Predictive value of positive lymph node ratio in patients with locally advanced gastric remnant cancer.

BACKGROUND: Traditional lymph node stage (N stage) has limitations in advanced gastric remnant cancer (GRC) patients; therefore, establishing a new predictive stage is necessary. AIM: To explore the predictive value of positive lymph node ratio (LNR) according to clinicopathological characteristics and prognosis of locally advanced GRC. METHODS: Seventy-four patients who underwent radical gastrectomy and lymphadenectomy for locally advanced GRC were retrospectively reviewed. The relationship between LNR and clinicopathological characteristics was analyzed. The survival analysis was performed using Kaplan-Meier survival curves and Cox regression model. RESULTS: Number of metastatic LNs, tumor diameter, depth of tumor invasion, Borrmann type, serum tumor biomarkers, and tumor-node-metastasis (TNM) stage were correlated with LNR stage and N stage. Univariate analysis revealed that the factors affecting survival included tumor diameter, anemia, serum tumor biomarkers, vascular or neural invasion, combined resection, LNR stage, N stage, and TNM stage (all P < 0.05). The median survival time for those with LNR0, LNR1, LNR2 and LNR3 stage were 61, 31, 23 and 17 mo, respectively, and the differences were significant (P = 0.000). Anemia, tumor biomarkers and LNR stage were independent prognostic factors for survival in multivariable analysis (all P < 0.05). CONCLUSION: The new LNR stage is uniquely based on number of metastatic LNs, with significant prognostic value for locally advanced GRC, and could better differentiate overall survival, compared with N stage.

ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

Faecalibacterium prausnitzii Abrogates Intestinal Toxicity and Promotes Tumor Immunity to Increase the Efficacy of Dual CTLA4 and PD-1 Checkpoint Blockade.

Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy; however, their application is limited by the occurrence of immune-related adverse events. The gut microbiota plays important roles in the response to and toxicity of immunotherapy and Faecalibacterium prausnitzii (F. prausnitzii) has been shown to possess immunomodulatory potential. Here, we found that patients receiving ICIs who developed colitis had a lower abundance of F. prausnitzii. In vivo, immunocompetent mice administered with dextran sodium sulfate and immunodeficient NSG mice with human peripheral blood mononuclear cell transfer were treated with ICIs to study ICI-induced colitis. Dual CTLA4 and PD-1 blockade exacerbated autoimmune colitis, activated an inflammatory response, and promoted myeloid cell infiltration, with higher percentages of macrophages, dendritic cells, monocytes, and neutrophils. F. prausnitzii administration mitigated the exacerbated colitis induced by ICIs. Concomitantly, F. prausnitzii enhanced the antitumor immunity elicited by ICIs in tumor-bearing mice while abrogating colitis. In addition, administration of F. prausnitzii increased gut microbial alpha diversity and modulated the microbial composition, increasing a subset of gut probiotics and decreasing potential gut pathogens. F. prausnitzii abundance was reduced in mice that developed ICI-associated colitis. Together, this study shows that F. prausnitzii administration ameliorates ICI-induced colitis, reshapes the gut microbial composition, and enhances the antitumor activity of immunotherapy. SIGNIFICANCE: F. prausnitzii alleviates colitis while enhancing the tumor-suppressive effects of immune checkpoint blockade, indicating that supplementation with F. prausnitzii could be a treatment strategy to mitigate immunotherapy toxicity in patients with cancer.

Fusobacterium nucleatum-derived succinic acid induces tumor resistance to immunotherapy in colorectal cancer.

Immune checkpoint blockade therapy with anti-PD-1 monoclonal antibody (mAb) is a treatment for colorectal cancer (CRC). However, some patients remain unresponsive to PD-1 blockade. The gut microbiota has been linked to immunotherapy resistance through unclear mechanisms. We found that patients with metastatic CRC who fail to respond to immunotherapy had a greater abundance of Fusobacterium nucleatum and increased succinic acid. Fecal microbiota transfer from responders with low F. nucleatum, but not F. nucleatum-high non-responders, conferred sensitivity to anti-PD-1 mAb in mice. Mechanistically, F. nucleatum-derived succinic acid suppressed the cGAS-interferon-ß pathway, consequently dampening the antitumor response by limiting CD8+ T cell trafficking to the tumor microenvironment (TME) in vivo. Treatment with the antibiotic metronidazole reduced intestinal F. nucleatum abundance, thereby decreasing serum succinic acid levels and resensitizing tumors to immunotherapy in vivo. These findings indicate that F. nucleatum and succinic acid induce tumor resistance to immunotherapy, offering insights into microbiota-metabolite-immune crosstalk in CRC.

Hairy gene homolog increases nasopharyngeal carcinoma cell stemness by upregulating Bmi-1.

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is overexpressed in various cancer types. We found that Bmi-1 mRNA levels were elevated in nasopharyngeal carcinoma (NPC) cell lines. In immunohistochemical analyses, high Bmi-1 levels were observed in not only 5 of 38 non-cancerous nasopharyngeal squamous epithelial biopsies, but also in 66 of 98 NPC specimens (67.3%). High Bmi-1 levels were detected more frequently in T3-T4, N2-N3 and stage III-IV NPC biopsies than in T1-T2, N0-N1 and stage I-II NPC samples, indicating that Bmi-1 is upregulated in advanced NPC. In 5-8F and SUNE1 NPC cells, stable depletion of Bmi-1 using lentiviral RNA interference greatly suppressed cell proliferation, induced G1-phase cell cycle arrest, reduced cell stemness and suppressed cell migration and invasion. Likewise, knocking down Bmi-1 inhibited NPC cell growth in nude mice. Both chromatin immunoprecipitation and Western blotting assays demonstrated that Hairy gene homolog (HRY) upregulated Bmi-1 by binding to its promoter, thereby increasing the stemness of NPC cells. Immunohistochemistry and quantitative real-time PCR analyses revealed that HRY expression correlated positively with Bmi-1 expression in a cohort of NPC biopsies. These findings suggested that HRY promotes NPC cell stemness by upregulating Bmi-1, and that silencing Bmi-1 can suppress NPC progression.

A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells.

To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.

m6A demethylase ALKBH5 suppression contributes to esophageal squamous cell carcinoma progression.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant gastrointestinal cancer with a high recurrence rate and poor prognosis. Although N6-methyladenosine (m6A), the most abundant epitranscriptomic modification of mRNAs, has been implicated in several cancers, little is known about its participation in ESCC progression. We found reduced expression of ALKBH5, an m6A demethylase, in ESCC tissue specimens with a more pronounced effect in T3-T4, N1-N3, clinical stages III-IV, and histological grade III tumors, suggesting its involvement in advanced stages of ESCC. Exogenous expression of ALKBH5 inhibited the in vitro proliferation of ESCC cells, whereas depletion of endogenous ALKBH5 markedly enhanced ESCC cell proliferation in vitro. This suggests ALKBH5 exerts anti-proliferative effects on ESCC growth. Furthermore, ALKBH5 overexpression suppressed tumor growth of Eca-109 cells in nude mice; conversely, depletion of endogenous ALKBH5 accelerated tumor growth of TE-13 cells in vivo. The growth-inhibitory effects of ALKBH5 overexpression are partly attributed to a G1-phase arrest. In addition, ALKBH5 overexpression reduced the in vitro migration and invasion of ESCC cells. Altogether, our findings demonstrate that the loss of ALKBH5 expression contributes to ESCC malignancy.

Liver-specific over-expression of Cripto-1 in transgenic mice promotes hepatocyte proliferation and deregulated expression of hepatocarcinogenesis-related genes and signaling pathways.

In this study, we investigated the role of embryonic gene Cripto-1 (CR-1) in hepatocellular carcinoma (HCC) using hepatocyte-specific CR-1-overexpressing transgenic mice. The expression of truncated 1.7-kb CR-1 transcript (SF-CR-1) was significantly higher than the full-length 2.0-kb CR-1 transcript (FL-CR-1) in a majority of HCC tissues and cell lines. Moreover, CR-1 mRNA and protein levels were significantly higher in HCC tissues than adjacent normal liver tissues. Hepatocyte-specific over-expression of CR-1 in transgenic mice enhanced hepatocyte proliferation after 2/3 partial hepatectomy (2/3 PHx). CR-1 over-expression significantly increased in vivo xenograft tumor growth of HCC cells in nude mice and in vitro HCC cell proliferation, migration, and invasion. CR-1 over-expression in the transgenic mouse livers deregulated HCC-related signaling pathways such as AKT, Wnt/ß-catenin, Stat3, MAPK/ERK, JNK, TGF-ß and Notch, as well as expression of HCC-related genes such as CD5L, S100A8, S100A9, Timd4, Orm2, Orm3, PDK4, DMBT1, G0S2, Plk2, Plk3, Gsta1 and Gsta2. However, histological signs of precancerous lesions, hepatocyte dysplasia or HCC formation were not observed in the livers of 3-, 6- or 8-month-old hepatocyte-specific CR-1-overexpressing transgenic mice. These findings demonstrate that liver-specific CR-1 overexpression in transgenic mice deregulates signaling pathways and genes associated with HCC.

Diagnostic value of different color ultrasound diagnostic method in endometrial lesions.

BACKGROUND: Endometrial lesions include endometrial cancer and inferior fibroids. Among them, endometrial cancer as a malignant tumor seriously endangers the life and health of patients. Ultrasonography is an important means of diagnosing female reproductive system diseases, and it is of critical value for the early diagnosis of endometrial cancer. However, different ultrasound inspection programs have achieved different results. It is of great significance to choose a suitable inspection program. AIM: To explore the diagnostic efficacy of different ultrasonic examination methods in clinical endometrial lesions. METHODS: The 140 patients with endometrial lesions who were treated in our hospital from April 2018 to October 2019 were used as the research subjects. All patients underwent transvaginal color ultrasound and transabdominal color ultrasound. We compared the diagnostic coincidence and image display effects of the two different examination methods, and the endometrial thickness, blood flow, uterine effusion and resistance index of different diseases were observed by transvaginal color ultrasound. RESULTS: The diagnostic coincidence rate of all types of diseases of transvaginal color ultrasound was significantly higher than that of transabdominal color ultrasound (P = 0.001, 0.005, 0.001 and 0.001). In addition, the excellent and good rate of image display of transvaginal color ultrasound was higher than that of transabdominal color ultrasound (P = 0.001). There were significant differences in endometrial thickness in patients with different types of endometrial lesions through the transvaginal color examination (P = 0.001). The incidence rate of uterine effusion in patients with endometrial carcinoma was significantly higher than that in patients with other types of endometrial lesions (P = 0.001), and the rate of the blood flow was the highest (P = 0.001). The comparison of blood flow resistance index indicated that the blood flow resistance index in endometrial cancer patients was the lowest, which shows that the difference was statistically significant (P = 0.001). CONCLUSION: The overall diagnostic efficacy of transvaginal color ultrasound in the clinical diagnosis of endometrial lesions is better than that of transabdominal color ultrasound, which held higher diagnostic coincidence rate and image display effect. There were significant differences in the thickness of the endometrium and the blood flow in different types of lesions.

MST4 negatively regulates the EMT, invasion and metastasis of HCC cells by inactivating PI3K/AKT/Snail1 axis.

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a poor prognosis due to the high incidence of invasion and metastasis-related progression. However, the underlying mechanism remains elusive, and valuable biomarkers for predicting invasion, metastasis, and poor prognosis of HCC patients are still lacking. Methods: Immunohistochemistry (IHC) was performed on HCC tissues (n = 325), and the correlations between MST4 expression of the clinical HCC tissues, the clinicopathologic features, and survival were further evaluated. The effects of MST4 on HCC cell migratory and invasive properties in vitro were evaluated by Transwell and Boyden assays. The intrahepatic metastasis mouse model was established to evaluate the HCC metastasis in vivo. The PI3K inhibitor, LY294002, and a specific siRNA against Snail1 were used to investigate the roles of PI3K/AKT pathway and Snail1 in MST4-regulated EMT, migration, and invasion of HCC cells, respectively. Results: In this study, by comprehensively analyzing our clinical data, we discovered that low MST4 expression is highly associated with the advanced progression of HCC and serves as a prognostic biomarker for HCC patients of clinical-stage III-IV. Functional studies indicate that MST4 inactivation induces epithelial-to-mesenchymal transition (EMT) of HCC cells, promotes their migratory and invasive potential in vitro, and facilitates their intrahepatic metastasis in vivo, whereas MST4 overexpression exhibits the opposite phenotypes. Mechanistically, MST4 inactivation elevates the expression and nuclear translocation of Snail1, a key EMT transcription factor (EMT-TF), through the PI3K/AKT signaling pathway, thus inducing the EMT phenotype of HCC cells, and enhancing their invasive and metastatic potential. Moreover, a negative correlation between MST4 and p-AKT, Snail1, and Ki67 and a positive correlation between MST4 and E-cadherin were determined in clinical HCC samples. Conclusions: Our findings indicate that MST4 suppresses EMT, invasion, and metastasis of HCC cells by modulating the PI3K/AKT/Snail1 axis, suggesting that MST4 may be a potential prognostic biomarker for aggressive and metastatic HCC.

The Yunnan national medicine Maytenus compound inhibits the proliferation of hepatocellular carcinoma (HCC) by suppressing the activation of the EGFR-PI3K-AKT signaling pathway.

Objective: To investigate the effects of Maytenus compound on the proliferation of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to explore the underlying mechanism. Methods: The half maximal inhibitory concentration (IC50) values of Maytenus compound in HepG2 and BEL-7402 cells were determined by the MTS assay. HepG2 and BEL-7402 cells were treated with different concentrations of Maytenus compound. MTS assays, colony formation assays and cell cycle analyses were performed to clarify the inhibitory effect of Maytenus compound on the proliferation of HepG2 and BEL-7402 cells in vitro. After subcutaneous injection of HepG2 cells, nude mice were randomly divided into a vehicle control group and a drug intervention group, which were intragastrically administered ddH2O or Maytenus compound, respectively. The inhibitory effect of Maytenus compound on the proliferation of HepG2 cells in vivo was analyzed using subcutaneous tumor growth curves, tumor weight, the tumor growth inhibition rate and the immunohistochemical detection of BrdU-labeled cells in S phase. The organ toxicity of Maytenus compound was initially evaluated by comparing the weight difference and organ index of the two groups of nude mice. The main proteins in the EGFR-PI3K-AKT signaling pathway were detected by Western blot after Maytenus compound intervention in vivo and in vitro. Results: Maytenus compound showed favorable antiproliferation activity against HepG2 and BEL-7402 cells with IC50 values of 79.42±11.71 µg/mL and 78.48±8.87 µg/mL, respectively. MTS assays, colony formation assays and cell cycle analyses showed that Maytenus compound at different concentration gradients within the IC50 concentration range significantly suppressed the proliferation of HepG2 and BEL-7402 cells in vitro and inhibited cell cycle progression from G1 to S phase. Additionally, Maytenus compound, at an oral dose of 2.45 g/kg, dramatically inhibited, without obvious organ toxicity, the proliferation of subcutaneous tumors formed by HepG2 cells in nude mice. In addition, the tumor growth inhibition rate for Maytenus compound was 66.94%. Furthermore, Maytenus compound inhibited the proliferation of liver orthotopic transplantation tumors in nude mice. Western blot analysis showed that Maytenus compound significantly downregulated the expression of p-EGFR, p-PI3K, and p-AKT and upregulated the expression of p-FOXO3a, p27, and p21 in vivo and in vitro. Conclusion: Maytenus compound significantly inhibited the proliferation of HCC cells in vitro and in vivo. The downregulation of the EGFR-PI3K-AKT signaling pathway and subsequent inhibition of cell cycle progression from G1 to S phase is one of the possible mechanisms. Maytenus compound has a high tumor growth inhibition rate and has no obvious organ toxicity, which may make it a potential anti-HCC drug, but the results from this study need to be confirmed by further clinical trials in HCC patients.

MST4 inhibits human hepatocellular carcinoma cell proliferation and induces cell cycle arrest via suppression of PI3K/AKT pathway.

Objective: MST4 has exhibited functions in regulating cell polarity, Golgi apparatus, cell migration, and cancer. Mechanistically, it affects the activity of p-ERK, Hippo-YAP pathway and autophagy. The aim of this study is to further examine the functions of MST4 in hepatocellular carcinoma (HCC) and the underlying mechanism. Methods: The expression level of MST4 in HCC and noncancer adjacent liver tissues was determined by qRT-PCR and immunohistochemistry staining. Wild-type MST4 (MST4) and a dominant-negative mutant of MST4 (dnMST4) were overexpressed in HCC cell lines, respectively. CCK-8 assay, EdU incorporation assay, and soft agar assay were used to determine cell proliferation in vitro. The xenograft mouse model was employed to determine HCC cell growth in vivo. Cell cycle analysis was performed by PI staining and flow cytometry. The expression of key members in PI3K/AKT pathway was detected by Western blot analysis. Results: In our study, we reported new evidence that MST4 was frequently down-regulated in HCC tissues. Gain-of-function and loss-of-function experiments demonstrated that MST4 negatively regulated in vitro HCC cell proliferation. Additionally, MST4 overexpression suppressed Bel-7404 cell tumor growth in nude mice. Further experiments revealed that the growth-inhibitory effect of MST4 overexpression was partly due to a G1-phase cell cycle arrest. Importantly, mechanistic investigations suggested that dnMST4 significantly elevated the phosphorylation levels of key members of PI3K/AKT pathway, and the selective PI3K inhibitor LY294002 can reverse the proliferation-promoting effect of dnMST4. Conclusions: Overall, our results provide a new insight into the clinical significance, functions and molecular mechanism of MST4 in HCC, suggesting that MST4 might have a potential therapeutic value in the HCC clinical treatment.

Loss of PDK4 expression promotes proliferation, tumorigenicity, motility and invasion of hepatocellular carcinoma cells.

Although the roles and underlying mechanisms of other PDK family members (i.e., PDK1, PDK2 and PDK3) in tumor progression have been extensively investigated and are well understood, the functions and underlying molecular mechanisms of pyruvate dehydrogenase kinase 4 (PDK4) in the tumorigenesis and progression of various cancers [including hepatocellular carcinoma (HCC)] remain largely unknown. In this study, we examined the expression profile of PDK4 in HCC clinical tissue specimens and the roles of PDK4 in the proliferation, tumorigenicity, motility and invasion of HCC cells. The immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) results revealed that PDK4 was significantly downregulated in the cohort of HCC clinical specimens. Additionally, PDK4 protein was found in both the nucleus and cytoplasm of HCC cells based on an immunofluorescence (ICC) assay, and PDK4 protein was also found in the nucleus and cytoplasm of cancer cells contained in HCC clinical specimens based on IHC. The CCK-8 assay and cell colony formation assay demonstrated that stable depletion of endogenous PDK4 by lentivirus-mediated RNA interference (RNAi) markedly promoted the proliferation of HCC cell lines (i.e., BEL-7402 and BEL-7404 cells) in vitro, while PDK4 silencing significantly enhanced the tumorigenic ability of BEL-7404 cells in vivo. In addition to enhance proliferation and tumorigenesis induced by PDK4 silencing, additional studies demonstrated that knockdown of PDK4 led to increase migration and invasion of BEL-7402 and BEL-7404 cells in vitro. Taken together, these findings suggest that the loss of PDK4 expression contributes to HCC malignant progression.

miR-19 regulates the expression of interferon-induced genes and MHC class I genes in human cancer cells.

MicroRNA-19 (miR-19) is identified as the key oncogenic component of the miR-17-92 cluster. When we explored the functions of the dysregulated miR-19 in lung cancer, microarray-based data unexpectedly demonstrated that some immune and inflammatory response genes (i.e., IL32, IFI6 and IFIT1) were generally down-regulated by miR-19 overexpression in A549 cells, which prompted us to fully investigate whether the miR-19 family (i.e., miR-19a and miR-19b-1) was implicated in regulating the expression of immune and inflammatory response genes in cancer cells. In the present study, we observed that miR-19a or miR-19b-1 overexpression by miRNA mimics in the A549, HCC827 and CNE2 cells significantly downregulated the expression of interferon (IFN)-regulated genes (i.e., IRF7, IFI6, IFIT1, IFITM1, IFI27 and IFI44L). Furthermore, the ectopic miR-19a or miR-19b-1 expression in the A549, HCC827, CNE2 and HONE1 cells led to a general downward trend in the expression profile of major histocompatibility complex (MHC) class I genes (such as HLA-B, HLA-E, HLA-F or HLA-G); conversely, miR-19a or miR-19b-1 inhibition by the miRNA inhibitor upregulated the aforementioned MHC Class I gene expression, suggesting that miR-19a or miR-19b-1 negatively modulates MHC Class I gene expression. The miR-19a or miR-19b-1 mimics reduced the expression of interleukin (IL)-related genes (i.e., IL1B, IL11RA and IL6) in the A549, HCC827, CNE2 or HONE1 cells. The ectopic expression of miR-19a or miR-19b-1 downregulated IL32 expression in the A549 and HCC827 cells and upregulated IL32 expression in CNE2 and HONE1 cells. In addition, enforced miR-19a or miR-19b-1 expression suppressed IL-6 production by lung cancer and nasopharyngeal carcinoma (NPC) cells. Taken together, these findings demonstrate, for the first time, that miR-19 can modulate the expression of IFN-induced genes and MHC class I genes in human cancer cells, suggesting a novel role of miR-19 in linking inflammation and cancer, which remains to be fully characterized.

Dulcitol suppresses proliferation and migration of hepatocellular carcinoma via regulating SIRT1/p53 pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) spreads further with continuance and increasing incidence due to its high-grade malignancy and metastasis. More effectual strategies on blocking proliferation and metastasis of cancer cells should be studied in HCC. Dulcitol, a natural product extracted from euonymus alatus, was reported that it could induce apoptosis of C6 glioma cells. However, the underlying mechanism of Dulcitol on HCC remains unclea. PURPOSE: In this study, we aimed to reveal the effect and potential mechanisms of Dulcitol on hepatocellular carcinoma in vitro and in vivo. Study design and methods The cell proliferation and apoptosis were evaluated by MTT, Ki-67 and Hoechst 33258/PI double staining. The migratory and invasive abilities of HepG2 cells were measured by wound-healing and transwell assays. Pathological changes of tumor tissue were observed by HE staining and IHC methods. The expression levels of protein were detected using Western Blot analysis. RESULTS: The results showed that Dulcitol inhibited HepG2 cells proliferation by down-regulating the protein expression of SIRT1, Bcl-2, along with up-regulating p53, acetylated-p53 (K382), cleaved-caspase9, cleaved-caspase3, Bax, and cytochrome c in a dose-dependent manner. Furthermore, Dulcitol surpressed the migration and invasion of HepG2 cells through decreasing the levels of MMP-2, uPA and MMP-9 and increasing E-cadherin associated with tumor invasion. In vivo, Dulcitol distinctly inhibited the growth of HepG2 cancer xenograft tumors via inhibiting SIRT1/p53 pathway. CONCLUSIONS: Our findings suggested that Dulcitol acted as a SIRT1 inhibitor, inducing apoptosis and inhibiting proliferation, migration and invasion of HepG2 cells and its modulatory mechanism seemed to be associated with regulation of MMPs, SIRT1/p53 pathways.

Loss of Cirbp expression is correlated with the malignant progression and poor prognosis in nasopharyngeal carcinoma.

Purpose: The correlation of cold-inducible RNA-binding protein (Cirbp) expression with clinicopathological features including patient prognosis in nasopharyngeal carcinoma (NPC) was investigated. Methods: The expression of Cirbp in NPC cell lines and tissue specimens was examined by qRT-PCR or immunohistochemistry (IHC). Results: Immunohistochemistry (IHC) results showed that high Cirbp expression was detected in 61 of 61 non-cancerous nasopharyngeal squamous epithelial biopsies, whereas the significantly reduced expression of Cirbp was observed in NPC specimens. In addition, IHC assay for Cirbp protein illustrated that the cells of 177 NPC samples and nasopharyngeal squamous epithlial cells displayed strong signals in nuclei and faint signals in cytoplasm, whereas Cirbp protein is mainly detected in the cell's cytoplasm in many other cancers. More importantly, TNM classification displayed that the low expression of Cirbp was more frequently observed in T3-T4, N2-N3, M1 and III-IV NPC biopsies, and undifferentiated carcinoma (UDC) than T1-T2, N0-N1, M0 and I-II tumors, and differentiated nonkeratinizing carcinoma (DNKC), suggesting that Cirbp loss is a key molecular event in advanced cases of NPC. Kaplan-Meier survival analysis indicated that NPC patients showing lower Cirbp expression had a significantly shorter overall survival time than those with high Cirbp expression. Multivariate analysis suggested that the level of Cirbp expression was an independent prognostic indicator for NPC survival. Finally, we revealed a significant positive association between Cirbp expression and E-cadherin, and a notable negative correlation between Cirbp expression and Ki67 labeling index in NPC biopsies. Conclusion: Collectively, these findings demonstrate that loss of Cirbp expression is correlated with malignant progression and poor prognosis in NPC.

miR-26a promotes hepatocellular carcinoma invasion and metastasis by inhibiting PTEN and inhibits cell growth by repressing EZH2.

A previous study revealed that therapeutic miR-26a delivery suppresses tumorigenesis in a murine liver cancer model, whereas we found that forced miR-26a expression increased hepatocellular carcinoma (HCC) cell migration and invasion, which prompted us to characterize the causes and mechanisms underlying enhanced invasion due to ectopic miR-26a expression. Gain-of-function and loss-of-function experiments demonstrated that miR-26a promoted migration and invasion of BEL-7402 and HepG2 cells in vitro and positively modulated matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, and MMP-10 expression. In addition, exogenous miR-26a expression significantly enhanced the metastatic ability of HepG2 cells in vivo. miR-26a negatively regulated in vitro proliferation of HCC cells, and miR-26a overexpression suppressed HepG2 cell tumor growth in nude mice. Further studies revealed that miR-26a inhibited cell growth by repressing the methyltransferase EZH2 and promoted cell migration and invasion by inhibiting the phosphatase PTEN. Furthermore, PTEN expression negatively correlated with miR-26a expression in HCC specimens from patients with and without metastasis. Thus, our findings suggest for the first time that miR-26a promotes invasion/metastasis by inhibiting PTEN and inhibits cell proliferation by repressing EZH2 in HCC. More importantly, our data also suggest caution if miR-26a is used as a target for cancer therapy in the future.

Direct conversion of pig fibroblasts to chondrocyte-like cells by c-Myc.

Unexpectedly, we found that c-Myc-expressing porcine embryonic fibroblasts (PEFs) subcutaneously implanted into nude mice formed cartilage-like tissues in vivo, while previous studies revealed the direct conversion of mouse and human somatic cells into chondrocytes by the combined use of several defined factors, including c-Myc, which prompted us to explore whether PEFs can be reprogrammed to become pig induced chondrocyte-like cells (piCLCs) via ectopic expression of c-Myc alone. In this study, c-Myc-expressing PEFs, designated piCLCs, which exhibited a significantly enhanced proliferation ability in vitro, displayed a chondrogenic phenotypes in vitro, as shown by the cell morphology, toluidine blue staining, alcian blue staining and chondrocyte marker gene expression. Additionally, piCLCs with a polygonal chondrocyte-like morphology were readily and efficiently converted from PEFs by enforced c-Myc expression within 10 days, while piCLCs maintained the chondrocytic phenotype and normal karyotype during long-term subculture. piCLC-derived single clones with a chondrogenic phenotype in vitro exhibited homogeneity in cell morphology and staining intensity compared with mixed piCLCs. Although the mixtures of cartilaginous tissues and tumorous tissues accounted for ~12% (6/51) of all xenografts (51), piCLCs generated stable, homogenous, hyaline cartilage-like tissues without tumour formation at 45 out of the 51 injected sites when subcutaneously injected into nude mice. The hyaline cartilage-like tissues remained for at least 16 weeks. Taken together, these findings demonstrate for the first time the direct induction of chondrocyte-like cells from PEFs with only c-Myc.

[Analysis on prescription rules of Chen Baogui's prescriptions for treating epigastric fullness based on data mining].

To summary and analyze the prescription rules of Professor Chen Baogui, a famous traditional Chinese medicine (TCM) doctor, for treating epigastric fullness. Professor Chen Baogui's prescriptions for treating epigastric fullness were collected and the treatment data were input into traditional Chinese medicine inheritance support system (TCMISS) to analyze the rules of the prescriptions by using data mining methods. Based on the screened 214 cases, the treatment experience of Professor Chen Baogui for treating epigastric fullness was summarized and analyzed. It was found that Professor Chen gave priority to recuperation of Qi activity. The results of four properties and five tastes showed Professor Chen's medication compatibility rules: one was simultaneous use of cold and warm drugs, and the other was simultaneous use of pungent drugs for dispersion and bitter drugs for purgation. In drug use, the basic prescriptions had the efficacy of promoting Qi circulation and regulating viscera function, additionally with the drugs with functions of eliminating digestion and inducing stagnation, activating blood circulation to dissipate blood stasis, replenishing Qi and nourishing Yin, tranquilizing mind, strengthening muscles and bones according to the TCM syndrome type. The clinical experience of Professor Chen for treating epigastric fullness was objectively summarized with the help of TCMISS, which was significant for analyzing and inheriting academic thinking and medication experience from famous TCM doctors.

Influences of adding negative couplings between cliques of Kuramoto-like oscillators.

We study the dynamics in a clustered network of coupled oscillators by considering positive and negative coupling schemes. Second order oscillators can be interpreted as a model of consumers and generators working in a power network. Numerical results indicate that coupling strategies play an important role in the synchronizability of the clustered power network. It is found that the synchronizability can be enhanced as the positive intragroup connections increase. Meanwhile, when the intragroup interactions are positive and the probability p that two nodes belonging to different clusters are connected is increased, the synchronization has better performance. Besides, when the intragroup connections are negative, it is observed that the power network has poor synchronizability as the probability p increases. Our simulation results can help us understand the collective behavior of the power network with positive and negative couplings.