The exploration of surgery and survival prediction in patients with peritoneal metastasis from gastric adenocarcinoma based on the SEER database.

Background: As one of the most common diseases in terms of cancer-related mortality worldwide, gastric adenocarcinoma (GA) frequently develops peritoneal metastases (PMs) in advanced stages. Systemic therapy or optimal supportive care are recommended for advanced GA; however, patients frequently develop drug resistance. Surgical resection is not recommended for stage IV patients, and there have been some controversies regarding the role of it in GA patients with PMs. The aim of the study was to preliminarily evaluate the possible effect of surgical treatments on patients with only PMs from GA. Methods: Data were collected from the Surveillance, Epidemiology and End Results (SEER) database (year 2000-2022). A propensity score matching (PSM) was performed to reduce the influence of selection bias and confounding variables on comparisons. Then Cox proportional hazard regression, Kaplan-Meier analysis, and log-rank test were performed to assess the efficacy of surgical treatment in patients with PMs from GA. Results: A total of 399 patients diagnosed with PMs from GA were enrolled for our analysis, of which, 180 (45.1%) patients did not receive surgery and 219 (54.9%) patients received surgery. Multivariate Cox regression analysis before PSM indicated higher rates of overall survival (OS) outcome for patients who had received surgery [hazard ratio (HR) =0.4342, 95% confidence interval (CI): 0.3283-0.5742, P<0.001]. After PSM, a total of 172 patients were enrolled, with 86 in each group. Multivariate Cox analysis showed that surgery was the independent factor reflecting patients' survival (HR =0.4382, 95% CI: 0.3037-0.6324, P<0.001). Subgroup survival analysis revealed that surgery may bring advantages to patients with grades I-IV, stages T1-T4, stage N0, and tumor size less than 71 mm (P<0.05). We also found that the OS of chemotherapy patients who had undergone surgery was better than that of chemotherapy patients who had not undergone surgery (P<0.01). Conclusions: Based on the SEER database, surgery has better OS for patients only with PMs from GA. Patients without lymph node metastasis and those who received chemotherapy before may benefit from surgery. These specific groups of patients may have surgery as an option to improve the prognosis.

Nomogram model for predicting esophsagogastric varices in hepatocellular carcinoma with cirrhosis.

BACKGROUND/AIMS: The prognosis for hepatocellular carcinoma (HCC) with cirrhosis is poor. The risk of death also increases in patients with esophagogastric varices (EGV). Based on routine clinical features and related noninvasive parameters, a nomogram prediction model was developed in this study to facilitate the early identification of EGV in HCC patients. METHODS: A retrospective cohort analysis of patients with HCC in the Renmin Hospital of Wuhan University from 2020 to 2021 was performed. Clinical and noninvasive parameters closely related to EGV risk were screened by univariate and multivariate logistic regression analysis and integrated into a nomogram. The nomogram was validated internally and externally by calibration, receiver operating characteristic curve and decision curve analysis (DCA). RESULTS: A total of 165 patients with HCC-related cirrhosis were recruited. In the raining cohort, multivariate logistic regression analysis identified platelet (PLT) [odds ratio (OR), 0.950; 95% confidence interval (CI), 0.925-0.977; P < 0.001], D-dimer (OR. 3.341; 95% CI, 1.751-6.376, P < 0.001), spleen diameter (SD) (OR, 2.585; 95% CI, 1.547-4.318; P < 0.001) as independent indicators for EGV. The nomogram for predicting EGV risk was well calibrated with a favorable discriminative ability and an area under curve of 0.961. In addition, the nomogram showed better net benefits in the DCA. The results were validated in the validation cohort. CONCLUSIONS: The proposed nomogram model based on three indicators (PLT, D-dimer and SD) showed an excellent predictive effect, leading to the avoidance of unnecessary esophagogastroduodenoscopy.

ROS-Related miRNAs Regulate Immune Response and Chemoradiotherapy Sensitivity in Hepatocellular Carcinoma by Comprehensive Analysis and Experiment.

Reactive oxygen species (ROS) plays an essential role in the development of cancer. Here, we chose ROS-related miRNAs for consensus clustering analysis and ROS score construction. We find that ROS is extremely associated with prognosis, tumor immune microenvironment (TIME), gene mutations, N6-methyladenosine (m6A) methylation, and chemotherapy sensitivity in hepatocellular carcinoma (HCC). Mechanistically, ROS may affect the prognosis of HCC patients in numerous ways. Moreover, miR-210-3p and miR-106a-5p significantly increased the ROS level and stagnated cell cycle at G2/M in HCC; the results were more obvious in cells after ionizing radiation (IR). Finally, the two miRNAs suppressed cell proliferation, migration, and invasion and promoted apoptosis in huh7 and smmc7721 cells. It indicated that ROS might affect the prognosis of HCC patients through immune response and increase the sensitivity of HCC patients to radiotherapy and chemotherapy.

ß-Catenin-CCL2 feedback loop mediates crosstalk between cancer cells and macrophages that regulates breast cancer stem cells.

Breast cancer is the most frequently diagnosed cancer among women worldwide. Though advances in diagnosis and treatment have prolonged overall survival (OS) for patients with breast cancer, metastasis remains the major obstacles to improved survival for breast cancer patients. The existence of breast cancer stem cells (BCSCs) is a major reason underlying cancer metastasis and recurrence. Therefore, understanding the molecular pathways sustaining BCSC properties and targeting BCSCs will ultimately improve breast cancer treatments. In this study, we found that activation of ß-Catenin directly regulated CCL2 expression at the transcriptional level, and in turn promoted macrophages infiltration and M2 polarization. Moreover, macrophages co-cultured with breast cancer cells showed a significant increase in CCL2 expression and promoted ß-Catenin-induced BCSCs properties, whereas depletion of CCL2 by adding neutralizing antibodies suppressed BSCSs properties. In addition, we found that ß-Catenin-mediated CCL2 secretion recruited macrophages into tumor microenvironment and promoted breast cancer growth and metastasis in vivo. Clinically, we observed a significant positive correlation between ß-Catenin, CCL2 and CD163 expression, and increased expression of ß-Catenin, CCL2 and CD163 predicted poor prognosis in breast cancer. Furthermore, pharmacological inhibition of CCR2 and ß-Catenin synergistically suppressed BCSC properties and breast cancer growth. Collectively, our findings suggested that ß-Catenin-mediated CCL2 secretion forms a paracrine feedback loop between breast cancer cells and macrophages, which in turn promotes BCSC properties and supports breast cancer growth and metastasis. Targeting ß-Catenin/CCL2 signaling might be an effective strategy for breast cancer therapy.

Comprehensive Analysis of the Immune Infiltrates and PD-L1 of m6A RNA Methylation Regulators in Hepatocellular Carcinoma.

Recently, N 6-methyladenosine (m6A) RNA methylation in eukaryotic mRNA has become increasingly obvious in the pathogenesis and prognosis of cancer. Moreover, tumor microenvironment is involved in the regulation of tumorigenesis. In our research, the clinical data, including 374 tumor and 50 normal patients, were obtained from The Cancer Genome Atlas (TCGA). Then 19 m6A regulators were selected from other studies. Hepatocellular carcinoma (HCC) patients were clustered in cluster1/2, according to the consensus clustering for the m6A RNA regulators. We found that m6A regulators were upregulated in cluster1. The cluster1 was associated with higher programmed death ligand 1 (PD-L1) expression level, higher immunoscore, worse prognosis, and distinct immune cell infiltration compared with cluster2. Five risk signatures were identified, including YTH N6-methyladenosine RNA-binding protein 1, YTHDF2, heterogeneous nuclear ribonucleoprotein C, WT1-associated protein, and methyltransferase-like 3, based on univariate Cox and least absolute shrinkage and selection operator regression analysis. High-risk group and low-risk group HCC patients were selected based on the risk score. Similarly, the high-risk group was extremely associated with higher PD-L1 expression level, higher grade, and worse overall survival (OS). Also, cluster1 was mainly enriched in high-risk group. Receiver operating characteristic (ROC) and a nomogram were used to predict the ability and the probability of 3- and 5-year OS of HCC patients. The time-dependent ROC curve (AUC) reached 0.77, 0.67, and 0.68 at 1, 3, and 5 years in the training dataset. Also, AUC areas of 1, 3, and 5 years were 0.7, 0.63, and 0.55 in the validation dataset. The gene set enrichment analysis showed that MTOR signaling pathway and WNT signaling pathway were correlated with cluster1 and high-risk group. Collectively, the research showed that the m6A regulators were significantly associated with tumor immune microenvironment in HCC. Risk characteristics based on m6A regulators may predict prognosis in patients with HCC and provide a new therapeutic target for improving the efficacy of immunotherapy.

MicroRNA-223-3p Protect Against Radiation-Induced Cardiac Toxicity by Alleviating Myocardial Oxidative Stress and Programmed Cell Death via Targeting the AMPK Pathway.

Objectives: Radiotherapy improves the survival rate of cancer patients, yet it also involves some inevitable complications. Radiation-induced heart disease (RIHD) is one of the most serious complications, especially the radiotherapy of thoracic tumors, which is characterized by cardiac oxidative stress disorder and programmed cell death. At present, there is no effective treatment strategy for RIHD; in addition, it cannot be reversed when it progresses. This study aims to explore the role and potential mechanism of microRNA-223-3p (miR-223-3p) in RIHD. Methods: Mice were injected with miR-223-3p mimic, inhibitor, or their respective controls in the tail vein and received a single dose of 20 Gy whole-heart irradiation (WHI) for 16 weeks after 3 days to construct a RIHD mouse model. To inhibit adenosine monophosphate activated protein kinase (AMPK) or phosphodiesterase 4D (PDE4D), compound C (CompC) and AAV9-shPDE4D were used. Results: WHI treatment significantly inhibited the expression of miR-223-3p in the hearts; furthermore, the levels of miR-223-3p decreased in a radiation time-dependent manner. miR-223-3p mimic significantly relieved, while miR-223-3p inhibitor aggravated apoptosis, oxidative damage, and cardiac dysfunction in RIHD mice. In addition, we found that miR-223-3p mimic improves WHI-induced myocardial injury by activating AMPK and that the inhibition of AMPK by CompC completely blocks these protective effects of miR-223-3p mimic. Further studies found that miR-223-3p lowers the protein levels of PDE4D and inhibiting PDE4D by AAV9-shPDE4D blocks the WHI-induced myocardial injury mediated by miR-223-3p inhibitor. Conclusion: miR-223-3p ameliorates WHI-induced RIHD through anti-oxidant and anti-programmed cell death mechanisms via activating AMPK by PDE4D regulation. miR-223-3p mimic exhibits potential value in the treatment of RIHD.

Dihydroartemisinin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cell by Upregulating Tumor Necrosis Factor via JNK/NF-κB Pathways.

BACKGROUND: Dihydroartemisinin (DHA) is a predominant compound in Artemisia annua L., and it has been shown to inhibit tumorigenesis. METHODS: In this study, the antitumor potential of DHA was investigated in the MHCC97-L hepatocellular carcinoma cell line. Cells were treated at various concentrations of DHA, and then the cell cycle, viability, and DNA synthesis were measured to evaluate cell proliferation. Furthermore, the expression of genes and proteins related to proliferation and apoptosis was measured to determine the effects of DHA. Finally, the mechanism was investigated using RNA-sequencing to identify differentially expressed genes and signaling pathways, and JNK/NF-κB pathways were evaluated with Western blotting. RESULTS: Cells were treated with a concentration range of DHA from 1 to 100 µM, and cell proliferation was suppressed in a dose-dependent manner. In addition, the genes and proteins involved in typical cellular functions of MHCC97-L cells were significantly inhibited. DHA treatment downregulated the angiogenic gene ANGPTL2 and the cell proliferation genes CCND1, E2F1, PCNA, and BCL2. DHA treatment significantly upregulated the apoptotic genes CASP3, CASP8, CASP9, and TNF. Global gene expression profiles identified 2064 differentially expressed genes (DEGs). Among them, 744 were upregulated and 1320 were downregulated. Furthermore, MAPK, NF-kappa B, and TNF pathways were enriched based on the DEGs, and the consensus DEG was identified as TNF using a Venn diagram of those pathways. DHA promoted phosphorylation of JNK, inhibited nuclear p65, and then significantly induced TNF-α synthesis. CONCLUSION: DHA inhibited cell proliferation and induced apoptosis in human hepatocellular carcinoma cells by upregulating TNF expression via JNK/NF-κB pathways.

LncRNA LINC00460 promotes tumor growth of human lung adenocarcinoma by targeting miR-302c-5p/FOXA1 axis.

Accumulating evidence has shown that long non-coding RNAs (lncRNAs) had malfunctioning roles in the development of human cancers, especially lung adenocarcinoma (LC). In the present study, we aimed to investigate the role and potential mechanism of lncRNA long intergenic non-protein coding RNA 460 (LINC00460) in LC progression using human tissues and cell lines. We observed that LINC00460 was increased in lung adenocarcinoma tissues and cells in comparison to their corresponding controls. Moreover, overexpression of LINC00460 indicated the poor prognosis of lung adenocarcinoma patients. In addition, silencing LINC00460 was able to suppress lung adenocarcinoma cell growth in vitro and in vivo. Rescue assay confirmed that LINC00460 contributed to lung adenocarcinoma progression by regulating miR-302c-5p/FOXA1 signal pathway. In conclusion, LINC00460 promotes LC progression by competitively binding miR-302c-5p and regulating FOXA1 signal pathway. Our findings reveal that LINC00460 may be a potential prognostic biomarker and a candidate target for LC therapy.

Effect of lenalidomide on the human gastric cancer cell line SGC7901/vincristine Notch signaling.

AIM OF STUDY: To examine the function of lenalidomide (LEN) on the human multidrug resistance (MDR)-type gastric cancer line SGC7901/vincristine (VCR) via regulating Notch signaling. MATERIALS AND METHODS: Quantitative polymerase chain reaction was used for checking the genes of Notch, DNA methyltransferase (DNMT), RBP-J, Hes1/5, Deltex1, MDR/multidrug resistant protein (MRP); the cell proliferation and cell death were detected by cell counting kit-8 (CCK8) staining, Ki-67 expression, and propidium-iodide staining, and methylated DNA immunoprecipitation assay (MeDIP) was used for checking the 5 mC enrichment, indicating the DNA methylation of the Notch2 gene loci. RESULTS: LEN reduced the mRNA expression of Notch2 (P < 0.01) and increased the expression of the DNMT3A (P < 0.001) in SGC7901/VCR cell, suggesting the involvement of epigenetic regulation by DNMT3A on Notch2 gene expression. Consistently, Notch2 gene expression showed no obvious change between the LEN treatment and the control when the DNMT3A was knockdown using the interference of shRNA. The modulation of DNA methylation process on gene expression was then confirmed by 5 mC enrichment on Notch2 gene loci after LEN treatment. Furthermore, LEN could suppress the downstream genes in Notch2 signaling including RBP-J (P < 0.05), Hes1 (P < 0.001), and Deltex1 (P < 0.01). Due to the changes of gene expression pattern in Notch pathway, LEN showed a phenotype of cell proliferation suppression using CCK8 staining. Meanwhile, the expression of the genes associated with MDR and MRP was also significantly decreased (MDR, P < 0.01; MRP, P < 0.001) after LEN treatment. Therefore, inhibition of cell proliferation by LEN via Notch2 signaling combined with the MDR/MRP expression modulation contributes to the efficacy of LEN on the gastric cancer cell line SGC7901/VCR. CONCLUSION: The data implicate that LEN would be an effective chemical for the therapy of drug-resistant human gastric cancer cell and the gastric cancer patients.

Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells.

AIM: To investigate whether autophagic cell death is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells, and to explore the underlying mechanism. METHODS: Human hepatocellular carcinoma cells were treated with hyperthermia and ionizing radiation. MTT and clonogenic assays were performed to determine cell survival. Cell autophagy was detected using acridine orange staining and flow cytometric analysis, and the expression of autophagy-associated proteins, LC3 and p62, was determined by Western blot analysis. Intracellular reactive oxygen species (ROS) were quantified using the fluorescent probe DCFH-DA. RESULTS: Treatment with hyperthermia and ionizing radiation significantly decreased cell viability and surviving fraction as compared with hyperthermia or ionizing radiation alone. Cell autophagy was significantly increased after ionizing radiation combined with hyperthermia treatment, as evidenced by increased formation of acidic vesicular organelles, increased expression of LC3II and decreased expression of p62. Intracellular ROS were also increased after combined treatment with hyperthermia and ionizing radiation. Pretreatment with N-acetylcysteine, an ROS scavenger, markedly inhibited the cytotoxicity and cell autophagy induced by hyperthermia and ionizing radiation. CONCLUSION: Autophagic cell death is involved in hyperthermic sensitization of cancer cells to ionizing radiation, and its induction may be due to the increased intracellular ROS.

Long non-coding RNA CASC2 regulates cell biological behaviour through the MAPK signalling pathway in hepatocellular carcinoma.

Long non-coding RNAs have previously been demonstrated to play important roles in regulating human diseases, especially cancer. However, the biological functions and molecular mechanisms of long non-coding RNAs in hepatocellular carcinoma have not been extensively studied. The long non-coding RNA CASC2 (cancer susceptibility candidate 2) has been characterised as a tumour suppressor in endometrial cancer and gliomas. However, the role and function of CASC2 in hepatocellular carcinoma remain unknown. In this study, using quantitative real-time polymerase chain reaction, we confirmed that CASC2 expression was downregulated in 50 hepatocellular carcinoma cases (62%) and in hepatocellular carcinoma cell lines compared with the paired adjacent tissues and normal liver cells. In vitro experiments further demonstrated that overexpressed CASC2 decreased hepatocellular carcinoma cell proliferation, migration and invasion as well as promoted apoptosis via inactivating the mitogen-activated protein kinase signalling pathway. Our findings demonstrate that CASC2 could be a useful tumour suppressor factor and a promising therapeutic target for hepatocellular carcinoma.

High expression of G-protein signaling modulator 2 in hepatocellular carcinoma facilitates tumor growth and metastasis by activating the PI3K/AKT signaling pathway.

The aim of this study was to investigate the role of G-protein signaling modulator 2 in the carcinogenesis and progression of hepatocellular carcinoma. We previously showed that G-protein signaling modulator 2 was upregulated in hepatitis B virus-related hepatocellular carcinoma tissues through a hierarchical clustering analysis. With this study, we first assessed the expression pattern of G-protein signaling modulator 2 in hepatocellular carcinoma specimens and adjacent noncancerous tissues; clinical data were analyzed, along survival times, utilizing the Kaplan-Meier method. Moreover, the functions of G-protein signaling modulator 2 were examined using small-interfering RNAs in vitro. The results showed that G-protein signaling modulator 2 was clearly overexpressed in hepatocellular carcinoma tissues and cell lines and that the G-protein signaling modulator 2 expression level was related to tumor size and hepatitis B virus infection. Furthermore, G-protein signaling modulator 2 knockdown studies suggested that G-protein signaling modulator 2 accelerates cell growth, cell cycle, migration, and invasion and inhibits apoptosis, acting as an oncogene in hepatocellular carcinoma. Western blotting indicated that silencing of G-protein signaling modulator 2 in HepG2 and SMMC-7721 cells increased the expression levels of Bax, caspase-3, and E-cadherin, while notably suppressing the cyclin-dependent kinase 4, cyclin-dependent kinase 6, CyclinD1, Snail1, Vimentin, and matrix metallopeptidase 9 expression levels, compared with that in the control groups. In addition, we found that G-protein signaling modulator 2 can affect the expression of key proteins involved in protein kinase B activation. In conclusion, high expression of G-protein signaling modulator 2 was involved in the pathological processes of hepatocellular carcinoma through activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which may provide an attractive potential diagnostic biomarker and therapeutic target for treatment of hepatocellular carcinoma.

Long non-coding RNA AK058003, as a precursor of miR-15a, interacts with HuR to inhibit the expression of γ-synuclein in hepatocellular carcinoma cells.

Long non-coding RNAs (lncRNAs) have been identified as critical players in multiple cancers and lncRNAs are tightly linked to cancer progression. However, only little amount of lncRNAs have been identified to participate in the molecular mechanisms of the progression of hepatocellular carcinoma. In this study, we found that lncRNA-AK058003 is down-regulated in hepatocellular carcinoma tissues and it is associated with the relapse and metastasis of the cancer. Furthermore, lncRNA-AK058003 acts as a tumor suppressor, suppressing hepatocellular carcinoma cell proliferation and metastasis in vitro and in vivo. lncRNA-AK058003 can reduce mRNA stabilizing protein HuR, which results in the inhibition of the expression of γ-synuclein. In addition, a bioinformatics study indicated that γ-synuclein is a target of miR-15a. To verify whether lncRNA-AK058003 plays a role in miR-15a-mediated inhibition of γ-synuclein, we demonstrated that lncRNA-AK058003 is very likely to be a precursor of miR-15a. Collectively, lncRNA-AK058003 can reduce the expression of mRNA stabilizing protein HuR and act as a precursor of miR-15a to suppress γ-synuclein-mediated cell proliferation and the metastasis of hepatocellular carcinoma.

Twist mediates an aggressive phenotype in human colorectal cancer cells.

Epithelial-mesenchymal transition (EMT) is a crucial process providing cancer cells with the ability to migrate and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype and chemoresistance. Twist is a transcription factor that regulates EMT in a various cancer cells, including colorectal cancer (CRC). Our study was done to determine the role of Twist in mediating aggressive phenotype in CRC. Human CRC cell lines were transduced with a retroviral Twist construct or vector control. Migration and invasion abilities were determined in vitro using modified Boyden chamber assays. Mammosphere formation assay was performed to detect CSC characteristics. EMT and CSC markers were detected using western blotting and RT-PCR. Chemosensitivity to oxaliplatin of the transfected cells were determined by the MTT assay. Human CRC specimens were stained for Twist and P-gp expression. Twist overexpression triggered EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration, invasion and mammosphere formation abilities. In addition, Twist-overexpressing CRC cells were more chemo-resistant to oxaliplatin than control cells. Furthermore, Twist over-expression increased P-gp expression in CRC cells, which is a transmembrane glycoprotein conferred multidrug-resistance phenotype to various cancer cells. Importantly, Twist and P-gp were expressed correlatively in human CRC specimens. Thus, Twist is a potential therapeutic target in metastatic CRC.

Effect of silencing NEK2 on biological behaviors of HepG2 in human hepatoma cells and MAPK signal pathway.

To investigate the expression level of NEK2 in 40 tissue specimens of primary liver cancer and to search for clues whether the effect of NEK2 depletion plays a role on biological behaviors of HepG2 cells and the relevant molecular mechanism are the objectives of this study. Real-time PCR and immunohistochemistry assessed expression level of NEK2 in specimens of cancerous tissues and carcinoma-adjacent tissues. The NEK2 expression level in HepG2, Huh7, SMMC, and 7402 cells was detected by real-time PCR and western blot to screen experimental cell line. To assess the expression levels of NEK2 mRNA and protein, an effective siRNA transfected into the HepG2 cells was designed. CCK8 and colony-forming assays were performed to verify short-term and long-term proliferative activities, respectively. Capacity of apoptosis and cell cycle changes were assessed by flow cytometry. Ability of transference and invasion was measured by Transwell Chambers. Western blot approach was used to determine the protein expression levels. There was significantly high expression level of NEK2 in cancerous tissues compared to adjacent tissues. The expression of NEK2 was higher in HepG2 cells than other cell lines. Real-time PCR and western blot shown there were obviously down-regulated NEK2 expression in the NEK2-siRNA group compared to control groups. The capacity of amplification and invasion was inhibited distinctly, and FCM revealed the apoptosis rate was increased and G1 phase was arrested in NEK2-siRNA group. Western blot indicated that low expression of NEK2 in HepG2 cells could increase the expression levels of Bax, caspase-3, P21, and TIMP-1, but significantly suppressed the c-myc, c-jun, Bcl-2, cyclinD1, CDK4, MMP2, and MMP9 expression levels and the phosphorylation levels of ERK, JNK, and P38 compared with the control groups. Our findings demonstrated that NEK2 could be a valuable carcinogenic factor and a promising therapeutic target for primary liver cancer; NEK2 may regulate proliferation, apoptosis, and other biological behaviors of HepG2 cells via MAPK signal pathway.

Long noncoding RNAs in hepatitis B virus-related hepatocellular carcinoma.

AIM: To study the expression of long noncoding RNAs (lncRNAs) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: The lncRNA profiles between HBV-related HCC tissues and corresponding normal liver tissues were generated using microarray analysis. Datasets were analyzed using multiple algorithms to depict alterations in gene expression on the basis of gene ontology (GO), pathway analysis, and lncRNA levels. RESULTS: The microarray revealed that 1772 lncRNAs and 2508 mRNAs were differently expressed. The pathway analysis demonstrated that the cell cycle, cytokine-cytokine receptor interaction, chemokine signaling pathway, and phosphoinositide 3-kinase-protein kinase B signaling pathway may play important roles in HCC. Several GO terms, such as cell cycle, DNA replication, immune response, and signal transduction, were enriched in gene lists, suggesting a potential correlation with HBV-related HCC. The upregulated large intergenic noncoding RNA ULK4P2 was physically combined with enhancer of zeste homolog 2. Therefore, the lncRNAs may participate in regulating HBV-related HCC. CONCLUSION: lncRNAs play important roles in HCC, future studies should verify whether large intergenic noncoding ULK4P2 functions by combining with enhancer of zeste homolog 2 in HCC.

Inhibition of 12-lipoxygenase reduces proliferation and induces apoptosis of hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND: 12-lipoxygenase (12-LOX) has been reported to be an important gene in cancer cell proliferation and survival, and tumor metastasis. However, its role in hepatocellular carcinoma (HCC) cells remains unknown. METHODS: Expression of 12-LOX was assessed in a diethyl-nitrosamine-induced rat HCC model, and in SMMC-7721, HepG2 and L-02 cells using immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). GST-π and Ki-67 were determined in vivo by immunohistochemical staining. Apoptosis was evaluated by TUNEL assay. Cell viability and apoptosis were determined by MTT assay and flow cytometry, respectively. Apoptosis-related proteins in SMMC-7721 and HepG2 cells were detected by Western blotting. RESULTS: Immunohistochemical staining and RT-PCR showed that 12-LOX was over-expressed in rat HCC and two HCC cell lines, while the expression was inhibited by baicalein, a specific inhibitor of 12-LOX. Baicalein inhibited cell proliferation and induced apoptosis in rat HCC and both cell lines in a dose- and time-dependent manner. Our in vivo study demonstrated that baicalein also reduced neoplastic nodules. Mechanistically, baicalein reduced Bcl-2 protein expression coupled with a slight increase of the expression of Bax and activation of caspase-3. Furthermore, baicalein inhibited the activation of ERK-1/2 (phosphorylated). Interestingly, the effects of baicalein were reversed by 12(S)-HETE, a metabolite of 12-LOX. CONCLUSIONS: Inhibition of 12-LOX leads to reduced numbers of HCC cells, partially caused by increased apoptosis. 12-LOX may be a potential molecular target for HCC prevention and treatment.

5-Lipoxygenase contributes to the progression of hepatocellular carcinoma.

5-Lipoxygenase (5-LOX) has been implicated in the development and progression of lung, pancreatic and esophageal cancers. However, its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore the role of 5-LOX in the pathogenesis of HCC. The expression of 5-LOX was detected in human HCC, HepG2 cells and diethylnitrosamine (DEN)-induced rat HCC using immunohistochemistry (IHC) staining or reverse transcriptase-polymerase chain reaction. Apoptosis in rat HCC was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Cell viability and apoptosis were determined in HepG2 cells by MTT assay and flow cytometry, respectively. IHC staining showed that the 5-LOX protein was highly expressed in human HCC, HepG2 cells and rat HCC, but not in the normal liver tissues. 5-LOX mRNA expression in human and rat HCC was also significantly increased compared to normal liver tissues. Zileuton, a 5-LOX inhibitor, reduced the nodule incidence and the mean number of nodules per nodule-bearing liver in DEN-induced rats. Further study using TUNEL assay showed that zileuton treatment induced apoptosis in the liver as the result of inhibition on 5-LOX levels. This result is consistent with our observation of significantly higher apoptotic indices in rats treated with DEN/zileuton, which were significantly higher compared to those from the control groups. In addition, zileuton reduced cell viability and induced apoptosis in a concentration- and time-dependent manner as detected using HepG2 cells in our in vitro analysis. In conclusion, 5-LOX is expressed in HCC, and the inhibition of 5-LOX blocks the development of HCC via the induction of apoptosis in tumor cells.

Hepatic oval cells activated by hepatocyte apoptosis in diethylnitrosamine-induced rat liver cirrhosis.

OBJECTIVE: To investigate whether hepatic oval cells are activated in diethylnitrosamine (DEN)-induced rat liver cirrhosis, and to explore its mechanism. METHODS: Liver cirrhosis was induced in rats (n=8) by weekly intraperitoneal injections of DEN at a dose of 50mg/kg body weight for 12 weeks followed by a 2-week wash out period. Rats (n=5) that received isovolumic vehicle served as the control group. Liver pathology was examined. Apoptotic hepatocytes were identified and quantified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Oval cells were detected using immunohistochemical staining for pyruvate kinase type M2 (M2PK) and cytokeratin 19 (CK19). The work was carried out at Renmin Hospital of Wuhan University, Wuhan, Hubei, China from February to December 2009. RESULTS: Liver cirrhosis developed in rats subjected to DEN administration. The TUNEL and morphology assay showed that a substantial number of hepatocytes underwent apoptosis. The apoptotic index in rats subjected to DEN administration (0.75 0.15) was much higher than normal control rats (0.10 0.05). Both CK19 and M2PK were moderately expressed in the rat liver cirrhosis, and the expression was dispersed or forming small cords in the liver; but the expression was hardly detected in the liver tissue of normal control rats. CONCLUSION: In the DEN-induced rat liver cirrhosis, oval cells are activated and stimulated to proliferation, the mechanism of which may be related to substantial hepatocyte apoptosis in the model.