- Open Access
MiR-218 produces anti-tumor effects on cervical cancer cells in vitro
© The Author(s). 2018
- Received: 10 June 2018
- Accepted: 30 September 2018
- Published: 12 October 2018
As indoleamine-2,3-dioxygenase 1 (IDO1) is critical in tumor immune escape, we determined to study the regulatory mechanism of miR-218 on IDO1 in cervical cancer.
Real-time PCR (RT-qPCR) was carried out to measure the expression of miR-218. RT-qPCR and Western blot were performed to detect the expression of IDO1 in cervical cancer. Dual-luciferase reporter assay was used to determine the binding of miR-218 on the IDO1 3′UTR. Cell viability, apoptosis, and related factors were determined using cell counting kit-8 (CCK-8), Annexin-V/PI (propidium) assay, enzyme-linked immunosorbnent assay (ELISA), RT-qPCR, and Western blot assays after miR-218 mimics has been transfected to HeLa cervical cancer cells.
MiR-218 was downregulated in cervical cancer. The expression of miR-218 was negatively correlated with IDO1 in cervical cancer tissues and cells. IDO1 is a direct target of miR-218. MiR-218 overexpression was found to inhibit cell viability and promoted apoptosis via activating the expression of Cleaved-Caspase-3 and to inhibit the expression of Survivin, immune factors (TGF-β, VEGF, IL-6, PGE2, COX-2), and JAK2/STAT3 pathway.
MiR-218 inhibits immune escape of cervical cancer cells by direct downregulating IDO1.
- Immune escape
- Cervical cancer
Cervical cancer is one of the most common malignancies in the female reproductive system. Globally, the cancer has the second highest death rate among other cancer deaths among females [1, 2]. Advances in diagnostic technology allow many cervical cancer patients to be diagnosed and treated at an early stage [3, 4]. However, the mortality of cervical cancer is still high, accounting for approximately 50% [5, 6]. Studies have shown that tumor occurrence is closely related to the immune system, which has immune surveillance functions [7, 8]. The immune system can recognize and specifically eliminate “non-self” cells through immune mechanisms to resist the occurrence and development of tumors . However, in some cases, via tumor immune escape process, malignant cells could result in occurrence, development, metastasis, and recurrence of tumors . In addition, as the tumor grows, it can form a microenvironment that helps the tumor escape immune surveillance . The tumor cells secrete immunosuppressive factors, to name a few, transforming growth factor-β(TGF-β). Interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), prostaglandin 2 (PGE2), and cytochrome c oxidase subunit II (COX-2), etc., induce normal host cells to undergo immunosuppression, reduce immune function, and cause immune escape [12–15].
As a heme-containing enzyme, indoleamine-2,3-dioxygenase (IDO) is a negative immunoregulatory factor that not only decomposes tryptophan into multiple metabolites, therefore preventing an effective immune response of T cells, but also induces regulatory T cells (Tregs)-mediated immune escape [16, 17]. IDO was observed to be expressed in some primary tumors, for example, gastric cancer, colon cancer, and renal cell carcinoma [17, 18]. The expression of IDO1 in cervical cancer, breast cancer, ovarian cancer, endometrial cancer, colon cancer, and brain tumors predicts less satisfied clinical prognosis [19, 20]. In a study of non-small cell lung cancer, ovarian cancer and other tumor tissues, IDO1 was found to be associated with malignancy degrees of the tumors . Studies have confirmed that IDO was overexpressed in primary myeloblasts, leading to a significant lack of tryptophan, and that IDO overexpression was also found to inhibit the proliferation of T cells, and this is because T cells in G1 phase are highly sensitive to tryptophan deficiency [19, 22]. On the other hand, the metabolites of tryptophan can cause apoptosis of T cells, leading to immune escape [23, 24]. Thus, inhibiting the proliferation of tumor cells by suppressing the function of IDO1 has drawn much research attention [25–27].
MicroRNA-218 (miR-218) is a tumor-suppressive miRNA in cancers. MiR-218 inhibits cell proliferation of glioma cells , osteogenic differentiation in synovial mesenchymal stem cells , and tumor angiogenesis in prostate cancer . MiR-218 is downregulated in renal cell carcinoma (RCC) tissue, and cell proliferation is suppressed by miR-218 overexpression in RCC cells . The bioinformatics analysis shows that IDO1 has binding targets of miR-218; we decided to study the mechanism of miR-218 functioning on IDO1 in the tumor immune escape of cervical cancer.
Therefore, we aimed to measure the effects of miR-218 on cervical cancer. This study will help to understand the principle of tumor development, to improve the immune status of the body, to reverse the tumor escape, and to design new treatment strategies.
Patients and tissues
Clinical pathological features of patients with cervical cancer
Cell culture and transfection
Human cervical epithelial cells (HcerEpic cells) and cervical cancer cells (HeLa, SiHa, C-33 and Caski cells) were purchased from Shanghai Institute of Cell Biology and cultured with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA), which contained 10% fetal bovine serum (FBS; Gibco, USA), 1% penicillin, and streptomycin (Invitrogen, USA)with 5% CO2 at 37 °C. The cells cultured to logarithm phase were used in following experiments. The expression levels of miR-218 and IDO1 were first detected in above cell lines. The MiR-218 mimics (Mimics group) and NC control sequence (NC group) were synthesized by GenePharm (Shanghai, China) and then respectively transfected to HeLa cervical cancer cells using lipofectamine 2000 (Invitrogen, USA) as a transfection reagent. Cells with non-treatment were treated as control (Cntl group). Next, the expression levels of miR-218 and IDO1 were detected in Cntl, NC, and Mimics groups.
Bioinformatics and dual-luciferase reporter assays
The potential target sequences of miR-218 in 3′-UTR fragment of IDO1 were predicted with reference to TargetScan website (http://www.targetscan.org/vert_72/). Next, a direct combination of miR-218 and IDO1 was verified by dual-luciferase reporter assay. Using the GeneTailor Site-Directed Mutagenesis System (Invitrogen, USA), the binding sequence of miR-218 on the 3′-UTR fragment of IDO1 was intentionally mutated. The IDO1-3′-UTR sequence or mutated IDO1-3′-UTR sequence (IDO1-3′-UTR mut) was then ligated to pmirGLO firefly and rinilla dual-luciferase reporter vector (Promega, USA). IDO1-3′-UTR or IDO1-3′-UTR mut recombinant luciferase reporter plasmid was co-transfected with miR-218 mimics. Finally, the luciferase activities were measured using the Dual-Glo™ Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s protocols.
Cell counting kit-8 (CCK-8) assay
The effect of miR-218 mimics transfection on cell viability of HeLa cells was determined by CCK-8 assay (Beyotime, China). Cells in the Cntl, NC, and Mimics groups were respectively seeded into 96-well plates at a density of 5 × 103 cells/well, with each experiments being repeated five times. After being incubated at 37 °C for 24 h, 20 μL CCK-8 reagent was added into each well for another 1 h of incubation at 37 °C. Next, optical density (OD) values were read at 450 nm using a microplate reader (Thermo, USA).
Annexin-V/PI (propidium) assay
The effect of miR-218 mimics transfection on apoptosis of HeLa cells was determined by Annexin-V/PI assay (Roche, USA) according to the protocols of the manufacturer. Cells in Cntl, NC, and Mimics groups were respectively seeded in 6-well plates (5 × 104 cells/well) and then put into reaction with 5 μl Annexin-V and 5 μl PI in the dark at 37 °Cfor 5 min. The apoptosis rates were analyzed using a flow cytometer (BD, USA) and Cell Quest software.
Enzyme-linked immunosorbnent assay (ELISA)
The quantities of TGF-β, VEGF, IL-6, and PGE2 in the Cntl, NC, and Mimics groups were determined by ELISA kits (R&D, Minneapolis, USA) according to the manufacturer’s instructions. Samples were added into 96-well plate and incubated at 37 °C for 90 min, and biotinylated antibodies were then added into the plate and incubated for another 60 min. Next, avidin peroxidase complex (ABC) was added and incubated for 30 min prior to TMB (tetramethylbenzidine) coloration. Finally, OD values were read at 450 nm by a microplate reader (Thermo, USA), and the quantities were calculated by standard curve.
The primer sequences applied in the study
The protein levels of IDO1 were detected in cervical cancer tissues and cervical cancer cells (HeLa, SiHa, C-33, and Caski cells). In addition, the protein levels of IDO1, Survivin, Cleaved-caspase-3, Pro-caspase-3, TGF-β, VEGF, IL-6, COX-2, Janus kinase 2 (JAK2), phosphorylated-JAK2 (p-JAK2), signal transducers and activators of transcription 3 (STAT3), and phosphorylated-STAT3 (p-STAT3) were detected in the Cntl, NC, and Mimics groups. The proteins were first extracted by RIPA and quantified by BCA (Pierce, USA). Twelve percent of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to isolate the proteins, which were then transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher, USA). Next, the membranes were blocked in 5% non-fat dry milk for 1 h at 37 °C and incubated first with specific primary antibodies (Abcam, USA) overnight at 4 °C and then with secondary antibodies conjugated with horseradish peroxidase (CST, 7074, 1:5000, USA) at 37 °C for 1 h. GAPDH was used as loading control. The proteins were detected by enhanced chemiluminescense (ECL; Pierce, USA) and analyzed by Bio-Rad ChemiDoc XRS densitometry with Image Lab™ Software version 4.1 (Bio-Rad, USA).
SPSS 18.0 statistical package with mean ± standard deviations (mean ± SD) was used to conduct statistical analysis. One-way analysis of variance (ANOVA) followed with Dunnett’s test was used to compare the differences. P < 0.05 was considered as significantly different.
MiR-218 was downregulated and negatively correlated with IDO1 in cervical cancer tissues and cells
Subsequently, we detected the mRNA expression levels of IDO1 and miR-218 in several cervical cancer cells such as HeLa, SiHa, C-33, and Caski cells. RT-qPCR results showed that the mRNA levels of IDO1 significantly increased, while miR-218 remarkably decreased in HeLa, SiHa, C-33, and Caski cervical cells, compared with cervical epithelial HcerEpic cells (P < 0.05, Fig. 1f, g). The protein levels of IDO1 were detected by Western blot, and the results showed similar pattern to that of the mRNA assay (P < 0.05, Fig. 1h, i).Expression changes of IDO1 and miR-218 were the greatest in HeLa cells among others. Therefore, HeLa cell line was used for miR-218 mimics transfection.
MiR-218 directly targets the 3′-UTR fragment of IDO1
Using bioinformatics analysis of TargetScan, we found potential target sequences of miR-218 in the 3′-UTR fragment of IDO1 (Fig. 1j). Three bases in the target sequence were then mutated to obtain mutated 3′-UTR sequence of IDO1. Luciferase activity assay was used to observe the effect of miR-218 on the firefly luciferase activity of pmirGLO-UTR. The results showed that the co-transfection of miR-218 and IDO1-3′-UTR resulted in a significant downregulation of the luciferase activity of the reporter vector, and a statistically significant difference was identified compared to Cntl+IDO1-3′-UTR group (P < 0.01, Fig. 1k). However, no significant change in luciferase activity was found in the miR-218+IDO1-3′-UTR mut co-transfection group. The interaction between miR-218 and IDO1-3′-UTR affected the expression level of the reporter gene, suggesting that miR-218 might act on the 3′-UTR region of the IDO1 gene, regulating the expression of IDO1.
MiR-218 overexpression inhibited cell viability and promoted cell apoptosis of cervical cancer cells
MiR-218 overexpression inhibited cell viability and promoted cell apoptosis of cervical cancer cells by regulating apoptosis-related factors
MiR-218 overexpression inhibited cell viability and promoted cell apoptosis of cervical cancer cells by regulating immune escape-related factors
MiR-218 overexpression inhibited cell viability and promoted cell apoptosis of cervical cancer cells via JAK2/STAT3 pathway
In tumor immune escape, IDO1 is a critically negative immunoregulatory factor, which induces the development, metastasis, and recurrence of tumors . In this study, we adopted bioinformatics method and dual-luciferase reporter assay to confirm the direct targeting of miR-218 on the 3′-UTR of IDO1. Thus, miR-218 might have critical regulatory functions on the molecular mechanism of IDO1 in cervical cancer. Thirty-seven cervical cancer tissues were collected and detected, and we found that miR-218 was upregulated, while IDO1 was downregulated and that the two were negatively correlated with each other. In addition, high-level IDO1 and low-level miR-218 were correlated to advanced FIGO stages and low differentiated histological features of cervical cancers, as well as low survival rates in 2 years. Therefore, we studied the expression of miR-218 and IDO1 in some commonly used cervical cancer cells (HeLa, SiHa, C-33, and Caski cells), and the results showed the miR-218 was inhibited and the IDO1 levels were promoted. By transfecting miR-218 mimics into HeLa cervical cancer cells, we observed that the overexpression of miR-218 significantly suppressed the expression of IDO1, inhibited cell viability, and promoted apoptosis of cervical cancer cells.
Subsequently, variations of critical effectors in apoptosis caused by miR-218 overexpression were measured. Caspase-3, the direct execution factor to initiate apoptosis, is a cysteine protease protein specifically cleaving peptide bonds after aspartic acid residues. Caspase-3 usually acts as pro-caspase-3 in normal conditions; however, it will be phosphorylated and activated when apoptosis occurs. Survivin is a new member of the apoptotic inhibitor protein family, and it is also a tumor-specific apoptosis inhibitor that is expressed only in tumors and embryonic tissues. Survivin directly acts on Caspase by mainly inhibiting the activity of Caspase-3. In our study, miR-218 overexpression was observed to inhibit the expression of Survivin and promoted the activity of Caspase-3.
Researchers found that the body’s immune suppression was associated with a variety of cytokines . TGF-β inhibits the immune system so as to protect tumor cells from being killed by immune cells . The increase of VEGF levels in tumors reduces the immune function and promotes the formation of tumor interstitial blood vessels, allowing the tumors to grow rapidly [35, 36]. IL-6 stimulates the proliferation, differentiation, and function of cells involved in the immune response . PGE2 is an important cell growth and regulation factor that can dilate blood vessels and can produce both immunosuppressive and anti-inflammatory effects. Under normal physiological conditions [38, 39], COX-2 is not expressed. However, in pathological conditions such as inflammation and tumors, COX-2 expression will be increased after being induced by pro-inflammatory mediators, for example, inflammatory stimulators, injuries, and carcinogens, participating in various pathological and physiological processes [40–42]. During our research, miR-218 overexpression inhibited the expression and function of these inflammatory factors TGF-β, VEGF, IL-6, PGE2, and COX-2 in cervical cancer cells.
JAK2/STAT3 signaling pathway and immune escape signaling pathway are important pathways of inflammatory response . Many cytokines transfer from the outside to the nucleus via JAK2/STAT3 pathway and mediate the response of cells under the corresponding conditions . STAT3 can mediate the onset of inflammatory reactions via IL-6/JAK pathway . In addition, STATs can also modulate inflammatory/immune responses by regulating COX-2 and IDO, becoming intermediate bridges linking tumors and inflammatory responses . In our study, the activation of JAK2 and STAT3 were significantly inhibited by miR-218 overexpression.
. The expression of miR-218 was negatively correlated with IDO1 in cervical cancer. IDO1 is a direct target of miR-218. Overexpression of miR-218 exerted anti-tumor effects on cervical cancer through promoting apoptosis and inhibiting the expression of inflammatory factors. The inhibition of JAK2/STAT3 signaling pathway contributed to the anti-tumor effect produced by miR-218. This study will provide effective solutions for biological treatment of cervical cancer and other tumors.
Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.
LZ and HT provided substantial contributions to the conception and design. YL and DT contributed to the data acquisition, data analysis, and its interpretation. YL is responsible for drafting the article or critically revising it for important intellectual content. All authors gave their final approval of the version to be published. DT agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Consent for publication
The authors declare that they have no competing interests.
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