CXC chemokine ligand‐13 promotes metastasis via CXCR5‐dependent signaling pathway in non‐small cell lung cancer

Abstract The CXC chemokine ligand‐13 (CXCL13) is a chemoattractant of B cells and has been implicated in the progression of many cancers. So far, CXCL13 and its related receptor CXCR5 have been proved to regulate cancer cell migration as well as tumour metastasis. However, the role of CXCL13‐CXCR5 axis in metastasis of lung cancer is still poorly understood. In this study, we found that CXCL13 and CXCR5 were commonly up‐regulated in lung cancer specimens compared with normal tissues among different cohorts. Our evidence showed that CXCL13 obviously promoted migration of lung cancer cells, and this effect was mediated by vascular cell adhesion molecule‐1 (VCAM‐1) expression. We also confirmed that CXCR5, the major receptor responsible for CXCL13 function, was required for CXCL13‐promoted cell migration. We also test the candidate components which are activated after CXCL13 treatment and found that phospholipase C‐β (PLCβ), protein kinase C‐α (PKCα) and c‐Src signalling pathways were involved in CXCL13‐promoted cell migration and VCAM‐1 expression in lung cancer cells. Finally, CXCL13 stimulated NF‐κB transcription factor in lung cancer cells, contributing to VCAM‐1 expression in translational level. These evidences propose a novel insight into lung cancer metastasis which is regulated by CXCL13.


| INTRODUC TI ON
Lung cancer is the major cause responsible for cancer death worldwide. Most of lung cancer patients are diagnosed as non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD) and lung squamous-cell carcinoma (LUSC). 1 The golden treatment regimens such as chemotherapy and radiotherapy have improved prognosis of lung cancer over the past decades, survival rate remains low; the 5-year survival rate is about 15% while the patients are diagnosed with lung cancer. The low survival rate of lung cancer is caused by distant metastasis occurred in late-stage. However, the mechanisms of metastasis in NSCLC have not yet been fully illustrated. Cancer metastasis is a complex, multi-step process, in which cells escape from the primary origin and form metastases at a distant site. 2 Primary cancer cells loss their cell adhesion phenotype, polarity, rearrange cytoskeleton and adjacent extracellular matrix (ECM) to invade the surrounding stroma. 3 Primary tumour release cancer cells into the circulation long before diagnosis. To establish disseminated cancer cells that may finally progress to metastases, circulating cancer cells must first transmigrate endothelial capillary walls and successfully adapt to the new environmental stress. 4 Transendothelial migration (TEM) of monocytes is the process by which monocytes or leukocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall, then enter the underlying tissue. 5 Similar to leukocyte extravasation, lung metastasis requires cancer cells to cross the lung endothelium. 6 Vascular cell adhesion molecule 1 (VCAM-1) has been implicated in early leukocyte transmigration. 5 More and more is being learnt about the key role played by VCAM-1 in tumorigenesis and metastasis. 7 VCAM-1 expression correlates with glioblastoma grade, 8 as well as with poor survival in lung cancer 9 and epithelial ovarian cancer. 10 Previous research has indicated that VCAM-1 contributes to breast cancer cells metastasizing to lung. 11 Moreover, VCAM-1 overexpression in breast cancer promotes bone metastasis by activating osteoclastogenesis and subsequent early relapse. 12 These findings establish VCAM-1 as a promising target for the prevention and inhibition of metastasis.
Several studies have reported that chemokines have a key role in regulating the homing of leukocytes as well as cancer cells. 13 We found that expression levels of CXCL13 and CXCR5 were highly correlated with lung cancer progression. 14 Chemokines have revealed that they have crucial roles in the metastasis and progression of different cancers. In breast cancer, the CXCL12/CXCR4 axis has been extensively investigated in regard to the homing of cancer cells to metastatic sites and promoting metastasis. 15,16 Metastatic breast cancer cells express high levels of CXCR4; its ligand CXCL12 is highly secreted by stromal cells within these tissues. 17 CXCL13, also known as B-cell-attracting chemokine 1 (BCA-1), and its receptor CXCR5 help to regulate lymphocyte migration and promote inflammation. 18,19 The binding of CXCL13 to CXCR5 elicits the activation of PI3K/Akt, MAPK, integrin-β3/Src/FAK and the DOCK2/Rac/JNK pathways, which regulate cell survival, invasion, and proliferation, respectively. 20 Recently, the CXCL13-CXCR5 axis has been implicated in the progression of various tumours, including colon cancer, 21 hepatocellular carcinoma, 22 chronic lymphocytic leukaemia, 23 breast cancer, 23 neuroblastoma 24 and prostate cancer. 25 Much clinical evidence highlights the crucial roles of the CXCL13-CXCR5 axis in the regulation of tumour growth, progression and metastasis in the tumour microenvironment. 26 However, the role of the CXCL13-CXCR5 axis in lung cancer is poorly discussed.
Here, we found that expression levels of CXCL13 and CXCR5 were highly correlated with lung cancer progression. Also, CXCL13/ CXCR5 axis contributed to cell motility in lung cancer cells, which was caused by VCAM-1 expression. Finally, PLCβ, PKCα and c-Src signalling pathways were involved in CXCL13-promoted cell migration and VCAM-1 expression in lung cancer cells. Our present work provides novel application of CXCL13/CXCR5 axis in lung cancer therapeutic strategy.

| Meta-analysis of associations between CXCL13, CXCR5 and VCAM-1 expression in NSCLC
The data sets used in meta-analysis were selected from Lung Cancer Explorer (LCE; http://qbrc.swmed.edu/lce/), which contains data sets of gene profiling of tumour patients and healthy subjects obtained from Gene Expression Omnibus (GEO), The Cancer Genomics Atlas (TCGA) and other published literatures. Data sets of lung cancer tissues containing adenocarcinoma (LUAD) and squamous-cell carcinoma (LUSC) were selected from LCE. For the meta-analysis of CXCL13 and CXCR5 expression levels in selected data sets, the sample size of each group (ADC, SCC, normal) more than 20 was required to assure robust analysis results. The correlation analysis of CXCL13, CXCR5 and VCAM-1 were performed with Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cance r-pku.cn/) web-based tool. 27 The RNA profiling data of CXCL13, CXCR5 and VCAM-1 were performed with Correlation Analysis provided by GEPIA.

| Cell viability assays
Cells (7 × 10 3 ) were seeded in 96-well plates prior to the indicated treatment. Cell viability was assayed using CCK8 reagent as described 28 and was determined as the percentage of the control.
Samples were protected from light during the assays, and the incubation time for CCK8 was set to 4 hours. Each condition was performed in eight replicate wells, and the data present were from at least three independent experiments.

| Transwell migration and invasion assay
All cell migration and invasion assays were performed using Transwell The experimental protocol for the invasion assay was as described above for the cell migration assay. Each experiment was performed with triplicate wells and was repeated at least three times.

| Western blot analysis
The cellular lysates collected from lung cancer cells were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with NAP-BLOCKER ™ in TBS [2X] (G-Biosciences) for 1 hour at room temperature and then probed with rabbit anti-human antibodies against (1:1000) for 1 hour at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody (1:5000) for 1 hour at room temperature. The blots were visualized with enhanced chemiluminescence substrate and photographed using a charge-coupled device camera-based detection system (UVP Inc). Quantitative data were obtained using ImageJ software (National Institutes of Health).

| Quantitative real-time PCR
qPCR analysis was carried out using TaqMan ® one-step PCR Master Mix (Applied Biosystems). One hundred ng of total cDNA was added per 25-µL reaction with sequence-specific primers and TaqMan ® probes. Sequences for all target gene primers and probes were purchased commercially (β-actin was used as internal control; Applied Biosystems). Quantitative RT-PCR assays were carried out in triplicate on StepOnePlus sequence detection system. The cycling conditions were 10-minutes polymerase activation at 95°C followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted as C T ).

| Immunofluorescence
Cells were seeded on chamber slides before immunofluorescence staining. After treatment with indicated condition as described in figure legends, the cells were washed with PBS and fixed in 3.7% formaldehyde for 10 minutes at room temperature. Cells were washed three times with PBS and blocked with 4% BSA for 15 minutes.
Cells were then incubated with the p65 primary antibody (1:100) for 1 hour at room temperature, washed again and incubated with FITCconjugated secondary antibody (1:100) for 1 hour. Finally, cells were washed, mounted with DAPI containing solution, and photographed with a Nikon Ti2 microscopy System (Nikon).

| Transfection of shRNAs and siRNAs
The lung cancer cells were transfected with shRNAs or siRNAs using Lipofectamine 3000 (Invitrogen) according to the manufacturer's recommendations. After 24 hours post-transfection, the cells were performed with cell migration, qPCR, Western blot, Immunofluorescence or luciferase reporter assays as described in

| Luciferase reporter assay
The luciferase activity in the transfected cells was measured using a Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Lung cancer cells were transfected with NF-κB vectors using Lipofectamine 3000 (Invitrogen) for 24 hours, the cells were treated with inhibitors or vehicle control for 30 minutes and then exposed to CXCL13 (30 ng/mL) for 24 hours. Transactivation was determined by monitoring the firefly luciferase levels in the pGL2 vector. The luciferase assay was performed by adding lysis buffer (100 μL) and harvesting the cells through centrifugation (16200 g for 5 minutes). The supernatant was transferred to fresh tubes, and 20 μL of cell lysate was added to 80 μL of fresh luciferase assay buffer in an assay tube. The luciferase activity was measured using a microplate luminometer. Luciferase activity was normalized to transfection efficiency based on the cotransfected β-galactosidase expression vector.

| Statistics
The values given will be the means ± SD The significance of difference between two experimental groups was analysed by Student's t test, and multiple group comparisons were performed using oneway analysis of variance (one-way ANOVA) with Fisher LSD post hoc tests. Between-group differences will be considered significant if the P value is <.05.

| CXC chemokine ligand-13 and CXCR5 is highly expressed in lung cancer specimens
CXC chemokine ligand-13 has been implicated in the progression of various cancers. 26 The evidence also shows that the CXCL13-CXCR5 pathway is important in prostate cancer cell invasion and migration. 25 However, little is known about the CXCL13-CXCR5 axis and its roles in lung cancer. We first examined the expression profile of CXCL13 and CXCR5 in lung cancer tissues by using online lung cancer-specific database-the Lung Cancer Explorer (LCE). 29 Surprisingly, with meta-analysis in all lung cancer tissues containing adenocarcinoma (LUAD) and squamous-cell carcinoma (LUSC), we found consistent up-regulation of CXCL13 and CXCR5 in multiple lung cancer studies ( Figure 1A,B), suggesting the impact of CXCL13-CXCR5 axis in lung cancer progression.

| CXC chemokine ligand-13 promotes cell mobility in lung cancer cells
To determine whether CXCL13 affected migration of lung cancer cells, a Transwell migration and invasion assays were used. The

| Vascular cell adhesion molecule-1 expression contributes to CXCL13-induced cell migration in lung cancer cells
We next explored the candidate molecules that regulated cell migration in lung cancer cells. The data showed that VCAM-1 was obviously induced after incubation with CXCL13 ( Figure 3A). Knockdown of VCAM-1 expression in lung cancer cells confirmed that CXCL13-promoted cell migration was mediated through VCAM-1 ( Figure 3F-H). Finally, we manipulated online gene expression profiling tool GEPIA 27 to assess the correlation between CXCL13 and VCAM-1 in lung cancer tissues. The data indicated that VCAM-1 expression levels were highly correlated with CXCL13 in specimens of LUAD and LUSC ( Figure 3I,J). In summary, these evidences propose that VCAM-1 is required for cell migratory potential in response to CXCL13 incubation.

| CXCR5 mediates cell migration and VCAM-1 expression in response to CXCL13 incubation in lung cancer cells
CXCR5, a member of the G-protein coupled receptors (GPCR) family, is required for CXCL13 biological functions. 30 The CXCL13-CXCR5 axis is implicated in regulating lymphocyte migration and promoting inflammation. 18,19 Therefore, we hypothesized that CXCL13induced lung cancer cell migration may be regulated by CXCR5. Lung

| Phospholipase Cβ/PKCα/c-Src signalling pathways are required for CXCL13-promoted cell migration and VCAM-1 in lung cancer cells
The intracellular signalling pathways that activate transcription factors, and subsequently up-regulate gene expression, are crucial for cellular biological functions. The chemokine receptor elicits multiple signalling pathways including the PLC/PKC, c-Src, JAK/STAT, PI 3 K/Akt and MAPK pathways. 31 Here, we investigated whether CXCL13 activated these signalling pathways in lung cancer cells. The results indicated that pre-treatment with PLCβ, PKCα and c-Src inhibitors (U73122, GF109203X and PP2) reversed CXCL13-promoted cell migration and VCAM-1 in lung cancer cells ( Figure 5A,B). Moreover, incubation with CXCL13 obviously induced phosphorylation of PLCβ, PKCα and c-Src ( Figure 5C). Finally, we certified our finding by using siRNA to suppress these pathways activation, and the data showed that transfection with F I G U R E 3 Vascular cell adhesion molecule-1 expression is responsible for CXCL13-promoted cell migration in lung cancer. A, The screening of candidate molecules associated with cell migration ability. The CL1-0 lung cancer cells were incubated with CXCL13 (30 ng/ mL) for 24 h, follow by collecting RNA sample, then subjecting to qPCR analysis to validate gene expression of candidate molecules which associated with cell migration ability. B and C, The CL1-0 lung cancer cells were incubated with different concentrations (0, 3, 10 and 30 ng/ mL) of CXCL13 for 24 h, the RNA or cell lysate were collected, then subjected to analyse gene expression level of VCAM-1 by qPCR and Western blot. D and E, The CL1-0 lung cancer cells were treated with CXCL13 (30 ng/mL) for different time interval (0, 6, 12 and 24 h), the RNA or cell lysate were collected and then subjected to analyse gene expression level of VCAM-1 by qPCR and Western blot. F, The CL1-0 lung cancer cells were transfected with VCAM-1 siRNA for 24 h, and then, the expression level of VCAM-1 was confirmed by Western blot. G, The CL1-0 lung cancer cells transfected with VCAM-1 siRNA were subjected to analyse cell mobility in the presence of CXCL13 (30 ng/ mL) by Transwell migration assay. H, The CL1-0 lung cancer cells transfected with CXCL13 shRNA were performed with qPCR to detect VCAM-1 expression level. I and J, The correlation analysis data of CXCL13 and VCAM-1 by using GEPIA bioinformatics tool. The boxplot analysis used log 2 (TPM + 1) for log-scale. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group F I G U R E 4 CXCR5 mediates the VCAM-1 expression and cell migration in response to CXCL13 incubation. A, The CL1-0 lung cancer cells were transfected with CXCR5 siRNA for 24 h, and the knockdown efficiency of CXCR5 was confirmed by Western blot. B and C, The CL1-0 lung cancer cells transfected with CXCR5 siRNA were further performed with cell migration assay B, in the presence of CXCL13 (30 ng/mL) for 24 h, as well as qPCR C, to detect VCAM-1 expression. D and E, The CL1-0 lung cancer cells were pre-incubated with CXCR5 neutralized antibody (0.5 and 1 μg/mL) for 1 h and then performed with cell migration assay D, and qPCR E, in the presence of CXCL13 (30 ng/mL) for 24 h to evaluate cell migratory ability and VCAM-1 expression, respectively. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group PLCβ, PKCα and c-Src siRNA apparently reversed cell migration and VCAM-1 expression after CXCL13 incubation ( Figure 5D-G). These results suggest that the PLCβ/PKCα/c-Src signalling pathways contribute to CXCL13-promoted cell migration in lung cancer cells.

| NF-κB transcription factor is responsible for CXCL13-induced cell migration in lung cancer cells
NF-κB is a crucial transcription factor and its activation is correlated with cancer cell migration and invasion. 32,33 It is also a key downstream effector of chemokine receptor signalling. 31 Figure 7B). Finally, to further examine the transcriptional activation of NF-κB is responsible for CXCL13 effects, the luciferase reporter assay was performed. The NF-κB activity was significantly increased after CXCL13 treatment ( Figure 7C).
However, in the presence with pathway inhibitors or siRNAs of CXCR5, PLCβ, PKCα and c-Src, the NF-κB activity was reversed after CXCL13 treatment ( Figure 7D,E). In conclusion, these evidences suggest that the CXCR5, PLCβ, PKCα and c-Src signalling pathways mediate NF-κB activation in lung cancer cells treated with CXCL13.

| D ISCUSS I ON
CXC chemokine ligand-13 and its corresponding receptor CXCR5 have been widely reported in various cancers. 34 However, the prognostic value and pathological role of CXCL13/CXCR5 in lung cancer is still in its infancy. Here, we report the prognostic relevance of CXCL13/CXCR5 and found that they are commonly overexpressed among different subtypes of lung cancer. Previous report showed that high levels of CXCR5 and CXCL13 were up-regulated in tissues and sera of lung cancer patients respectively. 35 Here, we used online database to perform meta-analysis, and results indicated CXCL13 and CXCR5 were highly expressed in different data sets.
This evidence suggests that CXCL13/CXCR5 axis activation is common feature in lung cancer progression. Recently, the therapeutic applications by targeting CXCL13/ CXCR5 axis have been summarized. 34 The strategies by using RNA interference and pharmacological intervention have been investigated in several studies and showed promising effects. Our present work reveals that CXCL13/ CXCR5 axis could be a novel therapeutic target against lung cancer.
CXC chemokine ligand-13, first identified as BLC (B-lymphocyte chemoattractant) or BCA-1 (B cell-attracting chemokine 1), a homeostatic chemokine which attracting B lymphocytes while exhibiting a minor effect on T cells and macrophages. 36 Previous studies have proved that CXCL13 promotes cancer cells migration via different biological molecules, for example matrix metalloproteinase-9 (MMP9) 37 and MMP13. 38 Here, we found that VCAM-1, a cell adhesion molecule associated with metastasis, which was up-regulated in response to CXCL13 in lung cancer cells. VCAM-1 expression is reported to be closely implicated in the metastasis of many cancer cells.
In breast cancer cells, VCAM-1 expression was up-regulated in the selected cancer cells which metastasized to lung. 39 In glioblastoma, VCAM-1 expression correlated with the clinicopathological grade of cancer. 8 VCAM-1 expression was also found to be up-regulated in lung cancers. 9 Here, we also investigated the correlation between CXCL13 and VCAM-1 in tissues of lung cancer patients. The highly correlation between CXCL13 and VCAM-1 supports our finding in in vitro experiments. As previous study, the indication of VCAM-1 is a critical component requiring for lung cancer invasion and further metastasis. 9 Our finding here supposes that CXCL13/CXCR5 axis is a major regulator of VCAM-1 up-regulation in lung cancer.
The signal activation downstream of CXCL13/CXCR5 axis has been discussed in several reports, and however, the whole scenario of CXCL13/CXCR5 is still poorly understood. In this study, we found PLCβ/PKCα/c-Src signal cascade was activated after CXCL13 treatment in lung cancer cells. Furthermore, this activation was required for VCAM-1 expression and then cell migration. As the G F I G U R E 5 Phospholipase Cβ/PKCα/c-Src signalling pathways are required for CXCL13-promoted cell migration and VCAM-1 in lung cancer cells. A and B, The CL1-0 lung cancer cells were pre-treatment with pathway inhibitors of PLCβ, PKCα or c-Src (U73122, 0.5 μmol/L; GF109203X, 3 μmol/L; PP2, 5 μmol/L) for 1 h and then performed with cell migration assay A, and qPCR B, in the presence of CXCL13 (30 ng/mL) for 24 h to evaluate cell migratory ability and VCAM-1 expression, respectively. C, The CL1-0 lung cancer cells were incubated with CXCL13 (30 ng/mL) for different time course (0, 10, 15, 30 and 60 min), the cell lysates were collected, and activation of PLCβ, PKCα and c-Src signalling pathways was evaluated by monitoring phosphorylation of these proteins. The total proteins were used as internal control. D, The CL1-0 lung cancer cells were transfected with PLCβ, PKCα or c-Src siRNAs for 24 h, and the knockdown efficiency was confirmed by using Western blot. E, The CL1-0 lung cancer cells transfected with PLCβ, PKCα or c-Src siRNAs were further evaluated cell migration ability E, and VCAM-1 expression F and G, by using Transwell migration assay, qPCR and Western blot, respectively, in the presence of CXCL13 (30 ng/mL) for 24 h. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group protein-coupled receptors (GPCRs), which forming chemokine li- The transcription factor NF-κB has been widely discussed in cancer progression and targeting NF-κB as a therapeutic strategy has been explored extensively in the past decades. NF-κB has been F I G U R E 6 NF-κB activation is responsible for CXCL13-induced cell migration and VCAM-1 expression in lung cancer cells. A-C, The CL1-0 lung cancer cells were pre-treatment with pathway inhibitors of NF-κB (PDTC, 5 μmol/L; TPCK, 5 μmol/L) for 1 h and then performed with cell migration assay A,, qPCR B, and Western blot C, in the presence of CXCL13 (30 ng/mL) for 24 h to evaluate cell migratory ability and VCAM-1 expression, respectively. D, The CL1-0 lung cancer cells were incubated with CXCL13 (30 ng/mL) for different time course (0, 10, 15, 30 and 60 min), the cell lysates were collected, and activation of NF-κB signalling cascade was evaluated by monitoring phosphorylation of IKKα/β, IκBα and p65 proteins. The total proteins were used as internal control. E and F, The CL1-0 lung cancer cells were transfected with dominant mutants of IKKα and IKKβ for 24 h and then performed with cell migration assay E, and qPCR F, in the presence of CXCL13 (30 ng/mL) for 24 h to evaluate cell migratory ability and VCAM-1 expression, respectively. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group implicated in various cellular functions such as cell proliferation, apoptosis, angiogenesis, cell migration and invasion. 45 Previous review has summarized that NF-κB is a downstream signalling effector of G protein-coupled receptor (GPCR), which is regulated by affecting upstream signal components or other transcription factors that coordinate with NF-κB. 46 However, the activation of NF-κB by CXCR5, a member of GPCR super family, has never been reported yet. Our results indicated that NF-κB signal pathway was activated by CXCL13/CXCR5 axis. Interestingly, previous reports have provided evidence that NF-κB transcriptional activation was required for CXCL13 release or CXCR5 expression, 47,48 revealing the pivotal role of NF-κB in CXCL13/CXCR5 axis activation. Combined with our F I G U R E 7 NF-κB activation is downstream effector of CXCR5/PLCβ/PKCα/c-Src signal cascade which contributes to VCAM-1 expression and cell migration in lung cancer cells. (A and B) The CL1-0 lung cancer cells were pre-incubated with CXCR5 neutralized antibody (1 μg/ mL), U73122 (0.5 μmol/L), GF109230X (3 μmol/L) or PP2 (5 μmol/L) for 1 h, follow by incubated with CXCL13 (30 ng/mL) for 24 h. Then, immunofluorescence staining was performed with anti-p65 antibody. Nuclei were counterstained with DAPI. Representative microscopy images were shown A. The NF-κB activation was monitored by phosphorylation of p65. The p65 and β-Actin were used as internal control B. C, The CL1-0 lung cancer cells were transfected with NF-κB luciferase reporter vector for 24 h, follow by incubated with CXCL13 (30 ng/mL) for further 24 h. Then, luciferase activity assay was performed to examine NF-κB activation. D, The CL1-0 lung cancer cells were transfected with NF-κB luciferase reporter vector for 24 h, follow by incubated with CXCR5 neutralized antibody (1 μg/mL), U73122 (0.5 μmol/L), GF109230X (3 μmol/L) or PP2 (5 μmol/L) for 1 h, then exposed to CXCL13 (30 ng/mL) for further 24 h. Finally, the luciferase activity was performed to examine NF-κB activation. E, The CL1-0 lung cancer cells were cotransfected with NF-κB luciferase reporter vector in the presence of CXCR5, PLCβ, PKCα or c-Src siRNAs for 24 h, follow by incubated with CXCL13 (30 ng/mL) for further 24 h. Then, luciferase activity assay was performed to examine NF-κB activation. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group finding here, the autocrine loop mediated by CXCL13/CXCR5 and NF-κB may contribute to enhancement of cell migration effect.
In conclusion, our present study suggests that CXCL13/CXCR5 axis is up-regulated in lung cancer cells, which promotes VCAM-1 expression and further cell migration. We also decipher the molecular mechanism involved in regulation of this effect, which is achieved by PLCβ/PKCα/c-Src signal cascade and NF-κB transcription factor.
Based on our finding here, the clinical relevance of CXCL13/CXCR5 in lung cancer progression and development of novel therapeutic targets should be addressed in the future.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and analysed during the current study are available from the corresponding author on reasonable request.