2023 Volume 46 Issue 12 Pages 1797-1804
Gestational diabetes mellitus (GDM) is an important cause of the increase in incidence rate and mortality of pregnant women and perinatal infants. This study aimed to analyze the role of fentanyl, a μ-opioid agonist, in the GDM progression. The high glucose (HG) treatment HTR8/SVneo cells was used as a GDM model in vitro. The cell viability was assessed with cell counting kit-8 assay. The apoptosis rate was analyzed with flow cytometry and the transwell assay was conducted to test the cell migration and invasion. RT-quantitative PCR (qPCR) assay was performed to determine the relative expressions of related genes. The N6-Methyladenosine (m6A) levels were analyzed with MeRIP analysis. The tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and IL-10 levels of the cells were analyzed with commercial kits. The results showed that fentanyl increased the cell viability, migration and invasion, and IL-10 levels, and declined the apoptosis rate, TNF-α and IL-1β levels of the HG stimulated HTR8/SVneo cells. The chemokine ligand 5 (CCL5) was over expressed in GDM tissues and HG stimulated HTR8/SVneo cells, which was depleted after fentanyl treatment. Over expressed CCL5 neutralized the fentanyl roles in the HG stimulated HTR8/SVneo cells. The methyltransferase-like protein 14 (METTL14) levels was decreased in HG stimulated HTR8/SVneo cells, which was up-regulated after fentanyl treatment. Additionally, METTL14 silenced prominently decreased the m6A and mRNA levels, along with the mRNA stability of CCL5. In conclusion, fentanyl promoted the growth and inhibited the apoptosis of the HG stimulated HTR8/SVneo cells through regulating the METTL14 mediated CCL5 levels.
Gestational diabetes mellitus (GDM), as a common complication of pregnancy, is an important cause of the increase in incidence rate and mortality of pregnant women and perinatal infants, and seriously threatens maternal and infant health.1) In recent years, with the increase of average age of pregnancy and obesity rate, the incidence of GDM has gradually increased.2) The International Diabetes Federation (IDF) survey showed that in 2013, more than 1 million GDM patients were in China and seriously endangered maternal and infant health.3) With the continuous in-depth clinical research on GDM, it was found that placental dysfunction plays an important role in the occurrence of GDM. In the whole process of pregnancy, the proliferation and apoptosis of placental trophoblast will not only affect the physiological function of placenta, but also affect the maternal and infant safety and pregnancy outcome in the whole process of pregnancy to a certain extent.4,5) The development of extravillous trophoblastic (EVT) cells was crucial for this process. EVT is an important component of embryo, which invade the maternal decidua and endometrium to remodel spiral arteries.4) If the function of EVT cell is either excessive or deficient, it can result in several complications to pregnancy, including GDM.6,7) Therefore, regulating the biological behavior of placental trophoblast may be a potential method for the treatment of GDM.
It is reported that 160 kinds of RNA modifications have been found, including mRNA, tRNA, ribosomal RNA (rRNA) and non coding RNA. Among them, N6-Methyladenosine (m6A) is the most common internal chemical modification in mammalian cells.8) M6A in transcription can change the secondary structure of RNA to promote the binding of regulatory proteins to affect its shearing, translation and cytoplasmic degradation, thus determining its translation potential.9) In addition, m6A and related enzymes regulate gene expression and are widely involved in a variety of biological processes such as cell renewal, differentiation, immunity, proliferation and metabolism. Its dynamic changes affect cell growth, development and metabolism.10) At this stage, the research on m6A mainly focuses on tumor, and there is less research on metabolic diseases. Wang et al. indicated that the decrease of m6A of placental mRNAs was closely related in GDM progression in Chinese women, which implied that abnormal m6A levels might be the key in GDM.11)
Fentanyl is a μ-opioid agonist. As a strong narcotic analgesic, fentanyl is widely used in surgical procedures.12) Additionally, fentanyl is one of the best pain reliever for breakthrough cancer pain in advanced cancer patients.13) In clinical treatment, the dosage of fentanyl used for anesthesia or postoperative analgesia is usually 0.7 to 1.5 µg/kg. However, recent study demonstrated that low dose of fentanyl (5–500 ng/mL) plays a regulatory role in cell growth and development. Accumulating researches have demonstrated that fentanyl inhibits malignant behavior of cancer cells as well as cancer development, such as cell growth, cycle, metastasis, and chemosensitivity.14,15) In short, the potential therapeutic effects of fentanyl in cancer treatment has been demonstrated. However, Therefore, we speculate whether fentanyl can regulate the biological behavior of placental trophoblast cells, thereby affecting the progression of GDM. Here, our study aimed to investigate the effects of fentanyl on the growth and development of the human placenta trophoblast cells HTR8/SVneo.
Placenta tissues samples from the GDM patients and healthy pregnant women were obtained from Shanxi Provincial Maternity and Child Health Hospital. Pregnant women were diagnosed as GDM by oral glucose tolerance test (OGTT) at 24–28 weeks of gestation: fasting ≥5.1 mmol/L; 1 h blood glucose ≥10.0 mmol/L; 2 h blood glucose ≥8.5 mmol/L. After the delivery of pregnant women, the tissue at the placenta’s maternal lobule was cut Immediately, washed with phosphate buffered saline (PBS) washing solution, and stored in an −80 °C refrigerator for further experiments.
Cell Culture and High Glucose (HG) TreatmentHuman trophoblast cell line HTR-8/SVneo was gained from ATCC (Manassas, VA, U.S.A.). The HTR-8/SVneo cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with addition of 10% fetal bovine serum in a moist environment (5% CO2 at 37 °C) for 24 h. Then the cells were divided into 2 groups, the negative control group (NC, treated with 5 mM of glucose) and HG group (treated with 25 mM of glucose). Additionally, the cells were stimulated with (6.5, 12.5, 25, 50, 100, 200 ng/mL) fentanyl for 48 h.
Cell TransfectionThe chemokine ligand 5 over-expressed vector (CCL5), small interfering RNA methyltransferase-like protein 14 (si-METTL14), METTL14 over-expressed vector (oe-METTL14) and the negative controls (empty vector and si-NC) were provided by GenePharma (Shanghai, China) and transfected into the cells with Lipofectamine 2000 following the manufacturer’s instructions. The sequence of si-METTL14 1# is as follows: GCAGCACCUCGA UCAUUUATT and the sequence of si-METTL14 2# is as follows: GGAUGAAGGAGAGACAGAUTT. The sequence of si-NC is as follows: UUCUCCGAACG UGUCACGUTT.
Cell Viability DeterminationHTR-8/SVneo cells were plated into a 96-well plate (2 × 104 cells/well). After 48 incubation, HTR-8/SVneo cells were treated with cell counting kit-8 (CCK-8) solution (Sigma-Aldrich, St. Louis, MO, U.S.A.) for 4 h. Next, the plate was supplemented with 150 µL dimethyl sulphoxide after medium removal and the absorbance was analyzed at 450 nm using a microscope.
Ethical ApprovalThis study protocol was approved by the Ethics Committee of Shanxi Provincial Maternity and Child Health Hospital (No. IRB-KY-2020-011).
Flow Cytometry AnalysisThe apoptosis rate of the cells was examined using an Annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit (Procell, Wuhan, China). In short, cells were collected and resuspended The Annexin V-FITC as well as propidium iodide (PI) were utilized to stained the cells in the darkness. Immediately, the apoptotic cells were obversed and quantified with flow cytometry (FACS Calibur, BD Biosciences, Franklin Lakes, NJ, U.S.A.) following the manufacturer’s instructions.
Transwell AnalysisThe migrated and invaded cells were assessed using transwell assays utilizing transwell plates (Corning Costar, Corning, NY, U.S.A.). After treated with trypsin, the cells were resuspended with 200 µL serum-free medium and transferred into the upper chamber. Meanwhile, the lower chamber was added with 500 µL complete medium supplemented with 10% fetal bovine serum (FBS). After 25 h incubation, the cells were discarded with the cotton swab. For invaded cells determination, the transwell plates were pretreated with Matrigel chambers. Finally, the cells were fixed with methanol, dyed by crystal violate solution and counted under a microscopy in five random fields to analyzed the cell migration and invasion.
Real-Time Quantitative PCR (RT-qPCR) AnalysisAfter TRIzol reagent (Beyotime, Nantong, China) treatment, the RNAs were obtained. Afterwards, a cDNA Synthesis Kit (TransGen, Beijing, China) was purchased and performed for reverse transcription. After obtaining cDNA, RT-qPCR was carried out on an Applied Biosystems 7500 real-time PCR system utilizing the SYBR Green Kit (TransGen). Finally, the 2−ΔΔCt method was exploited for the relative expression calculation with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference.
Determination of Tumor Necrosis Factor-α (TNF-α), Interleukin 1β (IL-1β), and IL-10The TNF-α, IL-1β, and IL-10 levels of the cells were analyzed with commercial kits, which were purchased form the Nanjing Jiangcheng Bioengineering Institute (Nanjing, China). All experimental steps were carried out according to the instructions of the kits.
Methylated RNA Immunoprecipitation (MeRIP) AnalysisThe Magna MeRIP m6A kit (IVDSHOW, Hebei, China) was purchased and performed for MeRIP assay. Briefly, the extracted RNAs extracts were sonicated into fragments. The RNAs were mixed with magnetic beads containing m6A antibody or immunoglobulin G (IgG) for 4 h. After that, the immunoprecipitated RNAs were collected and RT-qPCR assay was conducted to analyzed the m6A levels of ERI1 in enriched RNAs.
Statistical AnalysisSPSS 25.0 was selected to perform the statistical analysis. The statistical data was displayed as the mean ± standard deviation (S.D.). Student’s t-test was used for the difference analysis in the two groups and one way ANOVA followed Tukey’s test were used for the difference analysis in multiple groups. p < 0.05 was considered as significant. All experiments were repeated at least three times.
The structural formula of fentanyl was displayed in Fig. 1A. After different concentration (6.5, 12.5, 25, 50, 100, 200 ng/mL) of fentanyl treatment, we found 6.5, 12.5, 25, 50 ng/mL fentanyl treatment exhibited no influence on the cell viability of the HTR-8/SVneo cells, while 100, 200 ng/mL fentanyl treatment dramatically depleted the cell viability (Fig. 1B). Hence, we selected 50 ng/mL fentanyl for the next experiments.
A: The structural formula of fentanyl. B: The cell viability of the HTR-8/SVneo cells after different concentration (6.5, 12.5, 25, 50, 100, 200 ng/mL) of fentanyl treatment. ** p < 0.01, *** p < 0.001.
In the HTR-8/SVneo cells, HG stimulation prominently declined the cell viability (Fig. 2A), induced the apoptosis (Figs. 2B, C), and depleted the migrated (Figs. 2D, E) and invaded (Figs. 2F, G) cells. Meanwhile, Fentanyl treatment prominently enhanced the cell viability as well as the migrated and invaded cells, and declined the apoptosis rated in the HG-stimulated HTR-8/SVneo cells. Additionally, the TNF-α and IL-1β levels were prominently enhanced and IL-10 levels were depleted after HG stimulation in the HTR-8/SVneo cells, while Fentanyl treatment reversed the HG effects on TNF-α, IL-1β, and IL-10 levels (Fig. 2H).
The HG stimulated HTR-8/SVneo cells were treated with 50 ng/mL of fentanyl. A: Cell viability was assessed by CCK-8 assay. B, C: Annexin V-FITC apoptosis detection kit was performed for cell apoptosis determination. D–G: The migrated and invaded cells were analyzed by transwell assay. H: The TNF-α, IL-1β and IL-10 levels of the cells were analyzed with commercial kits. ** p < 0.01, *** p < 0.001. ##p < 0.01, ###p < 0.001.
Subsequently, GSE154414 dataset was obtained in the GEO database for the selection of differently expressed genes in GDM, which displayed as the volcano plot (Fig. 3A). The up-regulated genes, CCL5, RC3H1 and CGB3, was selected to perform the RT-qPCR in the HG stimulated HTR-8/SVneo cells. We found CCL5 and RC3H1 was prominently up-regulated and CCL5 levels were the highest (Fig. 3B). Additionally, the CCL5 was prominently up-regulate in the GDM placenta tissues (Fig. 3C) as well as HG stimulated HTR-8/SVneo cells (Fig. 3D). Fentanyl treatment prominently decreased it in the cells.
A: The volcano plot of the differently expressed genes in GDM. B: The ERI1, GUCY1B3 and DCD levels in the HG stimulated HTR-8/SVneo cells were assessed with RT-qPCR. The CCL5 levels in the GDM placenta tissues (C) as well as HG and fentanyl treated HTR-8/SVneo cells (D) were assessed with RT-qPCR. ** p < 0.01, *** p < 0.001, ###p < 0.001.
Next, after CCL5 over-expressed vector transfection, the CCL5 levels were prominently enhanced at both mRNA (Fig. 4A) and protein (Fig. 4B) levels. Then, we found CCL5-overexpressed prominently depleted the cell viability (Fig. 4C), enhanced the apoptosis rate (Figs. 4D, E), declined the migrated (Figs. 4F, G) and invaded cells (Figs. 4H, I), and elevated the TNF-α and IL-1β levels and depleted the IL-10 (Fig. 4J) in the HG stimulated HTR-8/SVneo cells treated with fentanyl.
CCL5 mRNA (A) and protein (B) levels were assessed after CCL5 transfection. The HG stimulated HTR-8/SVneo cells were treated with 50 ng/mL of fentanyl and CCL5 over-expressed vector. C: Cell viability was assessed by CCK-8 assay. D-E: Annexin V-FITC apoptosis detection kit was performed for cell apoptosis determination. F-I: The migrated and invaded cells were analyzed by transwell assay. J: The TNF-α, IL-1β and IL-10 levels of the cells were analyzed with commercial kits. *** p < 0.001, ##p < 0.01, &&p < 0.01, ###p < 0.001.
As displayed in Fig. 5A, the m6A content was prominently depleted after HG stimulation, while fentanyl treatment prominently elevated it. Then we found that in the HTR-8/SVneo cells, METLL14 mRNA levels was prominently depleted after HG stimulation, which was elevated after fentanyl treatment (Fig. 5B). Meanwhile, the m6A levels of CCL5 was prominently depleted after HG stimulation and elevated after fentanyl treatment (Fig. 5C). The METTL14 was prominently depleted after si-METTL14 transfection, and enhanced after METTL14 overexpressed vector transfection (Figs. 5D, E). Besides, METTL14-silenced aggravated the effects of HG stimulation on the m6A levels of CCL5, while METTL14-overexpressed reversed it (Fig. 5F). Next, we found that METTL14-silenced neutralized the fentanyl effects on the m6A (Fig. 5G) and mRNA (Fig. 5H) levels of CCL5 in the HG stimulated HTR-8/SVneo cells. Finally, we found the stability of CCL5 was enhanced after HG stimulation, while fentanyl treatment depleted it. What is more, METTL14-silenced neutralized the fentanyl roles (Fig. 5I).
A: The m6A levels in the HG stimulated HTR-8/SVneo cells were detected after fentanyl treatment. B: The mRNA levels of m6A related enzymes were analyzed using RT-qPCR after fentanyl treatment. C: The m6A levels of CCL5 in the HG stimulated HTR-8/SVneo cells were detected after fentanyl treatment. The validation of transfection efficiency of si-METTL14 and METTL14 overexpressed plasmid was detected by RT-qPCR (D) and Western blot (E) assays. F: The m6A levels of CCL5 were analyzed after si-METTL14 and METTL14 transfection. The m6A (G), mRNA levels (H) and stability (I) of CCL5 in the HG stimulated HTR-8/SVneo cells after fentanyl and si-METTL14 treatment. *** p < 0.001. ##p < 0.01, ###p < 0.001. &&p < 0.01.
Here, we found that fentanyl effectively promoted the cell viability, migration and invasion, and depleted the apoptosis in the HG stimulated HTR-8/SVneo cells. Mechanistically, fentanyl declined the CCL5 levels via modulating the METTL14-meditated m6A levels in the HG stimulated HTR-8/SVneo cells.
Studies have demonstrated that anesthetics can promote the growth of tumor cells by reducing the body’s cellular immune response and inhibiting the body’s immune surveillance and immune attack on tumors.16) However, some scholars believed that anesthetics can induce the apoptosis of tumor cells.17) Hence, the specific mechanism of anesthetics on the cell growth needs to be further explored. Fentanyl, an anesthetic commonly used in surgery, has been confirmed to participate in the modulation of the occurrence and development of tumor.18,19) In addition to its role in cancer, fentanyl also been proved to be involved in other diseases. Xu et al. demonstrated that fentanyl relieved the ischemia-reperfusion induced cardiomyocyte apoptosis through modulating the Bcl-2 and Bax levels.20) Wang and Chen found that fentanyl effectively relieved severe acute pancreatitis-induced injury of pancreas and heart injuries through modulating the nuclear factor-κB signaling.21) However, as far as we know, the role of fentanyl in the GDM progression remains unclear. In this study, we initially found that it can alleviate HG induced enhancement of apoptosis and reduction of cell growth, migration and invasion in the HTR-8/SVneo cells. These findings indicated that fentanyl might has therapeutic effect on GDM.
CCL5 is a member of the C-C chemokine family and is mainly expressed in CD8 + T cells.22) CCL5 has the ability to recruit, activate and stimulate T cells, thereby mediating innate and adaptive immune responses.23) Previous researches have demonstrated that CCL5 play an important role in the occurrence, development and complications of autoimmune diseases such as rheumatoid arthritis,24) diabetes,25) autoimmune thyroid diseases,26) suggesting that CCL5 and its major receptors are closely related to the occurrence and development of autoimmune diseases. Additionally, CCL5 was also proved to participate in the cancer progression, such as prostate cancer,27) breast cancer,28) Gastric Cancer29) and so on. However, the role of CCL5 in GDM remains unclear. This study found that CCL5 was over expressed in GDM. Fentanyl treatment decreased the CCL5 levels and CCL5 over expressed neutralized the fentanyl effects on the cell viability, migration and inflammation of the HG stimulated HTR-8/SVneo cells. These results indicated that CCL5 may be the target of fentanyl in GDM progression.
Subsequently, we explored the m6A methylation levels in the HG stimulated HTR-8/SVneo cells. And found fentanyl prominently decreased the m6A methylation levels. Through the analysis of mRNA levels of m6A related enzymes, METTL14 was confirmed to be regulated by fentanyl. M6A methylation is the most common post transcriptional modification in eukaryotes, which widely exists in many organs such as liver, kidney and brain.30) METTL14 is one of the important components of M6 methyltransferase complex, and its methylation transfer ability in vitro is higher than METTL3.31) Many reports have demonstrated that METTL14 participate in the diseases development through regulating the m6A levels of related genes. For example, Zhang et al. confirmed METTL14 enhanced the m6A modification of miR-19a, thus inducing the growth of atherosclerotic vascular endothelial cells in atherosclerosis.32) Yao et al. found that METTL14 inhibited the stomach adenocarcinoma progression via increasing the mRNA m6A variation of phosphatase and tensin homologue.33) As far as we know, the METTL14 role in GDM has not been reported yet. This study found that HG treatment decreased the m6A content and METTL14 levels in the HTR-8/SVneo cells, while fentanyl treatment enhanced them. This indicated that fentanyl regulated the M6A methylation through targeting METTL14. Besides, we further confirmed that METTL14 over expressed enhanced the m6A levels, mRNA levels and stability of CCL5 in the HG stimulated HTR-8/SVneo cells, implying that fentanyl regulated the CCL5 levels in GDM via modulating the METTL14 mediated m6A methylation modification.
To sum up, this study demonstrated that fentanyl alleviated the GDM progression through regulating the METTL14 mediated CCL5 levels. Additionally, by exploring specific mechanisms of fentanyl in HG stimulated HTR-8/SVneo cells, our findings may pave the way for investigating new therapeutic strategies against GDM.
The authors declare no conflict of interest.
