Long non-coding RNA CCL14-AS suppresses invasiveness and lymph node metastasis of colorectal cancer cells by regulating MEP1A.

BACKGROUND: Long non-coding RNAs (lncRNAs) play important roles in the biology of colorectal cancer (CRC). There are several lncRNAs associated with invasion and metastasis have been characterized in CRC. However, studies focusing on the precise molecular mechanisms by which lncRNAs function in lymph node (LN) metastasis in CRC are still limited. METHODS: In this study, by analyzing TCGA dataset, we identified that AC244100.2 (termed CCL14-AS), a novel lncRNA enriched in the cytoplasm, was negatively correlated with LN metastasis and unfavorable prognosis of CRC. In situ hybridization was used to examine CCL14-AS expression in clinical CRC tissues. Various functional experiments including migration assay and wound-healing assay were used to investigate the effects of CCL14-AS on CRC cells migration. The nude mice popliteal lymph node metastasis model assay further confirmed the effects of CCL14-AS in vivo. RESULTS: CCL14-AS expression was significantly downregulated in CRC tissues compared to adjacent normal tissues. In addition, low CCL14-AS expression was correlated with advanced T classification, LN metastasis, distant metastasis, and shorter disease-free survival of CRC patients. Functionally, CCL14-AS overexpression inhibited the invasiveness of CRC cells in vitro and LN metastasis in nude mice. On the contrary, knockdown of CCL14-AS promoted the invasiveness and LN metastasis abilities of CRC cells. Mechanistically, CCL14-AS downregulated the expression of MEP1A via interacting with MEP1A mRNA and reduced its stability. Overexpression of MEP1A rescued the invasiveness and LN metastasis abilities in CCL14-AS-overexpressing CRC cells. Moreover, the expression levels of CCL14-AS was negatively correlated with that of MEP1A in CRC tissues. CONCLUSIONS: We identified a novel lncRNA, CCL14-AS, as a potential tumor suppressor in CRC. Our findings supported a model in which the CCL14-AS/MEP1A axis serves as critical regulator in CRC progression, suggesting a novel biomarker and therapeutic target in advanced CRC.

Inhibition of human lung cancer cells by anti-p21Ras scFv mediated by the activatable cell-penetrating peptide.

Activatable cell-penetrating peptide (ACPP) is a tumour-targeting cell-penetrating peptide. Here, we used ACPP to carry anti-p21Ras scFv for Ras-driven cancer therapy. The ACPP-p21Ras scFv fusion protein was prepared by a prokaryotic expression system and Ni-NTA column purification. The human tumour cell lines A549, SW480, U251 and Huh7 and the normal cell line BEAS 2B were used to study the tumor-targeting and membrane-penetrating ability of ACPP-p21Ras scFv. The antitumour activity of ACPP-p21Ras scFv on A549 cells and H1299 cells in vitro was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, scratch wound healing, plate cloning and apoptosis assays. The penetration pathway of ACPP was determined by enhanced green fluorescent protein. The ACPP-p21Ras scFv fusion protein was successfully obtained at a concentration of 1.8 mg/ml. We found that ACPP-p21Ras scFv could penetrate tumour cell membranes with high expression of matrix metalloproteinase-2 (MMP-2), effectively inhibit the migration and proliferation of A549 cells and H1299 cells, and promote the apoptosis of A549 cells and H1299 cells. The membrane penetration experiment demonstrated that ACPP could enter A549 cells by direct penetration. The ability of ACPP to penetrate the membrane was affected by the addition of a membrane affinity inhibitor and a change in the potential difference across the cell membrane but not by the addition of endocytosis inhibitors and a change in temperature. The ACPP-p21Ras scFv fusion protein can penetrate tumour cells with MMP-2 expression and has antitumour activity against A549 cells and H1299 cells in vitro. This molecule is expected to become a potential antitumour drug for Ras gene-driven lung cancer.

RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Anti-colorectal cancer effects of anti-p21Ras scFv delivered by the recombinant adenovirus KGHV500 and cytokine-induced killer cells.

BACKGROUND: Colorectal cancer (CRC) is the most common type of gastrointestinal cancer. CRC gene therapy mediated by adenovirus holds great promise for the treatment of malignancies. However, intravenous delivery of adenovirus exhibits limited anti-tumor activity in vivo when used alone. METHODS: In this study, the antitumor activity of the recombinant adenovirus KGHV500 was assessed with the MTT, TUNEL, Matrigel invasion and cell migration assays. To enhance the intravenous delivery of KGHV500 in vivo, cytokine-induced killer (CIK) cells were used as a second vector to carry KGHV500. We explored whether CIK cells could carry the recombinant adenovirus KGHV500 containing the anti-p21Ras single chain fragment variable antibody (scFv) gene into tumors and enhance antitumor potency. RESULTS: Our results showed that KGHV500 exhibited significant antitumor activity in vitro. In the nude mouse SW480 tumor xenograft model, the combination of CIK cells with KGHV500 could induce higher antitumor activity against colorectal cancer in vivo than that induced by either CIK or KGHV500 alone. After seven days of treatment, adenovirus and scFv were detected in tumor tissue but were not detected in normal tissues by immunohistochemistry. Therefore, KGHV500 replicates in tumors and successfully expresses anti-p21Ras scFv in a colorectal cancer xenograft model. CONCLUSIONS: Our study provides a novel strategy for the treatment of colorectal cancer by combining CIK cells with the recombinant adenovirus KGHV500 which carried anti-p21 Ras scFv.

[Chemical constituents of Scrophularia ningpoensis root].

OBJECTIVE: To investigate the chemical constituents from Scrophularia ningpoensis root. METHODS: The compounds were isolated and purified by silica gel and Sephadex LH-20 column chromatography. The structures of these compounds were elucidated on the basis of spectroscopic analysis. RESULTS: Eleven compounds were isolated and identified as vanillin (1), eugenol (2), isoeugenol methyl ether(3), ferulic acid(4), benzoic acid(5), syringic acid(6) ,rhamnopyranosyl vanilloyl(7), syringic acid-4-O-alpha-L-rhamnopyr-anoside(8), beta-sitosterol(9), adenosine (10) and dibutyl phthalate(11). CONCLUSION: Compound 1-3 and 6-8 are obtained from this plant for the first time.

Resveratrol suppresses hepatic fatty acid synthesis and increases fatty acid ß-oxidation via the microRNA-33/SIRT6 signaling pathway.

Hyperlipidemia is a strong risk factor for numerous diseases. Resveratrol (Res) is a non-flavonoid polyphenol organic compound with multiple biological functions. However, the specific molecular mechanism and its role in hepatic lipid metabolism remain unclear. Therefore, the aim of the present study was to elucidate the mechanism underlying how Res improves hepatic lipid metabolism by decreasing microRNA-33 (miR-33) levels. First, blood miR-33 expression in participants with hyperlipidemia was detected by reverse transcription-quantitative PCR, and the results revealed significant upregulation of miR-33 expression in hyperlipidemia. Additionally, after transfection of HepG2 cells with miR-33 mimics or inhibitor, western blot analysis indicated downregulation and upregulation, respectively, of the mRNA and protein expression levels of sirtuin 6 (SIRT6). Luciferase reporter analysis provided further evidence for binding of miR-33 with the SIRT6 3'-untranslated region. Furthermore, the levels of peroxisome proliferator-activated receptor-γ (PPARγ), PPARγ-coactivator 1α and carnitine palmitoyl transferase 1 were increased, while the concentration levels of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element-binding protein 1 were decreased when SIRT6 was overexpressed. Notably, Res improved the basic metabolic parameters of mice fed a high-fat diet by regulating the miR-33/SIRT6 signaling pathway. Thus, it was demonstrated that the dysregulation of miR-33 could lead to lipid metabolism disorders, while Res improved lipid metabolism by regulating the expression of miR-33 and its target gene, SIRT6. Thus, Res can be used to prevent or treat hyperlipidemia and associated diseases clinically by suppressing hepatic fatty acid synthesis and increasing fatty acid ß-oxidation.

Resveratrol improves palmitic acid­induced insulin resistance via the DDIT4/mTOR pathway in C2C12 cells.

The present study aimed to establish a model of palmitic acid (PA)­induced insulin resistance (IR) in C2C12 cells and to determine the mechanism underlying how resveratrol (RSV) improves IR. C2C12 cells were divided into the control (CON), PA, PA + RSV, PA + RSV + DNA damage­inducible transcript 4 (DDIT4)­small interfering (si)RNA and PA + RSV + MHY1485 (mTOR agonist) groups. Glucose contents in culture medium and triglyceride contents in cells were determined. Oil red O staining was performed to observe the pathological changes in the cells. Reverse transcription­quantitative PCR and western blotting were conducted to evaluate the mRNA and protein expression levels, respectively, of DDIT4, mTOR, p70 ribosomal protein S6 kinase (p70S6K), insulin receptor substrate (IRS)­1, PI3K, AKT and glucose transporter 4 (GLUT4). Compared with in the CON group, glucose uptake was decreased, cellular lipid deposition was increased, phosphorylated (p)­IRS­1, p­mTOR and p­p70S6K protein expression levels were increased, and p­PI3K, p­AKT, GLUT4 and DDIT4 protein expression levels were decreased in the PA group. By contrast, compared with in the PA group, culture medium glucose content and cellular lipid deposition were decreased, p­PI3K, p­AKT, GLUT4 and DDIT4 protein expression levels were increased, p­IRS­1 protein expression levels were decreased, and mTOR and p70S6K mRNA and protein expression levels were decreased in the PA + RSV group. Compared with in the PA + RSV group, DDIT4 protein and mRNA expression levels were reduced in the PA + RSV + DDIT4­siRNA group, but showed no change in the PA + RSV + MHY1485 group. Following transfection with DDIT4­siRNA or treatment with MHY1485, the effects of RSV on improving IR and lipid metabolism were weakened, mTOR and p70S6K protein expression levels were upregulated, p­PI3K, p­AKT and GLUT4 protein expression levels were down­regulated, p­IRS­1 protein expression levels were upregulated, and culture medium glucose content and cellular lipid deposition were increased. In conclusion, RSV may improve PA­induced IR in C2C12 cells through the DDIT4/mTOR/IRS­1/PI3K/AKT/GLUT4 signaling pathway, as well as via improvements in glucose and lipid metabolism.

Isolation, molecular characterization, and functional analysis of the vacuolar Na+/H+ antiporter genes from the halophyte Karelinia caspica.

The full-length cDNAs of two Karelinia caspica genes, KcNHX1 and KcNHX2, were isolated by RACE and RT-PCR based on the conserved regions of Na(+)/H(+) antiporter (NHX) genes from other halophyte species. The cloned KcNHX1 cDNA contained 2,022 nucleotides with an open reading frame (ORF) of 1,620 bp and the KcNHX2 cDNA contained 1,976 nucleotides with an ORF of 1,653 bp. The deduced amino acid sequences indicated that both genes were homologous to NHXs from other higher plants. To investigate the possible roles of KcNHX1 and KcNHX2 in the salt stress response of K. caspica and the underlying regulatory mechanisms, RNAi vectors were constructed and transformed into K. caspica to specifically silence endogenous KcNHX1 and KcNHX2. The physiological results showed that silencing KcNHX1 in K. caspica led to reduced salt tolerance in high concentrations of NaCl, suggesting that KcNHX1 plays an essential role in the response of K. caspica to salt stress. However, the inhibition of KcNHX2 seemed to have little influence on the salt resistance of transgenic plants, indicating that KcNHX2 may be relevant for functions other than salt tolerance in K. caspica.

BR2 cell penetrating peptide effectively delivers anti-p21Ras scFv to tumor cells with ganglioside expression for therapy of ras-driven tumor.

PURPOSE: Cell membrane penetrating peptide BR2 can bind with ganglioside and introduce foreign drugs into tumor cells. In this study, we employed BR2 to carry the broad-spectrum anti-p21Ras scFv prepared in our laboratory into ganglioside expressing tumor cells for therapy of ras-driven tumors. METHODS: BR2-p21Ras scFv gene was cloned to prokaryotic expression vector and expressed in E. coli BL21, then the fusion protein was purified with HisPur Ni-NTA. The immunoreactivity of the fusion protein with p21Ras was detected by ELISA and western blotting. The membrane-penetrating and immune co-localization with p21Ras of the fusion protein were determined by immunofluorescence. The antitumor activity was investigated using MTT, wound healing, colone formation, and apoptosis assays in vitro. RESULTS: BR2-p21Ras scFv fusion protein was successfully expressed and purified. We found that the fusion protein could specifically penetrate into human tumor cell lines which express ganglioside including human neuroblastoma cell line SK-N-SH, human colon cancer cell line HCT116 and human glioma cell line U251. After entering tumor cells the fusion protein bonded specifically with p21Ras. In vitro experiments revealed that it could significantly inhibit the proliferation, migration, and colone formation of HCT116, SK-N-SH, and U251 cells and promote the apoptosis of these tumor cells. CONCLUSIONS: BR2-p21Ras scFv can penetrate ganglioside expressing tumor cells and inhibit the growth of ras-driven tumor by binding with p21Ras, and producing an inhibitory effect. It is suggested that BR2-p21Ras scFv is a potential ras-driven tumor therapeutic antibody.

Circulating levels of DDIT4 and mTOR, and contributions of BMI, inflammation and insulin sensitivity in hyperlipidemia.

Evidence shows a high incidence of insulin resistance, inflammation and excess body mass index (BMI) in adults with hyperlipidemia. The present study aimed to determine the circulating levels of DNA damage inducible transcript 4 (DDIT4) and mTOR and assess the contributions of lipids, inflammatory markers, insulin sensitivity and BMI in hyperlipidemia. The study subjects were divided into a hyperlipidemia group and a normal control group (n=55 per group). Sex, age, blood pressure, waist circumference (WC), height, weight and BMI were recorded. Fasting venous blood samples were collected and an automatic biochemical analyzer was used to detect fasting blood glucose (FBG), fasting insulin (FINS), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Quantitative ELISA kits were used to determine the levels of DDIT4, mTOR and inflammatory markers and calculate the homeostatic model assessment of insulin resistance (HOMA-IR). Compared with the normal control group, the hyperlipidemia group had significantly increased blood pressure, WC, weight, BMI, FBG, FINS, HOMA-IR, mTOR and inflammatory markers, but significantly reduced DDIT4. A concurrent correlation analysis showed that insulin resistance was positively correlated with blood pressure, BMI, lipid profiles (TG, TC, LDL-C), mTOR and inflammatory markers, but negatively correlated with HDL-C and DDIT4. Lipid profiles were positively correlated with BMI, mTOR and inflammatory markers, but negatively correlated with DDIT4. A factor analysis identified four domains in hyperlipidemia (inflammation-lipid 1 domain, 44.429%; overweight domain, 21.695%; insulin sensitivity domain, 11.782%; lipid 2 domain, 6.723%). In conclusion, people with hyperlipidemia have elevated mTOR and reduced DDIT4 and are accompanied by abnormal indicators such as insulin sensitivity, BMI and inflammatory factors. The identified domains may be applied to predict the outcomes of cardiovascular diseases and metabolic diseases in the future.

RNA Interference Induces BRCA1 Gene Methylation and Increases the Radiosensitivity of Breast Cancer Cells.

Purpose: To investigate the relationship between breast cancer susceptibility gene-1 (BRCA1) gene methylation and the radiosensitivity of breast cancer. Materials and Methods: The authors studied three breast cancer cell lines: MDA-MB-435, MDA-MB-231, and MCF-7 cells. They constructed five short hairpin RNAs (shRNAs) and five small interfering RNAs to target selected promoter loci and initiate sequence-specific methylation in breast cancer cells. Pyrosequencing was used to analyze the state of DNA methylation. Quantitative real-time polymerase chain reaction was used to detect BRCA1 mRNA expression and RNA-directed DNA methylation (RdDM)-related gene expression. Western blotting was performed to analyze BRCA1 protein expression. Colony formation assays and γ-histone H2A foci formation assays were conducted to assess the surviving fraction (SF) and double-strand break (DSB) repair ability of cells after irradiation. Results: The authors constructed five strains of lentivirus vectors and five plasmid vectors targeting BRCA1 promoter region. In MDA-MB-435 cells, lentivirus-mediated RNA interference targeting Site 1 of BRCA1 increased the methylation levels of BRCA1 and reduced BRCA1 mRNA and protein expression. The SF and the ability to repair DNA DSBs were reduced in the combined LV-BRCA1RNAi-Site 1 infection and irradiation group. Conclusions: The authors' findings suggest that the shRNA suppressed the expression levels of the BRCA1 gene and reduced the SF and DNA repair ability of cells after irradiation through RdDM. In summary, the radiosensitivity of breast cancer cells may correlate with BRCA1 methylation. Advances in Knowledge: The authors first utilized a lentivirus-based shRNA-mediated specific-sequence DNA methylation of the BRCA1 gene mediated by RdDM.

ZNF24 regulates the progression of KRAS mutant lung adenocarcinoma by promoting SLC7A5 translation.

Background: Clinical treatment of RAS mutant cancers is challenging because of the complexity of the Ras signaling pathway. SLC7A5 is a newly discovered downstream gene of the Ras signaling pathway, but the regulatory mechanism is unclear. We aimed to explore the molecular mechanism and role in KRAS mutant lung adenocarcinoma progression. Methods: Key gene that regulated SLC7A5 in KRAS mutant lung adenocarcinoma was screened by RNA sequencing and bioinformatics analysis. The effect of this gene on the expression of SLC7A5 was studied by RNAi. The regulatory mechanism between the two genes was investigated by immunofluorescence, CoIP, pulldown and yeast two-hybrid assays. The location of the two genes was determined by inhibiting Ras and the downstream pathways PI3K-AKT and MEK-ERK. By in vivo and in vitro experiments, the effects of the key gene on the biological functions of KRAS mutant lung adenocarcinoma were explored. Results: We found a novel gene, ZNF24, which upregulated SLC7A5 protein expression rather than mRNA expression in KRAS mutant lung adenocarcinoma. Endogenous protein interactions occurred between ZNF24 and SLC7A5. Ras inhibition reduced the expression of ZNF24 and SLC7A5. ZNF24 and SLC7A5 are located downstream of the MEK-ERK and PI3K-AKT pathways. In vivo and in vitro functional experiments confirmed that the ZNF24-SLC7A5 signaling axis promoted the proliferation, invasion and migration of KRAS mutant lung adenocarcinoma. Conclusions: ZNF24 promoted the growth of KRAS mutant lung adenocarcinoma by upregulating SLC7A5 protein expression, which suggested that ZNF24 is a new biomarker of KRAS mutant tumors and could be a new potential therapeutic target for Ras-driven tumors.

Inhibition of glioma by adenovirus KGHV500 encoding anti-p21Ras scFv and carried by cytokine-induced killer cells.

Ras gene mutation or overexpression can lead to tumorigenesis in multiple kinds of cancer, including glioma. However, no drugs targeting Ras or its expression products have been approved for clinical application thus far. Adenoviral gene therapy is a promising method for the treatment of glioma. In this study, the human glioma cell line U251 was co-cultured with recombinant adenovirus KGHV500, and the anti-tumor effects of KGHV500 were determined by MTT, scratch test, Transwell invasion, and apoptosis assays. Then, KGHV500 was delivered via the intravenous injection of CIK cells into glioma xenografts. Tumor volume, ki67 proliferation index, apoptosis levels, and anti-p21Ras scFv expression were tested to evaluate targeting ability, anti-tumor efficacy, and safety. We found that the KGHV500 exhibited anti-tumor activity in U251 cells and increased the intracellular expression of anti-p21Ras scFv compared with that in the control groups. CIK cells delivered KGHV500 to U251 glioma cell xenografts and enhanced anti-tumor activity against glioma xenografts compared to that produced by the control treatment. In conclusion, targeting Ras is a useful therapeutic strategy for gliomas and other Ras-driven cancers, and the delivery of anti-p21Ras scFv by recombinant adenovirus and CIK cells may play an essential role in the therapy of Ras-driven cancers.

Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer.

Background: Oncolytic adenovirus-mediated gene therapy is an emerging strategy for cancer treatment. However, oncolytic adenoviruses are mainly administered locally at tumor site. Intravenous administration of oncolytic adenovirus for cancer gene therapy is a problem that needs to be solved urgently. Methods: We constructed recombinant oncolytic adenovirus KGHV500 carrying anti-p21Ras scFv and employed CIK cells to deliver KGHV500. TUNEL, wound healing, MTT, and Transwell invasion assays were used to determine the anti-tumor efficacy of KGHV500 on liver cancer cells in vitro. Nude mouse xenograft model was used to examine the anti-tumor efficacy of CIK cells combined with KGHV500 in vivo. Furthermore, KGHV500 accumulation in different organs was detected to assess the safety. Results: KGHV500 inhibited the migration, proliferation, invasion, and induced the apoptosis of liver cancer cells. CIK cells carrying KGHV500 reached tumor site and exerted much better anti-tumor efficacy than CIK cells or KGHV500 alone in nude mouse xenograft model. Moreover, we detected KGHV500 and anti-p21Ras scFv in different organs of nude mice, with little effects on the organs. Conclusions: We develop a novel strategy for the treatment of Ras-driven liver cancer by combining CIK cells with oncolytic adenovirus expressing anti-p21Ras scFv. Intravenous injection of CIK cells carrying KGHV500 in vivo significantly inhibits tumor growth, has little effect on normal organs, and is relatively safe.

[Comparative analysis of endogenous hormones between two species of Ammopiptanthus seedlings around germination and under Pb2+ stress].

The changes in three endogenous hormones, phytohormones gibberrelic acid (GA3), indoles-3-acid (IAA) and abscisic acid (ABA), were studied around germination and under tress of different density of Pb2+ between two species of Ammopiptanathus. It was found that (1) around germination, in Xinjiang Ammopiptanthus the rate IAA decreased 77.80%, and the rate of ABA decreased 98.90%; and in Mongolia Ammopiptanthus the rate of IAA decreased 75.80%, the rate of ABA decreased 66.20%, and the GA3 contents in both had no big change. (2) With the increase in Pb2+ concentration (20-1 500 mg x L(-1)), the IAA decreased significantly; and only under the high density (more than 1 000 mg x L(-1)) of Pb2+, the GA3 was affected by it; the ABA did not change regularly. So the three endogenous hormone levels of Mongolia Ammopiptanthus were higher than those of Xinjiang Ammopiptanthus. (3) the distributions of Pb2+ in Ammopiptanthus seedlings are the root > stems > leaves. (4) the stress of high concentration of Pb2+ damages irreversibly the leaf cells of Ammopiptanthus. This study can provide the basis of reference data for further research on the growth characteristics, resilience and the mechanism of specific broad-leaved evergreen shrubs-Ammopiptanthus in desertification area.

Comprehensive treatment of rare multiple endocrine neoplasia type 1: A case report.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) is a rare hereditary disorder caused by mutations of the MEN1 gene. It is characterized by hyperparathyroidism and involves the pancreas, anterior pituitary, duodenum, and adrenal gland. Here, we report a 40-year-old male patient with MEN1 who first manifested as thymic carcinoid, then primary hyperparathyroidism and prolactinoma, and a decade later pancreatic neuroendocrine tumor. CASE SUMMARY: The patient underwent a thymectomy because of the thymic carcinoid 10 years prior and a prolactinoma resection 2 years prior. His sister suffered from prolactinoma. His parents displayed a typical triad of amenorrhea, galactorrhea, and infertility. Computed tomography revealed a strong signal in the upper portion of the left lobes and posterior portion of the right lobes of the thyroid and irregular soft tissue densities of the pancreatic body. Positron emission tomography/computed tomography imaging further showed strong 18F-flurodeoxyglucose uptake in the tail of the pancreatic body and segment IV of the liver. The patient underwent pancreatic body tail resection, pancreatic head mass enucleation, and ultrasound-guided radio-frequency ablation for liver cancer. Pathology results reported neuroendocrine tumor grade 2. Whole exome sequencing revealed a verified pathogenic mutation c.378G>A (p.Trp126*) in the MEN1 gene. The diagnosis of MEN1 was confirmed. At the 1.5-year follow-up, the patient appeared healthy without any sign of reoccurrence. CONCLUSION: The present case may add some insight into the diagnosis and treatment of patients with MEN1.

The immunoreactivity of the anti-p21Ras single-chain fragment variant KGH-R1 and its predicted binding sites to p21Ras.

Aim: Previously, we constructed a novel anti-p21Ras single-chain antibody fragment, KGH-R1-single-chain fragment variant (ScFv). However, the immunoreactivity of this antibody toward p21Ras is still unclear. Materials & methods: ELISAs, immunohistochemistry, western blotting and immunofluorescence were used to identify the immunoreactivity of KGH-R1-ScFv toward p21Ras. An in silico approach was used to determine the protein structures of KGH-R1-ScFv and p21Ras and then to predict the site involved in the binding of KGH-R1-ScFv to p21Ras. Results: KGH-R1-ScFv had a specific immune reaction with prokaryotically expressed p21Ras, human tumor cells and tumor tissues with RAS mutations or overexpression of RAS. Molecular docking showed that KGH-R1-ScFv could stably interact with wild-type and mutant p21Ras and the binding sites were in the complementarity-determining regions of KGH-R1-ScFv. Conclusion: KGH-R1-ScFv shows specific immunoreactivity toward wild-type and mutant p21Ras as well as the corresponding tumors, which suggests that KGH-R1-ScFv shows potential as a therapeutic antibody for therapy of RAS-driven tumors.

P21-activated kinase 5 is overexpressed during colorectal cancer progression and regulates colorectal carcinoma cell adhesion and migration.

P21-activated kinase 5 (PAK5) is the recently identified member of the group B p21-activated kinase (PAK) family which are effectors of the small GTPase Cdc42 and Rac1, known to regulate cell motility and activate cell-survival signaling pathways. However, overexpression of PAK5 has not been associated with any cancers so far. Interestingly, we found that PAK5 was overexpressed in a variety of colorectal carcinoma (CRC) cell lines in a Western-blotting examination. Therefore, in this study, we aim to examine the PAK5 expression during CRC progression and to answer if PAK5 is involved in malignant progression of CRC. By immunohistochemistry, our results showed that PAK5 expression was increased with CRC progression through the adenoma to carcinoma sequence, with the most significant increases in invasive and metastatic CRCs (p < 0.0001). Furthermore, increased PAK5 expression was also found with the development of CRC from lower Duke's grades to higher ones (p < 0.01). Moreover, PAK5 was also increased from well to poorly differentiated CRCs (p < 0.01). Using gain and loss of function experiments, we found that PAK5 reduced CRC cell adhesion but promoted their migration on collagen type I. Taken together, our study demonstrated that PAK5 expression increased significantly with malignant progression of CRC and that PAK5 might promote CRC metastasis by regulating CRC cell adhesion and migration.

MicroRNA-527 inhibits TGF-ß/SMAD induced epithelial-mesenchymal transition via downregulating SULF2 expression in non-small-cell lung cancer.

OBJECTIVE: To explore the potential mechanism which miR-527 targeting the heparan sulfate 6-O-endosulfatase (SULF2) regulates TGF-ß/SMAD signaling pathway induced epithelial-mesenchymal transition (EMT) in non-small-cell lung cancer (NSCLC). METHODS: 38 pairs of lung tumor biopsies and corresponding paracancerous biopsies were obtained from NSCLC patients with surgical resection, normal human bronchial epithelial BEAS-2B cells and five NSCLS cell lines were applied for our study. miR-527 and SULF2 expression were determined by qRT-PCR and immunohistochemistry. MiR-527 and SULF2 biological link were predicted by Targetscan.org and tested by dual luciferase. Cells proliferation and apoptosis were respectively detected by EDU staining and flow cytometry. Cells migration was examined by transwell and scratch-wound assay. Expression of proteins related to EMT and TGF-ß/SMAD signaling pathway, such as E-cadherin, N-cadherin, p-Samd3 and p-Smad2, was detected by western blot. RESULTS: miR-527 expression was decreased in lung tumor tissues and NSCLS cell lines, conversely, SULF2 expression was significantly increased. In addition, we found that miR-527 targeted 3'-untranslated regions (3'-UTR) of SULF2 and mediated its expression. Overexpression of miR-527 evidently suppressed NSCLC proliferation, invasion and EMT via TGF-ß/SMAD signaling pathway. Moreover, the silence of SULF2 exhibited a similar effect. CONCLUSION: miR-527 targeting SULF2 down-regulated SULF2 expression, concurrently, suppressed NSCLC epithelial-mesenchymal transition and invasion via inhibiting TGF-ß/SMAD signaling pathway.