was placed on the lower chamber. After 24 h of transfection, cells that passed from the upper chamber onto the lower chamber were fixed with methanol, stained with crystal violet and imaged under a light microscope.

Five-week-old female nude mice were purchased from the National Laboratory Animal Center (Beijing, China) and maintained under specific pathogen-free conditions. Subsequently, the mice were randomly separated into two groups. Cells (1 × 10⁶) of the LINC00514 overexpression group and NC group were subcutaneously injected into the right axillary of nude mice. Tumor volume was measured every 4 days, and weight was measured at the end of the experiment.

To further evaluate the effects of LINC00514, we carried out pulmonary metastasis analysis. PC cells (1 × 10⁶) were injected into nude mice via the caudal vein. After 30 days, the mice were euthanized. The lungs of mice were removed to observe tumor metastasis. All experiments were approved by the Animal Research Ethics Committee of Nanchang University.

The PARIS Kit (Life Technologies) was used to isolate nuclear and cytoplasmic RNAs according to the manufacturer’s protocol. Reverse transcription of extracted RNAs and RT-PCR were conducted as described before.

The online software StarBase3.0 http://​starbase.​sysu.​edu.​cn/​was used to predict the binding sites of LINC00514 to miRNA-28-5p. Wild-type LINC00514 and mutant LINC00514 of the putative binding sites were cloned into a luciferase vector (Promega) and cotransfected with miR-28-5p mimics into PC cells via LF-3000 transfection reagent. After 48 h, cells were harvested for luciferase activity analysis.

Wt-miR-28-5p and NC-miRNA were labeled with biotin and transfected into BxPC-3 and SW1990 cells. The cell lysates were incubated with streptavidin magnetic beads at 4 °C for 4 h. After that, the beads were rinsed with precooled lysis buffer and salt buffer. The pull-down RNAs were extracted to detect LINC00514 levels.

The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA) was used to conduct the RIP assay according to the manufacturer’s protocol. The cells were lysed and incubated with Ago₂ and IgG. Then, cell lysates were mixed with anti-Ago₂ and anti-IgG in RIP buffer (Millipore). Precipitated RNAs were collected for RT-PCR analysis.

Protein was extracted from cells and transferred to polyvinylidene difluoride (PVDF) membranes after 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After that, membranes were blocked with 5% nonfat milk and incubated overnight at 4 °C with primary antibodies. After rinsing the membranes three times with PBS, a secondary antibody labeled with horseradish peroxidase was used to incubate membranes for 2 h at room temperature. Antibodies against E-Cadherin, N-Cadherin, and Vimentin were all purchased from CST company, and β-actin and Rap1b antibodies were purchased from Abcam. The dilution ratio was determined according to the instructions.

First, we analyzed the LINC00514 profile in PC. We found that LINC00514 was remarkably increased in PC tissues compared with the corresponding normal tissues (Fig. 1a). The upregulated expression of LINC00514 was significantly associated with the LINC00514 level and tumor size, lymph node metastasis and clinical stage (Fig. 1b-d), while no significant correlation was found between LINC00514 expression and age, gender or tumor differentiation (Table 2). Additionally, LINC00514 expression was increased in PC cell lines compared with the normal pancreatic epithelial cell line (Fig. 1e). Furthermore, Kaplan–Meier survival curves revealed that high LINC00514 expression was related to a lower overall survival rate compared with that of the low LINC00514 level group (Fig. 1f). Overall, LINC00514 was increased in PC and might be associated with clinical progression and a poor prognosis of PC patients.

To investigate whether LINC00514 is involved in cell proliferation, migration and invasion, we carried out gain- and loss-of-function assays. The LINC00514 overexpression plasmid and LINC00514 shRNA were stably transfected into BxPC-3 and SW1990 cells, with a scramble plasmid and shRNA used as negative controls (Fig. 2a-b). According to the results of CCK-8 and colony formation assays, LINC00514 overexpression significantly promoted cell (BxPC-3 and SW1990) proliferation capacity, while suppression of LINC00514 remarkably inhibited these processes (Fig. 2c-e). Moreover, transwell assays were utilized to prove that LINC00514 increased cell migration and invasion capabilities (Fig. 2f-g). For further confirmation, western blotting was performed to measure the expression of EMT markers in both BxPC-3 and SW1990 cells. As expected, E-cadherin was observed to be strikingly downregulated by LINC00514 overexpression, whereas N-cadherin and Vimentin were obviously upregulated, and the sh-LINC00514 group showed the opposite results (Fig. 2h). In summary, LINC00514 promoted PC cell proliferative, migratory and invasive capacities.

To further identify the bioeffects of LINC00514 on tumor growth, we constructed a subcutaneous xenograft tumor model. BxPC-3 cells transfected with LINC00514 shRNA compared with NC shRNA or transfected with the LINC00514 overexpression plasmid and compared with the empty plasmid were subcutaneously injected into nude mice. The tumors were measured every 4 days after injection. After euthanizing the mice, we obtained images of the tumors (Fig. 3a-b). Compared with those of the NC group, the volume and weight of tumors in the LINC00514 shRNA group were significantly reduced, while the opposite results were observed in the LINC00514 overexpression group (Fig. 3c-f). QRT-PCR was used to assess the transfection efficiency (Fig. 3g-h). Then, we further investigated the role of LINC00514 in PC metastasis in vivo. Nude mice were injected with BxPC-3 cells transfected with LINC00514 shRNA compared with NC shRNA or the LINC00514 overexpression plasmid compared with the empty plasmid into the tail vein. Images of pulmonary metastasis were acquired at the endpoint (Fig. 3i-j). There was an obviously lower incidence of pulmonary metastasis and a smaller number of metastatic tumors per lung in the sh-LINC00514 group compared with the NC group, whereas the LINC00514 overexpression group showed the opposite results (Fig. 3k-l).

To explore the underlying molecular mechanism of the oncogenic effects of LINC00514 on PC, we determined the subcellular localization of LINC00514. The results showed that LINC00514 was mostly distributed in the cytoplasm (Fig. 4a-b), which suggested that LINC00514 might exert its biological function by sponging miRNA. StarBase3.0 was utilized to identify a candidate microRNA (miR-28-5p) and predict the potential downstream targets of LINC00514 (Fig. 4c). The luciferase reporter assay results confirmed that the luciferase activity of WT-LINC00514 was clearly decreased by miR-28-5p mimics, while the luciferase activity of Mut-LINC00514 did not change significantly (Fig. 4d-e). In addition, the RIP assay further revealed that LINC00514 and miR-28-5p were enriched in beads conjugated to Ago₂ compared with the IgG group (Fig. 4f-g). Furthermore, overexpression of WT-LINC00514, but not Mut-LINC00514, decreased miR-28-5p expression in BxPC-3 and SW1990 cells (Fig. 4h). Additionally, overexpressing LINC00514 dramatically decreased miR-28-5p levels in both BxPC-3 and SW1990 cells, while silencing LINC00514 increased miR-28-5p levels, with NC shRNA used as an internal reference (Fig. 4i-j). Then, we further detected miR-28-5p expression in tumor tissue. The results revealed a lower level of miR-28-5p in PC tissue than in normal tissue and a negative correlation between LINC00514 expression and miR-28-5p levels (Fig. 4k-l). To obtain more evidence, in vivo experiments were performed. We detected the expression level of miR-28-5p in the tumors we collected before, and the results showed that miR-28-5p expression was higher in the cell lines with LINC00514 knockdown, while miR-28-5p was lower in the cells with LINC00514 overexpression (Fig. 4m-n). LINC00514 promoted cell proliferation, migration and invasion at least partially by sponging miR-28-5p (Fig. 4o-r). In summary, LINC00514 accelerates PC progression by sponging miR-28-5p.

The posttranscriptional function of miRNAs is usually to inhibit protein synthesis by base pairing with the 3′-untranslated region [17]. Next, to ascertain the detailed regulatory mechanism of LINC00514 in PC, we searched StarBase3.0 and observed that Rap1b was predicted to be a downstream target of miR-28-5p (Fig. 5a). Mut-Rap1b or WT-Rap1b and miR-28-5p or NC-miRNA were transfected into BxPC-3 and SW1990 cells. A luciferase reporter assay and RIP assay were used to confirm the hypothesis that Rap1b is a direct target of miR-28-5p (Fig. 5b-e). Then, we found that Rap1b expression was downregulated by LINC00514 silencing, while cotransfecting miR-28-5p and sh-LINC00514 inhibited the effect of LINC00514 knockdown on Rap1b at both the transcriptional and translational levels (Fig. 5f-g).

Then, we detected Rap1b levels in tumor tissue. Rap1b was obviously increased in PC tissue compared with adjacent normal tissue, and there was a positive relationship between Rap1b expression and LINC00514 levels, while a negative correlation was observed between Rap1b expression and miR-28-5p levels (Fig. 5h-j). In addition, we also detected the expression of Rap1b in vivo, and the transfection efficiency was examined previously. As expected, the expression of Rap1b was clearly decreased in BxPC-3 cells by LINC00514 knockdown, while increased expression was observed in LINC00514-overexpressing cells (Fig. 5k-l). Thus far, we have proven that Rap1b is a direct target of miR-28-5p. Thereafter, functional experiments were carried out to investigate the bioeffects of Rap1b. The results demonstrated that Rap1b silencing remarkably suppressed the promoting effects of the miR-28-5p inhibitor on cell proliferation, migration and invasion capacities (Fig. 5m-p).

Finally, we explored the role of LINC00514 mediated by Rap1b in promoting tumor growth. We knocked down Rap1b in BxPC-3 and SW1990 cells to determine whether Rap1b inhibition can reverse the oncogenic effects of LINC00514. Based on the rescue assays, Rap1b inhibition partially inhibited the effect of LINC00514 overexpression on cell proliferation, migration and invasion (Fig. 6a-d). In conclusion, these results collectively demonstrated that LINC00514 acted as a key tumor promotor of PC by competitively binding to miR-28-5p and then upregulating the expression of Rap1b.