A novel protein CYTB-187AA encoded by the mitochondrial gene CYTB modulates mammalian early development.

The mitochondrial genome transcribes 13 mRNAs coding for well-known proteins essential for oxidative phosphorylation. We demonstrate here that cytochrome b (CYTB), the only mitochondrial-DNA-encoded transcript among complex III, also encodes an unrecognized 187-amino-acid-long protein, CYTB-187AA, using the standard genetic code of cytosolic ribosomes rather than the mitochondrial genetic code. After validating the existence of this mtDNA-encoded protein arising from cytosolic translation (mPACT) using mass spectrometry and antibodies, we show that CYTB-187AA is mainly localized in the mitochondrial matrix and promotes the pluripotent state in primed-to-naive transition by interacting with solute carrier family 25 member 3 (SLC25A3) to modulate ATP production. We further generated a transgenic knockin mouse model of CYTB-187AA silencing and found that reduction of CYTB-187AA impairs females' fertility by decreasing the number of ovarian follicles. For the first time, we uncovered the novel mPACT pattern of a mitochondrial mRNA and demonstrated the physiological function of this 14th protein encoded by mtDNA.

Exploring a specialized programmed-cell death patterns to predict the prognosis and sensitivity of immunotherapy in cutaneous melanoma via machine learning.

The mortality and therapeutic failure in cutaneous melanoma (CM) are mainly caused by wide metastasis and chemotherapy resistance. Meanwhile, immunotherapy is considered a crucial therapy strategy for CM patients. However, the efficiency of currently available methods and biomarkers in predicting the response of immunotherapy and prognosis of CM is limited. Programmed cell death (PCD) plays a significant role in the occurrence, development, and therapy of various malignant tumors. In this research, we integrated fourteen types of PCD, multi-omics data from TCGA-SKCM and other cohorts in GEO, and clinical CM patients to develop our analysis. Based on significant PCD patterns, two PCD-related CM clusters with different prognosis, tumor microenvironment (TME), and response to immunotherapy were identified. Subsequently, seven PCD-related features, especially CD28, CYP1B1, JAK3, LAMP3, SFN, STAT4, and TRAF1, were utilized to establish the prognostic signature, namely cell death index (CDI). CDI accurately predicted the response to immunotherapy in both CM and other cancers. A nomogram with potential superior predictive ability was constructed, and potential drugs targeting CM patients with specific CDI have also been identified. Given all the above, a novel CDI gene signature was indicated to predict the prognosis and exploit precision therapeutic strategies of CM patients, providing unique opportunities for clinical intelligence and new management methods for the therapy of CM.

Neuroprotective Effects of Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cell Extracellular Vesicles in Ischemic Stroke Models.

BACKGROUND: Stroke represents the second leading cause of death and the primary cause of long-term disability in humans. The transplantation of mesenchymal stem cells (MSC) reportedly improves functional outcomes in animal models of cerebral ischemia. Here, we evaluate the neuroprotective potential of extracellular vesicles secreted from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-EV) using preclinical cell-based and animal-based models of ischemic strokes. METHODS: hiPS-MSC-EV were isolated using an ultrafiltration method. HT22 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury for 2 h, followed by treatment with hiPS-MSC-EV (100 µg/mL). Male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) followed by an intravenous injection of hiPS-MSC-EV (100 µg) at three distinct time points. RESULTS: Our experimental approach revealed hiPS-MSC-EV promoted HT22 cell proliferation, reduced apoptosis, and altered cellular morphology following OGD/R. In addition, hiPS-MSC-EV reduced the volume of infarcts, improved spontaneous movement abilities, and enhanced angiogenesis by expressing the VEGF and CXCR4 proteins in the infarcted hemisphere of the MCAO-treated mouse model. CONCLUSION: Our findings provide evidence of the potential neuroprotective effects of hiPS-MSC-derived extracellular vesicles (hiPS-MSC-EVs) in both in vitro and in vivo mouse models of ischemic stroke. These results suggest that hiPS-MSC-EVs may play a role in neurorestoration and offer insights into potential cell-free strategies for addressing cerebral ischemia.

Identification of Two Subsets of Subcompartment A1 Associated with High Transcriptional Activity and Frequent Loop Extrusion.

Three-dimensional genome organization has been increasingly recognized as an important determinant of the precise regulation of gene expression in mammalian cells, yet the relationship between gene transcriptional activity and spatial subcompartment positioning is still not fully comprehended. Here, we first utilized genome-wide Hi-C data to infer eight types of subcompartment (labeled A1, A2, A3, A4, B1, B2, B3, and B4) in mouse embryonic stem cells and four primary differentiated cell types, including thymocytes, macrophages, neural progenitor cells, and cortical neurons. Transitions of subcompartments may confer gene expression changes in different cell types. Intriguingly, we identified two subsets of subcompartments defined by higher gene density and characterized by strongly looped contact domains, named common A1 and variable A1, respectively. We revealed that common A1, which includes highly expressed genes and abundant housekeeping genes, shows a ~2-fold higher gene density than the variable A1, where cell type-specific genes are significantly enriched. Thus, our study supports a model in which both types of genomic loci with constitutive and regulatory high transcriptional activity can drive the subcompartment A1 formation. Special chromatin subcompartment arrangement and intradomain interactions may, in turn, contribute to maintaining proper levels of gene expression, especially for regulatory non-housekeeping genes.

Degradation kinetics and formation of regulated and emerging disinfection by-products during chlorination of two expectorants ambroxol and bromhexine.

Ambroxol hydrochloride (AMB) and bromhexine hydrochloride (BRO) are classic expectorants and bronchosecretolytic pharmaceuticals. In 2022, both AMB and BRO were recommended by medical emergency department of China to alleviate cough and expectoration for symptoms caused by COVID-19. The reaction characteristics and mechanism of AMB/BRO with chlorine disinfectant in the disinfection process were investigated in this study. The reaction of chlorine with AMB/BRO were well described by a second-order kinetics model, first-order in both AMB/BRO and chlorine. The second order rate reaction constant of AMB and BRO with chlorine at pH 7.0 were 1.15 × 102 M-1s-1 and 2.03 × 102 M-1s-1, respectively. During chlorination, a new class of aromatic nitrogenous disinfection by-products (DBPs) including 2-chloro-4, 6-dibromoaniline and 2, 4, 6-tribromoaniline were identified as the intermediate aromatic DBPs by gas chromatography-mass spectrometry. The effect of chlorine dosage, pH, and contact time on the formation of 2-chloro-4, 6-dibromoaniline and 2, 4, 6-tribromoaniline were evaluated. In addition, it was found that bromine in AMB/BRO were vital bromine source to greatly promote the formation of classic brominated DBPs, with the highest Br-THMs yields of 23.8% and 37.8%, respectively. This study inspired that bromine in brominated organic compounds may be an important bromine source of brominated DBPs.

Extracellular vesicles from iPSC-MSCs alleviate chemotherapy-induced mouse ovarian damage via the ILK-PI3K/AKT pathway.

Chemotherapy can significantly reduce follicle counts in ovarian tissues and damage ovarian stroma, causing endocrine disorder, reproductive dysfunction, and primary ovarian insufficiency (POI). Recent studies have suggested that extracellular vesicles (EVs) secreted from mesenchymal stem cells (MSCs) exert therapeutic effects in various degenerative diseases. In this study, transplantation of EVs from human induced pluripotent stem cell-derived MSCs (iPSC-MSC-EVs) resulted in significant restoration of ovarian follicle numbers, improved granulosa cell proliferation, and inhibition of apoptosis in chemotherapy-damaged granulosa cells, cultured ovaries, and in vivo ovaries in mice. Mechanistically, treatment with iPSC-MSC-EVs resulted in up-regulation of the integrin-linked kinase (ILK) -PI3K/AKT pathway, which is suppressed during chemotherapy, most likely through the transfer of regulatory microRNAs (miRNAs) targeting ILK pathway genes. This work provides a framework for the development of advanced therapeutics to ameliorate ovarian damage and POI in female chemotherapy patients.

WRN promotes bone development and growth by unwinding SHOX-G-quadruplexes via its helicase activity in Werner Syndrome.

Werner Syndrome (WS) is an autosomal recessive disorder characterized by premature aging due to mutations of the WRN gene. A classical sign in WS patients is short stature, but the underlying mechanisms are not well understood. Here we report that WRN is indispensable for chondrogenesis, which is the engine driving the elongation of bones and determines height. Zebrafish lacking wrn exhibit impairment of bone growth and have shorter body stature. We pinpoint the function of WRN to its helicase domain. We identify short-stature homeobox (SHOX) as a crucial and direct target of WRN and find that the WRN helicase core regulates the transcriptional expression of SHOX via unwinding G-quadruplexes. Consistent with this, shox-/- zebrafish exhibit impaired bone growth, while genetic overexpression of SHOX or shox expression rescues the bone developmental deficiency induced in WRN/wrn-null mutants both in vitro and in vivo. Collectively, we have identified a previously unknown function of WRN in regulating bone development and growth through the transcriptional regulation of SHOX via the WRN helicase domain, thus illuminating a possible approach for new therapeutic strategies.

Peptide hormone ELABELA promotes rat bone marrow-derived mesenchymal stem cell proliferation and migration by manipulating the cell cycle through the PI3K/AKT pathway under the hypoxia and ischemia microenvironment

BACKGROUND: Mesenchymal stem cells (MSCs) are emerging as a potential candidate for stem cell transplantation to repair myocardial tissue in myocardial infarctions (MI). However, there are some pivotal limitations such as poor survival and low migration capacity of MSCs in hypoxic and ischemic microenvironments of MI. Our previous work verified that ELABELA (also abbreviated as ELA), a peptide hormone, could play a role as a growth factor and prolong the life span of rat bone marrow-derived mesenchymal stem cells (RAT BM-MSCs) under hypoxic and ischemic conditions. Nevertheless, the influence of ELA on the cell cycle, proliferation, and migration remains elusive. This study will further explore the improvement of the biological functions of ELA-treated RAT BM-MSCs, so as to provide a reference for improving the efficacy of RAT BM-MSCs in MI. METHODS: Rat BM-MSCs were isolated from 80 to 120 g Sprague Dawley rats by flushing femurs and tibias under the aseptic condition. RAT BM-MSCs of the third passage were divided into control group, hypoxic/ischemic (H/I) group, ELA group, ELA-LY group and LY group. RAT BM-MSCs were cultured under normoxia in control group. In H/I group, RAT BM-MSCs were exposed to hypoxia (1% O2) and serum deprivation for 24 h. RAT BM-MSCs in ELA group were treated with 5 µM ELA prior to the H/I exposure for 24 h. The PI3K/AKT inhibitor, LY294002 (50 µM), was used in ELA-LY group and LY group to observe the effect of ELA on PI3K/AKT activation. Cell proliferation ability was examined by CCK-8. Cell cycle was assessed with flow cytometry. Cell migration was evaluated by Transwell assay. Expression levels of total-AKT, phosphorylated-AKT, and cell cycle-associated proteins were examined by Western blotting. RESULTS: ELA-treated RAT BM-MSCs exhibited significantly higher proliferation ability, cell viability, and migration under H/I conditions. The cell cycle analysis showed that an increased proportion of cells in the S and G2/M phases of the cell cycle were observed in ELA-treated RAT BM-MSCs. The addition of ELA activated the PI3K/AKT signaling pathway. Additionally, upon treating with the inhibitor of the PI3K/AKT signaling pathway, ELA-triggered proliferation, cell viability, and migration were abrogated. CONCLUSIONS: ELA can be used to enhance the proliferation ability, cell viability, and migration of RAT BM-MSCs through the PI3K/AKT signaling pathway and alleviate cell cycle arrest at the G0/G1 phase under hypoxic and ischemic injury. Thus, this study provides a promising strategy that ELA may help to optimize the mesenchymal stem cell-based therapy in MI.

LINC00184 plays an oncogenic role in non-small cell lung cancer via regulation of the miR-524-5p/HMGB2 axis.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. We aimed to investigate the role of LINC00184 in NSCLC. Migration, proliferation and invasion of NSCLC cells were analysed using the wound healing assay, cell counting kit-8 assay and transwell assay, respectively. Apoptosis and cell cycle were assessed using flow cytometry. Online bioinformatics tools were utilized to predict downstream microRNAs (miRNA) or genes related to LINC00184 expression. The RNA pull-down experiment and luciferase reporter assay were performed to verify the predictions thereof. LINC00184, miR-524-5p, and high mobility group 2 protein (HMGB2) expression levels in NSCLC tissues and cell lines were detected using quantitative real-time polymerase chain reaction. An NSCLC mouse model was constructed for in vivo experiments. LINC00184 overexpression was observed in NSCLC tissues and cell lines and was found to be correlated with poor prognosis. LINC00184 knockdown inhibited cell proliferation, migration and invasion, induced cell cycle arrest and accelerated apoptosis in NSCLC cell lines. LINC00184 suppressed tumour growth and proliferation in NSCLC mouse models and directly targeted the miR-524-5p/HMGB2 axis. Moreover, the expression levels of LINC00184 and HMGB2 were negatively correlated with miR-524-5p expression, whereas LINC00184 expression was positively correlated with HMGB2 expression. LINC00184 affected the cell cycle, proliferation, apoptosis, migration and invasion in NSCLC via regulation of the miR-524-5p/HMGB2 axis.

Pten Regulates Cardiomyocyte Differentiation by Modulating Non-CG Methylation via Dnmt3.

The regulation of cardiomyocyte differentiation is a fundamental aspect of cardiac development and regenerative medicine. PTEN plays important roles during embryonic development. However, its role in cardiomyocyte differentiation remains unknown. In this study, a low-cost protocol for cardiomyocyte differentiation from mouse embryonic stem cells (ESCs) is presented and it is shown that Pten deletion potently suppresses cardiomyocyte differentiation. Transcriptome analysis shows that the expression of a series of cardiomyocyte marker genes is downregulated in Pten-/- cardiomyocytes. Pten ablation induces Dnmt3b expression via the AKT/FoxO3a pathway and regulates the expression of a series of imprinted genes, including Igf2. Double knockout of Dnmt3l and Dnmt3b rescues the deficiency of cardiomyocyte differentiation of Pten-/- ESCs. The DNA methylomes from wild-type and Pten-/- embryoid bodies and cardiomyocytes are analyzed by whole-genome bisulfite sequencing. Pten deletion significantly promotes the non-CG (CHG and CHH) methylation levels of genomic DNA during cardiomyocyte differentiation, and the non-CG methylation levels of cardiomyocyte genes and Igf2 are increased in Pten-/- cardiomyocytes. Igf2 or Igf1r deletion also suppresses cardiomyocyte differentiation through the MAPK/ERK signaling pathway, and IGF2 supplementation partially rescues the cardiomyocyte differentiation. Finally, Pten conditional knockout mice are generated and the role of PTEN in cardiomyocyte differentiation is verified in vivo.

ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways.

BACKGROUND: Mesenchymal stem cells (MSCs) have exerted their brilliant potential to promote heart repair following myocardial infarction. However, low survival rate of MSCs after transplantation due to harsh conditions with hypoxic and ischemic stress limits their therapeutic efficiency in treating cardiac dysfunction. ELABELA (ELA) serves as a peptide hormone which has been proved to facilitate cell growth, survival, and pluripotency in human embryonic stem cells. Although ELA works as an endogenous ligand of a G protein-coupled receptor APJ (Apelin receptor, APLNR), whether APJ is an essential signal for the function of ELA remains elusive. The effect of ELA on apoptosis of MSCs is still vague. OBJECTIVE: We studied the role of ELABELA (ELA) treatment on the anti-apoptosis of MSCs in hypoxic/ischemic (H/I) conditions which mimic the impaired myocardial microenvironment and explored the possible mechanisms in vitro. METHODS: MSCs were obtained from donated rats weighing between 80~120 g. MSCs were exposed to serum-free and hypoxic (1% O2) environments for 24 h, which mimics hypoxic/ischemic damage in vivo, using serum-containing normoxic conditions (20% O2) as a negative control. MSCs that were exposed to H/I injury with ELA processing were treated by 5 µM of ELA. Cell viability and apoptosis of MSCs were evaluated by CCK8 and flow cytometry, respectively. Mitochondrial function of MSCs was also assessed according to mitochondrial membrane potential (MMP) and ATP content. The protein expression of key kinases of the PI3K/AKT and ERK1/2 signaling pathways involving t-AKT, p-AKT, t-ERK1/2, and p-ERK1/2, as well as apoptosis-related protein expression of Bcl-2, Bax, and cleaved Caspase 3, were monitored by Western blot. RESULTS: We found that ELA treatment of H/I-induced MSCs improved overall cell viability, enhanced Bcl/Bax expression, and decreased Caspase 3 activity. ELA inhibited H/I-induced mitochondrial dysfunction by increasing ATP concentration and suppressing the loss of mitochondrial transmembrane potential. However, this anti-apoptotic property of ELA was restrained in APJ-silenced MSCs. Additionally, ELA treatment induced the phosphorylation of AKT and ERK, while the blockade of PI3K/AKT and ERK1/2 pathways with respective inhibitors, LY294002 and U0126, suppressed the action of ELA. CONCLUSION: ELA positively affected on the survival of MSCs and exhibited anti-apoptotic characteristics when exposed to hypoxic/ischemic condition in vitro. Also, the function of ELA was correlated with the APJ receptor, reduced mitochondrial damage, and activation of the PI3K/AKT and ERK1/2 signal axes.

Tambulin Targets Histone Deacetylase 1 Inhibiting Cell Growth and Inducing Apoptosis in Human Lung Squamous Cell Carcinoma.

There is an urgent unmet need to develop new therapeutics for lung squamous cell carcinoma (LSCC) as the current gold standard treatment regimens are dominated by chemotherapy. In this study, we observed the treatment effects of the natural compound tambulin on LSCC and explored its mechanism of action. LSCC cell lines H226 and H520 were cultured in vitro to observe the effects of tambulin on cell proliferation and apoptosis. Western blotting was used to detect the expression of histone deacetylase 1 (HDAC1) and apoptosis-related proteins. Cell derived xenografts (CDX) of H226 and H520 in nude mice were established to examine the inhibitory effects of tambulin in vivo. Results showed that tambulin inhibited the proliferation of H226 and H520 cells in a dose-dependent manner and inhibited the growth of CDX tumors. Tambulin also promoted the apoptosis of H226 and H520 cells, up-regulated the protein expression of cleaved caspase-3, cleaved caspase-9 and Bax, and down-regulated HDAC1 and Bcl-2 protein expression. In support of this, immunohistochemical analysis of CDX tumors from mice treated with tambulin showed increased expression of cleaved caspase-3 and Bax, while the expression of HDAC1 and Bcl-2 were decreased. What's more, when HDAC1 was over-expressed via adenovirus transduction in H226 or H520 cells, the effects of tambulin were significantly attenuated. Interestingly, we found that combining tambulin with cisplatin treatment in CDX models was more effective than single drug treatment, suggesting that tambulin may enhance the sensitivity of LSCC to cisplatin. Taken together, this study proves that tambulin has a definite therapeutic effect on LSCC. Mechanistically, tambulin downregulates HDAC1, which in turn regulates the Bcl-2/caspase signaling pathway and promotes cancer cell apoptosis.

Pten-mediated Gsk3ß modulates the naïve pluripotency maintenance in embryonic stem cells.

Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass of blastocysts, and they exist in different states of pluripotency-naïve and primed states. Pten is a well-known tumor suppressor. Here, we generated Pten-/- mouse ESCs with the CRISPR-Cas9 system and verified that Pten-/- ESCs maintained naïve pluripotency by blocking Gsk3ß activity. Serum/LIF and 2i (MAPK and GSK3 inhibitors) conditions are commonly used for ESC maintenance. We show that the Pten-inhibitor SF1670 contributed to sustaining mouse ESCs and that Pten activation by the S380A, T382A, and T383A mutations (Pten-A3) suppressed the pluripotency of ESCs. The in vivo teratoma formation ability of SF1670-treated ESCs increased, while the Pten-A3 mutations suppressed teratoma formation. Furthermore, the embryoid bodies derived from Pten-deficient ESCs or SF1670-treated wild-type ESCs showed greater expression of ectoderm and pluripotency markers. These results suggest that Pten-mediated Gsk3ß modulates the naïve pluripotency of ESCs and that Pten ablation regulates the lineage-specific differentiation.

Generation of a Bag1 homozygous knockout mouse embryonic stem cell line using CRISPR/Cas9.

Bag1 transcribes a multifunctional protein that participates in many important biological processes such as cell apoptosis, proliferation, differentiation and embryo development. Despite numerous published studies, the role of Bag1 in the context of embryonic stem (ES) cells, has not been explored. To investigate the function of Bag1 in ES cells, we generated mutant Bag1-/- ES cells using the CRISPR/Cas9 system. We established that the Bag1 double knockout ES cell line maintained their pluripotency, possessed a normal karyotype and the ability to differentiate into all three germ layers.

Effect and molecular mechanism of mir-146a on proliferation of lung cancer cells by targeting and regulating MIF gene.

OBJECTIVE: To discuss the effect and molecular mechanism of miR-146a on the proliferation of lung cancer cells by targeting and regulating the macrophage migration inhibitory factor (MIF) gene. METHODS: RT-PCR was employed to detect expression of miR-146a; immunohistochemistry was used to detect the expression of MIF. The luciferase reporter gene technique was adopted to verify that MIF was the specific reverse target gene of miR-146a and the liposome Lipofectamine™2000 was employed to transfer the modeled miR-146a mimics, and miR-146a negative control (NC) in NSCLC cells to detect the expression of MIF mRNA and protein. MTT assay was used to detect cell viability, cloning technique to detect cell proliferation ability, AnnexinV-PI to detect cell apoptosis, UV spectrophotometry to detect viability of cysteinyl aspartate specific proteinase 3 (Caspase 3), and western blot to detect expression of nuclear factor-κB (NF-κB) in cells. RESULTS: The expression of miR-146a in NSCLC lung tissues was lower than that in the normal lung tissues besides the lung cancer; while the expression of miR-146a in NSCLC cells was lower than that in normal human embryonic lung tissues. It was chosen as the subsequent cell line for its appropriate expression in A549. The expression of MIF protein in NSCLC lung tissues was higher than that in the normal lung tissues besides the lung cancer. The luciferase reporter gene proved that MIF was the reverse target gene of miR-146a. The miR-146a mimics were transfected into A549 cells through the liposome. Compared with NC group, the expression of MIF protein and mRNA was significantly decreased (P < 0.01), with the decrease in the cell viability (P < 0.01), the decrease in the number of clones (P < 0.01), cell apoptosis (P < 0.01), the increase in the activity of Caspase 3 (P < 0.01), and decrease in the phosphorylation of NF-κB p65 (P < 0.01). CONCLUSIONS: miR-146a has low expression in NSCLC tissues and cell lines, while MIF has the over expression in NSCLC tissues. The increased expression of miR-146a can inhibit the expression of MIF via the gene targeting and thus inhibit the proliferation of A549 cells and induce the apoptosis of cancer cells, which may be realized through NF-κB signaling pathway.

Hyperthermia inhibits hypoxia-induced epithelial-mesenchymal transition in HepG2 hepatocellular carcinoma cells.

AIM: To investigate the effect of hyperthermia on hypoxia-induced epithelial-mesenchymal transition (EMT) in HepG2 hepatocellular carcinoma (HCC) cells, and its mechanism. METHODS: Cells were treated with hyperthermia at 43 °C for 0.5 h, followed by incubation under hypoxic or normoxic conditions for 72 h. Cell morphology was observed. Expressions of E-cadherin and vimentin were determined by immunofluorescence assay or Western blot. The protein and mRNA expressions of Snail were also determined by Western blot and reverse transcription-polymerase chain reaction. Cell migratory capacity was evaluated. RESULTS: Hypoxia induced EMT in HepG2 cells, which was evidenced by morphological, molecular and functional changes, including the formation of a spindle shape and the loss of cell contact. The expression of E-cadherin was decreased but the expression of vimentin was increased; also, the migratory capability was increased by 2.2 ± 0.20-fold as compared with normoxia. However, those effects were inhibited by hyperthermia pretreatment. Furthermore, protein synthesis and mRNA expression of Snail in the cells were enhanced by hypoxia as compared with normoxia, and also significantly inhibited by hyperthermia pretreatment. CONCLUSION: Hyperthermia may inhibit hypoxia-induced EMT in HepG2 HCC cells, and the mechanism may involve inhibition of induced expression of Snail.