Clinical and Experimental Pharmacology and Physiology

M6A‐induced transcription factor IRF5 contributes to the progression of cervical cancer by upregulating PPP6C

AbstractCervical cancer (CC) is a gynaecological malignancy tumour that seriously threatens women's health. Recent evidence has identified that interferon regulatory factor 5 (IRF5), a nucleoplasm shuttling protein, is a pivotal transcription factor regulating the growth and metastasis of various human tumours. This study aimed to investigate the function and molecular basis of IRF5 in CC development. IRF5, protein phosphatase 6 catalytic subunit (PPP6C) and methyltransferase‐like 3 (METTL3) mRNA levels were evaluated by quantitative real‐time (qRT)‐polymerase chain reaction (PCR). IRF5, PPP6C, METTL3, B‐cell lymphoma 2 and Bax protein levels were detected using western blot. Cell proliferation, migration, invasion, angiogenesis and apoptosis were determined by using colony formation, 5‐ethynyl‐2′‐deoxyuridine (EdU), transwell, tube formation assay and flow cytometry assay, respectively. Glucose uptake and lactate production were measured using commercial kits. Xenograft tumour assay in vivo was used to explore the role of IRF5. After JASPAR predication, binding between IRF5 and PPP6C promoter was verified using chromatin immunoprecipitation and dual‐luciferase reporter assays. Moreover, the interaction between METTL3 and IRF5 was verified using methylated RNA immunoprecipitation (MeRIP). IRF5, PPP6C and METTL3 were highly expressed in CC tissues and cells. IRF5 silencing significantly inhibited cell proliferation, migration, invasion, angiogenesis and glycolytic metabolism in CC cells, while induced cell apoptosis. Furthermore, the absence of IRF5 hindered tumour growth in vivo. At the molecular level, IRF5 might bind with PPP6C to positively regulate the expression of PPP6C mRNA. Meanwhile, IRF5 was identified as a downstream target of METTL3‐mediated m6A modification. METTL3‐mediated m6A modification of mRNA might promote CC malignant progression by regulating PPP6C, which might provide a promising therapeutic target for CC treatment.

TPPP3 inhibits the proliferation, invasion and migration of endometrial carcinoma targeted with miR‐1827

AbstractObjective and designDatabase screening indicated that tubulin polymerization‐promoting protein 3 (TPPP3) was involved in pathogenesis of multiple cancer types. miR‐1827 has a potential role in a variety of human cancers. However, the role of TPPP3 and its underlying molecular mechanism in endometrial cancer (EC) has not been investigated. Herein, we aimed to reveal the role of TPPP3/miR‐1827 in EC progression.MethodsTumour tissue and whole blood samples were collected for the detection of TPPP3 expression. TPPP3 shRNAs and pcDNA‐TPPP3 were applied to knockdown or upregulate the TPPP3 expression, and miR‐1827 mimic was used to upregulate miR‐1827 level. CCK‐8 and colony assays were applied to estimate the cell proliferation. Wound healing and Transwell assays were conducted to assess the cell migration and invasion abilities. The dual‐luciferase reporter assay was conducted to validate the putative binding site between TPPP3 and miR‐1827. Expression of TPPP3, miR‐1827 and related proteins in cell lines, tissue and whole blood sample were detected using western blot, RT‐qPCR and immunofluorescence.ResultsTPPP3 was observed markedly elevated in EC patients and cells. TPPP3 knockdown displayed evident suppression in cell proliferation, migration and invasion in vitro and in vivo. Moreover, we identified TPPP3 as a direct and functional target gene of miR‐1827 in EC cells. The miR‐1827 induced regulatory effects on EC cells were partially reversed by TPPP3. Additionally, in vivo study confirmed the findings discovered in vitro.ConclusionTPPP3 exerted oncogenic roles in EC progression by sponging miR‐1827. This finding might provide potential targets for EC therapy.

CircSAMD11 facilitates progression of cervical cancer via regulating miR‐503/SOX4 axis through Wnt/β‐catenin pathway

AbstractCervical cancer (CC) is a common gynaecological malignant tumour with a high mortality rate. Circular RNAs (circRNAs) play a critical role in tumour occurrence and development. This study aimed to investigate the function and molecular basis of hsa_circ_0009189 (circSAMD11) in CC development. RNA levels were determined by qRT‐PCR, and protein expression was measured by western blot. Cell proliferation, migration, invasion and apoptosis were detected by Cell Counting Kit‐8 (CCK‐8), colony formation, Transwell and flow cytometry assays. The relationship between miR‐503 and circSAMD11/SOX4 was validated via dual‐luciferase reporter assay, RIP or RNA pull‐down assay. Xenograft assay was conducted to test tumour growth in vivo. CircSAMD11 and SOX4 levels were elevated, while miR‐503 level was reduced in CC tissues and cells. Knockdown of circSAMD11 suppressed CC cell proliferation, migration and invasion and accelerated apoptosis. CircSAMD11 was localised in cytoplasm and directly targeted miR‐503. Also, circSAMD11 sponged miR‐503 to modulate SOX4 expression. Additionally, circSAMD11 regulated CC progression via absorbing miR‐503 or modulating SOX4. Besides, depletion of circSAMD11 hindered tumorigenesis in vivo. CircSAMD11 contributed to CC progression by regulating miR‐503/SOX4 signalling and activating Wnt/β‐catenin pathway, which provides a promising therapeutic target for cervical cancer.

Tobramycin suppresses HUWE1 degradation to control MCL‐1 stability during tumour development

Abstract HUWE1 is an E3 ubiquitin ligase that is involved in cancer cell proliferation by regulating MCL‐1 stability. The HECT domain has been shown to be required for the ubiquitin ligase activity of HUWE1. To identify efficient drugs that impair the activity of HUWE1, and thus decrease MCL‐1 accumulation, we screened 2000 candidate compounds that might suppress HUWE1 activity. To evaluate these 2000 candidates, the HECT domain of HUWE1, which is the catalytic domain responsible for MCL1 ubiquitination, was selected as a conjugation site, and putative binding candidates were filtrated. Tobramycin emerged as one of the compounds that show efficient binding ability with the HECT domain of HUWE1. The surface plasmon resonance (SPR) results validated the specific binding of Tobramycin with the HECT domain. Subsequent analyses demonstrated its potential to inhibit cancer cell proliferation by binding to the HECT domain of HUWE1 and impeding the HUWE1‐mediated ubiquitination of MCL‐1. Consequently, the accumulation of MCL‐1 inhibited the proliferation of tumour cells, while the apoptosis rates were not significantly altered after Tobramycin treatment. In vitro experiments showed that Tobramycin could inhibit cell proliferation by regulating the G2/M transition in cancer cell models, including A549 and HeLaCaco2 cell lines. Our results indicated that Tobramycin could be a potential new probe to develop targeted therapies for the prevention or treatment of HUWE1‐overexpressing cancers.

MicroRNA‐367‐3p overexpression represses the proliferation and invasion of cervical cancer cells through downregulation of SPAG5‐mediated Wnt/β‐catenin signalling

AbstractMicroRNA‐367‐3p (miR‐367‐3p) has been previously reported as a cancer‐related miRNA that is dysregulated in various cancer types and functions either as an oncogenic or as tumour suppressive miRNA. However, whether miR‐367‐3p is dysregulated in cervical cancer and, further, whether it contributes to the development and progression of the disease remains unknown. Here, our results demonstrated that miR‐367‐3p expression was markedly decreased in both cervical cancer tissues and cell lines compared with corresponding controls. In vitro experiments revealed that miR‐367‐3p overexpression repressed the proliferation and invasion of cervical cancer cells. Notably, sperm‐associated antigen 5 (SPAG5) was identified as a target gene of miR‐367‐3p. Moreover, decreased expression of miR‐367‐3p was correlated with high expression of SPAG5 in cervical cancer tissue specimens. SPAG5 inhibition or miR‐367‐3p overexpression significantly downregulated Wnt/β‐catenin signalling in cervical cancer cells. However, the antitumour effect mediated by miR‐367‐3p overexpression was partially reversed by SPAG5 overexpression. Overall, these findings demonstrate that miR‐367‐3p overexpression restricts the proliferation and invasion of cervical cancer cells through targeting SPAG5 to downregulate Wnt/β‐catenin signalling, suggesting a mechanism for the tumour suppressive function of miR‐367‐3p in cervical cancer. Our study highlights the involvement of miR‐367‐3p/SPAG5/Wnt/β‐catenin signalling axis in regulating the malignant progression of cervical cancer.

LncRNA PTCSC3 inhibits the proliferation, invasion and migration of cervical cancer cells via sponging miR‐574‐5p

AbstractDysregulation of long non‐coding RNA papillary thyroid carcinoma susceptibility candidate 3 (lncRNA PTCSC3) has been found to correlate with various types of cancers. Quantitative RT‐PCR showed a down‐regulation of PTCSC3 in cervical cancer tissues compared with normal cervical tissues. The present study aimed to investigate the role of lncRNA PTCSC3 in cervical cancer and the underlying mechanisms. PTCSC3 was overexpressed in cervical cancer cell lines C‐33A and Hela by transfection with pcDNA3.1‐lncRNA PTCSC3 expressing plasmid. Overexpression of lncRNA PTCSC3 inhibited cell proliferation, induced cell cycle arrest, and suppressed cell invasion and migration using CCK8 assay, flow cytometry, Transwell assay and wound healing examination, respectively. Western blotting analysis showed that PTCSC3 overexpression decreased the expression of cyclinD1, matrix metalloproteinases 9 (MMP9), N‐cadherin and β‐catenin and increased E‐cadherin expression. Further, PTCSC3 negatively regulated miR‐574‐5p expression and dual‐luciferase assay verified the binding activity between miR‐574‐5p and lncRNA PTCSC3. Enforced up‐regulation of miR‐574‐5p abolished the inhibitory effect of lncRNA PTCSC3 on cervical cancer cell proliferation, invasiveness and mobility. Taken together, lncRNA PTCSC3 inhibited cell growth and metastasis via sponging miR‐574‐5p in cervical cancer. Therefore, we demonstrate the tumour‐suppressive function of lncRNA PTCSC3 in cervical cancer and suggest that PTCSC3 is a potential therapeutic target for cervical cancer.

Expressions of m6A RNA methylation regulators and their clinical predictive value in cervical squamous cell carcinoma and endometrial adenocarcinoma

AbstractThe mortality caused by cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) ranks second among female malignant tumour deaths, but their diagnostic and therapeutic targets are still limited. N6‐methyladenosine (m6A) is the most common and extensive modification in mRNA molecules, and its methylation regulators participate in regulating the occurrence and development of many tumours. However, whether m6A RNA methylation regulators can be used as independent prognostic indicators of CESC remains unknown. This study unveiled differential expression of 20 m6A RNA methylation regulators between normal and CESC tumour samples, which RNA sequence data and clinical information were obtained from TCGA database. As a result, five m6A RNA methylation regulators (FTO, HNRNPA2B1, RBM15, IGF2BP1, IGF2BP3) were identified to be significantly linked to CESC tumour status. After Lasso cox regression analysis, six m6A RNA methylation regulators (YTHDC2, YTHDC1, ALKBH5, ZC3H13, RBMX, YTHDF1) were chosen to construct a risk signature. CESC patients were then classified as high‐risk and low‐risk group based on the median risk score. The overall survival (OS) of the CESC patients in high‐risk group was significantly lower than that in low‐risk group, and the area under curve (AUC) is 0.718. Moreover, the risk model can be an independent prognosis factors for CESC patients and can predict OS of CESC patients with different clinical factors. In conclusion, m6A RNA methylation regulators are closely correlated with CESC clinical characteristics and the selected six m6A RNA methylation regulators may be useful for CESC patients personalized treatment.

N6‐methyladenosine methylation on FSCN1 mediated by METTL14/IGF2BP3 contributes to human papillomavirus type 16‐infected cervical squamous cell carcinoma

AbstractHuman papillomavirus (HPV) infection has been reported to be associated with N6‐methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV‐related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV‐positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV‐infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin‐bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3‐mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV‐positive CSCC patients. Finally, HPV16‐positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV‐positive CSCC cells. Their tumour‐suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3‐mediated FSCN1 m6A modification.

The anti‐tumourigenic effect of ellagic acid in SKOV‐3 ovarian cancer cells entails activation of autophagy mediated by inhibiting Akt and activating AMPK

Abstract This study investigated the effect of ellagic acid (EA) on SKOV‐3 cell growth and invasiveness and tested if the underlying mechanism involves modulating autophagy. Cells were treated with EA in the presence or absence of chloroquine (CQ), an autophagy inhibitor, compound C (CC), an AMPK inhibitor, or an insulin‐like growth factor‐1 (IGF‐1), a PI3K/Akt activator. EA, at an IC 50 of 36.6 µmol/L, inhibited cell proliferation, migration, and invasion and induced cell apoptosis in SKOV‐3 cells. These events were prevented by CQ. Also, EA increased levels of Beclin‐1, ATG‐5, LC3I/II, Bax, cleaved caspase‐3/8 and reduced those of p62 and Bcl‐2 in these cancer cells. Mechanistically, EA decreased levels of p‐S6K1 (Thr 389 ) and 4EBP‐1 (Thr 37/46 ), two downstream targets of mTORC1, and p‐Akt (Thr 308 ) but increased levels of AMPK (Thr 172 ) and p‐raptor (Ser 792 ), a natural inhibitor of mTORC1. CC or IGF‐1 alone partially prevented the effect of EA on cell survival, cell invasions, and levels of LDH, Beclin‐1, and cleaved caspase‐3. In conclusion, EA can inhibit SKOV‐3 growth, migration, and invasion by activating cytotoxic autophagy mediated by inhibition of mTORC1 and Akt and activation of AMPK.

Ginsenoside 20(S)‐Rg3 upregulates HIF‐1α‐targeting miR‐519a‐5p to inhibit the Warburg effect in ovarian cancer cells

Abstract The Warburg effect, one of the metabolic hallmarks of cancer, is responsible for rapid energy production through a high rate of aerobic glycolysis. Ginsenoside 20(S)‐Rg3 antagonizes the Warburg effect in ovarian cancer cells by upregulating some microRNAs, including miR‐519a‐5p, that target key enzymes involved in aerobic glycolysis. How 20(S)‐Rg3‐upregulated miR‐519a‐5p influences the Warburg effect in ovarian cancer cells remains poorly defined, however. Here we report that while overexpression of miR‐519a‐5p in ovarian cancer cells inhibited the Warburg effect, inhibition of miR‐519a‐5p negated the suppressive action of 20(S)‐Rg3 against the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and HK2 expression. We identified HIF‐1α as a direct target of miR‐519a‐5p via luciferase reporter assays and demonstrated the counteraction by overexpressed HIF‐1α of 20(S)‐Rg3‐suppressed Warburg effect. Further, 20(S)‐Rg3 decreased DNMT3A‐mediated DNA methylation in the promoter region of its precursor gene, leading to an increase in the level of miR‐519a‐5p. In conclusion, 20(S)‐Rg3 upregulates miR‐519a‐5p via reducing DNMT3A‐mediated DNA methylation to inhibit HIF‐1α‐stimulated Warburg effect in ovarian cancer.

Exendin‐4 exhibits a tumour suppressor effect in SKOVR‐3 and OVACR‐3 ovarian cancer cells lines by the activation of SIRT1 and inhibition of NF‐κB

Abstract This study investigated if EX‐527 has an anti‐tumour effect in SKOV‐3 and OVCAR‐3 ovarian cancer (OC) cell lines and if this effect involves the SIRT1/NF‐κB axis. Cells were cultured in the presence or absence of EX‐527, a selective SIRT‐1 inhibitor. Exendin‐4 significantly induced cell death in both cell lines and inhibited cell migration and invasion. Also, it decreased protein levels of Bcl‐2, MMP‐9, and ICAM‐1 and increased those of Bax, cyclin D1 and cleaved caspase‐3. Mechanistically, Exendin‐4 increased the activity and nuclear accumulation of SIRT1 and decreased nuclear levels of NF‐κB p65; acetylated levels of NF‐κB p65, and cytoplasmic levels of p‐IKKα and p‐IκBα. EX‐527 partially ameliorated the effect of Exendin‐4 on cell death, migration, and invasion, as well as on the expression of Bcl‐2, MMP‐9, Bax, cleaved caspase‐3 and ICAM‐1. In addition, EX‐527 did not affect the levels of nuclear p65 and p‐p65 (Ser536); p‐IκBα (Ser32) and p‐IKKαβ. In conclusion, Exendin‐4 can suppress OC by inhibiting NF‐kB through SIRT1 dependent and independent mechanisms.