MicroRNA‑628‑5p inhibits cell proliferation and induces apoptosis in colorectal cancer through downregulating CCND1 expression levels.

MicroRNA (miR)‑628‑5p serves as an antitumor gene in a variety of cancers; however, the role of miR‑628‑5p in colorectal cancer remains largely unclear. The purpose of this study was to investigate the role and mechanism of miR‑628‑5p in colorectal cancer. Reverse transcription‑quantitative PCR (RT‑qPCR), colony formation assays and flow cytometric analysis were used to determine the expression levels of miR‑628‑5p in colorectal cancer tissues and cell lines, and the proliferative ability of colorectal cancer cells. TargetScan version 7.2 and dual‑luciferase reporter assay were performed to predict and confirm miR‑628‑5p target genes. The expression levels of cyclin D1 (CCND1) and related genes were determined using RT‑qPCR or/and western blotting analysis. miR‑628‑5p mimics and CCND1 plasmids were used to overexpress miR‑628‑5p and CCND1; it was demonstrated that the expression levels of miR‑628‑5p were significantly downregulated in colorectal cancer tissues and cell lines. miR‑628‑5p mimic‑transfected cells inhibited the proliferation and induced apoptosis of HT‑29 cells. CCND1, a downstream effector of miR‑628‑5p, promoted the proliferation and suppressed apoptosis of HT‑29 cells, and the effects were reversed by miR‑628‑5p mimics. In conclusion, the present study suggested that colorectal cancer progression may be regulated through the miR‑628‑5p/CCND1 axis, and miR‑628‑5p could be used as a potential diagnostic and prognostic biomarker for colorectal cancer.

Introduction

Colorectal cancer is a digestive tract tumor that occurs in the colon, and its incidence is gradually increasing: In 2015, colorectal cancer was one of the most frequent tumors diagnosed in China, which severely impacted the health of Chinese patients (1). Colorectal cancer is the third most frequent cancer in men, and is the second most frequent cancer in women after breast cancer; it represents approximately 9% of deaths by cancer (2). There are many risk factors for colorectal cancer, including smoking, physical inactivity, being overweight and obesity, dietary patterns, drinking alcohol excessively and genetic and epigenetic factors (3–6). Earlystage colorectal cancer is confined to the mucosal layer or submucosa, and has a high curability rate, with >80% patients demonstrating a 5-year survival rate; however, a lower 5-year survival rate of <50% occurs in patients with later-stage colorectal cancer due to local lymph node metastasis and distant organ invasion (7). For patients with advanced colorectal cancer, tumor resection is not feasible, and chemotherapy and biotherapy have become the only treatment strategies (8). However, due to the development of chemotherapeutic resistance in cancer patients, these treatments are mostly ineffective.

MicroRNAs (miRNAs) are small-molecule, non-coding single-stranded RNAs of ~22 nucleotides in length that are encoded by an endogenous gene, and are mainly involved in the post-transcriptional regulation of gene expression (9). miRNAs directly bind to target genes through recognizing a complementary sequence in the 3′-untranslated region (3′-UTR) end of the target gene to downregulate the expression of the target gene and regulate the related signaling pathways (10). Previous studies have demonstrated that miRNAs serve an important role in the development of tumors; the aberrant overexpression or downregulation of multiple miRNAs are often detected in a variety of tumors, suggesting that miRNAs have different roles in different processes, such as tumor occurrence, development and metastasis (11,12). Previous studies have reported the abnormal expression of miRNA (miR)-628 in numerous malignant tumor tissues (13,14); however, there are few studies observing the effects of miR-628 in colorectal cancer.

Cyclins are widely distributed in eukaryotic cells and, among them, CCND1, encoding cyclin D1, is a highly conserved cell cycle family protein (15,16). Through responding to growth factor signaling, cyclin D1 promotes the cell cycle transition from the G1 to S phase, thus regulating cell cycle progression (17–19). In addition, having been identified as a proto-oncogene, the abnormal expression of cyclin D1 promotes cell colonization through regulating the cell cycle and arresting cells during synthesis (20). In colorectal cancer, bladder cancer, reproductive system tumors, gastric cancer and lung cancer, the expression levels of CCND1 were significantly higher compared with adjacent normal tissues, and were related to the pathological type and clinical stage of the tumor (21–24). In addition, previous studies demonstrated that the overexpression of CCND1 promoted cell invasion and migration in multiple tumors, such as breast cancer and gastric cancer, leading to a poor prognosis (25,26). A number of studies have reported that PAC and RAC1 may be involved in the development of colon cancer by targeting or regulating the expression of cyclin D1 (27,28). It has been reported that miR-365 inhibited cell cycle progression and promoted apoptosis of colon cancer cells by targeting cyclin D1 and Bcl-2 (29). Diospyros kaki Thunb (persimmon) may downregulate cyclin D1 as one of the potential anticancer targets via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells (30). A study reported that miR-519d reduces the 5-fluorouracil resistance in colorectal cancer cells by downregulating the expression of CCND1 (31).

The present study aimed to investigate the miR-628-5p expression profile in colorectal cancer and the regulatory mechanisms of miR-628-5p underlying colorectal cancer progression to help provide novel, effective treatment options for patients with colorectal cancer.

Materials and methods

Patient studies

The present study was approved by The First Affiliated Hospital of Hebei North University Ethics Committee (Zhangjiakou, China). Informed, written consent for the use of tissue for clinical research was obtained from all patients. A total of 30 patients with colorectal cancer (16 males, 14 females; age range, 28–64 years old) were recruited in The First Affiliated Hospital of Hebei North University between August 2017 to February 2018 for inclusion in the current study. Then, 30 surgically resected tissue specimens of primary colorectal cancer, confirmed by pathological examination, and corresponding paracancerous tissues (>5 cm obtained from the edge of the lump) were collected from The First Affiliated Hospital of Hebei North University. None of the patients received radiotherapy or chemotherapy prior to surgery.

Cell culture and reagents

Normal human colonic epithelial cells (FHC cells) and human colorectal cancer cell lines (SW480, SW620, HT-29, NCI-H508 and HCT15) were purchased from the American Type Culture Collection. Cells were cultured in DMEM (GE Healthcare BioSciences), supplemented with 10% FBS (GE Healthcare Bio-Sciences) and 1% streptomycin, the cells were incubated in a humidified 5% CO2 atmosphere at 37°C in an incubator for 72 h.

Cell transfection

A total of 40 pmol miR-628-5p mimic (Shanghai GenePharma Co., Ltd., 5′-AUGCUGACAUAUUUACUAGAGG-3′), mimic control (Shanghai GenePharma Co., Ltd., 5′-UUUGUACUACACAAAAGUACUG-3′), overexpressed CCND1 (Shanghai GeneChem Co., Ltd., 5′-AAAACAUAGAAAAAUUCAGCAA-3′) and their respective negative controls (NC; Shanghai GenePharma Co., Ltd., 5′-CAUGUGGUCUGUCGCAUAAUA-3′) were mixed in 50 µl serum-free medium, and 2 µl Lipofectamine® 3000 reagent (Thermo Fisher Scientific, Inc.) was used to transfect HT-29 cells (2×105 cells/well), according to the manufacturer's protocol. Cells were subsequently incubated at room temperature for 15 min. The lipid compounds were diluted in 300 µl serum-free medium and 600 µl medium containing FBS to produce a 1 ml mixture, and incubated with the cells in a humidified atmosphere at 37°C and 5% CO2 for subsequent experiments.

Cell Counting Kit (CCK)-8 assay

CCK-8 (Beyotime Institute of Biotechnology, C0038) was used to measure the cell viability according to the manufacturer's protocol. A total of 2×104 HT-29 cells/well in the exponential growth phase were plated into 96-well plates. Following 24, 48 or 72 h of transfection, 10 µl CCK-8 solution was added to each well, and cells were subsequently cultured at 37°C for 2 h. The optical density was measured at 450 nm using a Tecan Infinite M200 microplate reader to determine cell viability.

Colony formation assay

Following 24 h of transfection, a total of 800 HT-29 cells were plated into 6-well plates, with 3 replicate wells/treatment group. The cells were cultured in an incubator at 37°C with 5% CO2 for 2 weeks; the culture solution was changed every two days. Subsequently, the medium was aspirated and cells were fixed through the addition of 500 µl/well methanol solution for 15 min at room temperature. Methanol was discarded, and 1 ml 0.1% crystal violet dye solution (cat. no. C0121; Beyotime Institute of Biotechnology) was added to each well to stain the cells for 20 min at room temperature. Stained cells were visualized using an enzyme-linked spot image automatic analyzer (magnification, ×1).

Flow cytometric analysis of apoptosis

HT-29 cells (2×104 cells/well) were digested with 0.25% trypsin without EDTA (Gibco; Thermo Fisher Scientific, Inc.) and neutralized with 2% BSA solution (Sigma-Aldrich; Merck KGaA), and a total of 2×105 cells were collected into a 1.5 ml Eppendorf tube. Cells were collected by centrifugation (2,000 × g; 4°C) for 10 min and the supernatant was discarded. Cells were washed twice with pre-cooled PBS. The cells were subsequently stained with Annexin V-FITC and propidium iodide (PI) using the Annexin V-FITC Apoptosis Detection kit (cat. no K201-100; BioVision, Inc.), according to the manufacturer's protocol. Briefly, 500 µl 1X binding buffer and 5 µl Annexin V-FITC were added to the cell suspension and cells were incubated at 4°C for 30 min in the dark. PI (5 µl) was added and incubated at room temperature for 5 min. Apoptotic cells were subsequently analyzed using a flow cytometry and FlowJo software (version 10.0; FlowJo LLC).

Bioinformatics

TargetScan 7.2 software (http://www.targetscan.org/vert_72/) was used to predict target genes and potential binding sites for miRNAs.

Dual-luciferase reporter assay

Wild-type CCND1 3′-untranslated region (3′-UTR; CCND1-WT) and mutated CCND1 3′-UTR (CCND1-mut) were cloned into the pMIR-REPORT luciferase vector (Ambion; Thermo Fisher Scientific, Inc.). 293T cells (2×105 cells/well) were seeded in 6-well plates and transfected with cyclin D1-WT or cyclin D1-Mut using Lipofectamine® 3000 reagent (Thermo Fisher Scientific, Inc.) for 24 h. Following incubation, cells were collected and luciferase activity was detected using a Dual-Luciferase Reporter 1000 assay system (Promega Corporation), according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from HT-29 cells (2×105 cells/well in 6-well plates) using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The concentration and purity of RNA was determined using a NanoDrop™ 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). A total of 1 µg RNA was reverse-transcribed into cDNA using an RT cDNA kit (Thermo Fisher Scientific, Inc.) at 42°C for 60 min and 70°C for 5 min, and was subsequently maintained at 4°C. qPCR was subsequently performed using the SYBR® Green PCR Master mix (Roche Diagnostics) and the ABI 7500 RT-PCR Detection system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The primer pairs used for the qPCR are presented in Table I. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 94°C for 15 sec (denaturation), 60°C for 1 min (annealing), and 60°C for 1 min (elongation), with a final extension at 72°C for 10 min, followed by being maintained at 4°C. Expression levels were quantified using the 2ΔΔCq method (32), and mRNA expression was normalized to the internal reference gene GAPDH, whereas miR-628-5p expression was normalized to U6.

Table I.

Primers for reverse transcriptionquantitative PCR.

Gene	Primer sequence (5′→3′)
microRNA26a5p	F: GGGGGATGCTGACATATTTAC
	R: CAGTGCGTGTCGTGGAGT
CCND1	F: TATTGCGCTGCTACCGTTGA
	R: CCAATAGCAGCAAACAATGTGAAA
GAPDH	F: AGAAGGCTGGGGCTCATTTG
	R: AGGGGCCATCCACAGTCTTC
U6	F: CTCGCTTCGGCAGCACA
	R: AACGCTTCACGAATTTGCGT

F, forward; R, reverse; CCND1, cyclin D1.

Western blotting

HT-29 cells (2×106 cells) were washed three times with pre-cooled PBS, suspended in 1 ml PBS and transferred to a 1.5 ml Eppendorf tube. Cells were subsequently centrifuged (1,000 × g; 4°C; 5 min), the supernatant was discarded, and total protein was extracted using 200 µl RIPA assay buffer (Beyotime Institute of Biotechnology). Cells were centrifuged (4°C; 5 min), total protein was quantified using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.), and 30 µg protein/lane was separated via SDS-PAGE on a 10% gel. The separated proteins were subsequently transferred onto a PVDF membrane and blocked for 1 h at 37°C with 5% BSA solution (Sigma-Aldrich; Merck KGaA). The membranes were incubated with the following primary antibodies in 5% BSA overnight at 4°C: Rabbit anti-cyclin D1 (34 kDa; 1:1,000; cat. no. ab134175; Abcam), rabbit anti-Ki67 (359 kDa; 1:1,000; cat. no. ab16667; Abcam), rabbit anti-cleaved-caspase-3 (17 kDa; 1:1,000; cat. no. ab2302; Abcam), rabbit anti-Bax (21 kDa; 1:1,000; cat. no. ab32503; Abcam), rabbit anti-Bcl-2 (26 kDa; 1:1,000; cat. no. ab32124; Abcam) and mouse anti-β-actin antibody (42 kDa; 1:1,000; cat. no. ab8226; Abcam). Following the primary antibody incubation, membranes were incubated at room temperature for 2 h with a horseradish peroxidase (HRP)-conjugated secondary antibodies (mouse anti-rabbit IgG-HRP, 1:5,000; cat. nos. sc-516102/sc-2357; Santa Cruz Biotechnology, Inc.) diluted in 5% BSA. Protein bands were visualized by adding ECL coloring solution to the PVDF membrane and captured using ImageJ software (version 1.46; National Institutes of Health). Protein expression was quantified using Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.), with β-actin as the loading control.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 7.0 software (GraphPad Software, Inc.) and all data are presented as the mean ± SD from three independent experimental repeats. Statistical differences between groups were determined using one-way ANOVA followed by the Bonferroni's correction post hoc test for multiple comparisons. The Student's t-test was used to compare the difference between two groups (Fig. 1A). P<0.05 was considered to indicate a statistically significant difference.

Figure 1.

miR-6285-p expression levels are decreased in colorectal cancer in vivo and in vitro. (A and B) The expression levels of miR-628-5p were detected using reverse transcription-quantitative PCR in (A) colorectal cancer tissues and (B) colorectal cancer cell lines. **P<0.01 vs. adjacent tissue or FHC group. miR, microRNA.

Results

miR-628-5p expression levels are decreased in colorectal cancer in vivo and in vitro

To determine the role of miR-628-5p in human colorectal cancer, the expression levels of miR-628-5p in patients with colorectal cancer and common colorectal cancer cell lines were determined compared with adjacent benign tissue samples and normal colorectal cells, respectively. miR-628-5p expression levels were significantly decreased in the colorectal cancer samples compared with the normal colorectal tissue samples (P<0.01; Fig. 1A). Similarly, miR-628-5p expression levels were significantly decreased in all colorectal cancer cell lines (SW480, SW620, HT-29, NCI-H508 and HCT15) compared with the normal cell line, FHC (P<0.01; Fig. 1B). Due to miR-628-5p expression being the most significantly reduced in HT-29 cells, HT-29 cells were used in the subsequent experiments.

Overexpression of miR-628-5p suppresses proliferation and induces apoptosis of HT-29 cells

HT-29 cells were stably transfected with miR-628-5p mimics or mimic controls and the viability, proliferative and apoptotic abilities of HT-29 cells were determined using the CCK-8 assay, colony formation assay and flow cytometric analysis. HT-29 cells were successfully transfected with the miR-628-5p mimic; miR-628-5p mimic-transfected cells demonstrated significantly increased miR-628-5p expression levels compared with the mimic control and control group (P<0.01; Fig. 2A). Cell viability was significantly reduced in HT-29 cells transfected with miR-628-5p mimics compared with the mimic control and control group at 24, 48 and 72 h (Fig. 2B). Similar results were obtained in the colony formation assay: The proliferation of HT-29 cells was significantly suppressed when transfected with the miR-628-5p mimic compared with the mimic control and control group (P<0.01; Fig. 2C and D). Moreover, flow cytometric analysis revealed that HT-29 cells transfected with miR-628-5p mimics demonstrated significantly higher rates of apoptosis compared with the mimic control and control groups (P<0.01; Fig. 2E and F).

Figure 2.

miR-628-5p mimics suppress the proliferation and induce apoptosis of HT-29 cells. (A) Transfection efficiency of miR-628-5p mimics and its mimic control were confirmed using reverse transcriptionquantitative PCR. U6 served as an internal reference gene for miR-628-5p expression. (B) HT-29 cell viability was determined using the Cell Counting kit-8 in HT-29 cells transfected with miR-628-5p mimics or its mimic control. (C and D) HT-29 cell proliferation following transfection with miR-628-5p mimics or mimic control was (C) analyzed and (D) semi-quantified using a colony formation assay (magnification, ×1). (E and F) Apoptosis of HT-29 cells following transfection with miR-628-5p mimics or mimic control was (E) analyzed and (F) semi-quantified using flow cytometric analysis. *P<0.05, **P<0.01 vs. control group, #P<0.05, ##P<0.01 vs. mimic control group. miR, microRNA; PI, propidium iodide.

CCND1 is a bona fide target gene of miR-628-5p

The TargetScan database was used to identify potential direct targets for miR-628-5p according to the binding sites in the 3′-UTR. TargetScan-predicting algorithms identified that an area at positions 453–459 within the 3′-UTR of CCND1 was a potential target for miR-628-5p binding (Fig. 3A). To further determine whether CCND1 is a direct target for miR-628-5p, a wild-type and mutant dual-luciferase UTR vector in the miR-628-5p binding sites of the 3′-UTR of CCND1 were constructed. miR-628-5p significantly suppressed the luciferase reporter activity of the wild-type vector compared with the control and mutant vector; however, no significant difference in the luciferase activity of the mutant vector was observed compared with the control vector (P<0.01; Fig. 3B).

Figure 3.

CCND1 is a bona fide target gene for miR-628-5p. (A) CCND1 was identified as a downstream effector of miR-628-5p using TargetScan version 7.2 software. (B) Dual-reporter luciferase assay was used to confirm the binding of miR-628-5p to CCND1 in WT and mut vectors. **P<0.01 vs. control + CCND1, ##P<0.01 vs. miR-628-5p + CCND1-mut. miR/miRNA, microRNA; WT, wild-type; mut, mutant; CCND1, cyclin D1.

CCND1 expression levels are negatively correlated with miR-628-5p expression

The mRNA and protein expression levels of miR-628-5p and CCND1 were analyzed in HT-29 cells with miR-628-5p mimic and/or CCND1 overexpression. The expression levels of miR-628-5p were observed to be significantly increased in CCND-1overexpressed plus miR-628-5p mimic-transfected cells compared with cells overexpressing CCND1, while no significant difference between the miR-628-5p mimic group and CCND1+mimic group were observed (Fig. 4A). Transfection of the CCND1 vector significantly increased CCND1 expression levels compared with the control and NC groups, although this increase was significantly inhibited by cells transfected with the miR-628-5p mimic at both the mRNA (Fig. 4B) and the protein (Fig. 4C and D) expression levels (P<0.01). In addition, the mRNA and protein expression in the mimic group was obviously decreased than that in CCND1+mimic group (P<0.01; Fig. 4B-D).

Figure 4.

Cyclin D1 expression levels are negatively associated with miR-628-5p expression levels. mRNA expression level of (A) miR-628-5p and (B) CCND1 were detected using reverse transcriptionquantitative PCR in HT-29 cells transfected with cyclin D1 overexpression, miR-628-5p mimics, both or their respective NC. Protein expression levels of cyclin D1 were (C) detected and (D) semi-quantified using western blotting in HT-29 cells transfected with CCD1 overexpression, miR-628-5p mimics, both or their respective NC. Expression of each protein was normalized to the control protein, β-actin. **P<0.01 vs. control group, ##P<0.01 vs. NC, ^^P<0.01 vs. CCND1, ΔΔP<0.01 vs. CCND1 + mimic, CCND1, cyclin D1.

CCND1 is required for the proliferation and apoptosis of HT-29 cells and is regulated by the miR-628-5p mimic

The interaction between miR-628-5p and CCND1 was explored through co-transfecting the miR-628-5p mimic and the CCND1 vector into HT-29 cells. CCND1 overexpression in HT-29 cells significantly increased the cell viability compared with the control and NC group (Fig. 5A); cells transfected with the miR-628-5p mimic reduced this ability of CCND1 (P<0.01; Fig. 5A). Moreover, cells co-transfected with the miR-628-5p mimic and CCND1 overexpression demonstrated significantly reduced proliferative activity compared with the CCND1-overexpressed cells, although proliferation remained significantly higher compared with cells only transfected with miR-628-5p mimic (Fig. 5A). Similarly, the colony formation assay demonstrated that the significantly enhanced proliferative ability of CCND1-overexpressed cells compared with control and NC cells could be significantly reversed by the CCND1 plus mimic-transfected cells, and even further reduced by the miR-628-5p mimic-transfected HT-29 cells (P<0.01; Fig. 5B and C). The ability of HT-29 cells to undergo apoptosis was significantly higher in the CCND1-overexpressed plus miR-628-5p mimic co-transfected group compared to the overexpressed CCND1 group, and the apoptotic cells were significantly higher in the miR-628-5p mimic group compared with the CCND1-overexpressed plus miR-628-5p mimic co-transfected group. (P<0.01; Fig. 5D and E). Furthermore, CCND1 overexpression significantly increased the expression levels of Ki67 and Bcl-2 compared with the Control and NC groups, whereas the expression levels of cleaved-caspase-3 and Bax were significantly decreased (P<0.01; Fig. 6A-C). Meanwhile, CCND1 in combination with the miR-628-5p mimic partially reversed the effects of the CCND1 on the decreased expression levels of cleaved-caspase-3 (P<0.01) and Bax, and on the increased expression levels of Ki67 and Bcl-2 (P<0.01; Fig. 6A-C).

Figure 5.

CCND1 is required for the miR-628-5p mimic to inhibit the proliferation of HT-29 cells. (A) HT-29 cell proliferation following transfection with CCND1 overexpression, miR-628-5p mimics, both or their respective NCs were determined through Cell Counting kit-8 assays. (B and C) HT-29 cell proliferation following transfection with CCND1 overexpression, miR-628-5p mimics, both or their respective NC was (B) analyzed and (C) semi-quantified using colony formation assays (magnification, ×1). (D and E) HT-29 cell apoptosis following transfection with CCND1 overexpression, miR-628-5p mimics, both or their respective NC was (D) detected and (E) semi-quantified using flow cytometric analysis. *P<0.05, **P<0.01 vs. control group, #P<0.05, ##P<0.01 vs. NC, ^P<0.05, ^^P<0.01 vs. CCND1, ΔP<0.05, ΔΔP<0.01 vs. CCND1 + mimic. miR, microRNA; NC, negative control; PI, propidium iodide; CCND1, cyclin D1.

Figure 6.

CCND1 partially reverses the effects of miR-628-5p mimics on regulating proliferation- and apoptosis-associated proteins in HT-29 cells. (A) Protein expression levels of Ki67, cleaved-caspase-3, Bax and Bcl-2 were detected by western blotting in HT-29 cells transfected with CCND1 overexpression, miR-628-5p mimics, both or their respective NC. (B and C) Expression levels of each protein in (A) were semi-quantified and normalized to the loading control protein, β-actin. *P<0.05 and **P<0.01 vs. control group, #P<0.05 and ##P<0.01 vs. NC, ^P<0.05, ^^P<0.01 vs. CCND1, ΔΔP<0.01 vs. CCND1 + mimic. miR, microRNA; NC, negative control; CCND1, cyclin D1.

Discussion

Colorectal cancer is one of the most frequently occurring malignant tumors, and it is associated with a significantly high mortality rate (33); the American cancer society estimates that colorectal cancer accounted for about 9% of cancer mortalities in 2017 (34). However, the mechanism underlying the development of colorectal cancer is largely unclear, which poses great difficulty in designing treatments and improving prognosis (35). Despite an abundance of basic and clinical research conducted on colorectal cancer, the diagnosis and treatment of the disease remains within the scope of surgical chemotherapy and radiotherapy, the recurrence rate after surgery is high (36). Therefore, it is of great importance to discover novel colorectal cancer treatment targets and more effective treatment strategies.

Numerous aberrantly expressed miRNAs have been reported in colorectal cancer tissues (37). Some miRNAs are highly expressed in colorectal cancer tissues, and induce the development of sputum tumors through regulating its downstream target mRNA; for example, miR-21 was observed to induce the proliferation, invasion and metastasis of colorectal cancer cells (38), overexpression of miR-671-5p indicated a poor prognosis in colon cancer and accelerated proliferation, migration, and invasion of colorectal cancer cells (39). Some miRNAs also exhibit low expression levels in colorectal cancer tissues; the significant downregulation of miR-4262 expression levels in colorectal cancer tissue promotes apoptosis and inhibits the proliferation of colorectal cancer cells (40). In addition, low expression levels of miRNAs in colorectal cancer tissues have also been detected (41); in one study, miR-144 expression levels were significantly lower in colon cancer in vivo and in vitro (42), whereas in another, miR-143-3p expression levels were observed to be decreased in colorectal cancer (43). The present study revealed that the expression level of miR-628-5p was significantly decreased in colorectal cancer tissues and cell lines compared with primary tissues and normal cells. Therefore, it was hypothesized that miR-628-5p is a tumor suppressor for colorectal cancer. miR-628-5p decreased the tumorigenicity of epithelial ovarian cancer cells (44), and miRNA-628 inhibited the proliferation of acute myeloid leukemia cells (14). In addition, miR-628 was reported to suppress nonsmall cell lung cancer proliferation, migration and invasion (45). Similarly, the present study demonstrated that overexpressed miRNA-628-5p significantly inhibited colorectal cancer cell proliferation and promoted cell apoptosis. These data indicated that miR-628-5p may act as a biomarker for the diagnosis of patients with colorectal cancer.

Previous research has demonstrated that CCND1 serves an important role in promoting the development and progress of multiple types of human cancer, such as lung adenocarcinoma, glioma and renal cell cancer (46–49). A previous study indicated that miR-374a inactivated the phosphoinositide 3-kinase (PI3K)/AKT signaling axis through inhibiting CCND1 and suppressing the progression of colon cancer (49). Transmembrane protease, serine 4 (TMPRSS4) modulated both the invasion and proliferation of prostate cancer cells through targeting Slug and CCND1 (50). Notably, the present study identified CCND1 as a direct target for miR-628-5p binding. Through functional experiments, CCND1 overexpression markedly rescued the effects of overexpressed miR-628-5p on HT-29 cell proliferation and apoptosis. Moreover, a negative association was observed between CCND1 and miRNA-628-5p expression levels in HT-29 cells. Based on these findings, it is suggested that the miRNA-628-5p-induced suppression over colorectal cancer progression may be related to the downregulation of CCND1 expression.

The present study also examined the effect of miR-628-5p and CCND1 on cell proliferation and apoptosis related-protein. Ki67 is a nuclear antigen, which is expressed in proliferating cells from G1 to M-phase of the cell cycle (51). Several studies have shown Ki67 to be predictive of a variety of human malignancies, such as prostate cancer and breast cancer (52,53). Bcl-2 and Bax belong to the Bcl-2 gene family; Bcl-2 members are localized in the mitochondria and have either pro-apoptotic (Bax, Bak, Bid and Bim) or anti-apoptotic (Bcl-2, Bcl-xL and Bcl-W) functions (54). Activated Bax can induce the release of pro-apoptotic factors and cytochrome c (55). Caspase-3 is a member of the cysteine-aspartic acid protease family and plays a central role in cell apoptosis (56,57). The results of the present study indicated that CCND1 promoted the expression of Ki67 and Bcl-2, and inhibited Bax and Cleaved caspase-3, while miR-628-5p partially reversed the effect of CCND1 on these genes. It also indicated that miR-628-5p inhibited proliferation and induced apoptosis of colorectal cancer cell via regulating CCND1 expression.

Nevertheless, the present study has several limitations that hinder the interpretation of the results; for example, the function of overexpressed miR-628-5p in inhibiting colorectal cancer development through reducing proliferation and inducing apoptosis was only supported by in vitro studies. Additionally, the observed regulatory mechanism between miR-628-5p and CCND1 in colorectal cancer remains unclear, and it will require further investigations In conclusion, the results of the present study indicated that miR-628-5p may serve as a tumor suppressor, and could potentially be used as a promising biomarker for the prognosis of patients with colorectal cancer. In addition, miR-628-5p may serve as a potential therapeutic target for colorectal cancer treatment.