Background
Colorectal cancer (CRC) is recognized as the third highest cancer type in terms of incidence rate and the third most frequent cause of cancer death in both genders [
1‐
3]. Increasingly, CRC accounts for approximately 10% of all new cancer cases globally [
4,
5]. The current first-line treatments for CRC include surgical resection, chemotherapy, radiotherapy and targeted therapy [
6,
7]. Although various treatment approaches have shown obvious anticancer activity in CRC and improved the survival rate among cancer patients, the majority of patients have poor prognosis and cannot be fully cured due to inevitable recurrence [
8,
9]. Therefore, there is an urgent need to focus on improving the effectiveness and sensitivity of anticancer regimens, while an understanding of CRC pathogenesis and novel therapeutic targets may contribute to better selection of treatment approaches and reduced recurrence.
Growing research suggests that high levels of ribosomal protein expression are prognostic factors in certain tumors [
10‐
13]. In addition, tumor cells are characterized by a higher production of ribosomes, necessary to maintain enhanced growth and subsequent cell division. The increase in ribosome production is closely related to dysregulated ribosome biogenesis and alteration in hallmarks of cancer cells such as nucleoli number, size and shape [
14,
15].
The ribosome is a structurally and functionally conserved supramolecular ribonucleoprotein (RNP) complex, and its biogenesis is an energy-consuming and well-orchestrated process, requiring several assembly and maturation factors [
16]. It is well known that ribosomal proteins are synthesized in the cytoplasm, and subsequently combine with rRNA in nucleolus to provide a supply of ribosomal subunits [
17,
18]. In eukaryotic cells, the cytosolic 80S ribosome is composed of two subunits: the small 40S subunit and the large 60S subunit [
16]. eL31, a protein that is part of the 60S large ribosomal subunit, was demonstrated to be one of the constituent proteins of the ribosomal P-site [
19]. It is located at or near the vicinity of the peptidyl site of ribosomes, is an important constituent of peptidyltransferase center, and belongs to the eukaryotic ribosomal proteins [
20]. Furthermore, eL31 forms a rim around the polypeptide tunnel exit, which physically interacts with Zuo1 subunit of RAC (ribosome-associated complex) [
21]. Several lines of evidence recently presented that eL31 was involved in the proliferation of bicalutamide-resistant prostate cancer cells [
22,
23]. In addition to these, eL31 was recently identified as a novel Diamond Blackfan anemia (DBA) gene [
24]. On the other hand, HL31, which is highly homologous to eL31, has increased expression in colorectal tumors [
25]. However, the mechanistic role of eL31 in CRC remains poorly understood and warrants further investigation.
Here, we set out to characterize the function and mechanism of action of eL31 in CRC development in more detail. To that end, IHC analysis was employed to identify abundant eL31 expression in CRC tissues, which was associated with tumor size and prognosis of patients. Moreover, we found that eL31 depletion resulted in the restrain of cell growth and the promotion of cell apoptosis. Besides, the findings of in vivo experiments was consistent with the above results, indicating an inhibitory effect of eL31 depletion on CRC tumorigenesis. Additionally, the investigation on the regulatory mechanism of eL31 in CRC identified DEPDC1 as a potential downstream target of eL31. In summary, eL31 functions as tumor promotor in CRC, which may represent a novel therapeutic target for CRC.
Methods
Tissue specimens and cell lines
A tissue microarray (Cat. HColA180Su17, n = 101 CRC tissues and n = 77 morphological normal tissues) was provided by Shanghai Outdo Biotech Company. This study was approved by the Institutional Animal Care and Use Committee of Affiliated Hospital of Inner Mongolia Medical University and the Ethics committee of Affiliated Hospital of Inner Mongolia Medical University.
Human normal colorectal mucosal cell FHC and a panel of CRC cell lines including HCT 116, RKO and HT29 were obtained from Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China). The culture and treatments were performed as follows: HT29 cells were cultured in McCoy′s5A + 10%FBS medium, and the other three types of cells were cultured in 1640 medium with 10% FBS, placing in a 37 °C incubator containing 5% CO2.
Immunohistochemistry (IHC)
The tissue specimens were soaked with xylene and washed with alcohol (China National Pharmaceutical Group Co., Ltd, Beijing, China). After that, the slides were repaired with 1 × EDTA (Beyotime Biotechnology Co., Ltd, Shanghai, China) and blocked with 3% H
2O
2 and serum. Next, primary antibodies or secondary antibodies were used to incubate slides at 4 °C overnight, and then the slides were stained using DAB and hematoxylin (Baso DiagnosticsInc., Zhuhai, China). Finally, the slides were sealed with neutral resin (China National Pharmaceutical Group Co., Ltd, Beijing, China). The images were scored as negative (0), positive (1–4), + + positive (5–8), or + + + positive (9–12), based on the sum of the staining intensity (varied from weak to strong) and staining extent scores, which graded as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). Antibodies used were listed in Additional file
1: Table S1.
The Cancer Genome Atlas (TCGA) and GenomicScape database analysis
In this study, our expression profile analysis was based on the RNAseq counts data of 471 CRC and 41 normal tissues from The Cancer Genome Atlas website (TCGA,
https://tcga-data.nci.nih.gov/tcga/). In addition, we analyzed 76 samples with low eL31 and 101 samples with high eL31 from the GenomicScape website (
http://www.genomicscape.com) to investigate the relationship between patient overall survival and eL31 levels.
Plasmid construction and lentivirus transfection
eL31 and DEPDC1 RNAi/overexpression target sequences were designed by Shanghai Bioscienceres Co., Ltd. (Shanghai, China), subsequently inserted into the BR-V-108 vector through the restriction sites at both ends and transformed into TOP 10 E. coli competent cells (Tiangen, Beijing, China). The plasmids of positive recombinants were extracted with the EndoFree maxi plasmid kit (Tiangen, Beijing, China), and the concentration of which was determined in a spectrophotometer (Thermo_Nanodrop 2000). RKO or HCT-116 cells in logarithmic growth phase were transfected by adding 40 μL 1 × 108 TU/mL lentivirus, culturing in 1640 medium with 10% FBS in a 6-well dish (2 × 105 cells/well). The cell transfection efficiency and knockdown efficiency were evaluated.
Total RNAs were extracted according to the standard methods of TRIzol reagent (Sigma, St. Louis, MO, USA). cDNA synthesis and qRT-PCR were performed using Promega M-MLV Kit (Promega Corporation, Madison, Wisconsin, USA) and the SYBR Green Mastermixs Kit (Vazyme, Nanjing, Jiangsu, China). The mRNA level of GAPDH was used as an internal normalization control. The relative expression of mRNA was calculated by the 2
−△△Ct method. The primers sequences (5′-3′) were listed in Additional file
1: Table S2.
Western blot assay and Co-immunoprecipitation (Co-IP)
After transfection, RKO and HCT-116 were harvested and lysed with 1 × Lysis Buffer lysis (Cell Signal Technology, Danvers, MA), and quantified with BCA methods. At the same time, 10% SDS-PAGE was used to segregate total proteins and transferred into PVDF membranes, followed by blocking in TBST solution containing 5% non-fat milk and subsequently incubating with primary antibodies at room temperature for 1 h. Next, the membranes were incubated with secondary antibodies and washed with TBST solution for three times (10 min/time). Finally, the ECL + plusTM western blotting system kit was used for color rendering, and X-ray imaging was captured.
In Co-IP analysis, HCT 116 cells were lysed, and total proteins were collected. Then, 1.0–1.2 mg proteins were incubated with antibody overnight, followed by 2 h of incubation with 20 μL beads at 4 °C. After that, the cleared protein antibody beads complex was incubated at 95–100 °C for 10 min. Then the proteins in immunocomplex were separated by 10% SDS-PAGE as western blot assay. Finally, primary and secondary antibodies were added to identify interacting proteins. Antibodies used in western blot assay showed in Additional file
1: Table S1.
Cell proliferation detection
For MTT assay, RKO or HCT-116 cells were harvested, digested and resuspended into the cell suspension. 100 μL cell suspension was cultured in 96-well plates at the cell density of 2000 cells and determined for 5 days. 20 μL MTT (5 mg/mL) and 100 μL DMSO were added into 96-well plates. Optical density (OD) value at 490 nm was detected with microplate reader (Tecan infinite, Mannedorf Zurich, Switzerland).
For Celigo cell counting assay, RKO or HCT 116 cells were collected after transfecting indicated lentiviruses. The cells were cultured until the fusion reached 70—90%. Then the cells were seeded into 96-well plates at the density of 2,000 cells/well, placing in an incubator with 5% CO2 at 37 °C. The cell images were taken by Celigo image cytometer (Nexcelom Bioscience, Lawrence, MA, USA) and a continuous 5-day cell proliferation curve was drawn.
HCT 116 or RKO cells with indicated lentiviruses were digested and resuspended to seed into a 6-well plate (2 mL/well). Then the cells were cultured for 8 days and the medium was changed every 3 days to form colony. Visible clones in 6-well plate were recorded by fluorescence microscope (Olympus, Tokyo, Japan). Finally, the cells were washed with PBS, fixed with 1 mL 4% paraformaldehyde and stained by 500 μL Giemsa (Dingguo, Shanghai, China) and the number of colonies was recorded.
Cell migration assay
Wound-healing assay was performed to evaluate cell migration ability. RKO and HCT-116 cells, transfected with indicated lentiviruses, were seeded into a 96-well plate (5 × 104 cells/well). Then, the cells were incubated in an incubator with 5% CO2 at 37 °C. The cells were observed in a microscope at 24 h and 64 h. The experiment was repeated 3 times and the migration rate of cells was evaluated based on the scratch images.
For transwell assay, RKO and HCT-116 cells with indicated lentiviruses were cultured to reach the density of 1 × 105 cells/mL and loaded into the upper chamber containing serum-free medium. Then, the upper chamber was transferred to the lower chamber with 30% FBS and incubated for 72 h. Finally, 400 µL Giemsa was added for cell staining, and the cell migration ability was quantified.
Flow cytometry assay for cell apoptosis
Lentivirus-transfected RKO and HCT-116 cells were cultured in 6-well plates (2 mL/well) for 5 days. 10 μL Annexin V-APC was added for 10–15 min at room temperature in the dark. The cell apoptosis level was measured by using FACSCalibur (BD Biosciences, San Jose, CA, USA).
Human apoptosis antibody array
The effects of eL31 loss on apoptosis-related protein expression in HCT 116 cells were detected by the Human Apoptosis Antibody Array. After the cells were lysed, Handling Array membranes were blocked in 2 mL 1 × Wash Buffer II and incubated with cell lysates and 1 × Biotin-conjugated Anti-Cytokines overnight at 4 °C. Finally, the signals of membranes were tracked by chemiluminescence imaging system.
PrimeView human gene expression array
Total RNAs were extracted using the RNeasy kit (Sigma, St. Louis, MO, USA). The quality and integrity of obtained RNA were determined by Nanodrop 2000 (Thremo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 and Agilent RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA). According to the manufacturer’s instruction, we performed an Affymetrix human GeneChip PrimeView, and the outcomes were scanned by Affymetrix Scanner 3000 (Affymetrix, Santa Clara, CA, USA). The statistical significance of raw data was completed by using a Welch t-test with Benjamini–Hochberg FDR (|Fold Change|≥ 1.3 and FDR < 0.05 as significant). Functional enrichment analysis and interactive network analysis based on Ingenuity Pathway Analysis (IPA) (Qiagen, Hilden, Germany) were executed, and |Z—score|> 0 is considered valuable.
The construction of nude mouse tumor formation model
All animal experiments conformed to the European Parliament Directive (2010/63/EU) and were approved by the Institutional Animal Care and Use Committee of Affiliated Hospital of Inner Mongolia Medical University and the Ethics committee of Affiliated Hospital of Inner Mongolia Medical University. Four-week-old female BALB-c nude mice (Shanghai Weitong Lihua Animal Research Co., Ltd.) were used to establish a xenograft model. The mice were randomly divided into two groups (n = 5/group), and 4 × 106 well-grown HCT 116 cells with sheL31 and shCtrl were suspended in 100 μL of PBS and subcutaneously injected subcutaneously into nude mice. The tumor volume was tested during the entire feeding period. After 38 days, the mice were euthanized and the tumors were removed to weigh and photograph, and finally frozen in liquid nitrogen and stored at − 80 °C.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 8 (San Diego, CA, USA) and SPSS 19.0 (IBM, SPSS, Chicago, IL, USA). All data were represented as the mean ± SD from at least three repeated experiments. Student’s t-test (for comparisons of two groups) and one-way ANOVA (for multiple group comparisons) were used to analyze the statistical significance. The Spearman correlation analysis and Mann–Whitney U analysis were used to assess the association between eL31 expression and characteristics of CRC patients. P < 0.05 was considered to be significantly different.
Discussion
CRC is a common malignant tumor in the digestive department, with high morbidity and mortality, and is one of the major health threats to human beings [
1,
3]. There are multiple strategies for the management and treatment of CRC, such as surgical management, radiotherapy and chemotherapy [
7,
8]. The current main challenges of management and treatment of CRC are resistance to conventional agents and accompanying side-effects [
26]. As such, targeted therapy has attracted more attention in order to reduce adverse effects and overcome resistance [
27].
As part of the 60S large ribosomal subunit, an increasing body of evidence recently presented that eL31 is associated with cell cycle and modulate cancer development and progression [
22,
28]. However, the anti-tumor effects of eL31 on CRC had not been investigated in the existed publications. In this study, we investigated whether eL31 was capable of participating in the development of CRC. For this purpose, this study found that eL31 was upregulated in CRC and was significantly related to tumor size and patient’s prognosis. Concerning the implication of eL31 in CRC, in vitro data indicated an inhibitory effect of eL31 knockdown on cell proliferation and migration, as well as a promotion effect on apoptosis. Also, the effects of eL31 depletion on CRC tumorigenesis were studied in xenografted models. These findings from in vivo experiments further designated the function of eL31 in CRC, which was illustrated by the measurement of tumor growth index. Previous study revealed that eL31 is overexpressed in prostate carcinomas. More intriguingly, the protein levels of tumor suppressor p53 and its targets, p21 and MDM2, were increased in prostate cancer cells with downregulated eL31. Moreover, the degradation of p53 protein was curbed after eL31 knockdown. The suppression in growth and cell cycle from eL31 knockdown was partially recovered upon p53 siRNA transfection [
22]. From this, the author proposed that eL31 was linked to the development of prostate cancer via p53 pathway. We hypothesized that eL31 could use the above same mechanism in mediating CRC progression, which needs to be explored in depth in our further work. On the other hand, such kind of inhibition induced by eL31 depletion led to the downregulation of cancer-associated elements P-Akt, CCND1, CDK6 and PIK3CA. More importantly, we found that these alterations were related to DEPDC1 downregulation.
DEPDC1, a highly conserved 92-kDa protein, has been reported to be involved in multiple biological processes such as transcription regulation, cell mitosis, and apoptosis [
29‐
32]. DEPDC1 is related to cell cycle, mainly expressed during cell interphase, and plays a critical role in cell division in metaphase [
33‐
35]. In addition, DEPDC1 is linked to cell apoptosis, specifically, it could promote the NK-dependent degradation of BCL-2 family protein MCL1 to regulate microtubule-targeting chemotherapeutics, thereby inducing apoptosis [
30]. Of note, DEPDC1 was found to be highly expressed in bladder cancer [
36], hepatocellular carcinoma [
37], and multiple myeloma [
38], acting as a novel diagnostic marker or prognostic predictor, indicating that overexpression of DEPDC1 might play a role in the development and progression of cancers. It was worth mentioning that DEPDC1 was reported to be associated with CRC cell growth or apoptosis [
39‐
41]. In this direction, the present study was aimed to investigate the synergistic effects of eL31 and DEPDC1 on CRC development. In this study, we reported abundant DEPDC1 expression in CRC tissues. It was also concluded that DEPDC1 depletion mitigated the promotion effects of eL31 elevation on CRC.
Conclusion
In conclusion, our study highlighted that eL31 may work as a tumor promotor in CRC via targeting DEPDC1. Accordingly, the current idea was that eL31 might be used as a promising therapeutic target for CRC. In addition, the lack of in vivo data on eL31/DEPDC1 is a limitation in this study, and more studies are needed to support the promotion role of eL31/DEPDC1 in CRC. These knowledge will, in turn, provide a theoretical basis for new strategies for the diagnosis and treatment of CRC.
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