COL6A6 interacted with P4HA3 to suppress the growth and metastasis of pituitary adenoma via blocking PI3K-Akt pathway

The differently expressed genes and signaling pathway in PA

According to the above criteria, we finally chose 9 normal pituitary and 14 PA samples (GSE26966) for our re-analysis by Affymetrix Human Genome U133 Plus 2.0 Array. The results showed that the top 10 significantly downregulated genes (CSH1, CSH2, DLK1, NKX2-2, CSHL1, PTEN, POU1F1, COL6A6, GH1, and PCK1) associated with tumor metastasis were labeled in Figure 1A, 1B. Meanwhile, the expression of COL6A6 in PA tissues was the lowest (Figure 1B). Besides, the dotplot of GSEA showed that PI3K-Akt signaling pathway was notably activated in all comparisons (Figure 1C), and most of the genes associated with the PI3K-Akt pathway were downregulated (Figure 1D). Of note, P4HA3 acts as an upstream regulator gene of the PI3K-Akt pathway, was upregulated in PA tissues compared with the normal group (Figure 1D). Furthermore, COL6A6 was found to be closely related to P4HA3 in the networks of differentially expressed genes (Figure 1E).

Figure 1. The differentially expressed mRNAs and pathway in PA. (A) Volcano plot of differentially expressed genes in the whole cohort vs. normal. The red dots represent up-regulated genes and the green dots represent down-regulated genes; (B) Heatmap of top ten differentially expressed genes in tumor vs. normal; (C) The dotplot of significantly altered pathways in PA tissues analyzed by GSEA; (D) The heat map of differentially expressed mRNAs in the PI3K-Akt pathway analyzed from microarray GSE26966; (E) The correlative network of COL6A6 and differentially expressed mRNAs involved in the PI3K-Akt signaling pathway in PA.

Expression of COL6A6 and P4HA3 in PA and its correlation with tumorigenesis

Based on the regulated network of COL6A6 and P4HA3 in PA, the expression of COL6A6 and P4HA3 in PA and its correlation with tumorigenesis was investigated. As shown in Table 1, COL6A6 was a negative correlation with tumor size and high grade. RT-qPCR analysis results showed that COL6A6 was downregulated in PA tissues compared with the normal pituitary tissues (all P<0.001, Figure 2A), and P4HA3 was upregulated in PA tissues (P<0.001, Figure 2B), which consistent with the results of immunohistochemistry staining (Figure 2C, 2D). Furthermore, COL6A6 were markedly decreased, and P4HA3 upregulated in PA cell lines by western blotting, especially in AtT-20 and HP75 cells (all P<0.01, Figure 2E, 2F). Thus, low-expression of COL6A6 and P4HA3 overexpression may be associated with the progression of PA. Meanwhile, AtT-20 and HP75 cells were chosen for subsequent experiments.

Figure 2. The expression of COL6A6 and P4HA3 in PA tissues and cell lines. (A and B) RT-qPCR was used to detect the expression of COL6A6 and P4HA3 in PA tissues and normal pituitary tissues; (C and D) Immunohistochemistry staining was applied to detect the expression of COL6A6 and P4HA3 in PA tissues and normal pituitary tissues; (E and F) Western blotting was used to evaluate the expression of COL6A6 and P4HA3 in PA cell lines. ^***P<0.001, compared with normal pituitary tissues; ^##P<0.01, compared with AtT-20 cells; ^▲▲P<0.01, compared with HP75 cells.

Effect of COL6A6 on the growth and metastasis of PA cells in vitro and in vivo

To explore the effect of COL6A6 on the proliferation, invasion, migration, and apoptosis of AtT-20 and HP75 cells. Western blotting analysis results showed that the expression level of COL6A6 protein was increased or decreased in AtT-20 and HP75 cells when transferred with pcDNA-COL6A6 (both P<0.001) or si-COL6A6 (both P<0.01) compared with the NC group (Figure 3A). CCK-8 analysis results showed that overexpression of COL6A6 notably inhibited the proliferation ability of both AtT-20 and HP75 cells compared with the NC group (both P<0.05, Figure 3B). Besides, the effect of COL6A6 knockdown on the proliferation of both AtT-20 and HP75 cells were detected (both P<0.05, Figure 3B). Transwell and wound healing assay demonstrated that upregulation of COL6A6 remarkably inhibited invasion and migration capacity of both AtT-20 and HP75 cells (all P<0.01, Figure 3C–3G). Conversely, knockdown of COL6A6 promoted the invasion and migration of both At T-20 and HP75 cells (all P<0.01, Figure 3C–3G). Western blotting showed that COL6A6 elevated significantly suppressed the expression levels of N-cadherin and Vimentin proteins in AtT-20 and HP75 cells compared with NC group (all P<0.01, Figure 3H–3K), but enhanced the E-cadherin protein (all P<0.01, Figure 3H–3K). However, silencing of COL6A6 got opposite results (all P<0.01, Figure 3H–3K). These results strongly implied that overexpression of COL6A6 was suppressed the proliferation, invasion, migration, and EMT of AtT-20 and HP75 cells.

Figure 3. The effect of COL6A6 on the proliferation, invasion, migration, and EMT of AtT-20 and HP75 cells. (A) The expression of COL6A6 protein was determined by western blotting; (B) The proliferation ability of AtT-20 and HP75 cells were measured by CCK-8 assay; (C and D) The invasion of AtT-20 and HP75 cells were measured by Transwell assay (40×); (E–G) The migration ability of AtT-20 and HP75 cells were evaluated by wound healing assay (4×); (H–K) The expression of EMT related proteins in AtT-20 and HP75 cells were detected by western blotting. ^*P<0.05, ^**P<0.01, ^***P<0.001, compared with the NC group.

To further examine the effect of COL6A6 on the growth and metastasis of PA in vivo. Overexpression of COL6A6 noticeably inhibited the size of the tumor, tumor volume (all P<0.01) and tumor weight (all P<0.001) compared with the NC group (Figure 4A–4D). Moreover, western blotting analysis results showed that COL6A6 elevated markedly increased COL6A6 protein level in tumor tissues of PA mice model compared with the NC group (all P<0.01, Figure 4E, 4F). Immunohistochemistry results demonstrated that upregulation of COL6A6 decreased the expression of Ki-67 in xenograft tumor tissues (Figure 4G, 4H). Furthermore, compared with the NC group, overexpression of COL6A6 significantly suppressed the expression levels of N-cadherin and Vimentin protein, while the expression level of E-cadherin was upregulated (all P<0.01, Figure 4I, 4J). Therefore, our finding suggest that upregulation of COL6A6 may restrict PA growth and metastasis in vitro and in vivo.

Figure 4. The effect of COL6A6 on the growth and metastasis of xenograft tumor. (A and B) The tumor size was obtained from nude mice; (C and D) Tumor volume and tumor weight of nude mice were analyzed; (E and F) The expression of COL6A6 in xenograft tumor tissues were detected by western blotting; (G and H) The expression of Ki-67 in xenograft tumor tissues were detected by immunohistochemistry staining; (I and J) The expression of E-cadherin, vimentin, and N-cadherin were measured by western blotting. ^**P<0.01, ^***P<0.001, compared with NC group.

Effect of COL6A6 on the PI3K-Akt signaling pathway through interacting with P4HA3

To further clarify the mechanism of COL6A6 regulated the growth and metastasis of PA cells, we assessed the effect of COL6A6 interacted with P4HA3 on the PI3K-Akt pathway, which plays an important role in regulating tumor development, tumor cells invasion and EMT. Western blotting showed that COL6A6-elevated significantly decreased the expression of P4HA3 protein, as well as decreased the expression of p-PI3K and p-Akt, but the expression of PI3K and Akt remained unchanged (all P<0.01, Figure 5A, 5B). In addition, we further detected the expression p-PI3K and p-Akt in both AtT-20 and HP75 cells treated with the PI3K-Akt pathway activator (IGF-1) or P4HA3 overexpression, the results showed that P4HA3 upregulation or IGF-1 treatment abolished the inhibitory effect of COL6A6-elevated on the PI3K-Akt pathway (all P<0.01, Figure 5A, 5B). Furthermore, co-immunoprecipitation showed that COL6A6 was closely related to P4HA3 (Figure 5C, 5D). These data indicated that COL6A6 may serve an anti-oncogene role in the development of PA by interacting with P4HA3 and further blocking the PI3K-Akt pathway, while its mechanism needs further verification.

Figure 5. Effect of COL6A6 on the PI3K-Akt signaling pathway through interacting with P4HA3. (A and B) Western blotting was used to detect the expression of COL6A6, P4HA3, PI3K-Akt pathway-related protein in AtT-20 and HP75 cells; (C and D) Coimmunoprecipitation to validate the COL6A6-P4HA3 interaction. ^**P<0.01, ^***P<0.001, compared with NC group; ^##P<0.01, compared with COL6A6 group.

Effect of COL6A6 regulated growth and metastasis of PA cells through mediating P4HA3-PI3K-Akt pathway

Based on the effect of COL6A6 on the PI3K-Akt signaling pathway by interacting with P4HA3 in vitro, to further explore the role of COL6A6 and P4HA3 mediated the proliferation, invasion, migration, and apoptosis of both AtT-20 and HP75 cells by regulating PI3K-Akt pathway. CCK-8, Transwell and wound healing assay demonstrated that P4HA3 overexpression or IGF-1 treatment significantly abolished upregulation of COL6A6 exerted antitumor activity in both AtT-20 and HP75 cells (P_{proliferation}<0.05, P_invasion<0.01, P_migration<0.01, Figure 6A–6F). Besides, we found that IGF-1 addition or P4HA3-elevated reversed the inhibitory effect of COL6A6 on the EMT of both AtT-20 and HP75 cells (all P<0.001, Figure 6G). Furthermore, we observed that overexpression of COL6A6 significantly decreased the size of the tumor compared with the control group (all P<0.001, Figure 7A, 7B), while the size of the tumor with IGF-1 treatment was bigger than the COL6A6 overexpression group (all P<0.001). Meanwhile, IGF-1 rescued the antitumor activity of COL6A6 in subcutaneous xenograft models (Figure 7C–7F). Taken together, these data suggested COL6A6 elevated inhibited tumor growth and metastasis in PA via interacting with P4HA3 and blocking the PI3K-Akt pathway.

Figure 6. The effect of COL6A6 interacted with P4HA3 on the proliferation, invasion, migration, and EMT of AtT-20 and HP75 cells through regulating the PI3K-Akt pathway. (A and B: The proliferation ability of AtT-20 and HP75 cells were measured by CCK-8 assay; (C and E) The invasion of AtT-20 and HP75 cells were measured by Transwell assay (40×); (D and F) The migration ability of AtT-20 and HP75 cells were evaluated by wound healing assay (4×); (G) The expression of EMT related proteins in AtT-20 and HP75 cells were detected by western blotting. ^*P<0.05, ^**P<0.01, ^***P<0.001, compared with NC group; ^#P<0.05, ^##P<0.01, ^###P<0.001, compared with COL6A6 group.

Figure 7. The effect of COL6A6 on the growth and metastasis of xenograft tumor by mediating PI3K-Akt pathway. (A and B) The tumor size was obtained from nude mice; C and E: The tumor weight was measured; (D and F) The tumor volume curve of nude mice was analyzed. ^**P<0.01, ^***P<0.001, compared with control group; ^##P<0.01, ^###P<0.001, compared with COL6A6 group.

PA data collection

Microarray datasets of PA were searched and downloaded from the gene expression omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). We conducted a rigorous screening for these datasets and the following are inclusion criteria: (1) the human microarray datasets were genome-wide; (2) samples in each study should include cases and controls; (3) tumor samples are non-cell line samples; and (4) raw data or expression matrix were available. According to the above criteria, we finally chose GSE26966 (Contributed by Michaelis KA et al. [42]) for our re-analysis. The dataset contains 9 normal pituitary and 14 PA samples that were tested by Affymetrix Human Genome U133 Plus 2.0 Array. In the PA samples, there were 7 invasive samples, 7 non-invasive samples, 5 recurrent samples, and 9 non-recurrent samples.

Data pre-processing

R statistical software v3.4.1 was used to perform pre-processing and subsequent data analysis. We used the Robust Multichip Average (RMA) algorithm in the oligo package [43] to normalize the raw expression data and generate normalized gene expression intensity. We downloaded annotation platform GPL570 in GEO database for gene annotation and the methods see the previous publications [17, 18]. Finally, we got an expression matrix including 20307 genes and 23 samples.

Differential expression gene analysis

Differentially expressed gene analysis was performed using the empirical Bayes algorithm in the “limma” package [46] in R. Differences (up- or down-regulated) were considered statistically significant for the absolute value of log2(fold-changes) higher than 1 and false discovery rate (FDR) adjusted P values ≤ 0.05. Differentially expressed genes were calculated between tumor and normal samples in five comparisons (whole cohort, invasive, non-invasive, recurrent, and non-recurrent). The expression profiles of differentially expressed genes were showed by heatmap in “pheatmap” package. We chose “ward. D2” algorithm to perform hierarchical clustering for samples in different groups. Venn diagram was used to compare the differentially expressed genes between these groups.

Gene set enrichment analysis

We used javaGSEA desktop application v3.0 (http://software.broadinstitute.org/gsea/) [47] to perform gene set enrichment analysis (GSEA) of the five comparisons. Gene sets with less than 10 genes or more than 500 genes were excluded. The t-statistic mean of the genes was computed in each pathway using a permutation test with 1000 replications. Pathways with normalized enrichment scores (NESs) > 0 were considered up-regulated, and subsystems with NESs < 0 were considered down-regulated. Statistical significance was identified as FDR adjusted P values ≤ 0.05. We used “pathview” package [28] to display the enriched pathways and deregulated genes.

Protein-protein interaction networks

STRING web server (https://string-db.org/cgi/input.pl) [49] was used to construct protein-protein interaction (PPI) networks of screened SAA effect genes. The parameters were set as follows: (1) the network edges were set as the molecular action (line shape indicates the predicted mode of action); (2) all types of active interaction sources were chosen, including text mining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence; (3) the minimum required interaction score was set at the medium confidence of 0.4; and (4) the maximum number of interactors in the first shell was the total number of query proteins only and the maximum number of interactors in the second shell was set to none. The enriched KEGG pathways of the input genes were automatically exported from the webserver.

Tissue specimen

For the clinical aspect of the present study, 30 PA tissues and 15 normal pituitary tissues were collected. Tumor invasion was determined by Knosp garding scale. The present study complied with the principles of the Declaration of Helsinki and was approved by the regulations of the Ethics Committee of Kunming Medical University, as well as informed consent was obtained from patients. Samples were frozen in liquid nitrogen immediately and stored at -80°C for subsequent experiments.

Cell culture and transfection

PA cell lines (HP75, AtT-20, GT-1, and MMQ) were purchased from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences and cultured according to the manufacturer’s protocols. Media was supplemented with 1% penicillin-streptomycin and 10% FBS (Gibco, USA) at 37°C and 5% CO₂.

A total of 24 h prior to transfection, HP75 and AtT-20 cells were seeded in six-well plates at the optimum density and incubated overnight. pcDNA-COL6A6, sh-COL6A6, and control (blank plasmid) were transfected into both HP75 and AtT-20 cells using Lipofectamine^® 3000 reagent and Opti-MEM medium (Invitrogen Life Technologies, USA) according to the manufacturer’s protocol. The pcDNA-COL6A6, sh-COL6A6, and blank plasmid were purchased from Tolo Biotech (Shanghai, China).

RT-qPCR

Total RNA was isolated from 0.2 g tissues or 5×10⁵ cells using Trizol reagent (QIAGEN, Germany). Tissues were ground in liquid nitrogen before the addition of extraction buffers, while cells were directly mixed with extraction buffers. And then, performed revere-transcribed reactions using PrimeScript^TM one-step real-time quantitative polymerase chain reaction (RT-qPCR) kit (TaKaRa, Japan). Real-time PCR was carried out using the Power SYBR Green PCR Master Mix (TaKaRa, Japan) and examined using the Bio-Rad System (Bio-Rad, USA). The sequences of the primers used for qPCR are presented in Table 2. The PCR reactions were carried out as follows: 95°C for 60 s, followed by 40 cycles of 95°C for 10 s and 57.5°C for 30 s. The 2^-ΔΔCt methods were applied to calculate the relative expression levels of COL6A6 and P4HA3. qPCR was performed in triplicate.

CCK-8 assay

Cell Counting Kit-8 (Sigma, Japan) was used to assess the proliferation of both HP75 and AtT-20 cells. Both cells were seeded in 96-well plates at a density of 2.5×10⁴ cells per well and cultured with 5% CO₂ at 37°C incubator for 2 h to allow cells to adhere. A total of 10 μL CCK-8 solution was added to each well and mixed, and cells were incubated for a further 2 h. A dual-wavelength microplate reader was used to measure proliferation at 450 nm (Beckman Coulter, USA). The assay was performed in triplicate.

Flow cytometry analysis

Collected the samples mentioned above, washed with PBS, centrifuged at 800 ×g for 6 min, suspended in ice-cold 70% ethanol/PBS, centrifuged at 800 ×g for another 6 min, and suspended with PBS. Resuspended cells with 100 μL medium and added 5 μL of annexin V and 1 μL of propidium iodide according to the manufacturer’s instructions of Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher, USA), and incubated for 15 min at room temperature. BD LSR II flow cytometry was used to detect cells apoptosis (BD Biosciences, USA).

Transwell invasion assay

Matrigel (BD Biosciences, USA) was coated the upper surface of polycarbonate filters. A total of 1×10⁵ cells/ml of cells were plated in 200 μL of the serum-free medium in the upper layer of the Transwell chamber (Corning, USA) and 800 μL medium supplemented with 10 % FBS was added to the bottom chamber. After 24 h of incubation, the cells which had invaded were fixed using 4% paraformaldehyde, rinsed three times with PBS, stained with 0.1% crystal violet for 10 min and rinsed three times with PBS. For quantification, 5 randomly selected fields were analyzed (magnification ×20).

Wound healing assay

HP75 and AtT-20 cells (2×10⁵ cells/well) were treated with different reagents, seeded in six-well plates and cultured until they reached confluence. Wounds were made in the cell monolayer by making a scratch with a 20 μL pipette tip. Plates were washed once with fresh medium after 24 h in culture to remove non-adherent cells. Following this wash, plates were photographed under ×4 objective.

Western blotting

Total protein was extracted for western blotting analysis. The PVDF membranes were incubated overnight at 4°C with primary antibodies against COL6A6 (1:1000, PA5-60958, Thermo, USA), P4HA3 (1:1000, ab101657, Abcam, Cambridge, UK), E-cadherin (1:1000, ab1416, Abcam, Cambridge, UK), N-cadherin (1:1000, ab202030, Abcam, Cambridge, UK), Vimentin (1:1000, ab193555, Abcam, Cambridge, UK), p-PI3K(p85)(1:1000, #ab191606, Abcam, Cambridge, UK), PI3K(p85) (1:1000, #ab191606, Abcam, Cambridge, UK), and p-Akt (), Akt (), and subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (1:500, Abcam, Cambridge, UK). Signals were visualized using enhanced chemiluminescence reagent (Bio-Rad, USA).

Nude mice model

Female BALB/c nude mice (4~5 weeks old) were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences and maintained in a pathogen-free facility. The model was approved by the Kunming Medical University. HP75 or AtT-20 cells (5×10⁶) transfected with NC or pcDNA COL6A6 were suspended in serum-free DMEM medium and then inoculated in left armpit of nude mice (10 nude mice in each group) at 5-6 weeks old to establish heterotopic transplanted tumor models of PA. In rescue experiments, the nude mice model was treated with PI3K-Akt activator IGF-1 (4 mg/kg) or equivalent amount of PBS by vein injection every two days for 2 weeks. Tumor growth was recorded every three days by measuring tumor length and width. After 4 weeks incubation, the mice were killed and the mice tumor tissues to be collected for further evaluated.

Immunohistochemistry assay

The xenograft tissues were collected and fixed with formalin neutral solution of 10% volume fraction, paraffin-embedded and subsequently sectioned. A DAB horseradish peroxidase color development kit (Beyotime Institute of Biotechnology) alongside a Ki-67 antibody (1:2000, ab15580, Abcam, Cambridge, UK) were used to stain the samples at room temperature. Subsequently, slides were dyed with hematoxylin for 30 s, dehydrated and fixed, and then sealed with neutral glue. Stained images were observed and photographed under a fluorescence microscope (Olympus Corporation).

Statistical analysis

Continuous variables are presented as the mean ± SD. The experimental data and image preprocessing were analyzed by SPSS 20 statistical software (IBM, USA) and GraphPad Prism7.0 software (La Jolla, USA), respectively. Besides, student’s t-test was used to analyze differences between the two groups, and the differences between multiple groups were compared by one-way ANOVA. Moreover, P<0.05 was identified as a statistically significant difference.