The long noncoding RNA H19 is overexpressed in many cancers and acts as an oncogene. Here, we explored the role of H19 in breast cancer cells, including the effect of H19 on proliferation, migration, ...and invasion of breast cancer cells. We also investigated the relation of H19 to microRNA miR‐93‐5p and signal transducers and activators of transcription 3 (STAT3), the target gene of miR‐93‐5p. Ectopic expression of H19 in MCF‐7 cells and knockdown of H19 in MDA‐MB‐231 cells showed that overexpression of H19 promoted proliferation, migration, and invasion, whereas knockdown of H19 reduced proliferation, migration, and invasion in vitro. Dual‐luciferase reporter assays and RNA‐binding protein immunoprecipitation assays proved that H19 was a target of miR‐93‐5p. In addition, H19 antagonized the downregulation of miR‐93‐5p on its target STAT3 and antagonized miR‐93‐5p‐mediated cell proliferation. Our study revealed a new network in the expression of STAT3 involving H19 and miR‐93‐5p, which may contribute to a better understanding of breast cancer pathogenesis and provide new insights into the treatment of this disease.
H19 antagonized the downregulation of miR‐93‐5p on its target signal transducers and activators of transcription 3 (STAT3) and inhibited miR‐93‐5p‐induced cell proliferation. Our study revealed a new network in the expression of STAT3 involving H19 and miR‐93‐5p, which may contribute to a better understanding of breast cancer pathogenesis and provide new insights into the treatment of this disease.
Tumor cells metabolize more glucose to lactate in aerobic or hypoxic conditions than normal cells. Pyruvate kinase isoenzyme type M2 (PKM2) is crucial for tumor cell aerobic glycolysis. We ...established a role for let‐7a‐5p/Stat3/hnRNP‐A1/PKM2 signaling in breast cancer cell glucose metabolism. PKM2 depletion via small interfering RNA (siRNA) inhibits cell proliferation and aerobic glycolysis in breast cancer cells. Signal transducer and activator of transcription 3 (Stat3) promotes upregulation of heterogeneous nuclear ribonucleoprotein (hnRNP)‐A1 expression, hnRNP‐A1 binding to pyruvate kinase isoenzyme (PKM) pre messenger RNA, and the subsequent formation of PKM2. This pathway is downregulated by the microRNA let‐7a‐5p, which functionally targets Stat3, whereas hnRNP‐A1 blocks the biogenesis of let‐7a‐5p to counteract its ability to downregulate the Stat3/hnRNP‐A1/PKM2 signaling pathway. The downregulation of Stat3/hnRNP‐A1/PKM2 by let‐7a‐5p is verified using a breast cancer. These results suggest that let‐7a‐5p, Stat3, and hnRNP‐A1 form a feedback loop, thereby regulating PKM2 expression to modulate glucose metabolism of breast cancer cells. These findings elucidate a new pathway mediating aerobic glycolysis in breast cancers and provide an attractive potential target for breast cancer therapeutic intervention.
In this study, we explored the role of let‐7a‐5p and pyruvate kinase isoenzyme type M2 (PKM2) in the aerobic glycolysis of breast cancer cells. We found that let‐7a‐5p, signal transducer and activator of transcription 3 (Stat3), and heterogeneous nuclear ribonucleoprotein (hnRNP)‐A1 can form a feedback loop to regulate PKM2 expression, thereby regulating the glucose metabolism and growth of breast cancer cells. Thus, the let‐7a‐5p/Stat3/hnRNP‐A1/PKM2 signaling pathway may serve as a potential target for breast cancer treatment.
Although many methods and new therapeutic drugs have been developed, the overall survival rate and long‐term survival rate of patients with gastric cancer (GC) are still not satisfactory. In this ...study, we investigated the effects of microRNA miR‐133a‐3p and transcription factor FOXP3 on proliferation and autophagy of GC cells and their interactions. Our results showed that knockdown of FOXP3 increased the proliferation and autophagy of GC cells. The relationship between FOXP3 and autophagy has not been reported previously. In addition, FOXP3 could directly bind the promoter region of TP53 and inhibit its expression. miR‐133a‐3p increased the proliferation and autophagy via decreasing the protein level of FOXP3 by targeting its 3′‐UTR. Our research provides new insights into the development of GC and provides new ideas and theoretical basis for the clinical treatment of GC and the development of new drug targets.
miR‐133a‐3p increased the proliferation and autophagy via decreasing the protein level of FOXP3 by targeting its 3′‐untranslated region. Our research provides new insights into the development of gastric cancer and provides new ideas and theoretical basis for the clinical treatment of gastric cancer and the development of new drug targets.
Epithelial-mesenchymal transition (EMT) plays an important role in breast cancer cell metastasis. Both (megakaryoblastic leukemia)/myocardin-like 1 (MKL-1) and Signal transducer and activator of ...transcription 3 (STAT3) have been implicated in the control of cellular metabolism, survival and growth. Our previous study has shown that cooperativity of MKL-1 and STAT3 promoted breast cancer cell migration. Herein, we demonstrate a requirement for MKL-1 and STAT3 in miRNA-mediated cellular EMT to affect breast cancer cell migration. Here we show that cooperativity of MKL-1 and STAT3 promoted the EMT of MCF-7 cells. Importantly, MKL-1 and STAT3 promoted the expression of Vimentin via its promoter CArG box. Interestingly, miR-93-5p inhibits the EMT of breast cancer cells through suppressing the expression of MKL-1 and STAT3 via targeted their 3’UTR. These results demonstrated a novel pathway through which miR-93-5p regulates MKL-1 and STAT3 to affect EMT controlling breast cancer cell migration.
•Cooperativity of MKL-1 and STAT3 promoted the EMT of MCF-7 cells.•Cooperativity of MKL-1 and STAT3 promoted the expression of Vimentin via its promoter CArG box.•MiR-93-5p inhibits the EMT of MCF-7 cells through suppressing MKL-1 and STAT3 via targeted their 3’UTR.
High expression of estrogen receptor α (ERα) is associated with a poor prognosis that correlates closely with cellular proliferation in breast cancer. However, the exact molecular mechanism by which ...ERα controls breast cancer cell proliferation is not clear. Here we report that ERα regulates the cell cycle by suppressing p53/p21 and up‐regulating proliferating cell nuclear antigen (PCNA) and proliferation‐related Ki–67 antigen (Ki–67) to promote proliferation of MCF–7 cells. In addition, 17–β–estradiol (E2) enhances ERα‐induced proliferation of MCF–7 cells by stimulating expression of PCNA and Ki–67. Knockdown of ERα significantly affects PCNA/Ki–67 and p53/p21 expression. Furthermore, ERα inhibits the transcriptional activity of p53/p21 in an estrogen response element‐dependent manner. More importantly, we provide new evidence that ERα mediates proliferation of MCF–7 cells by up‐regulating miR–17 to silence the expression of p21. Thus, these data provide new insights into the underlying effect of ERα on breast cancer proliferation.
Model for how estrogen receptor α (ERα) regulates MCF‐7 cell proliferation by p53‐p21‐PCNA‐E2F1 pathway.
miR-5100 participates in the proliferation of lung cancer and pancreatic cancer cells, and participates in the differentiation of osteoblasts. However, the regulation of gastric cancer cells in ...gastric cancer cells remains unclear.
The blood of patients was collected to detect the expression level of miR-5100, and the apoptosis and autophagy levels of cells were detected using western blot, flow cytometry, and confocal. At the same time, in vitro tumor formation experiments in nude mice were used to verify the results of in vitro experiments.
The expression of miR-5100 is related to the prognosis of gastric cancer, miR-5100 can enhance the apoptosis level of gastric cancer cells and inhibit the occurrence of autophagy by targeting CAAP1. MKL1 can inhibit the apoptosis of gastric cancer cells and promote the occurrence of autophagy by targeting CAAP1. At the same time, MKL1 can also increase the expression of miR-5100.
Our research reveals the mechanism by which the MKL1/miR-5100/CAAP1 loop regulates apoptosis and autophagy levels in gastric cancer cells, and miR-5100 is expected to become a new potential target for gastric cancer treatment.
Aldolase A (ALDOA), an important metabolic enzyme in the glycolytic pathway, plays an important role in regulating tumour metabolism. In this study, we investigated the expression pattern of ALDOA in ...hepatocellular carcinoma (HCC) and its biological role in tumour progression. Bioinformatics analysis, western blot (WB) and RT-qPCR were performed to detect the relative expression of ALDOA in HCC tissues and cell lines. A loss-of-function approach was used to investigate the biological function of ALDOA. The role of ALDOA on glycolysis was assessed by WB, glucose and lactate assay kits and a nude mouse xenograft model. Luciferase reporter experiment, chromatin immunoprecipitation and WB were performed to elucidate the underlying molecular. The expression level of ALODA was up-regulated in HCC tissues and cell lines. High ALDOA levels were associated with poorer patient overall survival. Mechanistic studies suggest that ALDOA is a direct target of miR-34a-5p, which can inhibit glycolysis in hepatocellular carcinoma cells by targeting the 3'UTR of ALDOA. PINK1 antisense RNA (PINK1-AS) competitively sponged miR-34a-5p to increase ALDOA expression by antagonizing miR-34a-5p-mediated ALDOA inhibition. MKL-1 acted as a transcription factor to promote the expression of PINK1-AS and ALDOA, thus promoting the deterioration of HCC cells. This study shows that high expression of ALDOA contributes to the development and poor prognosis of hepatocellular carcinoma and will be a target and potential prognostic biomarker for the treatment of HCC.
Chemotherapy is a standard method in traditional treatment for gastric cancer. It is well known that the anti-tumor effects of chemotherapy are achieved mainly through the direct killing of cancer ...cells via apoptosis. However, chemotherapy often fails due to drug resistance. Therefore, non-apoptotic cell death induction by ferroptosis has recently been proposed as a new therapeutic modality to ablate cancer. In this study, we determined the role of MKL-1 in ferroptosis. In vitro and in vivo experiments showed that inhibition of MKL-1 expression significantly enhanced cell sensitivity to ferroptosis-inducing agents. It functions by targeting system Xc- to affect the synthesis of GSH in cells. Therefore, we developed an exosome-based therapeutic approach targeting MKL-1, which provides a novel insight into the treatment of gastric cancer. 1. Introduction Gastric cancer is one of the most common malignant tumors in the world. According to the latest data collected by GLOBOL CANCER, gastric cancer is the fifth most common cancer type and the third most fatal cancer type 1. Although the incidence of gastric cancer has declined globally over the past 50 years due to the prevention and treatment of Helicobacter pylori infection and improved dietary habits, mortality remains high among all cancers 2. For patients with early gastric cancer, surgical resection combined with chemotherapy has been shown to significantly improve the prognosis of some patients with gastric cancer 3. Unfortunately, many patients with gastric cancer are already in the middle-advanced stage or have metastasized when they are diagnosed, and cannot be treated by traditional surgery. Traditional apoptosis-based chemotherapy, targeted drugs or immunotherapy, moderately improve the prognosis of gastric cancer patients 4. The above retrospective analysis found that chemotherapy plays an important role in the treatment of gastric cancer. However, due to the existence of drug resistance mechanism of tumor cells, the efficacy of chemotherapeutic drugs will also be affected by drug resistance during the treatment of gastric cancer patients 5. Most studies have shown that chemotherapeutic drugs kill tumor cells mainly by inducing apoptosis 6. Therefore, ferroptosis is a novel non-apoptotic form of cell death induced by iron-dependent lipid peroxidation. Ferroptosis-based therapy holds promise as a new strategy for antitumor and improving drug resistance of tumor cells. Ferroptosis is a form of programmed death distinct from apoptosis. In the process of apoptosis, Caspase is activated. At the same time, the cell membrane was intact. During ferroptosis, the accumulation of reactive oxygen species (ROS) disrupts cell membrane integrity 7. It is also very different from cellular necrosis. Although the integrity of the cell membrane is lost during ferroptosis, it does not cause cell swelling 8. A growing number of studies have demonstrated that dysregulation of ferroptosis is highly correlated with human tumors 9. Therefore, the development of therapeutic methods for tumor cell ferroptosis has great application prospects. From the current research progress, multiple mechanisms are involved in the regulation of ferroptosis. According to its definition, it is mainly divided into three main pathways, including iron metabolism pathway, lipid peroxide production pathway and lipid peroxide decomposition pathway 10. However, insufficient progress has been made in the use of ferroptosis for the treatment of tumors. MKL-1 was initially identified as part of the RBM15-MKL-1 fusion protein, produced in conjunction with the translocated chromosome 22, which is implicated in acute megakaryocytic leukemia in infants and children 11. Typically, MKL-1 resides in the cytoplasm, where it binds to G-actin. However, upon cellular stimulation, the RhoA pathway is activated, leading to the polymerization of G-actin into filamentous actin, F-actin. This process releases MKL-1 from G-actin, allowing it to be transported into the nucleus. Within the nucleus, MKL-1 forms a complex with SRF, initiating the transcription of genes primarily by recognizing the CArG box within the promoter region of certain genes 12. As research has progressed, it has been discovered that MKL-1 plays a role in the regulation of tumor cell proliferation, migration, and apoptosis 13. Notably, a recent study demonstrated that oxidative stress amplifies the mesenchymal transition of human microvascular endothelial cells HMEC-1 via the MKL-1/RhoA/TGF-β pathway 14. Given that ferroptosis is intimately linked with oxidative stress-related diseases 15, these findings suggest a potential role for MKL-1 in the regulation of ferroptosis. Therefore, in this present study, ferroptosis, a novel form of programmed death based on iron and lipid peroxidation dependence, which we take as an entry point. To clarify the regulation of MKL-1 on the occurrence of ferroptosis in gastric cancer cells and the effect of its expression on ferroptosis inducers. On this basis, the upstream and downstream molecular mechanisms of MKL-1 regulating ferroptosis were further elucidated. And anti-tumor strategies based on ferroptosis therapy targeting MKL-1 were developed for gastric cancer. The results presented a theoretical and experimental basis for the development of novel antitumor drugs. 2. Materials and Methods 2.1 Cell culture The gastric cell lines GES-1, MGC-803, MKN-45, SGC-7901, HGC-27, AGS and embryonic kidney cell line HEK-293T were obtained from the Cell Storage Center of Wuhan University (Wuhan, China). Human umbilical cord-derived adipose-derived mesenchymal stem cells (hUC-MSCs) were derived from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in the following media: GES-1 and HEK-293T in DMEM supplemented with 10% FBS (Gibco, USA). MGC-803, MKN-45, SGC-7901 and HGC-27 in RPMI-1640 with 10% FBS. AGS cells were cultured in DMEM/F12 with 10% FBS, hUC-MSCs were cultured in Human umbilical cord blood mesenchymal stem cell complete medium (Pricella, China), supplemented with 1% penicillin/streptomycin (Meilunbio, China). Cell lines were tested and verified to be mycoplasma negative using MycoAlert™ PLUS (Lonza, Switzerland). All cells were incubated at 37°C in a humidified atmosphere of 5% CO2. 2.2 Clinical pathological tissue collection From 2019 to 2021, clinical pathology samples were obtained from Tongji Hospital. According to the Declaration of Helsinki, the collection of clinicopathological samples has been approved by the Ethics Committee of Tongji Hospital. Samples were plunged into liquid nitrogen immediately after surgery. Subsequently, the paraffin embedding of the samples was entrusted to Wuhan Servicebio Technology Co., Ltd. 2.3 Plasmid constructions The plasmid vector pCDH-CMV, pLKO.1-puro, pGL3-Promoter, pRL-CMV, VSVG and GAG-POL were obtained from Addgene (Addgene, USA). The coding sequence of MKL-1 was inserted into the plasmid pCDH-CMV and used to construct the stable overexpression plasmid pCDH-MKL-1 of MKL-1. Plasmid pLKO.1-puro was used to construct MKL-1 knockdown plasmids pLKO.1-sh-MKL-1-1, pLKO.1-sh-MKL-1-2 and pLKO.1-sh-MKL-1 -3. Plasmid pGL3-Promoter was used to construct luciferase reporter plasmids for the SLC3A2 and SLC7A11 promoters. Plasmid pRL-CMV was used as an internal control plasmid for dual-luciferase reporter activity assays. Plasmids VSVG and GAG-POL were used as lentiviral packaging plasmids. The primer sequences and shRNA sequences were entrusted to Shanghai Sangon Bioengineering Co., Ltd. (Sangong, China) to synthesize. 2.4 Lentivirus transduction Overexpression, knockdown plasmids and package plasmids were transfected into HEK-293T cells using the transfection reagent Lipofectamine 2000 (Invitrogen, USA). The virus-containing supernatants were harvested and filtered through the 0.45 μm filter after 48 h. Cells were plated in 6-well plates at 30-40% confluency per well and transfected with collected lentivirus. The transfected cells were selected by puromycin for two weeks. 2.5 Western Blot Cells were harvested and lysed with RIPA lysis buffer (with PMSF added, Meilunbio, China). The tissue samples were ground with a high-speed tissue grinder (Servicebio, China) lysed with RIPA lysis buffer. After lysis, the samples were disrupted with an ultrasonic cell disruptor, and the supernatant was collected by centrifugation for BCA (Meilunbio, China) protein quantification. The PAGE Gel Rapid Preparation Kit (Yeasen, China) was used to prepare SDS-PAGE gels. The extracted proteins were electrophoresed in SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, USA). The Blocking solution (Servicebio, China) was used to block the PVDF membrane. Subsequently, the PVDF membrane was immersed in the primary antibody and incubated overnight at 4°C. The next day, a secondary antibody was added for 2 hours at 37°C. After incubation, the membrane was washed with TBST and placed in ECLMeilunbio® Fector's Hypersensitive ECL Luminescent Solution (Meilunbio, China) for 5-10 s. The ChemiDoc XRS gel imaging system was used to collect images. The antibodies used in the Western Blot assay were: Anti-GAPDH (A19056, ABclonal, China), Anti-MKL-1 (77098, CST, USA), Anti-HRP Goat Anti-Rabbit IgG (H+L) (AS014, ABclonal, China), Anti-γ-GCS CST (30068, CST, USA), Anti-GSS (A14535, ABclonal, China), Anti-SLC3A2 (A3658, ABclonal, China), Anti-SLC7A11 (A15604, ABclonal, China), Anti-CD9 (A19027, ABclonal, China), Anti-CD63 (EPR5702, Abcam, USA), Anti-Calnexin (A4846, ABclonal, China). 2.6 qRT-PCR assay QIAGEN RNeasy Mini Kit (QIAGEN, Germany) was used to extract total RNA. The HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China) was used to reverse-transcribe cDNA. Taq Pro Universal SYBR qPCR Master Mix (Vazyme) was used to perform quantitative real-time RT-PCR analysis, with three replicate wells per sample.
Signal transducer and activator of transcription 3 (STAT3) controls cell survival, growth, migration, and invasion. Here, we observed that STAT3 exerted anti-apoptotic effects in breast cancer cells. ...On the other hand, miR-17-5p induced apoptosis in breast cancer cells, and overexpression of miR-17-5p sensitized MCF-7 cells to paclitaxel-induced apoptosis via STAT3. Overexpression of STAT3 in MCF-7 cells decreased paclitaxel-induced apoptosis, but STAT3 knockout abolished the miR-17-5p-induced increases in apoptosis. Finally, miR-17-5p promoted apoptosis by increasing p53 expression, which was inhibited by STAT3. These results demonstrate a novel pathway via which miR-17-5p inhibits STAT3 and increases p53 expression to promote apoptosis in breast cancer cells.
Cell division cycle-associated 5 (CDCA5) plays a critical role in the progression of various human cancers by regulating cell cycle-related proteins; however, the function of CDCA5 in breast cancer ...(BC) is poorly understood. The aim of the present study was to investigate the expression level of CDCA5 in BC and its effect on BC progression. CDCA5 was found to be highly expressed in patients with BC, as well as in BC cell lines. It was also found that a high CDCA5 expression in BC was significantly associated with a shorter survival rate. In addition, the expression level of CDCA5 was significantly increased in stem cells derived from suspension-cultured BC cells, as compared to adherent-cultured cells. CDCA5 knockdown in MCF7 and SKBR3 cells significantly reduced cell proliferation, migration and clone formation. At the same time, the stemness capacity of BC cells, determined by analyzing cancer stem cell marker expression and mammosphere formation, was also markedly diminished following the knockdown of CDCA5. In addition, in vivo experiments demonstrated that CDCA5 knockdown in MCF7 cells markedly reduced tumor growth. On the whole, the present study demonstrates that CDCA5 may be used as a prognostic biomarker and therapeutic target for BC.
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