PIMREG, a Marker of Proliferation, Facilitates Aggressive Development of Cholangiocarcinoma Cells Partly Through Regulating Cell Cycle-Related Markers.

BACKGROUND: Phosphatidylinositol binding clathrin assembly protein interacting mitotic regulator (PIMREG) is a protein associated with cell proliferation. Its aberrant expression was reported to be correlated with the development in multiple tumors. However, its role in cholangiocarcinoma (CAA) has not yet been evaluated in detail.
METHODS: Data were acquired from the public TCGA database for evaluating the expression pattern of PIMREG and assessing its clinical relevance as well as its correlation with overall survival. RBE and HUH28 cell lines were selected to perform loss- and gain-of-function of PIMREG assays respectively. Quantitative real-time PCR (RT-qPCR) and western blot analyses were used to measure the mRNA and protein levels of PIMREG. Cell Counting Kit-8, colony formation tests, and Transwell assays served to measure the effect of PIMREG on the proliferative, invasive and migratory capacities of CAA cells, appropriately. Gene set enrichment analysis (GSEA) was conducted to identify PIMREG associated gene set, which was further confirmed by western blot.
RESULTS: PIMREG was found to be highly expressed in CAA tissues and cell lines according to the public dataset and RT-qPCR analysis, and negatively related to the prognosis of patients with CAA. Moreover, knockdown of PIMREG suppressed and overexpression of PIMREG promoted the proliferation, invasion and migration of CAA cells. Furthermore, GSEA revealed that high PIMREG expression was positively associated with cell cycle signaling. And the next western blot analysis demonstrated that silencing PIMREG resulted in a reduction on the levels of p-CDK1, CCNE1, and CCNB1, whereas PIMREG overexpression led to an opposite result.
CONCLUSION: The results suggested that PIMREG facilitates the growth, invasion and migration of CAA cells partly by regulating the cell cycle relative biomarkers, revealing that PIMREG may be a crucial molecule in the progression of CAA.

Introduction

Cholangiocarcinoma (CAA) is an aggressive malignancy with rising morbidity and mortality,[1] which presents a poor prognosis, ranking the second most common in primary malignancy of the hepatobiliary system.[2] Due to the hidden location of the bile duct system, early diagnosis of CAA is very difficult, and most patients are diagnosed at an advanced stage and missed the opportunity of radical surgery.[3] Thus, early diagnosis is crucial to improve the prognosis of CAA. Moreover, it is verified that CAA is associated with cholelithiasis, chronic inflammation of the biliary tract, and cholechidrosis, but the specific etiology and pathogenesis are still unknown.[4] Some molecular markers were demonstrated to be correlated with poor prognosis and tumor progression of CAA, such as mucin antigen MUC1, MUC5 AC, fascin, and epidermal growth factor receptor (EGFR), unfortunately, most of them are not routinely used in clinical.[5-8] That is to say, at present, there is no definitive prognostic marker for CAA in clinical. Therefore, it is necessary to identify new biomarkers for early detection of CCA and improvement of prognosis.

Phosphatidylinositol binding clathrin assembly protein interacting mitotic regulator (PIMREG), also known as CATS, RCS1, and FAM64A, was discovered in screening proteins that interact with lymphoid myeloid clathrin assembly proteins in 2006.[9] Multiple northern blot analyses showed that PIMREG is predominantly expressed in thymus, spleen and colon.[9] PIMREG is known to control the metaphase-to-anaphase transition during the cell division and can be considered as a marker for proliferation, suggesting a role in cancer cell growth.[10] Furthermore, it has been reported that the protein levels of PIMREG are highly expressed in lymphoma, leukemia, and other cancer cell, but not in non-proliferating T-cells or human peripheral blood lymphocytes.[11] Moreover, it was demonstrated that PIMREG could promote breast cancer aggressiveness by activating NF-κB signaling, illustrating that it may be a valuable prognosticator for breast cancer.[12] Besides, Jiao et al reported that the high expression of PIMREG can be seen as an independent risk factor for the worse prognosis in pancreatic cancer.[13] However, the potential role of PIMREG in the prognosis and development of CAA has not yet been evaluated in detail.

In current report, we evaluated the expression of PIMREG in CAA and assessed the correlation between PIMREG expression and patients’ overall survival as well as with clinical characteristics. Furthermore, we performed a series of in vitro experiments to assess the effect of PIMREG on the cell proliferation, invasion and migration in CAA, and explored the potential mechanism. Our results suggested that PIMREG promoted the CAA cell proliferation, invasion and migration partly through regulating the cell cycle-related markers.

Materials and Methods

Bioinformatics Analysis Methods

The RNA transcriptome sequencing data for CAA and the corresponding clinical information were retrieved and downloaded from The Cancer Genome Atlas (TCGA, https://www.cancer.gov/tcga) data portal. And a total of 45 samples were enrolled in present study, including 36 CAA samples and 9 adjacent normal samples.[14] The RNA-Seq data of CAA were analyzed on the Illumina HiSeq miRNA Seq platform. Subsequently, identification of dysregulated genes associated with CAA was carried out using the R language package “edgeR” with thresholds of | log 2 (fold change [FC]) |  ≥  2 and p-value <0.01.[15]

A total of 30 CAA samples with complete clinical data in the TCGA cohort were divided into high- and low-groups on the basis of the median PIMREG expression. The Kaplan-Meier methods were used to assess the correlation between PIMREG and overall survival, with a log-rank method for comparison. Relationships between PIMREG and clinical characteristics were evaluated by chi-square tests. Gene Set Enrichment Analysis (GSEA) was conducted using GSEA 3.0 (http://www.broadinstitute.org/gsea/) to identify PIMREG associated gene sets.

Cell Culture and Treatment

The human CCA cell lines HuCCT1 and RBE were purchased from Shanghai Cell Bank, Chinese academy of medical sciences. The human CAA cell line HUH28 and human extrahepatic biliary epithelial cells (HEBEpics) were obtained from American ScienCell Research Laboratories. All the cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and antibiotics, and cultured in a 37°C incubator filled with 5% CO2. Small interference (si) RNAs against PIMREG (si-PIMREG#1: 5’-AGTGCTAGCATCAGATATTTGCTC-3’; si-PIMREG#2: 5’-TGACCTTGAGCCTTCTATTTGCTC-3’) and an unspecific scrambled siRNA (si-con: 5’-GATCTTGTAGAACTTGTACCTAGT-3’) were transfected into indicated cells to implement loss-of-function of PIMREG assays. The plasmid pcDNA3.1-PIMREG vector and pcDNA3.1 empty vector were transfected into indicated cells to perform gain-of-function of PIMREG assays. All the si-RNAs and plasmids were acquired form GeneChem Corporation (Shanghai, China). The Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to perform the transfection process following the manufacturer’s instructions. Cells were collected for subsequent experiments 48 hours after transfection. All cells used in this report were taken in logarithmic phase.

Quantitative Real-Time PCR (RT-qPCR)

Total RNA was extracted from cultured RBE, HuCCT1 and HUH28 cells using Trizol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. RNA was reverse transcribed to cDNA using a Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The cDNA was amplified using SYBR Premix Ex Taq (TaKaRa, Dalian, China). Real Time PCR was performed by an Applied Biosystem 7500 Thermocycler (Thermo Fisher Scientific, Carlsbad, USA). GAPDH served as an internal reference to normalize the mRNA level of PIMREG. The primer sequences are listed as below: PIMREG forward, 5’-TACCAGGGACTCGGAGGAAG-3’ and reverse, 5’-CTTCCTCCGAGTCCCTGGTA-3’; GAPDH forward, 5’-CCACCACACTGAATCTCCCC-3’ and reverse, 5’-AGTGATGGCATGGACTGTGG-3’. The themocycling conditions were: 5 minutes pre-treatment at 95°C, followed by 40 cycles at 95°C for 30 seconds, 60°C for 45 seconds and a final extension at 72°C for 30 minutes. Data were quantified using the comparative quantification cycle (Cq) method (2−ΔΔCq).

Western Blot

RIPA lysis buffer was used to prepare total lysate. A bicinchoninic acid (BCA) method (Thermo Fisher Scientific) was used to measure protein concentration. Equal amount of protein samples (20 μg) were added to electrophoresis chambers and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes. Subsequently, these membranes were blocked with 5% skimmed milk for an hour at room temperature, and incubated with antibodies against PIMREG (1:1000, ab118102, Abcam, Cambridge, UK), CDK1 (1:1000, AF0111, Beyotime, Nantong, Jiangsu, China), phosphor (p)-CDK1 (Thr14, 1:1000, AF5758, Beyotime), CCNE1 (1:1000, AF6384, Beyotime), CCNB1 (1:1000, AF6627, Beyotime) and GAPDH (1:1000, AF1186, Beyotime) at 4°C for 24 hours. Followed by washing in Tris-buffered saline with 0.1% Tween-20 thrice, the membranes were subjected to the corresponding horseradish peroxidase-conjugated secondary antibodies and incubated for another 1 hour. Signals were visualized by the enhanced chemiluminescence detection kit (Beyotime), and images of the blots were analyzed using the ImageJ software (NIH, Bethesda, MD, USA).

Detection of Cell Viability

The cell viability was determined using the Cell Counting Kit-8 reagent (Beyotime). The transfected cells at a density of 1000 cells per well were plated into 96-well plates and incubated with complete medium. The cell viability was measured every 24 hour. Before detecting, 10 µl of CCK-8 solution and 100 µl of DMEM were supplied to each well and incubated for another 1.5 hours. Finally, the absorbance was measured by a microplate reader at 450 nm.

Colony Formation Assay

RBE and HUH28 cells (400 cells per well) were plated in 6-well plates which contain 5 ml pre-heated medium after transfection, and then cultured in complete medium at 37°C for 12 days. After fixation with 4% paraformaldehyde for 30 minutes, the colonies were stained with 0.1% crystal violet. Following air-dried, the colonies were photographed and counted.

Transwell Assay

Cell invasive and migratory capacities were determined using 24-well Transwell chambers with or without 100 µl Matrigel (BD Biosciences) respectively. After transfection for 48 hours, 1×105 cells (migration assay 5×103 cells) were added to the upper chambers and the lower chambers were filled with 500 µl of complete medium. Following incubation for 24 hours, cells passed through the coated membrane to the lower surface, where cells were washed with phosphate buffered saline twice, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution for 30 minutes. Five images of different fields were captured, and the number of migrated or invaded cells was counted under a microscope (×100).

Statistical Analysis

Student’s t-test was performed to evaluate the significant difference between 2 groups. Differences among multiple groups were analyzed using 1-way analysis of variance followed by a Tukey’s post hoc tests. Significant differences were determined using SPSS 22.0 software (IBM SPSS, Armonk, NY, USA) and GraphPad Prism 6.0 software (San Diegl, CA, USA). P < 0.05 was considered to indicate a statistically significant difference. All experiments were repeated 3 times and presented as the mean ± standard deviation.

Results

PIMREG Is Significantly Upregulated in CAA

By analyzing a published RNA-Seq dataset from TCGA database, we observed that PIMREG was notably upregulated in CAA tissues (Tumor, n = 36), compared to adjacent normal tissues (Normal, n = 9; p < 0.01, Figure 1A). To help understand the role of PIMREG in CAA, RT-qPCR was performed in 3 CAA cell lines and a normal cell line HEBEpics. Similar results were observed in CAA cell lines by contrast with HEBEpics; the mRNA level of PIMREG was significantly elevated in the HUH28, HuCCT1, and RBE cell lines (p < 0.01, Figure 1B). As PIMREG was highly expressed in CAA tissues and cell lines, PIMREG seems to have a positive function on the progression of CAA.

Figure 1.

PIMREG was upregulated in CAA and led to an unfavorable overall survival. (A) The expression of PIMREG in CAA tissues (Tumor, n = 36) compared to adjacent tissues (Normal, n = 9), as measured using a published RNA-Seq dataset from TCGA database. (B) RT-qPCR analysis of PIMREG expression in 3 CAA cell lines (HuCCT1, RBE, and HUH28) compared with normal extrahepatic biliary epithelial cell line (HEBEpics). **p < 0.01 vs. HEBEpics. (C) Kaplan-Meier curves of CAA patients with high- and low-expression of PIMREG (n = 30; p = 0.037; log-rank test). CAA, cholangiocarcinoma.

High Expression of PIMREG Results in Poor Survival Rate of CAA Patients

To further explore the clinical relevance of PIMREG and its prognostic values in CAA, the clinical data obtained from TCGA database were divided into high- and low-expression of PIMREG groups on the basis of the median expression value. Chi-square analysis revealed that PIMREG expression was remarkably related to grade (p = 0.025, Table 1). However, no obvious correlation was identified between PIMREG expression with age, gender, pathologic-stage, pathologic-tumor (T), pathologic-node (N), or pathologic-metastasis (M) (p > 0.05). In addition, Kaplan–Meier methods indicated that patients with high PIMREG expression presented a poorer overall survival rate in comparison with the low PIMREG expression group (p = 0.037, Figure 1C). These outcomes suggested that targeting PIMREG may play an effective role in the prognosis of CAA.

Table 1.

Relationship Between PIMREG Level and Clinical Features in CAA Based on the Public RNA-Seq Dataset From TCGA Database.

Characteristics	Expression of PIMREG		P value
	Low	High
Age			0.390
<60	2	5
≥60	13	10
Gender			1.000
female	8	8
male	7	7
Grade			0.025*
G1+G2	9	3
G3	6	12
Pathologic-Stage			1.000
I+II	12	12
III+IV	3	3
Pathologic-T			0.598
T1+T2	12	14
T3+T4	3	1
Pathologic-N			1.000
N0	13	12
N1	2	3
Pathologic-M			1.000
M0	14	13
M1	1	2

*p < 0.05.

PIMREG Promotes CAA Cells Proliferation In Vitro

Before evaluating the effect of PIMREG on the CAA cells proliferation, the transfection efficiency was determined by RT-qPCR and western blot. After transfection with si-PIMREG#1 and si-PIMREG#2 in RBE and HUH28 cells, the mRNA expression of PIMREG was significantly reduced compared with relative si-con group (p < 0.01, Figure 2A), and the further analysis of PIMREG protein expression in RBE cells showed the same trends (p < 0.01, Figure 2B). On the other hand, the mRNA expression of PIMREG was elevated in pcDNA3.1-PIMREG transfected RBE and HUH28 cells compared with pcDNA3.1 empty vector group (p < 0.01, Figure 2C). Western blot analysis in HUH28 cells also showed that PIMREG protein expression was increased after overexpression of PIMREG (p < 0.01, Figure 2D).

Figure 2.

Detection of knockdown and overexpression of PIMREG efficiency. RT-qPCR (A) and western blot (B) analyses of PIMREG expression after transfection with si-con, si-PIMREG#1, and si-PIMREG#2 in indicated cells. **p < 0.01 vs. si-con. RT-qPCR (C) and western blot (D) analysis of PIMREG expression after transfection with pcDNA3.1 empty vector and pcDNA3.1-PIMREG in indicated cells. **p < 0.01 vs. vector.

Cell viability was determined by a CCK-8 assay in RBE and HUH28 cells at 24 h, 48 h, and 72 h after transfection. After downregulation of PIMREG, the RBE and HUH28 cells appeared a significant reduction on the optical density (OD) values at 48 h and 72 h points (p < 0.01, Figure 3A and B). Oppositely, these 2 cells presented a higher cell proliferative capacity when compared pcDNA3.1-PIMREG group with pcDNA3.1 empty vector group. (p < 0.01, Figure 3C and D).

Figure 3.

PIMREG promoted CAA cells proliferation in vitro. CCK-8 assays were performed to detect the RBE (A, C) and HUH28 (B, D) cells viability after PIMREG knockdown (A-B) or overexpression (C-D). assays were performed to detect the cells colony forming ability after PIMREG knockdown (E) or overexpression (F) . **p < 0.01 vs. si-con or vector group.

The effect of PIMREG on cell growth was further verified by colony formation tests. Results shown in Figure 3C revealed that colonies were remarkably diminished in RBE cells transfected with si-PIMREG#1 compared to cells transfected with scrambled RNA (p < 0.01, Figure 3E). On the other hand, we observed an enhancement in the colonization ability of HUH28 cells relative to the pcDNA3.1 empty vector group after transfection of pcDNA3.1-PIMREG (p < 0.01, Figure 3F). All these results illustrated that PIMREG plays an acceerative role on the cell growth in CAA.

PIMREG Promotes CAA Cells Invasion and Migration

Cell invasion and migration are critical steps in cancer metastasis, which are the major causes of death in patients with CAA. To determine the effect of PIMREG on the cells invasiveness and motility, Transwell methods were conducted. PIMREG-depleted RBE cells presented a significant decrease in both invasive and migratory abilities in comparison with si-con group (p < 0.01, Figure 4A). Conversely, overexpression of PIMREG showed an opposite effect, as we observed that the invaded and migrated HUH28 cells were more 4 than vector group (p < 0.01, Figure B). These outcomes clearly indicated that PIMREG might serve as a considerably promoting role on the movement of CAA cells.

Figure 4.

PIMREG facilitated CAA cells invasion and migration in vitro. (A) Representative micrographs (left) and the number of invaded and migrated RBE cells (right) were measured by Transwell assays after downregulation of PIMREG. (B) Representative micrographs (left) and the number of invaded and migrated HUH28 cells (right) were measured by Transwell assays after overexpression of PIMREG. **p < 0.01 vs. si-con group or vector group.

PIMREG Has a Positive Correlation With the Proliferation-Related Molecules

To explore how PIMREG affects the proliferation of CAA patients, GSEA was conducted in TCGA, and reveled that a significantly enriched gene set of “KEGG_CELL_CYCLE” was enriched in patients with high PIMREG expression group (p < 0.01, Figure 5A). Then we further conducted western blot to determine the effect of gain- and loss-of-function PIMREG on the cell cycle biomarkers CDK1, p-CDK1, CCNE1, and CCNB1. As shown in Figure 5B to D, the expression of PIMREG were observed to be positively correlated with the level of p-CDK1, CCNE1, and CCNB1, as we found that PIMREG knockdown resulted in a significant decrease on the protein levels of p-CDK1, CCNE1, and CCNB1, whereas PIMREG overexpression led to a remarkable increase on the expression of these markers (p < 0.01). While no significant change in CDK1 expression was observed after altering the expression of PIMREG. So we inferred that PIMREG play a cancerogenic role in CAA partly through regulating cell cycle biomarkers.

Figure 5.

Effects of PIMREG on cell cycle-related markers. (A) Enrichment plot of KEGG_CELL_CYCLE between high and low risk groups by GSEA using the TCGA dataset. (B-D) The effects of PIMREG knockdown and overexpression on the protein expression of CDK1, p-CDK1, CCNE1, CCNB1 were evaluated by western blot. (B) Representative protein bands. (C-D) Quantification of B. **p < 0.01 vs. si-con group or vector group. GSEA, gene set enrichment analysis.

Discussion

The present study aimed to evaluate the role of PIMREG on the CAA progression and the potential mechanism. The data revealed that PIMREG was significantly upregulated in CAA and resulted in a worse overall survival in CAA patients. Moreover, PIMREG expression was positively correlated with the phenotypic deterioration, manifested in promoting proliferation, invasion and migration of CAA cells. Furthermore, the results also demonstrated that PIMREG promotes the progression of CAA possibly through regulating the cell cycle-related markers. This study suggested that PIMREG may play a carcinogenic role in the progression of CAA and may be a potential target for CAA treatment in the future.

Cancer is a disease that involves dynamic changes in the genome.[16] Dysregulation of pro-cancer and anti-cancer factors can lead to uncontrolled cell growth, and plays a vital role in the development of tumors.[17] Through pan-cancer analysis, Hu et al identified 92 potential pan-cancer genes, including PIMREG, that were consistently upregulated across various tumors, and showed that high PIMREG expression led to a poorer survival rate in CAA, adrenocortical cancer, lung adenocarcinoma, ovarian cancer, pancreatic cancer, etc.[18] A recent study also demonstrates that PIMREG is significantly upregulated in clear cell renal cell carcinoma and correlated with overall survival and recurrence-free survival.[19] In addition, it was well clarified that PIMREG is upregulated in triple-negative breast cancer, and its high expression is related to poor prognosis of breast cancer.[20] In agreement with the above results, in our work, we also found that PIMREG was highly expressed in CAA tissues and cell lines based on TCGA database and in vitro experiments. Moreover, we observed that high PIMREG expression was associated with worse overall survival rate in CAA patients.

CAA is a highly aggressive and malignant tumor. It is divided into intrahepatic CAA and extrahepatic CAA according to the location of tumor, and the latter is relatively common.[21] Extrahepatic CAA is prone to invade intrahepatic bile duct and liver as well as peripheral tissue lymph nodes, which is the main factor leading to poor prognosis of patients.[22] Since PIMREG is significantly elevated in CAA, we wonder whether the high expression of PIMREG is related to the high malignant phenotype of CAA. Functional experiments demonstrated that PIMREG-depleted cells showed a lower capacity for proliferation, colony formation, invasion and migration, while overexpression of PIMREG could significantly enhance the cells growth, invasion and migration in CAA. A previous report also clarifies that PIMREG is involved in proliferation and metastasis in breast cancer, as with that silencing PIMREG suppressed the cell proliferation and migration,[23] while PIMREG overexpression promoted cell proliferation and migration,[24] which were consistent with our results. Besides, PIMREG can be considered as an indispensable molecular for fetal cardiomyocytes proliferation at the late embryonic stage.[25] In leukemia cell line, lentivirus-mediated PIMREG silencing also leads to reduced cell growth, cell cycle changes, and lower migratory capacity.[26] All these findings led to the conclusion that PIMREG may play a crucial role in the malignant phenotype of CAA.

PIMREG is considered as a cell cycle promoter located in the nucleus of rapidly proliferating cells,[26] and its protein level is cell cycle-dependent.[11,27] It was reported that PIMREG is essential for fetal cardiomyocytes proliferation, and its optimum expression and degradation are both required for the cell cycle to progress.[25] Coincidentally, by GSEA on the basis of mRNA expression data from TCGA, we also observed that high PIMREG expression was positively associated with cell cycle. Moreover, an immunohistochemical analysis of CAA tissue indicated that the upregulation of cell cycle relative makers includes p-CDK1, CCNB1, and CCNE1 in carcinoma cells.[28] CDK1 is a major regulator of cell cycle in all eukaryotes, with an estimated 8-13% of proteome phosphorylated.[29] CCNB1 controls the G2-M cell cycle transition, and its elevation causes premature entry into mitosis, abnormal cell proliferation, and neoplastic transformation.[30] High cyclin E1 expression causes accelerated G1/S phase, prolonged S phase, and increased chromosomal instability.[31] For in-depth investigating the underlying mechanism, we conducted western blot to determine the changes of these markers after altering the expression of PIMREG, and found that PIMREG was positively correlated with the protein levels of p-CDK1, CCNB1, and CCNE1, whereas no significant change was observed of the protein level of CDK1 when altering the expression of PIMREG. In summary, we infer that PIMREG might facilitate the growth of CAA cells through regulating cell cycle-related markers.

Taken together, the present study demonstrated that PIMREG is upregulated in CAA and related to the unfavorable survival of patients. To the best of our knowledge, this is for the first time illustrated that PIMREG significantly facilitates cell growth, invasion and migration partly by modulating the cell cycle-related biomarkers. These results supported the carcinogenic role of PIMREG on the progression of CAA and provided a potential target for CAA treatment in the future. However, the absence of in vivo data is a limitation of the present study. Further study on subcutaneous tumor formation in nude mice after PIMREG knockdown will provide better support for the results of present study, which is required for future studies.