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MicroRNAs (miRNAs) play a vital role in the progression of cancer, however, only limited data on miRNAs in rectal cancer are available. The aim of the present study was to investigate whether miR-195 could inhibit the progression of rectal cancer. The miR-195 mimic was transfected into 2 types of human rectal cancer cells (SW837 and SW1463). Cell viability and apoptosis were analyzed by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, and cell migration and invasion were assessed by scratch test and Transwell assay. The results revealed that insulin-like growth factor 1 (IGF1) was predicted as a potential target of miR-195 by Targetscan7.2, and the result was verified by dual-luciferase reporter assay. The co-transfection of IGF1 was performed to confirm the underlying mechanism of tumor suppressor of miR-195 in rectal cancer. The activation of PI3K/AKT signaling was determined by western blotting. The levels of miR-195 in SW837 and SW1463 cells were revealed to be lower than in human rectal mucosa epithelial cells. After the transfection with miR-195, the cell viability was decreased, while the apoptosis was significantly increased (SW837: 5.21% vs. 20.96%; SW1463: 4.19% vs. 25.22%). Moreover, cell migration and invasion were significantly inhibited in the mimic group. miR-195 specifically targeted IGF1, however, the co-transfection of IGF1 could partially reverse the inhibitory effects of miR-195 on rectal cancer cells. It was also determined that the phosphorylation of PI3K and AKT were significantly inhibited in the mimic group. The tumor suppressive ability of miR-195 in rectal cancer cell proliferation and metastasis was mediated by blocking IGF1 expression and inhibiting the PI3K/AKT pathway.
Introduction
MicroRNAs (miRNAs) are small (18–24 nucleotides) non-coding RNA molecules that act as negative regulators in target gene expression at the post-transcriptional level. Accumulated evidence suggests that these small RNAs occupy a crucial place in the modulation of multiple cellular bioprocesses, for example, cell differentiation, proliferation and apoptosis (1). In addition, abnormal miRNA expression profiles can result in cell dysfunction and subsequently contribute to pathogeneses of various diseases, even cancer (2,3). Research has demonstrated that many miRNAs are specific target oncogenes or cancer suppressor genes and directly participate in the development of cancers (4–6). Notably, miRNAs are reported to play a vital regulatory role in almost every cancer type due to their abundance and cell-type specifcity (7,8).
Colorectal cancer (CRC) is a very common malignant tumor. The incidence of CRC is increasing and it has become the fourth main cause of cancer-associated mortality worldwide (9). In 2012, ~1.36 million people were diagnosed with CRC, and rectal cancer accounted for ~28% and was highly associated with a poor clinical outcome (10,11). In recent years, many differentially expressed miRNAs have been identified in regulating the progression of colon cancer (12,13), however, limited data on miRNAs in rectal cancer are available. Some studies revealed that rectal and colon cancers were two different tumor entities, therefore, they required different treatment strategies due to the differences in the disease-associated genetic and biological factors (14,15). It is also urgent to investigate the miRNA expression profiles in rectal cancer. Currently, Gaedcke et al (16) mapped the expression of 2,090 miRNAs using LNA-enhanced miRCURY microarrays, and revealed 49 differentially expressed miRNAs after conducting comparative analysis of tumor and matched mucosa samples of locally advanced rectal cancer patients. miR-195 was one of the significantly downregulated miRNAs in rectal cancer (16). Studies have demonstrated that miR-195 acts as a tumor suppressor in many types of cancer, for instance, Cai et al (17) indicated that by blocking the expression of ribosomal protein S6 kinase, 70 kDa, polypeptide 1 (RPS6KB1), miR-195 had a marked inhibitory effect on human prostate cancer cell metastasis and angiogenesis. Similarly, in papillary thyroid carcinoma (PTC), miR-195 specifically targeted fibroblast growth factor 2 (FGF2) and cyclin D1 to regulate the proliferative, migratory and invasive capacities of PTC cells (18). miR-195 inhibits the growth and metastasis of non-small cell lung cancer cells by targeting insulin-like growth factor 1 receptor (IGF1R) (19). However, there is a lack of a detailed understanding of the suppressive effects of miR-195 on rectal cancer progression and development. Therefore, the present study aimed to investigate the potential mechanism of the tumor suppressive effects of miR-195 in rectal cancer.
Materials and methods
Cell culture and transfection
Human rectal mucosa epithelial cell line (PriCells) and 2 types of human rectal cancer cell lines (SW837 and SW1463; ATCC) were cultured with Leibovitz's 15 (L-15) culture medium (ATCC), which was supplemented with 10% fetal bovine serum (FBS; ATCC) in 5% CO2 in an incubator at 37°C. The cells in logarithmic phase were harvested for subsequent experiments.
miR-195 mimic (100 pmol) (sense, 5′-UAGCAGCACAGAAAUAUUGGC-3′ and antisense, 5′-CAAUAUUUCUGUGCUGCUAUU-3′) and mimic control (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) were obtained from Shanghai GenePharma Co., Ltd. Full-length insulin-like growth factor 1 (IGF1 sense, 5′-GAATTCATGGGAAAAATCAGCAGTC-3′ and antisense, 5′-GATATCGCATGTCATTCTTCACTCTTT-3′) were cloned into a pcDNA3.1 vector (Takara Biotechnology Co., Ltd.), and an empty pcDNA3.1 was set as negative control (NC). Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used in cell transfection. After being transfected with miR-195mimic, mimic control, IGF1 or NC vectors for 48 h, the cells were transferred to complete medium containing puromycin.
Cell viability assay
Cell viability was detected according to the instructions of a Cell Counting Kit-8 assay (CCK-8; Beyotime Institute of Biotechnology). Cells were trypsinized to a suspension of 3×104 cells/ml and every 100-µl suspension was added into each well of a 96-well plate. The viabilities of SW837 and SW1463 cells were assessed at 24, 48 and 72 h using a microplate reader at 450 nm (Molecular Devices, LLC).
Flow cytometry for apoptosis
The effects of miR-195 on cell apoptosis in the SW837 and SW1463 cells were analyzed by flow cytometric assay. In brief, cells were resuspended with EDTA-free pancreatin and then centrifuged at 3,000 × g at room temperature to remove the supernatant. Cell apoptosis was determined using Annexin V-FITC Cell Apoptosis Assay Kit (Sigma-Aldrich; Merck KGaA). The cells were resuspended with 100 µl Annexin V-fluorescein isothiocyanate/propidium iodide/HEPES dye liquor (Annexin V-FITC/PI/HEPES, 1:2:50) and evenly oscillated. The apoptosis rate was determined using a flow cytometer (BD FACSCalibur; BD Biosciences).
Scratch test
The cells were seeded in 6-well plates (1×105 cells/well) and maintained in 5% CO2 at 37°C until cell confluence was reached. Then, a 200-µl pipette tip was used to scratch the cell monolayer. After washing with PBS to remove the scratched cells, the plates were maintained in 5% CO2 at 37°C. After 48 h of incubation, several random fields were photographed using an inverted microscope to measure scratch width.
Transwell assay
The invasive capacities of SW837 and SW1463 cells were assessed by Transwell assay. To be more specific, 8.0-µm pore Transwells (EMD Millipore) were inserted into 24-well plates. The diluted Matrigel (Shanghai YASEN Biotechnology Co., Ltd.) solution (1:8) was added into the upper chamber of the basement membrane. After the Matrigel dried at room temperature, the cells (2×104) were resuspended with L-15 medium but without FBS and seeded into the upper chamber, and normal culture media was placed in the lower chamber. After 24 h of incubation, the cells on upper surface of the membrane were removed gently using cotton swabs, while the invaded cells were fixed with 4% paraformaldehyde for 15 min and then stained using 0.05% crystal violet solution for another 15 min. A total of 5 randomly selected views were photographed under a wide-field microscope (Nikon Corporation).
Luciferase reporter assay
According to the computational analysis of Targetscan7.2 (
Real-time quantitative PCR (RT-qPCR)
Total RNAs from SW837 and SW1463 cells were isolated by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reversed-transcribed using M-MLV MicroRNA Reverse Transcription Kit (Promega Corporation). The relative levels of miRNA were determined using Bulge-Loop™ miRNA qRT-PCR Primer Set (Guangzhou RiboBio Co., Ltd.). The primers of U6 and miR-195 were obtained from Guangzhou RiboBio Co., Ltd. (MQPS0000002-1-100 and MQPS0000758-1-100). To determine the expression of target genes, the reversed transcription of cDNA was performed using First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) and reacted at 37°C for 60 min and at 70°C for 5 min. RT-qPCR was conducted on ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR green (Invitrogen; Thermo Fisher Scientific, Inc.). The thermocycling parameters were as follows: 94°C for 2 min, followed by 40 cycles at 95°C for 30 sec, and 60°C for 30 sec. The relative expression of miRNA and mRNA were determined by 2−ΔΔCq formula (20) and normalized to U6 and GAPDH. Primers are presented in Table I.
Western blotting
Cellular protein of each type of transfected cells was digested by Radio-Immunoprecipitation Assay buffer (RIPA; Sigma-Aldrich; Merck KGaA). The protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membranes, which were blocked with 5% non-fat milk in TBST buffer for 1 h at room temperature and then incubated with a primary antibody at 4°C overnight. After being washed by PBS, the membranes were mixed with secondary antibodies (1:2,000; ID product codes ab205718 and ab205719; Abcam). Band signals were visualized by ECL solution (Thermo Fisher Scientific, Inc.) and normalized to GAPDH. Densitometry was performed using Quantity One software version 2.4 (Bio-Rad Laboratories, Inc.). The following primary antibodies were used in our study: p-PI3K (1:1,000; 85 kDa; product no. 4228) PI3K (1:2,000, 85 kDa; product no. 5405), p-Akt (1:500; 60 kDa; cat. no. 9271), and Akt [1:2,000; 60 kDa, cat. no. 9272; all from Cell Signaling Technology (CST)] and GAPDH (1:10,000; 36 kDa; ID product code ab8245; Abcam).
Statistical analysis
All data are presented as the mean ± SEM. Data significance from different groups was analyzed by one-way ANOVA, followed by the Tukey's multiple comparison tests. A P<0.05 was considered to indicate a statistically significant difference.
Results
The transfection of miR-195 mimic enhances apoptosis rates in SW837 and SW1463 cells
To investigate the role of miR-195 in the rectal cancer development, SW837 and SW1463 cells were transfected with miR-195 mimic. As revealed in Fig. 1A, the miR-195 levels in human rectal cancer cells (SW837 and SW1463) were significantly lower than the human rectal mucosa epithelial cells (P<0.01). After transfection with miR-195 mimic, data in Fig. 1B revealed that miR-195 mimic functioned stably in SW837 and SW1463 cells. The effects of miR-195 on rectal cancer cells were assessed based on cell viability and apoptosis of SW837 and SW1463 cells, and as revealed in Fig. 1C, it was demonstrated that the transfection of miR-195 mimic significantly inhibited cell viability both in SW837 and SW1463 cells, and that miR-195 more strongly affected SW837 cell viability than SW1463 cells. Concurrently, apoptosis analysis revealed that the miR-195 mimic transfection significantly enhanced the SW837 cell apoptosis from 5.21% in the Blank group to 20.96% in the mimic group, and from 4.19 to 25.22% in SW1463 cells (P<0.01, Fig. 1D and E). After miR-195 was transfected, a higher cell inhibition rate in SW837 was observed compared with that in SW1463 cells, however, an opposite result was obtained in apoptosis, and this could be explained by the fact that cell proliferation and apoptosis have different mechanisms. In addition, the mRNA levels of B-cell lymphoma 2 (Bcl-2) and Bax were also examined to further confirm the effects of miR-195 on apoptosis of rectal cancer cells and the results are presented in Fig. 1F. The downregulated Bcl-2 and upregulated Bax also indicated that miR-195 was positively correlated with the apoptosis rate in SW837 and SW1463 cells. Thus, the upregulated miR-195 level inhibited the cell viability and survival rate in SW837 and SW1463 cells.
Increased miR-195 has an inhibitory effect on the migratory and invasive capacities of SW837 and SW1463 cells
Tumor metastasis relies on the migratory and invasive abilities of tumor cells, thus, a scratch test and Transwell assay were carried out to determine the cell migration and invasion after the cells had been transfected with miR-195 mimic. As demonstrated in Fig. 2A, the wound closure rate was reduced in the mimic group of both cell lines, compared with the Blank and mimic control groups (P<0.05). Fig. 2B revealed that the number of invaded cells was reduced in the mimic group of both cell lines, compared with the Blank and mimic control groups (P<0.05). These results indicated that miR-195 overexpression could significantly suppress cell migratory and invasive capacities of SW837 and SW1463 cells.
miR-195 directly regulates IGF1 by targeting its mRNA
To further study the potential mechanism of the inhibitory effects of miR-195 on rectal cancer cells, Targetscan7.2 computational analysis was performed to identify possible target genes of miR-195. As revealed in Fig. 3A, the fragment of IGF1-3′-UTR contained a binding-site of miR-195 IGF1, indicating that IGF1 could be directly regulated by miR-195. A dual-luciferase reporter system assay was performed to further verify the relationship between miR-195 and IGF1 (Fig. 3B). Although IGF1 is a secreted growth factor, a transfection (the overexpression plasmid) was performed instead of directly adding the growth factor in the culture, since the transfection technique is more stable. The co-transfection of miR-195 mimic could significantly inhibit the luciferase activity of WT luciferase vector, compared with the transfection of WT luciferase vector alone (P<0.01), however, the luciferase activity of the MUT luciferase vector was not affected. Collectively, the present findings indicated that IGF1 was an effective target of miR-195.
The co-transfection of IGF1 could partially reverse the suppressive effects of miR-195 on SW837 and SW1463 cell viability
To confirm the role of IGF1 in the suppressive effects of miR-195 on rectal cancer cell viability, IGF overexpression vector was co-transfected with miR-195 mimic into SW837 and SW1463 cells. According to Fig. 4A, the transfection of IGF1 significantly increased the expression of IGF1 under the regulation of miR-195 mimic in SW837 and SW1463 cells. In addition, it was also observed that the transfection of IGF1 could not only significantly enhance normal rectal cancer cell viability, but also significantly increased miR-195 mimic-inhibited cell viability in comparison to mimic+NC group (P<0.05, Fig. 4B). Therefore, the co-transfection of IGF1 was able to partially reverse the suppressive effects of miR-195 on rectal cancer cell viability.
IGF1 co-transfection could partially reverse the suppressive effects of miR-195 on cell migration and invasion in SW837 and SW1463 cells
The changes in the migration and invasion of SW837 and SW1463 cells was also assessed. As observed in Figs. 5 and 6, the transfection of IGF1 alone could significantly increase the migration rate (P<0.01). When IGF1 was co-transfected with miR-195 mimic, the migration rate was significantly enhanced in the mimic+IGF1 group (P<0.01). In addition, cell invasion results in Fig. 7 demonstrated that IGF1 transfection could significantly promote cell invasion of SW837 and SW1463 cells and that the number of invaded cells in the IGF1 group was significantly higher than the NC group (P<0.01), and that the co-transfection of IGF1 could also enhance the number of invaded cells under the regulation of miR-195, compared with mimic+NC group (P<0.01). Thus, the present findings indicated that the suppressive effects of miR-195 on rectal cancer cell migration and invasion may rely on blocking the expression of IGF1.
The PI3K/AKT pathway is involved in the tumor suppressive ability of miR-195 in rectal cancer
To further study whether the PI3K/AKT signaling pathway was involved in the mechanism of miR-195 suppressing the progression of rectal cancer, the phosphorylation levels of PI3K and AKT were detected under the regulation of IGF1 and miR-195. Fig. 8 revealed that the overexpression of miR-195 could induce a significant reduction of the protein levels of p-PI3K and p-AKT, while the protein levels of p-PI3K and p-AKT were significantly upregulated in the cells were co-transfected with IGF1 and miR-195 mimic, however, no difference in PI3K and AKT protein levels was observed (P<0.01). Therefore, the present findings indicated that the tumor suppressive ability of miR-195 was partially attributed to the suppression of the PI3K/AKT pathway.
Discussion
Although microRNA microarray analyses revealed that several miRNAs are aberrantly expressed in rectal cancer (21,22), their functional effects and underlying mechanisms on rectal cancer development and progression remain elusive. Therefore, investigating the mechanism of participation of these miRNAs in rectal cancer progression is required in order to improve current knowledge on rectal cancer and to offer more effective diagnosis and therapy. In this present study, since miR-195 has been widely reported in numerous malignant tumors such as non-small cell lung (23), cervical (24) and breast cancer (25), the functional role of miR-195 was investigated in rectal cancer. The present results revealed the functional role and the potential mechanism of miR-195 in the progression of rectal cancer. Specifically, it was observed that miR-195 overexpression could significantly reduce rectal cancer cell proliferation and the survival rate. Then, IGF1 was confirmed to be an effective target of miR-195, suggesting that the suppressive effects of miR-195 may rely on controlling the expression of IGF1 in rectal cancer. In addition, it was also observed that the PI3K/AKT pathway also played a potential role during the process of the inhibition of miR-195 on the development of rectal cancer.
Previous studies revealed that miR-195 was located at chromosome 17p13.1 and has been extensively demonstrated as a tumor suppressor in many types of cancers (26). In 2017, Yan et al (27) revealed that miR-195 had the ability to inhibit tumor growth by the regulation of oncogene astrocyte elevated gene-1 (AEG-1) in hepatocellular carcinoma (HCC) cells. Research has also revealed a high association between low expression of miR-195 and epithelial-mesenchymal transition (EMT) in the progression of HCC, and accordingly, increasing miR-195 expression could strongly suppress the metastatic ability of HCC cells (28). These studies were consistent with the present results, since in this study, miR-195 was also considered as a novel cancer suppressor and potent metastatic inhibitor in rectal cancers. It was observed that miR-195 was significantly decreased, and its expression was negatively correlated with the cell survival rate, migratory and invasive capacities in rectal cancer. Thus, it was speculated that miR-195 may be a novel therapeutic target in rectal cancer.
It is considered that miRNAs directly regulate gene expression by binding to their target gene mRNAs (29). IGF1 was reported to be a key modulator in tissue growth and development, and some studies have demonstrated a positive correlation between the expression and activity of IGF1 and the risk of breast cancer (30,31). In addition to breast cancer, upregulated IGF1 contributed to the pathogenesis of endometrial cancer (EC) and increased the risk of colorectal cancer via the insulin signaling pathway (32,33). In addition, a recent study indicated that a high level of IGF1 could contribute to gastric cancer cell proliferation, metastasis, and EMT by promoting interferon-induced transmembrane protein (IFITMs) production (34). miR-195 was revealed to inhibit the growth and metastasis of non-small cell lung cancer cells by targeting, and by binding to IGF1R, and IGF1 functions as a secreted growth factor (19). IGF1 signaling may play a crucial role in the regulation of miR-195. In the present study, IGF1 was confirmed as an effective target of miR-195 by performing computational analysis and verification of luciferase reporter assay. However, it may be a limitation that IGF1R was not studied further in this research. The present results revealed that miR-195 potently suppressed the mRNA and protein levels of IGF1, while the co-transfection of IGF1 significantly enhanced rectal cancer cell proliferative, migratory and invasive capacities. Therefore, it was surmised that the suppressive effects of miR-195 on rectal cancer growth and metastasis may rely on the regulation of IGF1 expression. Furthermore, it was also revealed that the phosphorylation levels of PI3K and AKT had significant but different associations with the expression levels of miR-195 and IGF1. Research has revealed that the activation of PI3K/Akt signaling played a vital role in the development and progression of various types of cancers (35,36). In 2014, Johnson et al (37) revealed that inhibiting the upstream components of the PI3K/Akt pathway could effectively inhibit CRC tumor growth and metastatic capability and sensitize cancer cells to chemotherapy (38). Therefore, the suppressive effects of miR-195 on rectal cancer were attributed to, at least partially, the regulation of PI3K/Akt pathway. Notably, such a result would be more convincing by studying the negative regulation of miR-195 inhibitor on rectal cancer cells. However, since miR-195 was suppressed in rectal cancer cells, the overexpression of miRNA was commonly treated as the classical method to study the mechanism. The inhibition of miR-195 should be further studied in a future study.
In conclusion, the present findings indicated that the transfection of miR-195 mimic can decrease cell viability, enhance apoptosis and inhibit the migratory and invasive capacities in rectal cancer by directly targeting the mRNA of IGF1. The PI3K/Akt pathway also participates in the suppressive effects of miR-195 on the development and progression of rectal cancer, however, the underlying mechanism remains to be further determined. The close association between miR-195 and IGF1 provides a candidate target for rectal cancer treatment.
Table I.Primers for RT-qPCR.
| Gene name | Primer sequences |
|---|---|
| Bcl-2 | F: 5′-GCCTTCTTTGAGTTCGGTG-3′ |
| R: 5′-CAGAGACAGCCAGGAGAAATC-3′ | |
| Bax | F: 5′-GCAAACTGGTGCTCAAGG-3′ |
| R: 5′-CGCCACAAAGATGGTCAC-3′ | |
| IGF1 | F: 5′-CCTCGCATCTCTTCTACCTGG-3′ |
| R: 5′-CACTATTCCCGTCTGGGGC-3′ | |
| GAPDH | F: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ |
| R: 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ |
Bcl-2, B-cell lymphoma 2; IGF1, insulin-like growth factor 1; F, forward; R, reverse.
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