Introduction
Colorectal cancer (CRC), as the third most common cancer affecting the gastrointestinal tract, is a common type of cancer worldwide, and is associated with a high mortality rate due to its rapid progression and advanced tumor presentation at the time of diagnosis [
1‐
3]. CRC is becoming more prevalent in China [
4]. The vast majority of CRC patients have reached an advanced pathological stage before symptoms appear; therefore, that the survival rate of those diagnosed with CRC remains poor. The early detection of CRC is significantly beneficial to improve the probability of survival. Therefore, a better understanding of the mechanisms that result in CRC and the identification of a new molecular marker or factor that can be used to create novel diagnostic and therapeutic strategies is urgently needed for patients with CRC.
Long non-coding RNAs (lncRNAs, > 200 nucleotides in length) are a class of newly discovered non-coding RNA molecules with limited or no protein-coding capacity [
5,
6]. Recently, increasing evidence has shown that lncRNAs have important biological functions are closely related to human diseases, especially cancer [
7‐
10]. They may function as oncogenes [
11,
12] or anti-oncogenes [
13,
14] similar to protein-coding genes, and their dysregulated expression is significantly correlated with carcinogenesis [
15]; therefore, they may be considered to be promising candidate biomarkers for diagnosing cancer and may also represent therapeutic target in the future.
The underlying molecular mechanisms by which lncRNAs exert their functions are complex, diverse and exist at various levels during the development of cancer, including the epigenetic, transcriptional, post-transcriptional and translational levels [
16‐
18]. For instance, lncRNA HOTAIR is upregulated in CRC and may be a critical element in metastatic progression as a result of its interaction with PRC2 (Polycomb Repressive Complex 2) [
19]. Our previous studies also showed that HOTAIR could function as a competing endogenous RNA to regulate HER2 expression by sponging miR-331-3p in gastric cancer [
20]; lncRNA SPRY4-IT1 could be a growth regulator, and promoted the proliferation of ER (−) human breast cancer cells by upregulating the expression of ZNF703 [
21].
Loc554202, is a 2166-bp transcript on human chromosome 9p21.3, which is the host gene of miR-31 and dysregulated in breast [
22,
23] and lung [
24] cancer cells, although the importance of its function has not yet been established. Little is known concerning the potential role of Loc554202 in the development and progression of CRC. To detect the functions and molecular mechanisms of Loc554202 in CRC, we conducted a qRT-PCR analysis of Loc554202 expression in human CRC tissues and cell lines, and showed that the expression of Loc554202 was significantly downregulated in cancerous tissue samples and CRC cell lines compared with matched normal samples. Its downregulation was closely associated with an advanced pathological stage and a large tumor size. Further functional studies of Loc554202 indicated that enhanced expression of Loc554202 could decrease cell proliferation and induce apoptosis
in vitro and
in vivo.
Materials and methods
Tissue collection
Colorectal cancer tissues and normal tissues were obtained from 48 patients who had undergone surgical resection of colorectal cancer between 2010 and 2012 at Second Affiliated Hospital of Nanjing Medical, China. No local or systemic treatment had been administered to these patients prior to the operation. All tissue samples were washed with sterile phosphate-buffered saline before being snap frozen in liquid nitrogen and then stored at −80 °C until required for the analyses. The pathological stage, grade and nodal status were appraised by an experienced pathologist. The clinicopathological characteristics, including the tumor, node metastasis (TNM) staging are summarized in Table
1. The non-tumorous tissues were 5 cm from the edge of the tumor, and there were no obvious tumor cells present in these regions as determined by the pathologist. All of the experiments were approved by the Research Ethics Committee of Nanjing Medical University, China. Written informed consent was obtained from all patients.
Table 1
Correlation between Loc554202 expression and clinicopathological characteristics of CRC patients
Age (years) | | | | | 0.560 |
<60 | 21 | 43.8 % | 11 | 10 | |
≥60 | 27 | 56.2 | 13 | 14 | |
Gende | | | | | 0.430 |
Male | 29 | 60.4 % | 15 | 14 | |
Female | 19 | 39.6 % | 9 | 10 | |
Maximum tumor size | | | | | 0.007* |
<5 cm | 28 | 58.3 % | 19 | 9 | |
≥5 cm | 20 | 41.7 % | 5 | 15 | |
Location | | | | | 0.440 |
Colon | 19 | 39.6 % | 10 | 9 | |
Rectum | 29 | 60.4 % | 14 | 15 | |
Depth of tumor | | | | | 0.108 |
T1 and T2 | 18 | 37.5 % | 11 | 7 | |
T3 and T4 | 30 | 62.5 % | 13 | 17 | |
Tumor stage | | | | | 0.028* |
I and II | 26 | 54.2 % | 17 | 9 | |
III and IV | 22 | 45.8 % | 7 | 15 | |
Lymph node metastasis | | | | | 0.698 |
Negative | 25 | 52.1 % | 12 | 13 | |
Positive | 23 | 47.9 % | 12 | 11 | |
Ethics statement
The study was approved by the Ethics Committee of Nanjing Medical University and was performed in compliance with the Declaration of Helsinki. Written informed consent was obtained for the use of all patient samples. All experimental animals were housed under specific pathogen-free conditions. All experimental procedures were approved by the Institutional Review Board of Nanjing Medical University. All procedures were performed in accordance with the Nanjing Medical University Guide for the Care and Use of Laboratory Animals which was formulated by the National Society for Medical Research.
Cell lines and culture conditions
The human colorectal cancer cell lines (HCT116, DLD1, SW480, RKO, HT-29) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The human colonic epithelial cells HCoEpiC were obtained from American Type Culture Collection (Manassas, VA). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) in humidified air at 37 °C with 5 % CO2. All media were supplemented with 10 % fetal bovine serum (10 % FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Shanghai, China).
RNA extraction and qRT-PCR analyses
Total RNA was extracted from tissues or cultured cells with the TRIZOL reagent (Invitrogen Life Technologies) according to the manufacturer’s protocols. For qRT-PCR, RNA reverse transcribed to cDNA from 1 μg of total RNA was reverse transcribed in a final volume of 20 μl using random primers and a Reverse Transcription Kit (Takara, Dalian, China). According to the manufacturer’s instructions, the reverse transcription was performed at 37 °C for 15 min, then at 85 °C for 5 s. qRT-PCR analyses were performed using a standard protocol from Power SYBR Green (Takara, Dalian, China). All protocols were performed according to the manufacturer’s instructions. The Δct values were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used for the studies are shown in Additional file
1: Table S1. The qRT-PCR assays and data collection were performed using an ABI 7500 instrument. Each sample was analyzed in triplicate.
Treatment of HCT116 and DLD1 cells with 5-aza-2-deoxy-cytidine (5-aza-CdR)
CRC cells (2.5 × 105) were seeded into six-well cultureplates and exposed to 0, 5 or 10 μM 5-aza-CdR (Sigma-Aldrich, USA). Cells were harvested after 72 h for qRT-PCR to detect the expression level of Loc554202.
Transfection of colorectal cancer cells
Small interfering RNA (siRNA) and nonspecific control siRNA were synthesized (Carlsbad, California, USA) and transfected into cells using Lipofectamine 2000 (Invitrogen, USA). To overexpress Loc554202, the full length coding sequence for Loc554202 was amplified and subcloned into the pcDNA 3.1(+) vector (Invitrogen) according to the manufacturer’s instructions. HCT116 and DLD1 cells were transfected with a negative control vector or the Loc554202-expressing plasmid according to the manufacturer’s protocol. Cells were harvested after 48 h for qRT-PCR and western blot analyses. The sequences of the siRNAs are described in Additional file
2: Table S2.
Cell proliferation assays
Forty-eight hours after pCDNA-Loc554202 transfection, 3,000 cells per well were allowed to grow in 96-well plates with five replicate wells. After 6 h of culture, as well as at 24, 48, 72 and 96 h after atarting the culture, the cells were treated with 100 μg 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by adding it to the medium. The cells were incubated at 37 °C for another 4 h, then the medium was removed, and dimethylsulfoxide (DMSO) was added for 10 min to lyse the cells. Finally, the absorbance was measured at 490 nm. All experiments were performed in triplicate.
Cells were trypsinized into single-cell suspensions 48 h after transfection. For the colony formation assay, 1,000 cells were plated into each well of a six-well plate and were maintained in media containing 10 % FBS to allow colony formation, with the medium being repalaced every four days. After two weeks, colonies were fixed with methanol and stained with 0.1 % crystal violet (Sigma) in PBS for 15 min. The visible colonies were manually counted. Triplicate wells were measured for each treatment group.
Flow cytometry
Cells transiently transfected with pCDNA-Loc554202 were harvested 48 h after transfection by trypsinization, washed with ice-cold phosphate-buffered saline, and fixed with 75 % ethanol overnight. The cells used for the cell-cycle analysis were stained with propidium oxide (100 μg/mL) using the Cycle Test Plus DNA Reagent Kit (BD Biosciences) and were analyzed by flow cytometry (FACScan;BD Biosciences) using an instrument equipped with the CellQuest software program (BD Biosciences). The percentages of cells in the G0–G1, S, and G2–M phases were counted and compared. The cells used for the apoptosis analysis were harvested 48 h after transfection, and were stained for 15 min with fluorescein isothio-cyanate (FITC)-Annexin V and propidium iodide (PI) in the dark at room temperature, according to the manufacturer’s recommendations. The cells were then examined by flow cytometry (FACScan; BD Biosciences) and the CellQuest software program (BD Biosciences) and were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells. The percentage of early apoptotic cells was compared with the control groups from each experiment.
All of the samples were assayed in triplicate.
TUNEL
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was performed with an apoptosis detection kit (KeyGEN BioTECH, China) according to the manufacturer’s instructions. Randomly selected fields without significant necrosis in 10 high-power fields (6400) were assessed for TUNEL-positive cells. The TUNEL index was calculated based on the number of total nuclei and the number of cells with green nuclei.
Western blot analysis and antibodies
Cells were lysed using the mammalian protein extraction reagent, RIPA (Beyotime), supplemented with a protease inhibitor cocktail (Roche) and PMSF (Roche). Protein were separated by 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to 0.22 mm nitrocellulose (NC) or polyvinylidene difluoride membranes (Sigma). The membranes were washed, blocked, and incubated with specific primary antihuman antibodies. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG. An ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad). The mean ± SD values were calculated from three individual experiments. A GAPDH antibody was used as a control, and the anti-cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax (all 1:1000) antibodies were purchased from Cell Signaling Technology, Inc. (CST).
Four-week-old male BALB/c nude mice were obtained from the Shanghai Laboratory Animals Center of the Chinese Academy of Sciences (Shanghai, China). The mice were housed under pathogen-free conditions with a 12 h light/dark schedule, were fed an autoclaved diet ad libitum, and were injected subcutaneously with 5 × 106 cells to assess the tumor formation. Tumor growth was examined every three days, and tumor volumes were calculated using the following formula: 0.5 × length × width2. At two weeks after the cell injection, the mice were killed, and the subcutaneous weight of each tumor was measured and the tumors were used for further analyse. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Nanjing Medical University.
Immunohistochemical (IHC) analysis
Tumor tissue samples were immunostained for cleaved caspase-3 as described previously. The expression was considered to be positive when 50 % or more cancer cells were stained. The anti-cleaved caspase-3(1:50) antibody was purchased from CST.
Statistical analysis
All data were expressed as the means ± SD (standard deviation, SD), and were analyzed using Student’s t test between to compare two groups of in vitro and in vivo data using the SPSS 17.0 software program. A value of P < 0.05 was considered to be statistically significant.
Discussion
Over the past few decades, accumulating evidence has shown that long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of cancer [
6,
25,
26], and can provide a novel platform for the diagnosis and treatment of this disease. With the advances in tiling array and novel sequencing technologies, it has been revealed that only 2 % of the total human genome sequences encode proteins, whereas the remainder can be divided into two groups according length, including short ncRNAs (<200 nt) and long ncRNAs (>200 nt) [
27,
28]. The lncRNAs are important new members of the ncRNA family, which are greater than 200 nt in length and can be transcribed by RNA polymerase II (RNA pol II). Recent, evidence has indicated that many lncRNAs can be considered as vital regulators of tumorigenesis [
29,
30], as were lncRNA SPRY4-IT1 [
21] and HOTAIR [
20] in our previous studies. Therefore, the identification of cancer-associated lncRNAs and investigation of their molecular and biological functions are important to provide new insights into the diagnosis and treatment of cancer, including colorectal cancer.
A previous study demonstrated that Loc554202 as the host gene of miR-31 regulates the proliferation and migration of breast cancer cells [
22], and CpG island methylation plays an important role in silencing the Loc554202 genes [
23]. However, the functions of Loc554202 in CRC were previously unknown. In this study, we provided the first evidence that lncRNA Loc554202 was significantly downregulated in colorectal cancer tissues compared with adjacent normal tissues, and low expression of the lncRNA in CRC patients was associated with an increased tumor size and advanced TNM stage. The expression level of miR-31 was no significant change after transfecting CRC cells with pCDNA-Loc554202, which eliminated the influence of miR-31 may exist on overexpression of Loc554202. Additionally, DNA methylation could restrain the expression of Loc554202. Our subsequent studies showed that overexpression of Loc554202 decreased cell proliferation and caused a dramatic decrease in colony formation in the HCT116 and DLD1 cells. This result was confirmed by knocking down Loc554202 in both CRC cell lines. These findings suggest that Loc554202 might be a novel clinical marker for the prognosis of CRC and might represent a target for therapy.
To further clarify the functions of Loc554202 in CRC, such as whether its expression influenced cell cycle progression and apoptosis, we next used a flow cytometry assay to detect the cell cycle progression and apoptosis in HCT116 and DLD1cells after treatment with pCDNA-Loc554202 or an empty vector. The results demonstrated that Loc554202 overexpression promoted significant arrest in the G0/G1-phase and an obvious increase in apoptosis. These observations were verified by means of TUNEL staining, immunohistochemical (IHC) assays and in a mouse xenograft model. We also found that silencing Loc554202 expression mildly inhibited apoptosis in HCT116 and DLD1 cells in vitro. These results revealed that the anti-proliferative effects of Loc554202 in the CRC cells were mediated by its inhibition of cell cycle progression and induction of apoptosis.
Although lncRNAs have been shown to have vital biological functions in various malignant tumors, their precise regulatory mechanisms remain largely unknown, although many studies have focused on lncRNA-mediated effects on cell apoptosis. For instance, lncRNA MEG3 inhibits non-small cell lung cancer (NSCLC) cell proliferation and induces apoptosis by affecting p53 expression [
31], and lncRNA ANRIL promotes NSCLC cell proliferation and inhibits apoptosis by silencing KLF2 and P21 expression [
32]. However, the most important pathway discovered so far is the activation of specific caspase cleavage cascades.
To further confirm the role of caspase activation in Loc554202 induced apoptosis, we found that pretreatment of cells with the pan-caspase inhibitor, Z-VAD-FMK, decreased the Loc554202 induced apoptosis rate, as detected by flow cytometry. Likewise, the outcomes of qRT-PCR and western blot analyses showed that the mRNA and the protein levels of the pro-apoptotic proteins were significantly increased in pCDNA-Loc554202 treated cells, whereas the anti-apoptotic protein was decreased. These data indicate that Loc554202 induces CRC cell apoptosis at least partly through the activation of specific caspase cleavage cascades.
In summary, we have shown that Loc554202 is downregulated in colorectal cancer tissues, and we provided the first evidence that Loc554202 exerts critical effects on CRC cells by affecting both the cell cycle and apoptosis. In addition, CpG island methylation plays an important role in silencing the Loc554202 gene. Finally, we showed that Loc554202 regulated cell apoptosis at least partly through the activation of specific caspase cleavage cascades. Together, our findings suggest that lncRNA Loc554202 acts as a tumor-inhibiting factor in CRC, and could be a candidate prognostic biomarker or a target for new cancer therapies. However, further studies in a larger number of samples and investigations of the other possible mechanisms of action are required.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DJ designed the study, detected the cells biological function, conducted the qRT-PCR assays, carried out the Western blotting assays, performed the statistical analysis, and drafted the manuscript. LBB and WJP performed the TUNEL assays, provided the tissue samples and the clinical data and helped to draft the manuscript. WJ, SYG, LYF, and ZY participated in the design of the study. WJR, FYR, WZX and DW helped to acquire experimental data. WKM conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.