Yinghui Huang

Purpose: The study aims to lay a foundational probe for the thorough application microfluidic chips in brain function research with microfluidic chips. Neuron slide culture is a common culture method in vitro, and the microfluidic chip with the artificial network pattern not only can realize neuron cells 3 D culture in vitro, but also limit the extension space of neurite outgrow. Materials and Methods: In order to analyze the differences of hippocampal cells neurite growth length between the 3 D chips and the common 2 D culture, the experiments utilized statistical analysis method analyzing the length of the hippocampus neuron neurite of 3 days, 5 days and 7 days, respectively, with the common glass slide 2 D culture method and the microfluidic chip 3 D culture in vitro. Results: The results showed that there was no significant difference in the neurite length after 3 days. However, there was a significant difference after 5 days and 7 days. It can be seen that the microfluidic chip with artificial network pattern has limitations to the growth of neurite after 5 days. Conclusions: We concluded that the growth state of hippocampal cells in the restricted 3 D space is different from that of conventional 2 D culture.It showed that the artificial network pattern design has limited the growth space of the dendrites but also affected its growth.

Hypermethylation of gene promoter has been indicated for the contribution of gene silencing, and DNA demethylating drugs, such as 5‐aza‐2'‐deoxycytidine (DAC), has been used clinically for cancer treatment. However, the reason why a proportion of genes with hypermethylated promoter exhibit high expression levels remains unclear and this drug is not much successful as expected in use. Furthermore, CpG islands (CGIs) are found to be located in not only promotors, but also in gene bodies. By RNA‐seq and reduced representation bisulfite sequencing, we found the mismatch between the level of promoter methylation and gene expression. By chromatin Immunoprecipitation‐quantitative polymerase chain reaction and luciferase reporter assay, we identified putative promoters in gene body, and proved the activities of putative promoters were affected by the methylation level of the CGI nearby. DAC can reverse the DNA hypermethylation at promoter CGIs effectively but not the CGIs in gene body. We also found that TET1 could demethylate CGIs both in promoter and gene body. Furthermore, we revealed a novel mechanism that H3K36me3 could affect the activity of putative promoter, and 5hmC recruited MeCP2 and CREB1 as a coactivator to SETD2 promoter, to enhance its gene expression and result in increased H3K36me3 in gene body. Our results concluded that putative promoters existed in the gene bodies, and TET1 could influence the transcriptional activity of putative promoters by intragenic demethylation.

Breast cancer is the most prevalent malignancy in women, which remains untreatable once metastatic. The treatment of advanced breast cancer is restricted due to chemotherapy resistance. We previously investigated anti-cancer potential of a tumor selective oncolytic adenovirus along with cisplatin in three lung cancer cells; A549, H292, and H661, and found it very efficient. To our surprise, this virotherapy showed remarkable cytotoxicity to chemo-resistant cancer cells. Here, we extended our investigation by using two breast cancer cells and their resistant sublines to further validate CRAd’s anti-resistance properties. Results of in vitro and in vivo analyses recapitulated the similar anti-tumor potential of CRAd. Based on the molecular analysis through qPCR and western blotting, we suggest upregulation of coxsackievirus-adenovirus receptor (CAR) as a selective vulnerability of chemotherapy-resistant tumors. CAR knockdown and overexpression experiments established its important involvement in the success of CRAd-induced tumor inhibition. Additionally, through transwell migration assay we demonstrate that CRAd might have anti-metastatic properties. Mechanistic analysis show that CRAd pre-treatment could reverse epithelial to mesenchymal transition in breast cancer cells, which needs further verification. These insights may prove to be a timely opportunity for the application of CRAd in recurrent drug-resistant cancers.

Accumulating evidence suggests that Ras GTPase‐activating protein SH3 domain‐binding protein 1 (G3BP1) is very crucial to regulate tumorigenesis and metastasis. Recently, many research works have suggested that G3BP1 is overexpressed in many human cancers including esophageal cancer. Nevertheless, the functional roles of G3BP1 in esophageal cancer are still unknown. Here, the results suggested that silencing of G3BP1 inhibited proliferation, migration, and invasion of esophageal cancer cells, whereas overexpression of G3BP1 led to opposite effects on the growth and metastasis. Surprisingly, G3BP1‐depletion had no effect on cell death but caused the arrest of cell cycle in the G₀/G₁ phase and increased the levels of p53 and p21. In addition, loss of G3BP1 led to a significant elevation of E‐cadherin and decrease of N‐cadherin, Vimentin, Snail, MMP‐9, and MMP‐2. Mechanistically, loss of G3BP1 dramatically suppressed Wnt‐stimulated T‐cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor activity and downregulated its target genes including c‐Myc, Axin2, and cyclin D1. Moreover, knockdown of G3BP1 downregulated the expression levels of p‐PI3K, p‐AKT, and p‐GSK‐3β, but the total PI3K, AKT, and GSK‐3β were not changed. Furthermore, our data proved that the promoting effects of G3BP1‐overexpression on cell proliferation, migration, and invasion could be rescued by PI3K inhibitor LY294002 treatment. Collectively, our results here elucidate that G3BP1‐depletion suppresses proliferation, migration, and invasion capabilities of esophageal cancer cells via the inactivation of Wnt/β‐catenin and PI3K/AKT signaling pathways. Furthermore, our findings imply that G3BP1 can participate in the regulation of esophageal cancer progression, and will be taken as a promising target to treat esophageal cancer.

Cisplatin is ranked as one of the most powerful and commonly prescribed anti-tumor chemotherapeutic agents which improve survival in many solid tumors including non-small cell lung cancer. However, the treatment of advanced lung cancer is restricted due to chemotherapy resistance. Here, we developed and investigated survivin promoter regulating conditionally replicating adenovirus (CRAd) for its anti-tumor potential alone or in combination with cisplatin in two lung cancer cells, H23, H2126, and their resistant cells, H23/CPR, H2126/CPR. To measure the expression of genes which regulate resistance, adenoviral transduction, metastasis, and apoptosis in cancer cells, RT-PCR and Western blotting were performed. The anti-tumor efficacy of the treatments was evaluated through flow cytometry, MTT and transwell assays. This study demonstrated that co-treatment with cisplatin and CRAd exerts synergistic anti-tumor effects on chemotherapy sensitive lung cancer cells and monotherapy of CRAd could be a practical approach to deal with chemotherapy resistance. Combined treatment induced stronger apoptosis by suppressing the anti-apoptotic molecule Bcl-2, and reversed epithelial to mesenchymal transition. In conclusion, cisplatin synergistically increased the tumor-killing of CRAd by (1) increasing CRAd transduction via enhanced CAR expression and (2) increasing p53 dependent or independent apoptosis of lung cancer cell lines. Also, CRAd alone proved to be a very efficient anti-tumor agent in cancer cells resistant to cisplatin owing to upregulated CAR levels. In an exciting outcome, we have revealed novel therapeutic opportunities to exploit intrinsic and acquired resistance to enhance the therapeutic index of anti-tumor treatment in lung cancer.

The design of pattern that limit the hippocampal cells growth is an important explore for realizing a simplified artificial neuronal network in vitro. In this study, we examined the pattern in micro-fluidic chip to stipulate the hippocampal cells adhesion, growth and the formation of a functional neuronal network in vitro. Patch clamp recording technique was used to detect the growth situation and biological function of the haippocampal cells on the micro-fluidic chip which could simulate environment in vivo. We show that the number of neurons cultured is about 5000-6000 cells on the micro-fluidic chip, which is conductive to the hippocampal cells growth. The result of patch clamp recording technique show the signals of sodium and potassium channels, meanwhile, it also reveal the signals of synaptic connection. These findings involve placing cells in specific locations to create organized structures, and explore the spread function of synaptic on the micro-fluidic chip.

Purpose: Carboplatin is a first-line treatment for ovarian cancer. However, most patients develop resistance and undergo disease recurrence. This study aims to explore the relationship between the expression of X-linked inhibitor of apoptosis protein (XIAP) and carboplatin sensitivity in ovarian cancer. Patients and methods: We examined the expression of XIAP in ovarian cancer by immunochemistry. Next, we investigated the role of XIAP in regulating carboplatin sensitivity in ovarian cancer ES2 and 3AO cells through Cell Counting Kit-8 cell viability assay and fluorescein isothiocyanate-Annexin V/propidium iodide apoptosis assay. Expression of apoptotic effectors was measured by Western blot. Results: The immunochemistry results showed that high XIAP expression levels inversely correlated with carboplatin response (P=0.03) and progression-free survival (P=0.0068) in patients with ovarian cancer. Knockdown of XIAP repressed the cell viabilities in the carboplatin-treated cells and increased carboplatin-induced caspase activation. In summary, our data show that XIAP mediates carboplatin sensitivity of ovarian cancer. Conclusion: In summary, our data show that XIAP mediates carboplatin sensitivity of ovarian cancer and XIAP may be a novel target for the treatment of carboplatin-resistant ovarian cancer.

Cell fusion plays a crucial role in cancer progression and leads to massive aberrant changes in chromosome and gene expression involved in tumor metastasis. Cancer cells can fuse with many cell types, including stromal cells, epithelial cells, macrophages, and endothelial cells. Mesenchymal stem cells (MSCs) have been reported to migrate and incorporate into tumor sites during cancer progression. However, the underlying mechanism of stem cell fusion in tumor metastasis has not been fully deciphered. In this research, we established a cell fusion model between lung cancer cells and MSCs in vitro. We found that the hybrid cells showed enhanced metastatic capacity with increased expression of MMP‐2 and MMP‐9, whereas the proliferation ability was inhibited and cell cycle was blocked in the G₀/G₁ phase with elevated expression of p21, p27, and p53. Moreover, the hybrid cells lost epithelial morphology and exhibited an epithelial‐mesenchymal transition (EMT) change with downregulation of E‐cadherin and upregulation of N‐cadherin, Vimentin, α‐SMA and Fibronectin1. Meanwhile, the expressions of EMT transcription factors, including Snail1, Slug, Twist1, Zeb1, and Zeb2, were also increased in hybrid cells. More important, the fusion hybrids acquired stem cell‐like properties, which exhibited increased expression stem cell transcription factors Oct4, Sox2, Nanog, Kif4 as well as Bmi1. Taken together, our results suggested that cell fusion between lung cancer cells and MSCs offered enhanced metastatic capacity and characteristics of cancer stem cell by undergoing EMT. This study will contribute to explaning the origin of lung cancer stem cells and to elucidate the role of cell fusion in cancer metastasis.

Ten‐eleven translocation 1 (TET1), a widely reported DNA demethylation protein, has been associated with tumorigenesis and metastasis. However, whether TET1 is an oncogene or tumor suppressor gene has been controversial; the mechanism of how TET1 affects cancer progression remains unclear. The current study aims to investigate how TET1 is changed in the tumor microenvironment and to explore the mechanisms of how TET1 affects colon cancer progression. Because hypoxia prevails on solid tumors, we established an important connection between hypoxia and DNA demethylation in tumorigenesis. By qPCR and RNA interference (RNAi) technology, we found that hypoxia increased TET1 expression with a hypoxia‐inducible factor‐1‐alpha (HIF‐1α)‐dependent manner. By CHIP‐qPCR and pyrosequencing technology, we demonstrated that TET1 regulated the target gene expression of HIF‐1α through HIF‐1α binding to hypoxia‐responsive elements (HREs), and HIF‐1α binding to HREs depended on CpG methylation levels. By Cell Counting Kit‐8 (CCK‐8) and transwell assay, we showed that loss of TET1 did not affect cell proliferation but inhibited migration. We also identified two novel gene mutants of TET1 in 120 paired tumor/normal tissue specimens by DNA sequencing and found that TET1 E2082K mutant blocked the TET1‐enhanced cell migration. Our results showed that the downregulation of TET1 rescued the abnormally high levels of gene expression resulting from hypoxia in tumors and reduced the migration activity of tumor cells, suggesting a therapeutic role by interference with TET1 in colon cancer treatment. By demonstrating that hypoxia upregulated TET1 and that TET1 drove HIF‐1α‐responsive genes, we showed that an epigenetic mechanism and tumor microenvironment‐driven models coexisted and mutually affected colon cancer.

Exosomes are dish-shaped phospholipid bilayer-enclosed membranous vesicles with diameter range of 30–150 nm. Exosomes as stable vesicles contain various bioactive molecules derived from the cells of parental origin, including proteins, lipids, DNA fragments, mRNAs, miRNAs, lncRNAs etc. Exosomes are secreted by almost all kinds of cells. Here, we focus on the application of exosomes in precision oncology. Nowadays, accumulating evidence suggests that cancer cells release the excessive amounts of exosomes compared to normal cells. The communications through exosomes between cancer cells and cancer/normal cells could make the exchange of genetic information and reprogramming of the recipient cells. Besides, the tumor-derived exosomes contribute the establishment of pre-metastatic niche and distant tissue site for metastasis of cancer cells. That may leads to cancer initiation, progression, metastasis of cancer cells and escaping from immune cell surveillance. In this review, we summarize the definition, composition and the potential diagnostic/therapeutic applications of exosomes. We introduce that there are several kinds of proteins enriched in exosomes such as TSG101, HSP70, CD9 and so on, which could be as the biomarkers to monitor the qualification and purification of exosomes we purified, in which play a pivotal role in exosome research. We describe the ways of recipient cells uptaking exosomes into themselves and the methods of exosome purification. Furthermore, we focus on non-invasive liquid biopsies in oncology, relying on analyzing material including molecules as well as whole cells that are found in bodily fluids such as blood and urine. Liquid biopsies are now in quick development and have been being an intriguing topic. We discuss the potential role of circulating tumor cells, circulating tumor-derived DNA/RNAs, and exosomes in liquid biopsy of cancer. We discriminate their characteristics and focus on exosomes that are used in liquid biopsy. Because the biomolecules in exosomes are derived from their parental cells, these contents are resembled between with each other. On this basis, it is conformed that they can reflect the dynamic information for genetic or signaling alteration of their cells of origin. We also highlighted its advanced progression in cancer diagnosis and therapeutic targets. It has been assumed that their contents such as nucleotides or proteins are abnormally enriched in tumor-derived ones compared to the normal, such as glypican-1 in pancreatic cancer and CD63 in the melanoma, which have the potential to be diagnostic biomarkers in cancers. The advanced studies have shown the difference between exosomes derived from tumor cells and normal cells, which may mediate different signaling pathways of the recipient cells. These facts indicate that we could transform exosomes to make it a medium to mediate agents to the recipient cells or a tool for treatment. For example, exosomes can serve as specific delivery vehicles loaded siRNAs/shRNAs targeted to mRNAs, proteins or small molecules, as a tool of gene therapy. We also point out that we are still in early step of understanding the nature and extent of the involvement of exosomes in physiology and therapeutic targets. We highlight that these will shed light on the new strategy for cancer diagnosis and therapy. The advanced studies in exosomes we presented may make greater contribution to overcome a variety of cancer in the era of precision oncology.