Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Development of an integrated and project-based laboratory course in upper-level biochemistry and molecular biology.

An integrated and projected-based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project-based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning "genetic manipulation" and "recombinant DNA technology" to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease. Students could master applied and advanced techniques, such as cell culture, transfection, inducing exogenous fusion protein expression, purifying protein and its concentration assay, quantitative polymerase chain reaction, and western bot analysis. This laboratory exercise links laboratory practices with the methods of current basic research. Students need to complete the experimental design report and laboratory report, which could be advantageous for improving their ability to write lab summaries and scientific papers in the future. The reliability and validity analyses were conducted on the questionnaire, and we examined students' satisfaction with the course and their gains from the course. The student feedback was generally positive, indicating that the exercise helped consolidate theoretical knowledge, increase scientific research enthusiasm, and provide a powerful tool to be a better person and make informed decisions.

Tea domain transcription factor TEAD4 mitigates TGF-ß signaling and hepatocellular carcinoma progression independently of YAP.

Tea domain transcription factor 4 (TEAD4) plays a pivotal role in tissue development and homeostasis by interacting with Yes-associated protein (YAP) in response to Hippo signaling inactivation. TEAD4 and YAP can also cooperate with transforming growth factor-ß (TGF-ß)-activated Smad proteins to regulate gene transcription. Yet, it remains unclear whether TEAD4 plays a YAP-independent role in TGF-ß signaling. Here, we unveil a novel tumor suppressive function of TEAD4 in liver cancer via mitigating TGF-ß signaling. Ectopic TEAD4 inhibited TGF-ß-induced signal transduction, Smad transcriptional activity, and target gene transcription, consequently suppressing hepatocellular carcinoma cell proliferation and migration in vitro and xenograft tumor growth in mice. Consistently, depletion of endogenous TEAD4 by siRNAs enhanced TGF-ß signaling in cancer cells. Mechanistically, TEAD4 associates with receptor-regulated Smads (Smad2/3) and Smad4 in the nucleus, thereby impairing the binding of Smad2/3 to the histone acetyltransferase p300. Intriguingly, these negative effects of TEAD4 on TGF-ß/Smad signaling are independent of YAP, as impairing the TEAD4-YAP interaction through point mutagenesis or depletion of YAP and/or its paralog TAZ has little effect. Together, these results unravel a novel function of TEAD4 in fine tuning TGF-ß signaling and liver cancer progression in a YAP-independent manner.

The prognostic implications and tumor-suppressive functions of CYR61 in estrogen receptor-positive breast cancer.

Due to the therapeutic resistance of endocrine therapy and the limited efficacy of immune checkpoint inhibitors in estrogen receptor (ER)-positive breast cancer (BRCA), there is an urgent need to develop novel prognostic markers and understand the regulation of the tumor immune microenvironment (TIME). As a matricellular protein, CYR61 has been shown to either promote or suppress cancer progression depending on cancer types. However, how CYR61 functions in ER-positive BRCA remains elusive. In this study, we comprehensively analyzed the expression of CYR61 in BRCA based on the TCGA and METABRIC databases. Our findings showed that the expression of CYR61 is downregulated in different subtypes of BRCA, which is associated with elevated promoter methylation levels and predicts bad clinical outcomes. By comparing the high or low CYR61 expression groups of ER-positive BRCA patients, we found that CYR61 is intimately linked to the expression of genes involved in tumor-suppressive pathways, such as the TGF-ß and TNF signaling pathways, and genes related to cytokine-receptor interaction that may regulate cancer immunity. Moreover, reduced CYR61 expression is associated with an altered TIME that favors cancer progression. Finally, experimental analyses ascertained that CYR61 is downregulated in clinical BRCA tissues compared to matched normal breast tissues. Furthermore, CYR61 is able to impede the proliferation and colony formation of ER-positive BRCA cells. In summary, our study reveals that CYR61 could serve as a novel prognostic marker for ER-positive BRCA, and function as an inhibitor of cancer progression by both acting on cancer cells and remodeling the TIME.

Innovations in education of the medical molecular biology curriculum during the COVID-19 pandemic in China.

The COVID-19 pandemic is a huge challenge to education systems. Most governments around the world have temporarily closed schools, universities, and colleges. At the same time, teachers and students are encouraged to use the online and distance learning programs and platforms as an alternative. In the present study, we proposed a series of innovative solutions in Medical Molecular Biology education during the COVID-19 pandemic in China, including a flipped classroom model, live streaming course, chat Apps, and scientific papers on COVID-19 as additional learning material. Our results demonstrated that these innovations not only help teachers to maintain the teaching process as usual but also be useful for protecting students from psychological trauma. Our study indicates that online education with a well-designed workflow for conducting provides an alternative approach for teachers to maintain quality education during the onset of the emerging crisis.

KLF2 inhibits TGF-ß-mediated cancer cell motility in hepatocellular carcinoma.

Feedback regulation plays a pivotal role in determining the intensity and duration of TGF-ß signaling and subsequently affecting the pathophysiological roles of TGF-ß, including those in liver malignancy. KLF2, a member of the Krüppel-like factor (KLF) family transcription factors, has been implicated in impeding hepatocellular carcinoma (HCC) development. However, the underlying molecular mechanisms are not fully understood. In the present study, we found that TGF-ß stimulates the expression of KLF2 gene in several HCC cell lines. KLF2 protein is able to inhibit TGF-ß/Smad signaling in HCC cells as assessed by luciferase reporter assay. Further studies indicated that KLF2 inhibits the transcriptional activity of Smad2/3 and Smad4 and ameliorates TGF-ß-induced target gene expression, therefore creating a novel negative feedback loop in TGF-ß signaling. Functionally, stably expression of KLF2 in HCCLM3 cells attenuated TGF-ß-induced cancer cell motility in wound-healing and transwell assays by interfering with TGF-ß-mediated upregulation of MMP2. Together, our results revealed that KLF2 protein has a tumor-suppressive function in HCC through a negative feedback loop over TGF-ß signaling.

Cancer-associated adipocyte-derived G-CSF promotes breast cancer malignancy via Stat3 signaling.

Adipocyte is the most predominant cell type in the tumor microenvironment of breast cancer and plays a pivotal role in cancer progression, yet the underlying mechanisms and functional mediators remain elusive. We isolated primary preadipocytes from mammary fat pads of human breast cancer patients and generated mature adipocytes and cancer-associated adipocytes (CAAs) in vitro. The CAAs exhibited significantly different gene expression profiles as assessed by transcriptome sequencing. One of the highly expressed genes in CAAs is granulocyte colony-stimulating factor (G-CSF). Treatment with recombinant human G-CSF protein or stable expression of human G-CSF in triple-negative breast cancer (TNBC) cell lines enhanced epithelial-mesenchymal transition, migration, and invasion of cancer cells, by activating Stat3. Accordantly, targeting G-CSF/Stat3 signaling with G-CSF-neutralizing antibody, a chemical inhibitor, or siRNAs for Stat3 could all abrogate CAA- or G-CSF-induced migration and invasion of breast cancer cells. The pro-invasive genes MMP2 and MMP9 were identified as target genes of G-CSF in TNBC cells. Furthermore, in human breast cancer tissues, elevated G-CSF expression in adipocytes is well correlated with activated Stat3 signal in cancer cells. Together, our results suggest a novel strategy to intervene with invasive breast cancers by targeting CAA-derived G-CSF.

Contextual Regulation of TGF-ß Signaling in Liver Cancer.

Primary liver cancer is one of the leading causes for cancer-related death worldwide. Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine that signals through membrane receptors and intracellular Smad proteins, which enter the nucleus upon receptor activation and act as transcription factors. TGF-ß inhibits liver tumorigenesis in the early stage by inducing cytostasis and apoptosis, but promotes malignant progression in more advanced stages by enhancing cancer cell survival, EMT, migration, invasion and finally metastasis. Understanding the molecular mechanisms underpinning the multi-faceted roles of TGF-ß in liver cancer has become a persistent pursuit during the last two decades. Contextual regulation fine-tunes the robustness, duration and plasticity of TGF-ß signaling, yielding versatile albeit specific responses. This involves multiple feedback and feed-forward regulatory loops and also the interplay between Smad signaling and non-Smad pathways. This review summarizes the known regulatory mechanisms of TGF-ß signaling in liver cancer, and how they channel, skew and even switch the actions of TGF-ß during cancer progression.

CXXC5: A novel regulator and coordinator of TGF-ß, BMP and Wnt signaling.

CXXC5 is a member of the CXXC-type zinc-finger protein family. Proteins in this family play a pivotal role in epigenetic regulation by binding to unmethylated CpG islands in gene promoters through their characteristic CXXC domain. CXXC5 is a short protein (322 amino acids in length) that does not have any catalytic domain, but is able to bind to DNA and act as a transcription factor and epigenetic factor through protein-protein interactions. Intriguingly, increasing evidence indicates that expression of the CXXC5 gene is controlled by multiple signaling pathways and a variety of transcription factors, positioning CXXC5 as an important signal integrator. In addition, CXXC5 is capable of regulating various signal transduction processes, including the TGF-ß, Wnt and ATM-p53 pathways, thereby acting as a novel and crucial signaling coordinator. CXXC5 plays an important role in embryonic development and adult tissue homeostasis by regulating cell proliferation, differentiation and apoptosis. In keeping with these functions, aberrant expression or altered activity of CXXC5 has been shown to be involved in several human diseases including tumourigenesis. This review summarizes the current understanding of CXXC5 as a transcription factor and signaling regulator and coordinator.

WeChat: An applicable and flexible social app software for mobile teaching.

The rapid development and popularization of smart phones and mobile internet has created a new social lifestyle, and correspondingly prompts the transformation of network teaching from desktop computer to mobile teaching. This article has compared the pros and cons of Tsinghua Education Online and WeChat official account (WOA) in fulfilling teaching functions. We also described the construction of WOA platform with the example of WOA-based teaching in biochemistry and molecular biology. The platform can establish nearly 75 menu catalogs and 2,250 items, which is capable for the publication of any types of teaching materials and information. The WOA teaching is well accepted and becomes popular in China due to the free, interactive, attractive, adaptable, portable, sustainable, and more participatory teaching styles. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):555-560, 2018.

Roles of the MST1-JNK signaling pathway in apoptosis of colorectal cancer cells induced by Taurine.

The aim of this study was to observe the impact of the mammalian sterile 20-like kinase 1-c-Jun N-terminal kinase (MST1-JNK) signaling pathway on apoptosis in colorectal cancer (CRC) cells induced by Taurine (Tau). Caco-2 and SW620 cells transfected with p-enhanced green fluorescent protein (EGFP)-MST1 or short interfering RNA (siRNA)-MST1 were treated with Tau for 48 h. Apoptosis was detected by flow cytometry, and the levels of MST1 and JNK were detected by western blotting. Compared with the control group, 80 mM Tau could significantly induce apoptosis of CRC cells, and the apoptotic rate increased with increasing Tau concentration (P < 0.01). Meanwhile, the protein levels of MST1 and phosphorylated (p)-JNK in Caco-2 cells increased significantly (P < 0.01). The apoptotic rate of the p-EGFP-MST1 plasmid-transfected cancer cells was significantly higher than that of the control group (P < 0.05); however, the apoptotic rate of the p-EGFP-MST1+Tau group was increased further (P < 0.01). Silencing the MST1 gene could decrease the apoptotic rate of cancer cells, and Tau treatment could reverse this decrease. Blocking the JNK signaling pathway significantly reduced the Tau-induced apoptotic rate of CRC cells. Thus, the MST1-JNK pathway plays an important role in Tau-induced apoptosis of CRC cells.

Effect of taurine on cell proliferation and apoptosis human lung cancer A549 cells.

To investigate the effects of taurine on cell proliferation and apoptosis, the human lung cancer A549 cell line and xenograft tumors in nude mice were used. The effects of taurine on cell proliferation and apoptosis were observed at time points of 24, 48 and 72 h after treatment using an MTT assay to detect the survival rate, and flow cytometry to detect the apoptotic rate. Western blot analysis was performed to examine the levels of p53 upregulated modulator of apoptosis (PUMA), BCL2, apoptosis regulator (Bcl-2) and BCL2-associated X, apoptosis regulator (Bax) in A549 cells. The level of PUMA, Bax and Bcl-2 proteins in the mouse xenograft tumors treated with taurine and/or exogenous PUMA were assessed by immunohistochemistry, with taurine suppressing the proliferation of the human lung cancer A549 cell line in a concentration-dependent manner, and it significantly enhanced the apoptosis rate at all concentrations. Taurine induced the significant upregulation of PUMA and Bax, but led to downregulation of Bcl-2. In comparison to the control group, taurine treatment markedly reduced the volume and weight of A549-derived xenograft tumors in nude mice. Expression of PUMA and Bax were upregulated in the xenograft tumors following taurine treatment, whereas Bcl-2 was downregulated. In addition, the inhibitory effect of taurine and exogenous PUMA on tumor growth was significantly higher than that of a single treatment of taurine or exogenous PUMA. It can therefore be concluded that taurine can inhibit cell proliferation of the human lung cancer A549 cell line and the growth of the xenograft tumors, whereas PUMA serves an important role in taurine-induced growth suppression.

Effect of taurine on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells.

The aim of the present study was to observe the effect and molecular mechanism of taurine (Tau) on the cell proliferation and apoptosis of human hepatocellular carcinoma (HHCC) HepG2 cells. HHCC HepG2 cells were used as target cells, and the cell survival rate was assessed using a multi-time-step method. The p53 upregulated modulator of apoptosis (PUMA) gene was transiently transfected by lipofection and subsequently silenced with specific small interfering (si)RNA. The cell apoptosis rate was detected by flow cytometry, and protein expression levels were analyzed with western blotting. Addition of 20-160 mM Tau was shown to have a significant inhibitory effect on cell proliferation, while promoting the induction of HHCC HepG2 cell apoptosis (P<0.05). Transfection of the PUMA gene significantly enhanced the ability of Tau to inhibit proliferation and induce apoptosis of HepG2 cells. In addition, transfection of the PUMA gene increased the protein expression of B-cell lymphoma-2-associated X and reduced the expression of B-cell lymphoma-2 (P<0.05). Silencing the PUMA gene with specific siRNA was demonstrated to significantly reduce the ability of Tau to inhibit proliferation and induce the apoptosis of HHCC HepG2 cells (P<0.01). Therefore, the PUMA gene was shown to have an important role in mechanism underlying the effect that Tau exerts on cell proliferation and apoptosis in HHCC HepG2 cells.

Mechanism of taurine-induced apoptosis in human colon cancer cells.

Taurine (Tau) has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anti-cancer effect is not well understood. PUMA (p53-upregulated modulator of apoptosis) plays an important role in the process of apoptosis induction in a variety of human tumor cells in both p53-dependent and -independent manners. However, whether PUMA is involved in the process of Tau-induced apoptosis in cancer cells has not been well studied. In the present study, we treated human colorectal cancer cells HT-29 (mutant p53) and LoVo (wild-type p53) with different concentrations of Tau, which led to the repression of cell proliferation and induction of apoptosis in both cell lines. Meanwhile, we also observed the increased expression of PUMA and high Bax/Bcl-2 ratios. To determine the role of PUMA in Tau-induced apoptosis, we used small interfering RNA interference to suppress PUMA expression. As a result, apoptosis was decreased in response to Tau treatment. All these results indicated that PUMA plays a critical role in Tau-induced apoptosis pathway in human colorectal cancer cells. Demonstration of the molecular mechanism involved in the anti-tumor effect of Tau may be useful in the therapeutic target selection for p53-deficient colorectal cancer.

Effect of Mst1 overexpression on the growth of human hepatocellular carcinoma HepG2 cells and the sensitivity to cisplatin in vitro.

Mammalian STE20-like kinase 1 (Mst1) is the mammalian homologue of Drosophila Hippo, a major inhibitor of cell proliferation in Drosophila. It ubiquitously encodes serine threonine kinase that belongs to the family of protein kinases related to yeast STE20, and is involved in cell proliferation, apoptosis, oncogenesis, and organ growth. Recent studies have shown that Mst1 has tumor-suppressor function, and the deletion or mutation of Mst1 is reported to be associated with tumorigenesis. To investigate the effect of overexpression of Mst1 on the growth of human liver cancer cell line HepG2 cells and the sensitivity to cisplatin in vitro, here we constructed recombinant eukaryotic expression vector pEGFP-N1-Mst1 containing Mst1 gene, and transiently transfected into HepG2 cells. The effects of Mst1 overexpression on the cell proliferation and apoptosis, the phosphorylation status of Yes-associated protein, and the mRNA transcript levels of connective tissue growth factor (CTGF), amphiregulin (AREG), and birc5 (Survivin) were determined. Results showed that overexpression of Mst1 inhibited cell proliferation, induced apoptosis of HepG2 cells, promoted YAP (Ser127) phosphorylation, and downregulated the mRNA expression of CTGF, AREG, and Survivin. We also investigated the relationship between the expression and cleavage of Mst1 and cisplatin-induced cell death. We found that Mst1 overexpression could induce cisplatin chemosensitivity, and cisplatin could promote the cleavage of Mst1 without affecting the expression of Mst1. Overall, our results indicated that Mst1 might be a promising anticancer target.

Effect of diallyl trisulfide on human ovarian cancer SKOV- 3/DDP cell apoptosis.

AIM: To investigate the effects of diallyl trisulfide (DT) on apoptosis of cisplatin (DDP)-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP), and the role of p53 upregulated modulator of apoptosis (PUMA). METHODS: SKOV-3/DDP cells were randomly divided into control, DT, DPP and DPP+DT groups, which were treated with DT or combined DT and DDP. All cells were incubated for 48 h. and apoptosis rates were assessed by flow cytometry. mRNA and protein expression of PUMA, Bax and Bcl-2 was determined by RT-PCR and Western blot assays, respectively. RESULTS: Compared with control group, the apoptosis rates of SKOV-3/DDP cells in DT groups were obviously increased, with dose-dependence (P < 0.05), the mRNA and protein expressions of PUMA, Bax also being up-regulated (P < 0.05), while those of Bcl-2 were down-regulated (P < 0.05). Compared with DT groups, the apoptosis rate in the DDP+DT group was significantly increased (P < 0.05). After knockdown of PUMA with specific siRNA, the apoptosis rate of SKOV-3/DDP cells was obviously decreased (P < 0.05). CONCLUSION: DT can promote the apoptosis of SKOV-3/DDP cells with PUMA playing a critical role.

Inhibitory effect of p53 upregulated modulator of apoptosis targeting siRNA on hypoxia/reoxygenation-induced cardiomyocyte apoptosis in rats.

OBJECTIVES: The effect of p53 upregulated modulator of apoptosis (PUMA) in hypoxia/reoxygenation-induced cardiomyocyte injuries in rats was investigated. METHODS: PUMA-targeting (si-PUMA) and scramble siRNAs were designed and transfected into primarily rat cardiomyocytes in vitro. RESULTS: RT-PCR and Western blot analysis showed that 50 nmol/l of si-PUMA can specifically inhibit PUMA expression. MTT assay and lactate dehydrogenase activity detection showed that the cell survival rate in the si-PUMA group was enhanced and that the lactate dehydrogenase enzymatic activity dramatically decreased compared with the control group (p < 0.01). Spectrophotometry, as well as annexin V and propidium iodide staining, combined with flow cytometry, revealed that caspase-3 activity in the si-PUMA group was downregulated and the apoptotic rate was decreased (p < 0.01). RT-PCR also showed that Bax expression was downregulated and Bcl-2 expression was upregulated in the si-PUMA group, compared with the control group (p < 0.05). si-PUMA protects cardiomyocytes from apoptosis. CONCLUSION: PUMA mediates hypoxia/reoxygenation-induced cardiomyocyte apoptosis, which can be a potential target of gene therapy for ischemia/reperfusion cardiomyocyte injuries.

[Study on apoptosis of human stomach SGC-7901 cells induced by extracts of Solanum lyratum].

OBJECTIVE: To investigate the effects of extracts of Solanum lyratum (ESL) on the apoptosis of Human stomach cancer SGC-7901 cells. METHODS: Dried whole herbs of Solanum lyratum were extracted by boiling distilled water. SGC-7901 cells were randomly divided into control group, ESL-treated groups (12.5 g/L, 25 g/L, 50 g/L) and the positive control (25 mg/L DDP) group. The growth inhibitory rate was evaluated by MTT assay. Morphological changes of apoptosis were observed with fluorescence microscope. Cell apoptosis rate was determined by flow cytometry. Expressions of bcl-xl, Caspase-9 and bid mRNA were detected by semi-quantitive RT-PCR. The activity of Caspase-3 was detected by Fluorospectrophotometry. RESULTS: Compared with control group, the cell proliferation inhibitory rate and apoptosis rate of human stomach cancer SGC-7901 cells increased obviously (P < 0.05). There were obvious changes of morphology of the SGC-7901 cells as the nuclear shrinkage, chromatin condensation and margination; The expression of bcl-xl mRNA decreased obviously (P < 0.05), the expression of Caspase-9 and bid mRNA increased obviously respectively (P < 0.05), and displayed effect in a dose-dependent manner in the SGC-7901 cells of the ESL-treated groups. The activity of Caspase-3 in the SGC-7901 cells of the ESL-treated groups were higher than that of the control group significantly (P < 0.01). CONCLUSION: ESL can induce apoptosis and inhibit proliferation of the human stomach cancer SGC-7901 cells by regulating expression of bcl-xl, Caspase-9 and bid genes and strengthening the activity of Caspase-3.