Expression Of Pyruvate Kinase M2 mRNA Research Articles

PKM2 promotes myoblast growth and inosine monophosphate-specific deposition in Jingyuan chicken

Inosine monophosphate (IMP) is widely regarded as an important indicator for evaluating the flavour of poultry meat. However, little is known about the molecular mechanisms affecting the specific deposition of IMP. In this study, we functionally verified PKM2 (Pyruvate kinase M2), a candidate gene related to IMP synthesis, in order to reveal the important role of PKM2 in meat flavour and muscle development of Jingyuan chickens. The results showed that the IMP content in breast muscle of Jingyuan chickens was negatively correlated with PKM2 mRNA expression (r = −0.1710), while the IMP content in leg muscle was significantly positively correlated with PKM2 mRNA expression (r = 0.7350) (P < 0.05). During myogenesis, PKM2 promoted the proliferation rate of myoblasts and the expression of proliferation marker genes, inhibited the apoptosis rate and the expression of apoptosis marker genes, and decreased the expression of differentiation marker genes. Up-regulation of PKM2 enhanced the expression of key genes in the purine metabolic pathway and the de novo synthesis pathway of IMP, and suppressed the expression of key genes in the salvage pathway. ELISA assays showed that PKM2 decreased IMP and hypoxanthine (HX) contents, while adenosine triphosphate (ATP) and uric acid (UA) contents were clearly elevated. In summary, these studies revealed that PKM2 regulates myogenesis and specific deposition of IMP, which can be used to improve the quality of Jingyuan chicken meat.

Development of isoselenazolium chlorides as selective pyruvate kinase isoform M2 inhibitors

Alterations in cancer metabolic pathways open up an opportunity for targeted and effective elimination of tumor cells. Pyruvate kinase M2 (PKM2) is predominantly expressed in proliferating cells and plays an essential role in directing glucose metabolism in cancer. Here, we report the design of novel class of selective PKM2 inhibitors as anti-cancer agents and their mechanism of action. Compound 5c being the most active with IC50 = 0.35 ± 0.07 μM, also downregulates PKM2 mRNA expression, modulates mitochondrial functionality, induces oxidative burst and is cytotoxic for various cancer types. Isoselenazolium chlorides have an unusual mechanism of PKM2 inhibition, inducing a functionally deficient tetrameric assembly, while exhibiting a competitive inhibitor character. The discovery of robust PKM2 inhibitors not only offers candidates for anticancer therapy but is also crucial for studying the role of PKM2 in cancer.

The Role of TLR4 Inducing Macrophage Pyroptosis in the Pathogenesis of Severe Aplastic Anemia

Introduction：To investigate the expression level of macrophage pyroptosis in patients with severe aplastic anemia (SAA) and its effect on downstream effector T cells, the study explored the mechanism of Toll like receptor 4 (TLR4) inducing macrophage pyroptosis and maintaining immune homeostasis of SAA through pyruvate kinase M2 (PKM2) at cellular and molecular levels, which provided a theoretical basis for the targeted therapy of macrophages in SAA. Three groups of bone marrow macrophages of new diagnosed SAA patients, remission patients and healthy controls were induced and cultured in vitro. qRT-PCR and Western Blot were used to detect pyroptosis-related indicators IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD expression level; ELISA to detect the concentration of IL-1β and IL-18 in the culture supernatant.Results: 1. The mRNA and protein levels of macrophages IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD in the new diagnosed SAA group were significantly higher than those in healthy controls, and the IL-1β and IL-18 concentration in the macrophage culture supernatant increased significantly. The relative mRNA expression levels of macrophages IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD in the new diagnosed group were significantly negatively correlated with WBC, RBC, Hb and PLT. 2. The relative expression level of TLR4 mRNA in macrophages in the new diagnosed group was significantly higher than that in the remission group and healthy controls, and the protein level of TLR4 was significantly increased; at the same time, the relative expression level of TLR4 mRNA was positively correlated with IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD in macrophages. After knocking down TLR4 or adding TLR4 inhibitors, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD were significantly lower than those in healthy controls, and the concentration of IL-1β and IL-18 in the culture supernatant reduced significantly. 3. The expression rate of PKM2 in macrophages of new diagnosed group by flow cytometry was significantly higher than that in the remission group and heathy controls, and the levels of PKM2 mRNA and protein were significantly increased. At the same time, the relative expression level of PKM2 mRNA was positively correlated with the expression levels of IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD. After knocking down PKM2, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, Caspase-1, GSDMD were significantly lower than those healthy controls, and the concentration of IL-1β and IL-18 in the culture supernatant was also significantly reduced. After knocking down TLR4 or adding TLR4 inhibitor, PKM2 mRNA and protein expression levels in macrophages were significantly lower than those in healthy controls. 4. When CD8+ T cells were co-cultured with macrophages which were knocked down TLR4 or added with inhibitors, the expressions of perforin and granzyme B in CD8+ T cells were significantly reduced. CD8+ T cells were further co-cultured with K562 and the apoptosis level of K562 was reduced.Summary：The level of macrophage pyroptosis in SAA patients increased, and the expression levels of macrophages TLR4 and PKM2 increased and positively correlated with the level of pyroptosis. Inhibiting the expression of TLR4 and PKM2, respectively, reduced the level of macrophage pyroptosis, and at the same time inhibiting TLR4 expression reduced the expression of PKM2. Macrophages that inhibited TLR4 expression reduced the killing function of CD8+ T cells. Therefore, TLR4 may induce pyroptosis of macrophages in SAA patients through PKM2 and enhance the killing function of CD8+ T cells. DisclosuresNo relevant conflicts of interest to declare.

Expression and clinical significance of pyruvate kinase M2 in breast cancer: A protocol for meta-analysis and bioinformatics validation analysis.

Background:Breast cancer is a common malignant tumor in women. In recent years, its incidence is increasing year by year, and its morbidity and mortality rank the first place among female malignant tumors. Some key enzymes and intermediates in glycolysis are closely related to tumor development. Pyruvate kinase M2 (PKM2) is an important rate-limiting enzyme in glycolysis pathway. Meanwhile, it is highly expressed in proliferative cells, especially in tumor cells, and plays an important role in the formation of Warburg effect and tumorigenesis. Previous studies have explored the effects of PKM2 expression on the prognosis and clinical significance of breast cancer patients, while the results are contradictory and uncertain. This study uses controversial data for meta-analysis to accurately evaluate the problem. We collected relevant Oncomine and The Cancer Genome Atlas (TCGA) data to further verify the results. Through bioinformatics analysis, the mechanism and related pathways of PKM2 in breast cancer are explored.Methods:We searched Wanfang, Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure, the Chongqing VIP Chinese Science and Technology Periodical Database, PubMed, Embase, and Web of Science databases from inception to March 2021. The language restrictions are Chinese and English. The published literatures on PKM2 expression and prognosis or clinicopathological characteristics of breast cancer patients were statistically analyzed. Combined hazard ratios (HRs), odds ratios (ORs), and 95% confidence intervals (95% CIs) were used to evaluate the effects of PKM2 on the prognosis and clinicopathological features of breast cancer. Stata 14.0 software was applied for meta-analysis. Oncomine and TCGA databases were used to meta-analyze the differences of PKM2 mRNA expression between breast cancer and normal breast tissues. The expression of PKM2 protein was verified by Human Protein Atlas (HPA) database. The relationship between the gene and the survival of breast cancer patients was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA). The relationship between PKM2 gene and clinicopathological characteristics was analyzed by using LinkedOmics, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis was performed by using Metascape. Protein–protein interaction (PPI) network was constructed by String website.Results:The results of this meta-analysis will be submitted to a peer-reviewed journal for publication.Conclusion:This study provides high-quality medical evidence for the correlation between the expression of PKM2 and the prognosis and clinicopathological features of breast cancer. Through bioinformatics analysis, this study further deepens the understanding of the mechanism and related pathways of PKM2 in breast cancer.Ethics and dissemination:The private information from individuals will not be published. This systematic review also should not damage participants’ rights. Ethical approval is not available. The results may be published in a peer-reviewed journal or disseminated in relevant conferences.OSF REGISTRATION NUMBER:DOI 10.17605/OSF.IO/W52HB.

Pyruvate kinase M2 is a marker of poor prognosis in lung adenocarcinoma but not lung squamous cell carcinoma.

BackgroundTo explore the pyruvate kinase M2 (PKM2) expression profile as a prognostic marker of lung adenocarcinoma (LUAD) as well as lung squamous cell carcinoma (LUSC).MethodsRetrospective bioinformatics analysis of data from the Cancer Genome Atlas-Lung Cancer dataset and the Human Protein Atlas was performed. PKM2 mRNA expression was monitored using the Kaplan-Meier Plotter online database. GraphPad Prism 6.0 and the SPSS 19.0 software package were used for statistical analysis.ResultsPKM2 expression was found to be significantly higher in both LUAD and LUSC than in normal controls. Although increased PKM2 expression in LUAD was correlated with poor overall survival (OS) [hazard ratio (HR): 2.128; 95% CI: 1.754–3.653; P<0.001], recurrence-free survival (RFS) (HR: 1.524; 95% CI: 1.069–2.499; P=0.0237), and progression-free survival (PFS) (HR: 2.18; 95% CI: 1.58–3; P<0.001), no such associations were found in LUSC.ConclusionsPKM2 is a potential prognostic biomarker for LUAD but not for LUSC.

High expression of PKM2 synergizes with PD-L1 in tumor cells and immune cells to predict worse survival in human lung adenocarcinoma.

Background: Immunotherapy targeting PD-1/PD-L1 represents a breakthrough in the treatment of lung cancer. Pyruvate kinase M2 (PKM2) is not only a critical player in glycolysis, but also conducive to tumor progression and immune response. While both have been linked to lung adenocarcinoma (AC), the correlation and clinical significance of PKM2 and PD-L1 expression in human lung AC tissues remains not entirely explored.Methods: Expression of PKM2 and PD-L1 proteins were detected by immunohistochemistry in 74 lung AC cases and the corresponding noncancerous tissues. Simultaneously, multiplex immunofluorescence was used to detect PKM2, PD-L1, CK, CD3, and CD68 in the lung AC tissues. We measured expression patterns and co-localization of these markers, evaluating their association with clinicopathological features and overall survival. Validation of findings was conducted using mRNA expression data from The Cancer Genome Atlas (TCGA) of 515 lung AC cases.Results: High expression of PKM2 in tumor cells was significantly related with lymph node metastasis and TNM stage (p=0.035, p=0.017, respectively). Moreover, PKM2 expression in tumor cells was positively correlated with tumor PD-L1 expression. High expression of PKM2, PD-L1 in tumor cells and immune cells predicted high mortality rate and poorer survival rates, respectively. Additionally, multivariate Cox regression models indicated that high expression of PKM2 in tumor cells was an independent prognostic factor. Based on TCGA genomic data, high PKM2 mRNA expression was significantly associated with poorer survival (p=0.001).Conclusion: High expression of PKM2 synergizes with PD-L1 in tumor cells and immune cells to predict poorer survival rates in patients with lung AC.

Diagnostic value of pyruvate kinase M2 gene for papillary thyroid carcinoma in fine-needle aspiration specimens

Objective To investigate the diagnostic value of pyruvate kinase M2(PKM2)gene expression in papillary thyroid carcinoma(PTC). Methods Quantitative real-time PCR(RT-qPCR)was used to detect the expression of PKM2 mRNA in benign thyroid nodules, PTC, and normal thyroid tissues around nodules of fine-needle aspiration(FNA)specimens. Immunohistochemistry(IHC)was used to detect the expression of PKM2 protein in thyroid tissue after thyroidectomy. The receiver operating characteristic curve was constructed to evaluate the diagnostic value of PKM2 in PTC. Results The expression of PKM2 mRNA was detectable in FNA specimens of thyroid nodules, higher in PTC than those in normal thyroid tissue and benign thyroid nodules(P 0.05). The expression level of PKM2 mRNA in FNA specimens of thyroid nodules was paralleled with the expression level of PKM2 protein in postoperation pathological tissues. The accuracy, sensitivity, specificity of PKM2 gene in the diagnosis of PTC were 62.8%, 46.9%, and 95.7%, respectively. The accuracy and sensitivity of PKM2 combined with BRAFV600E were increased to 87.6% and 83.7%, significantly higher than those of single detection (P<0.05) . Conclusion Detection of PKM2 gene in FNA specimens is highly specific in the diagnosis of PTC, making it a valuable molecular marker for preoperative diagnosis. The combination of PKM2 and BRAFV600E detection shows a higher diagnosis efficiency. Key words: Papillary thyroid carcinoma; Pyruvate kinase M2; Fine-needle aspiration

PKM2 and HIF-1α regulation in prostate cancer cell lines.

Prostate cancer (PCA) is one of the most common cancer types in men, with cancer progression being linked to hypoxia and the induction of hypoxia-inducible factor (HIF).We investigated the expression of pyruvate kinase M2 (PKM2), its regulation by HIF isoforms 1α and 2α, and its role in HIF stabilization. We additionally examined cell survival in the prostate cancer cell lines PC3 and LNCaP under severe hypoxic (0.1% O2) and normoxic (20% O2) conditions. qRT-PCR showed higher up-regulation of PKM2 mRNA expression in LNCaP cells than in PC3 cells, while western blotting showed that PKM2 protein levels were up-regulated only in LNCaP cells. Inhibition of HIF-1α and HIF-2α by small interfering RNA (si-RNA) demonstrated HIF-1α dependent up-regulation of PKM2 at the mRNA and protein levels in LNCaP cells. PKM2 inhibition by si-RNA significantly decreased hypoxia-response element (HRE) activation in a gene reporter assay and down-regulated HIF-1α target vascular endothelial growth factor (VEGF) mRNA expression in PC3 cells, whereas HIF-1α protein levels were not significantly reduced. Additionally, PKM2 inhibition significantly reduced clonogenic survival in both cell lines in a colony formation assay. Prolyl hydroxylase 3 (PHD3) mRNA expression was up-regulated in both cell lines. It has been shown that PKM2 expression is regulated by HIF-1α and that PKM2 favors HIF-1α transactivation under mild (1% O2) but not severe (0.1% O2) hypoxic conditions, and some of our findings are consistent with these previous results. However, this mechanism was not fully observed in our studied cell lines, as PKM2 regulation and HIF-1α stabilization at the transactivation level occurred under severe hypoxic conditions. This discrepancy suggests that tumor tissue origin and cell type influence this model. Our findings expand the current knowledge of the mechanisms of PCA regulation, and would be important in developing novel therapeutic strategies.

External use of Ruyanneixiao cream efficiently blocks precancerous mammary lesions by interfering with glycolysis induced by inhibition of hypoxia inducible factor-1α, hexokinase 2, phosphofructokinase, and pyruvate kinase M2 expression

ObjectiveTo investigate the effect of Ruyanneixiao cream (RYNX) on the expression of hypoxia inducible factor-1α (HIF-1α), hexokinase 2 (HK2), phosphofructokinase (PFK), and pyruvate kinase M2 (PKM2) mRNA and protein in MCF-10AT cells and in an animal model of precancerous mammary lesions. MethodsFollowing treatment of MCF-10AT cells with RYNX, tamoxifen (TAM) and YC-1 for 48 h, HIF-1α, HK2, PFK, PKM2 mRNA and protein expression was analyzed. Fifty female SD rats were randomly divided into control, model, TAM, and high- and low-dose RYNX groups, with 10 rats in each group. A precancerous mammary lesion model was established for all groups except the control group. High- and low-dose RYNX cream containing TAM was coated on the breasts of animals in the corresponding groups. The rat mammary tissue was removed in the 10th week and HIF-1α, HK2, PFK, PKM2 mRNA and protein was analyzed. ResultsIn vitro analyses demonstrated that, compared with the matrix group, HIF-1α, HK2, PFK, PKM2 mRNA and protein expression was significantly decreased in the RYNX group (P < 0.05). Compared with the YC-1 + RYNX group, HK2, PFK, and PKM2 protein expression was significantly reduced in the RYNX group. HIF-1α, HK2, PFK, and PKM2 protein expression was increased significantly in the model group (P < 0.05) compared with the control group, while HIF-1α, HK2, PFK, and PKM2 mRNA and protein expression was significantly decreased in both the high- and low-dose RYNX groups (P < 0.05), with the effect being greater in the high-dose group. ConclusionRYNX can block precancerous breast lesions by decreasing the expression of HK2, PFK, and PKM2 mRNA and protein via inhibition of HIF-1α mRNA and protein overexpression in a dose-dependent manner.

Abstract 1567: Prostate cancer-derived exosomes alter the metabolic microenvironment of bone marrow pre-metastatic niche through PKM2 to promote bone metastasis

Abstract Tumor-derived exosomes are emerging mediators of tumor progression. We explored the role of prostate cancer (PCa)-derived exosomes (Ex) in PCa bone metastasis. Systemic pre-treatment of mice with PCa-Ex increased PCa tumor growth in bone, this was associated with increased early PCa cell seeding into the bone marrow. PCa-Ex had no direct impact on PCa growth or invasive ability in vitro; whereas, conditioned-media (CM) from PCa-Ex-treated stromal cells increased PCa cell growth, invasion and epithelial mesenchymal transition (EMT). PCa-Ex preferentially targeted bone marrow versus lung or liver stroma when injected IV. Proteomic analysis of PCa-Ex-treated cells revealed that PCa-Ex increased pyruvate kinase M2 (PKM2) protein expression in both ST2 stromal cells and primary bone marrow stromal cells, although PKM2 mRNA expression was not altered. PKM2 protein expression correlated with aggressiveness of PCa cell lines and was highest in serum of PCa patients with metastases compared to those with primary tumors. PCa-Ex induced both autophagy and β-hydroxybutarate (BHB) production from stromal cells. Knockdown of PKM2 in PCa cells resulted in production of PCa-Ex with low PKM2. The low PKM2 PCa-Ex had diminished ability to induce PCa cell growth, autophagy and BHB production. IV PCa-Ex and orthotopic tumors increased bone marrow BHB levels. PCa-Ex treatment of stromal cells induced the CM to (1) increase in oxygen consumption rate (OCR) by 80%; (2) extracellular acidification rate (ECAR) by 25%; and (3) increase ATP production in PCa cells. BHB induced OCR, but not ECAR in a dose responsive fashion. BHB administration induced a dose-responsive increase in intratibial tumor growth in mice. Our experiments indicate that primary tumor PCa-Ex can target the bone marrow microenvironment and educate stromal cells to promote PCa progression through increasing PKM2 expression in stromal cells resulting in ketone production that supports metastatic growth. Overall this demonstrates that primary tumor exosomes modulate the metabolic status of the distant pre-metastatic niche to promote metastasis. Citation Format: Jinlu Dai, Yi Lu, Jian Zhang, Evan Keller. Prostate cancer-derived exosomes alter the metabolic microenvironment of bone marrow pre-metastatic niche through PKM2 to promote bone metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1567.

Tanshinone ⅡA inhibits human esophageal cancer cell growth through miR-122-mediated PKM2 down-regulation

Pyruvate kinase M2 (PKM2) plays a pivotal role in the growth, survival and metabolic reprogramming of cancer cells. Here, we presented for the first time that tanshinone ⅡA inhibited human esophagus cancer cell growth through miR-122-mediated PKM2 down-regulation pathway. Tanshinone ⅡA inhibited cell proliferation and induced cell cycle arrest in S phase in human Ec109 cells. As expected, tanshinone ⅡA down-regulated PKM2 mRNA and protein expression in Ec109 cells. Given these findings, we further investigated microRNAs regulation of PKM2 and confirmed miR-122 for targeting PKM2. Moreover, we found that tanshinone ⅡA-induced up-regulation of miR-122 expression inhibited PKM2 expression in Ec109 cells. Meanwhile, tanshinone ⅡA inhibited proliferation through miR122-medated PKM2 down-regulation. It was demonstrated that the anticancer activity of tanshinone ⅡA was targeted at metabolic regulation of miR-122/PKM2 in human esophagus cancer cells. Taken together, our results revealed tanshinone ⅡA targeting at PKM2-mediated metabolic reprogramming play an important role in inhibition of esophageal cancer cell growth.