CD151-α3β1 Integrin Complexes are Prognostic Markers of Glioblastoma and Cooperate with EGFR to Drive Tumor Cell Motility and Invasion

Glioblastoma, one of the most aggressive forms of brain cancer, is featured by high tumor cell motility and invasiveness, which not only fuel tumor infiltration, but also enable escape from surgical or other clinical interventions. Thus, better understanding of how these malignant traits are controlled will be key to the discovery of novel biomarkers and therapies against this deadly disease. Tetraspanin CD151 and its associated α3β1 integrin have been implicated in facilitating tumor progression across multiple cancer types. How these adhesion molecules are involved in the progression of glioblastoma, however, remains largely unclear. Here, we examined an in-house tissue microarray-based cohort of 96 patient biopsies and TCGA dataset to evaluate the clinical significance of CD151 and α3β1 integrin. Functional and signaling analyses were also conducted to understand how these molecules promote the aggressiveness of glioblastoma at molecular and cellular levels. Results from our analyses showed that CD151 and α3 integrin were significantly elevated in glioblastomas at both protein and mRNA levels, and exhibited strong inverse correlation with patient survival (p \u3c 0.006). These adhesion molecules also formed tight protein complexes and synergized with EGF/EGFR to accelerate tumor cell motility and invasion. Furthermore, disruption of such complexes enhanced the survival of tumor-bearing mice in a xenograft model, and impaired activation of FAK and small GTPases. Also, knockdown- or pharmacological agent-based attenuation of EGFR, FAK or Graf (ARHGAP26)/small GTPase-mediated pathways markedly mitigated the aggressiveness of glioblastoma cells. Collectively, our findings provide clinical, molecular and cellular evidence of CD151-α3β1 integrin complexes as promising prognostic biomarkers and therapeutic targets for glioblastoma

Genome-Wide Identification and Expression Analysis of <i>C3H Zinc Finger</i> Family in Potato (<i>Solanum tuberosum</i> L.)

Transcription factors containing a CCCH structure (C3H) play important roles in plant growth and development, and their stress response, but research on the C3H gene family in potato has not been reported yet. In this study, we used bioinformatics to identify 50 C3H genes in potato and named them StC3H-1 to StC3H-50 according to their location on chromosomes, and we analyzed their physical and chemical properties, chromosome location, phylogenetic relationship, gene structure, collinearity relationship, and cis-regulatory element. The gene expression pattern analysis showed that many StC3H genes are involved in potato growth and development, and their response to diverse environmental stresses. Furthermore, RT-qPCR data showed that the expression of many StC3H genes was induced by high temperatures, indicating that StC3H genes may play important roles in potato response to heat stress. In addition, Some StC3H genes were predominantly expressed in the stolon and developing tubers, suggesting that these StC3H genes may be involved in the regulation of tuber development. Together, these results provide new information on StC3H genes and will be helpful for further revealing the function of StC3H genes in the heat stress response and tuber development in potato

Evaluating Effects of Regulated Deficit Irrigation under Mulched on Yield and Quality of Pumpkin in a Cold and Arid Climate

As the most effective irrigation method in arid and semi-arid regions, drip irrigation under mulch could general comprehension of the production efficiency of agricultural irrigation water, and reduce agriculture consumption of water resources. The paper has carried out an investigation over a two year period (2020&ndash;2021) in a semi-arid climate in the Hexi Oasis region of China, aiming at determining the influence of regulated deficit irrigation (RDI) under mulch on the growth, yield, water use efficiency (WUE), irrigation water use efficiency (IWUE) and quality of pumpkin at different growth stages. A total of nine treatments with three irrigation levels (75&ndash;85% field capacity, 65&ndash;75% field capacity, and 55&ndash;65% field capacity) have been used in four growing periods of pumpkin (seedling, vine extension, fruit expansion, and maturation stages). The results have shown that light water deficit treatment at the seedling stage had the highest water use efficiency (12.47 kg/m3) without significantly affecting yield (45,966.90 kg/ha), and improved pumpkin fruit quality. It was concluded that light water deficit at the seedling stage and adequate irrigation at other development stages was the optimal irrigation strategy for pumpkin growth. The results of this research provide theoretical and technical support for efficient water-saving plantation and industrialization of pumpkin in the Hexi Oasis

Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells

<div><p>Objective</p><p>To map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients.</p><p>Methods and Materials</p><p>Methylation patterns of HPV16 LCR in UM-SCC47, CaSki, and SiHa cells and HPV16-positiive OPSCC specimens were detected by bisulfite-sequencing PCR and TA cloning. For cells treated with 5-aza-2′-deoxycytidine and E6 and E7 knockdown, MTS and trypan blue staining, annexin-V and 7-AAD staining, and prodidium iodide were used to evaluate cell growth and cell proliferation, cell apoptosis, and cell cycle arrest, respectively. E6 and E7 mRNA and protein expression were analyzed by quantitative real-time PCR and immunocytochemistry, respectively.</p><p>Results</p><p>Hypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5′-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10).</p><p>Conclusions</p><p>Our study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.</p></div

Origin and pathological characteristics of HPV-positive cancer cell lines.

<p>Origin and pathological characteristics of HPV-positive cancer cell lines.</p

Demethylation of HPV16 LCR CpG sites and knockdown of E6 and E7 inhibit cell growth in HPV-positive cancer cells.

<p><b>A)</b> After demethylation of the CpG sites, cell growth was strongly inhibited in UM-SCC47 and CaSki cells, but not in SiHa cells. <b>B)</b> E6 and E7 expression was significantly decreased after siRNA transfection. <b>C)</b> Cell growth was inhibited at 96 h after E6 and E7 knockdown. All data are means ± SEM from 3 independent experiments in <b>A)</b> and <b>C)</b>, and means ± SD from 3 independent experiments in <b>B)</b>.</p

Structure of HPV16 LCR and location of the CpG sites.

<p><b>A)</b> The structure of HPV16 LCR and location of the CpG sites. Transcription factor binding sites are also shown. <b>B, C)</b> Nucleotide mutations at nt7435 (G→A) and nt31 (C→T) of UM-SCC47 cells affected the presence of the CpG sites.</p

Demethylation of HPV16 LCR CpG sites decreases E6 and E7 expression.

<p>Demethylation of the CpG sites induced by 0.5 μM 5-aza-dc for 96 h decreased E6 and E7 expression, which was detected by reverse transcription PCR <b>A)</b> and quantitative real-time PCR <b>B)</b>; E6 and E7 were clearly decreased in UM-SCC47 and CaSki cells, but there was no obvious change in SiHa cells. The values (mean ± SD) were obtained from at least 3 independent experiments. <b>C)</b> E6 and <b>D)</b> E7 protein after demethylation of the CpG sites was detected by immunocytochemistry; E6 and E7 oncoproteins were distributed in the cytoplasm and nucleus of UM-SCC47 and CaSki cells with a strong staining intensity (×100, bar = 100 μm), and both the percentages and intensity of stained cells were clearly decreased after demethylation (×100, bar = 100 μm).</p

Apoptosis and cell cycle arrest after E6 and E7 knockdown.

<p><b>A)</b> The number of apoptotic cells increased at 96 h after E6 and E7 knockdown in UM-SCC47, CaSki, and SiHa cells. The cells were arrested in G2/M phase in <b>B)</b> UM-SCC47 and <b>C)</b> CaSki cells, and in both G2/M and S phases in <b>D)</b> SiHa cells. All data are means ± SEM from 3 independent experiments.</p