LPAR5 confers radioresistance to cancer cells associated with EMT activation via the ERK/Snail pathway

To reveal the patterns of radiation affecting messenger RNA profiles for identifying the early responsive targets determining the sensitivity of cancer cells to radiotherapy, we performed transcriptome-wide RNA-sequencing (RNA-seq) assays in HeLa cells, including at 0.5 h and 2 h after 4 Gy γ-ray irradiation. We obtained sequencing information for 29,529 genes in total. In the 0.5 h vs. con group, there were 372 differentially expressed genes with statistical significance compared to the control cells, including 312 downregulated genes (83.8%) and 60 upregulated genes (16.1%) (Additional file 1: Fig. S1A and Additional file 2: Fig. S2A). The same analysis method found that in the 2 h vs. con group, 492 (59%) genes had a downward trend, and 345 (41%) genes had an upward trend (Fig. 1A and Additional file 1: Fig. S1B). Therefore, we chose genes with differentially downregulated expression as the research object. KEGG enrichment analysis showed that in the 0.5 h vs. con group, differentially expressed genes were enriched in tumor development-related pathways, such as the PI3K-AKT signaling pathway, PPAR signaling pathway and NOD-like receptor signaling pathway (Additional file 1: Fig. S2B). In the 2 h vs. con group, KEGG enrichment analysis showed that differentially expressed genes were enriched in the PI3K-AKT signaling pathway, MAPK signaling pathway and Wnt signaling pathway (Fig. 1B). Compared with the KEGG analysis of sequencing at two different time points, we found that the PI3K-Akt signaling pathway was significantly changed, so we chose the genes related to the PI3K-Akt signaling pathway as the selection range. We found the top 10 genes with downregulated differential changes in the 2 h vs. con group and compared the differential changes in these genes in the 2 h vs. 0.5 h group (Fig. 1C, D and Additional file 3: Fig. S3). Among them, only the LPAR5 gene was downregulated most significantly, indicating that it may have a radiation time effect relationship. GO analysis showed that the differentially expressed mRNAs in irradiated HeLa cells are involved in a variety of biological processes, including signal transduction, apoptotic processes, and G protein-coupled receptor signaling pathways, are distributed in various cellular components (membrane, cytoplasm, plasma membrane, nucleus), and play roles in multiple molecular functions (protein binding, metal ion binding, DNA binding) (Fig. 1E and Additional file 2: Fig. S2C). At the same time, we focused on the correlation of the G protein-coupled receptor signaling pathway in the GO analysis results. It has been reported that the LPAR5 protein is also involved in the regulatory mechanism of this pathway. Therefore, we chose LPAR5 as the study object for the next validation step.

We performed RT-qPCR analysis and confirmed that the expression of LPAR5 mRNA was suppressed by 4 Gy γ-ray irradiation in HeLa and A549 cells (Fig. 2). Compared with the unirradiated cells, the RNA expression of LPAR5 significantly decreased at 2 h and 4 h after 4 Gy irradiation and then gradually recovered (Fig. 2A, B). Western blot analysis also showed that in HeLa cells and A549 cells, the expression of LPAR5 protein decreased within 2–4 h after irradiation and thereafter gradually recovered, even increasing significantly at 24 h after irradiation (Fig. 2C–F). Therefore, ionizing radiation changes the expression of LPAR5 in bidirectional dynamic processes of the early downregulating phase and late upregulating phase. We further confirmed the dose-dependent inhibition of LPAR5 expression at 2 h after irradiation at the mRNA level detected by RT-qPCR (Fig. 2G , H) and the protein level detected by Western blotting analysis (Fig. 2I–L) in both HeLa (Fig. 2G, I and J) and A549 cells (Fig. 2H, K and L).

To explore the effects of LPAR5 on the proliferation phenotype of cancer cells, we knocked down LPAR5 in HeLa cells and A549 cells with specific siRNA molecules (Fig. 3A, B) and found that the proliferation of both cell lines was significantly suppressed (Fig. 3C, D). The suppressed cell growth was rescued by overexpression of exogenous siRNA-resistant LPAR5 vectors. Furthermore, knockdown of LPAR5 significantly suppressed the colony-forming ability of HeLa (Fig. 3E, F) and A549 (Fig. 3G, H) cells. On the other hand, knockdown of LPAR5 promoted the apoptosis of HeLa (Fig. 3I, J) and A549 cells (Fig. 3K, L), which was attenuated by overexpression of exogenous siRNA-resistant LPAR5 vectors. The wound healing assay indicated that knockdown of LPAR5 dramatically inhibited the migratory ability of both HeLa (Fig. 3M, N) and A549 cells (Fig. 3O, P).

To further explore the effects of LPAR5 on IR-induced apoptosis and inhibition of proliferation, we irradiated HeLa and A549 cells with LPAR5. As shown in Fig. 4A, B cell proliferation was suppressed by γ-ray irradiation, which was largely attenuated by overexpressing LPAR5. Similarly, overexpression of LPAR5 significantly attenuated the annihilation of the colony-forming ability of HeLa (Fig. 4C, D) and A549 (Fig. 4E , F) cells after irradiation. We also found that overexpression of LPAR5 significantly reduced the apoptosis induction of HeLa (Fig. 4G, H) and A549 (Fig. 4I, J) cells caused by γ-ray irradiation.

According to our data, LPAR5 is an important molecule affecting the migration of HeLa cells and A549 cells. To further clarify the role of LPAR5, we determined its potential action on EMT in cancer cells. Western blot analysis showed that compared with the control group, E-cadherin expression was upregulated in HeLa and A549 cells after knocking down LPAR5, while N-cadherin and vimentin expression was downregulated, which could be rescued by overexpressing siRNA-resistant LPAR5 (Fig. 5A–D). Conversely, overexpressing LPAR5 downregulated E-cadherin expression and upregulated N-cadherin and vimentin expression (Fig. 5E–H). These results indicated that LPAR5 promotes EMT. HeLa and A549 cells underwent EMT at 24 h after 4 Gy irradiation, as demonstrated by decreased E-cadherin expression and increased N-cadherin and vimentin expression (Fig. 5I–L). When LPAR5 was knocked down by specific siRNA in HeLa and A549 cells, IR-induced EMT was inhibited (Fig. 5I–L). The results indicated that LPAR5 plays a positive role in promoting IR-induced EMT.

To investigate the molecular pathway by which LPAR5 functions in IR-induced EMT, we tested the effect of LPAR5 on related EMT regulators and transcription factors. Western blot analysis showed that compared with the control group, p-ERK, MMP1, MMP9 and Snail expression was downregulated in HeLa and A549 cells after siRNA-mediated knockdown of LPAR5, while the expression of constitutive ERK was not affected (Fig. 6A–D). These alterations were rescued by overexpression of siRNA-resistant LPAR5. Overexpression of LPAR5 upregulated p-ERK, MMP1, MMP9 and Snail expression but not constitutive ERK expression (Fig. 6E–H). In addition, after pretreatment with the ERK inhibitor FR180204, compared with those in the LPAR5-overexpressing group, the expression levels of p-ERK and Snail were downregulated in HeLa (Fig. 6I, J) and A549 cells (Fig. 6K, L), while the expression of constitutive ERK was not affected. Pertusis toxin (PTX) is known to catalyze the ADP-ribosylation of the Gi subunits of the heterotrimeric G protein, thereby inhibiting the GPCR signaling pathway. After pretreatment with PTX, compared with the LPAR5-overexpressing group, the expression levels of p-ERK were downregulated in HeLa (Fig. 6M, N) and A549 cells (Fig. 6O, P), while the expression of constitutive ERK was not affected. These results suggest that LPAR5 activates the ERK cascade through Gi. Western blot analysis showed that compared with the control group, MMP1 and MMP9 expression was downregulated in HeLa (Fig. 6Q, R) and A549 (Fig. 6S, T) cells after siRNA-mediated knockdown of Snail. These results suggest that LPAR5 can strictly control the expression of Snail, a critical component in EMT programing through the ERK pathway. The expression levels of p-ERK, MMP1, MMP9 and Snail were upregulated in HeLa (Fig. 6U, V) and A549 cells (Fig. 6W, X) 24 h after 4 Gy irradiation and were attenuated by knocking down LPAR5. The expression of LPAR5 protein was significantly increased at 24 h after 4 Gy irradiation, which may contribute to the activation of p-ERK and Snail and consequently promote the EMT program.

The cell lines used in this study (human alveolar type II epithelial carcinoma cell line A549 and human cervical cancer cell line HeLa) were maintained in our laboratory. HeLa cells and A549 cells were grown in DMEM (HyClone) containing 10% FBS (HyClone and PAN) and 1% penicillin‒streptomycin. All cells were cultured at 37 °C under 5% CO₂ in a humidified incubator. Cells were subjected to ⁶⁰Co γ-ray irradiation at a dose rate of 85.69 cGy min^–1 at room temperature.

The full sequence of LPAR5 was cloned into pcDNA3.1 to generate an overexpression plasmid.

For LPAR5 knockdown, two synthesized duplex RNAi oligos targeting human mRNA sequences from Sigma were used (si-LPAR5-1: 5′- GCAGCUGCAUCUUCCUGAUGCUCAU-3′, si-LPAR5-2: 5′-CGCCUGCACUUGGUGGUCUACAGCU-3′). For Snail knockdown, two synthesized duplex RNAi oligos targeting human mRNA sequences from Sigma were used (si-Snail-1: 5′-CACUCAGAUGUCAAGAAGUTT-3′, si-Snail-2: 5′-CAGAUGUCAAGAAGUACCATT-3′). A scrambled duplex RNA oligo (5′-UUCUCCGAACGUGUCACGU-3′) was used as an RNA control. The cells were transfected using Lipofectamine 2000 reagent (Invitrogen) with vector control, plasmid construct, siRNA negative control (siNC), or siRNAs according to the manufacturer’s instructions.

Total RNA was isolated using TRIzol. One microgram of RNA was reverse-transcribed into cDNA with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Code No. FSQ-301) according to the manufacturer’s instructions. Quantitative real-time PCR analysis was performed with 1 μL of cDNA using HUNDERBIRD SYBR qPCR Mix (TOYOBO, Code No.QPS-201). β-actin was used as an endogenous control. β-actin primers used for qRT‒PCR, F: 5′-TTGCTGACAGGATGCAGAAG-3, R: 5′-ACTCCTGCTTGCTGATCCACAT-3′. LPAR5 primers used for qRT‒PCR, F: 5′-GAGGTCTCTGCTGCTGAT-3′; R: 5′-AGAACTGTTGGTTGAGGAG-3′.

Cells (1.5 × 10³) were seeded into 96-well plates and incubated at 37 °C in a humidified 5% CO₂ atmosphere. Cellular proliferation was measured with a Cell Counting Kit-8 (DOJINDO, SJ608). Briefly, 10 μL/well CCK8 solution was added at the indicated times, and then, the cells were incubated at 37 °C for 3 h. The absorbance at 450 nm was recorded by a microplate reader.

Cell migration was examined by wound-healing assays. After treatment of the cells, scratches were made using sterile 10-μL pipette tips, and bright-field microphotographs were taken at different times. The percentages of cell migration were quantitated by ImageJ software by measuring the width of the cell-free zone immediately after scratching.

To assess cell apoptosis, after treatment with trypsin, the cells were collected and washed twice with PBS. Then, 4 μL of PI (propidium iodide) and 4 μL of FITC (fluorescein isothiocyanate) (Dojindo, AD10) were added after the cells were pelleted and resuspended in 400 μL of 1 × binding buffer. Apoptosis was detected by flow cytometry after 15 min of incubation in the dark at room temperature.

Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quality and quantity were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN > 7.0. Approximately more than 200 µg of total RNA was subjected to isolation of poly(A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Following purification, the poly(A) mRNA fractions were fragmented into 100-nt oligonucleotides using divalent cations under elevated temperature. Fragments were converted to a final cDNA library in accordance with strand-specific library preparation by the dUTP method. The average insert size for the paired-end libraries was 100 ± 50 bp. Then, we performed paired-end 2 × 150 bp sequencing on an Illumina NovaSeq 6000 platform at LC-BIO Biotech, Ltd. (Hangzhou, China).

Data are presented as the mean ± SEMs or SDs. Statistical analyses were performed in GraphPad Prism 6 (GraphPad Software, Inc.) The unpaired two-tailed Student’s t test was used to compare differences between two groups with a significance of P < 0.05. One-way analysis of variance with multiple comparisons tests was used to compare three or more groups with a significance of P < 0.05.