Introduction
Colorectal cancer (CRC) is a frequently occurring malignant tumor occurring in the digestive system. According to the
Global Cancer Statistics 2018, globally there were 1.8 million newly diagnosed CRC cases and 881,000 CRC deaths in 2018, accounting for approximately 1/10 of all cancer cases and deaths. Overall, CRC ranked second as a cause of cancer death [
1,
2]. Despite the increasing number of new therapies for CRC, the treatment outcome for patients with advanced CRC is not satisfactory. Therefore, it is of great significance to develop new treatments.
Tumor cells are mainly characterized by uncontrolled growth, disturbed cell cycle, and indefinite proliferation ability, in which disturbance of the cell cycle may lead to tumorigenesis. Cell cycle-dependent protein kinases and cyclins precisely regulate the cell cycle, while changes in their concentrations can affect the cell cycle, thereby promoting cell proliferation or leading to apoptosis [
3‐
5]. Cell cycle-dependent protein kinase 1 (CDK1), which belongs to the serine/threonine-protein kinase family, is mainly active in the late G2 and early M phases, and high expression of active CDK1 can promote the G2/M transition and accelerate the growth of tumor cells [
6‐
8]. CDK1 plays an oncogenic role in various tumors, such as liver cancer, melanoma, bladder cancer, and breast cancer [
9‐
12], indicating its potential as a therapeutic target for treating different cancers. However, the regulatory mechanism of CDK1 in CRC has not been fully studied and needs further exploration.
MicroRNAs (miRNAs), noncoding small RNAs with high evolutionary conservation (with a length of approximately 19–23 nucleotides), modulate posttranscriptional gene expression and play a vital role in regulating human cancer and affect tumor cell proliferation, apoptosis, and migration [
13].
Recently, miR-378a-5p was shown to suppress renal cell carcinoma (RCC) development [
14], which is associated with patient prognosis; in addition, it has been reported to have an antiapoptotic function and regulate smooth muscle cell migration and invasion in breast cancer [
15]. Of note, miR-378a-5p has been reported to be highly correlated with CRC prognosis [
16]; however, there are few reports on the mechanism of action of miR-378a-5p in CRC, which requires further study.
To explore the role of miR-378a-5p in colorectal cancer and discover new treatments for colorectal cancer, we conducted this study. The present work discovered that miR-378a-5p showed low expression levels in CRC, which suppressed CRC cell proliferation and apoptosis resistance. Furthermore, miR-378a-5p downregulated CDK1 levels to suppress CRC development.
Materials and methods
Tissue sample collection
The present work gained approval from the Medical Ethics Committee of Yijishan Hospital of Wannan Medical College (Wuhu, China). Each case provided the informed consent for participation. A total of 108 paraffin sections were collected from the Pathology Department, Yijishan Hospital Affiliated to Wannan Medical College, and 22 CRC tissue samples with matched adjacent non-carcinoma tissue samples (around 5 cm apart from cancer edge) were acquired and frozen within liquid nitrogen at once.
Cell culture and transfection
Various types of CRC cells (SW480, HCT116, SW620, HT-29) were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China); meanwhile, the colon epithelial cells (NCM460) were obtained from the Affiliated Comprehensive Cancer Center of Drum-Tower Hospital of Medical School of Nanjing University. The CRC cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) within the incubator under 5% CO2 and 37 °C conditions at the Roswell Park Memorial Institute. Then, the Lipofectamine 3000 Kit (Invitrogen, Carlsbad, CA, USA) was used to transfect cells in accordance with manufacturer instructions. The small interfering RNA (siRNA) CDK1, together with the corresponding negative control (NC), was prepared by Guangzhou Ribo Biotechnology Co. Ltd (Guangzhou, China), and its sequences are listed below: siRNA1: GGAACTTCGTCATCCAAAT, siRNA2: GTACTGCAATTCGGGAAAT, siRNA3: GGTTATATCTCATCTTTGA. In addition, the CDK1 overexpression plasmid as well as the empty plasmid was provided by GenePharma Co. Ltd (Shanghai, China), whereas the miR-378a-5p inhibitor and miR-378a-5p mimic, together with corresponding NCs were provided by Guangzhou Ribo Biotechnology Co. Ltd. (Guangzhou, China).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
TRIzol reagent (Invitrogen, CA, USA) was used to extract the total tissue and cellular RNA in accordance with specific protocols. Total RNA was reverse transcribed to complementary DNA following the protocols of the RT Kit (Takara, Dalian, China). The RT reaction program used was 42 °C for 60 min and 70 °C for 10 min. Real-time fluorescence PCR was carried out with a qPCR Kit (Takara, Dalian, China). U6 and GAPDH were used as internal references for miR-378a-5p and CDK1, respectively. The primer sequences of miR-378a-5p and U6 were designed by Ruibo: GAPDH, forward: GAACGGGAAGCTCACTGG, reverse: GCCTGCTTCACCACCTTCT; CDK1, forward: GGTTCCTAGTACTGCAATTTTCG, reverse: TTTGCCAG.
The results were calculated by the 2−ΔΔCt method.
Cell counting kit-8 (CCK-8) assay
Cell viability was assayed using Cell Proliferation Kit (Keygen Biotechnology Co. Ltd. (Nanjing, China)). CRC cells were detached and inoculated into the 96-well plates at 2 × 104 cells/well, and the adhering cells after 12-h cell culture were transfected. At 12, 24, 36, 48, and 60 h of transfection, every group was added with 10 mL reagent to detect cell proliferation; then, cells were incubated for another 2 h, and the absorbance (OD) was detected at 450 nm.
5-Ethynyl-2’-deoxyuridine (EdU) assay
CRC cells (1 × 105/ well) at logarithmic stage were seeded into the 96-well plates to culture until the normal growth stage. Thereafter, cells were labeled with EdU and stained with Apollo and DNA as per the manufacturer’s instructions (Guangzhou Ribo Biotechnology Co. Ltd., Guangzhou, China); at last, the confocal laser scanning microscope was utilized to observe cells.
CRC cells (200 cells/well) after transfection were grown into the 6-well plates and further incubated for 2 weeks under 37 °C. At last, 4% paraformaldehyde was used to fix cells, while 0.1% (w/v) crystal violet was used to stain cells. The Image-Pro Plus 5.0 software was employed for counting cell colonies.
Wound-healing assay
The single straight wound was made using a sterilized 10-μL pipette tip at the bottom of 6-well plates full of transfected CRC cells with cell debris removed by washes with phosphate buffer saline (PBS). Then, every well was added with RPMI-1640 containing 2% FBS, followed by further cell culture under 5% CO2 and 37 °C conditions. The width of the scratch was measured with the ImageJ software and an inverted optical microscope. The scratch width measured initially was assigned to be the original scratch width, and the scratch width measured again after 48 h was used as final scratch width. Relative scratch width was calculated by the ratio of final scratch width to the original one.
Transwell assay
At 24 h after transfection of CRC cells, 100 μL of the cells diluted to 1 × 105 with serum-free RPMI-1640 medium was plated at apical side of a transwell chamber, whereas 600 μL of 20% RPMI-1640 medium was put at basolateral side. After 24 h of cell incubation under 37 °C and 5% CO2 conditions, the 4% paraformaldehyde was used to fix the chamber, while 0.1% crystal violet was used for staining. Thereafter, a cotton swab was used to remove upper chamber cells. Finally, migrating cells were photographed and counted with an inverted optical microscope.
Immunohistochemical (IHC) staining
CDK1 expression was determined with the CDK1 antibody (1:100, Abcam, USA). Xylene and alcohol were used for dewaxing, 3% H2O2 was used to remove endogenous catalase, and citrate buffer was added; the sample was placed in a microwave oven and cooked to expose the antigen site, after which serum blocking was performed. After incubation, the antibody and the secondary antibody were used to develop the color, and the samples were mounted. Each tissue section was rated using a light microscope based on the staining level (0, 1, 2, and 3 indicated negative, yellowish, light brown, and dark brown staining, respectively) and the extent of positivity (1, 2, 3, and 4 represented 0–25%, 26–50%, 51–75%, and 76–100%, respectively). Finally, the scores were added for comparison. All IHC sections were evaluated independently by two expert pathologists.
Dual-luciferase reporter gene assay
Luciferase reporter gene assay was performed to assess the direct binding of miR-378a-5p with CDK1. The CDK1 3′-untranslated region (UTR) that may bind to miR-378a-5p was mutated from GTCAGGAA to CAGTCCTT, and a mutant fragment and an unmutated fragment of the CDK1 3′-UTR were transfected to pmirGLO reporter plasmid, respectively. 293T cells were then inoculated in the 24-well plates, followed by co-transfection with equivalent unmutated or mutated pmirGLO and miR-378a-5p mimic or NC-mimic. Renilla luciferase was adopted to be endogenous control. After 24 h of cell culture, the Dual-luciferase Reporter Gene Assay Kit (Shanghai Beyotime Biotechnology Co. Ltd., Shanghai, China) was applied in detection.
Cell apoptosis detection
Flow Cytometric Kit (BD Biosciences, CA, USA) was utilized to measure cell apoptosis. In brief, cells after transfection were inoculated into the 12-well plates to further culture for another 12 h. Thereafter, cells were cultivated with serum-free medium for another 24 h for apoptosis induction; later, 0.25% trypsin was used to detach CRC cells, followed by 5–10 min centrifugation at 2000 rpm under ambient temperature. Thereafter, cells were harvested to suspend in the pre-chilled 1 × PBS (4 °C), followed by 5–10 min of centrifugation at 2000 rpm. Then, cells were washed and resuspended into 300 μL 1 × binding buffer, sufficiently blended using 5 μL Annexin-V-fluorescein isothiocyanate (FITC), followed by 15 min of incubation in dark under room temperature. Afterward, the cells were subjected to 5 min of staining using 5 μL propidium iodide (PI) solution before placing on a flow cytometer, and 200 μL 1 × binding buffer added. Finally, flow cytometry (BD Biosciences, CA, USA) was adopted to detect cell apoptosis.
Cell cycle detection
The cell cycle was determined using Flow Cytometry kit (Keygen Biotechnology Co., Ltd., Nanjing, China) according to specific protocols. In brief, after 24 h transfection, cells were rinsed by PBS, followed by 5 min of centrifugation at 2000 rpm. Then, cells were collected, and the cell density was regulated at 1 × 106/mL. Afterwards, 1 mL single-cell suspension was collected for centrifugation, and the supernatant was fixed with 500 μL of the 70% pre-chilled ethanol overnight at 4 °C. After PBS washes, 500 μL of PI/RNaseA staining solution was added to further incubate in dark for 1 h, and cell cycle was directly examined by flow cytometry (BD Biosciences, CA, USA).
Tumor xenograft mouse models
Four-week-old male nude mice reared in an SPF animal room (Spf Laboratory Animal Room of Wannan Medical College) were injected with 2*10^6 HT-29 cells in the left armpit. After observing the size of the tumor every day for 15 days, the nude mice were killed by cervical dislocation, and then, the tumors were removed and recorded.
Western blotting analysis
Radioimmunoprecipitation (RIPA) lysis buffer (Thermo Fisher Scientific, MA, USA) was utilized to extract total cellular protein from CRC cells transfected for 48 h. A bicinchoninic acid (BCA) protein detection kit (Thermo Fisher Scientific, MA, USA) was used to measure the protein content. SDS-PAGE (10%) (Bio-Rad Laboratories, Hercules, CA, USA) was used to separate proteins, after which the proteins were transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibody (1:1000) overnight at 4 °C, followed by incubation with the secondary antibody (1:5000) at room temperature for 2 h before exposure analysis. CDK1 (Abcam, USA) was assessed later, with β-actin as the internal reference (Cell Signalling Technologies, Beverly, MA, USA).
Statistical methods
Prism (GraphPad Prism 8) was utilized for statistical analysis. The results were expressed as mean ± SD and examined by t-test. A difference of p < 0.05 was deemed to be statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.
Discussion
CRC ranks 3rd in terms of its morbidity, second only to lung cancer and breast cancer. Recently, CRC morbidity has shown an increasing trend because of changes in lifestyle, increased obesity, and economic development [
17]. Therefore, it is particularly important to elucidate the pathogenesis of CRC to improve the diagnosis and treatment of CRC, thereby reducing its mortality.
Our previous study found that LINC00365 promotes the development of CRC and that the expression of CDK1 increases with the upregulation of LINC00365, revealing that CDK1 may be related to the development of CRC [
18]. In this experiment, it was found that CDK1 is closely related to the development of colorectal cancer. Overexpression of CDK1 promotes the proliferation, invasion, and migration of CRC cells, indicating that CDK1 has a promoting role in the development of CRC. Some researchers have found that CDK1 and cyclin B1 may be potential diagnostic biomarkers for rhabdomyosarcoma and hepatocellular carcinoma [
19,
20]. As discovered by Yamamura et al., phosphorylated CDK1 (p-CDK1), p-CDK2, cyclin B1, and cyclin E1 levels increase within cholangiocarcinoma tissues; in addition, p-CDK1 and cyclin B1 nuclear levels positively correlate with the clinical stage and with lymph node metastasis, while the expression of p-CDK 1 is related to poor patient survival [
21]. A recent study has also shown that CDK1 is closely related to autophagy [
22]. Therefore, CDK1 may be an important biomarker and therapeutic target for CRC.
Noncoding RNAs are tiny RNA molecules that function in transcription and other specific processes, but they cannot encode proteins [
23]. Among noncoding RNAs, miRNAs are overexpressed and mutated in a variety of malignant tumors, mainly by regulating the expression of mRNA and are considered to be important for the treatment of various diseases, especially cancer [
23‐
27]. In this study, it was found that the expression of miR-378a-5p in CRC tissues decreased, and by overexpression or knockdown of miR-378a-5p, it was found that miR-378a-5p inhibited CRC cell proliferation and migration and promoted cell apoptosis. Through bioinformatics prediction and dual luciferase reporter gene detection, CDK1 was found to be the target gene of miR-378a-5p, revealing that miR-378a-5p may regulate the development of CRC cells by regulating the expression of CDK1. There are many unique noncoding RNA sequences in cells, among which lncRNAs are considered competitive endogenous RNAs (ceRNAs) that interact with microRNAs and participate in the regulation of target gene expression [
28,
29]. Whether there is a lncRNA upstream of miR-378a-5p that participates in regulation is not yet known, and whether miR-378a-5p has the same regulatory effect in other tumors has not been studied. These questions will be explored in future studies.
In conclusion, we found that miR-378a-5p inhibited the proliferation of colorectal cancer cells, and CDK1 promoted the development of colorectal cancer. The findings in this work suggested that miR-378a-5p inhibits CRC cell proliferation by targeting CDK1, which can shed more light on CRC treatment.
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