Publications by authors named "Yuchi Li"

30 Publications

  • Page 1 of 1

The expression characteristics of FAM71D and its association with sperm motility.

Hum Reprod 2017 11;32(11):2178-2187

Guangdong Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, FuTian District, Shenzhen 518036, China.

Study Question: What are the features of FAM71D (Family with sequence similarity 71, member D) expression and is there an association between FAM71D expression and sperm motility?

Summary Answer: FAM71D, a novel protein exclusively expressed in the testis, is located in sperm flagella and is functionally involved in sperm motility.

What Is Known Already: Some testis-specific proteins have been reported as potential diagnostic biomarkers to evaluate the spermatogenesis process and sperm quality. We have identified a novel testis-specific protein, FAM71D, through microarray data analysis, yet little is known about its expression and function.

Study Design, Size, Duration: FAM71D mRNA and protein expression was quantified during mouse testis development. Its localization in germ cells was detected by dual-labeled immunostaining in testis sections and sperm smears. The clinical significance was assessed by comparing FAM71D expression in spermatozoa from normozoospermic controls and asthenozoospermic patients.

Participants/materials, Setting, Methods: Testes were dissected from C57BL/6 J male mice at postnatal ages of 1, 2, 3, 4, 6, 8 weeks and 6 months, and sperm was collected from cauda epididymides of adult mice by the swim-up method. Human spermatozoa were isolated from 100 human semen samples by density gradient Percoll centrifugation. RT-qPCR and western blot were performed to semi-quantify the expression of FAM71D in mouse testis, and in the ejaculated spermatozoa of normozoospermic controls and asthenozoospermic patients. Immunofluorescence staining was used to detect the localization of FAM71D. Co-immunoprecipitation assay was performed to evaluate the interaction between FAM71D and calmodulin. An antibody blocking assay was employed to assess the role of FAM71D in sperm motility.

Main Results And The Role Of Chance: Our results showed that FAM71D was exclusively expressed in the testis in an age-dependent manner. FAM71D expression exhibited dynamic change in the cytoplasm of spermatids during spermiogenesis and was finally retained in sperm flagella. FAM71D could interact with calmodulin. Use of anti-FAM71D antibody on sperm significantly decreased sperm motility. Expression level of FAM71D was markedly reduced in the ejaculated spermataozoa of asthenozoospermic patients (P < 0.05), and this was correlated with sperm progressive motility (r = 0.7435, P < 0.0001).

Large Scale Data: N/A.

Limitations, Reasons For Caution: The sample size was limited and it is necessary to verify the correlation of FAM71D expression with sperm motility in larger cohorts. Furthermore, our results were descriptive and follow-up studies would be needed to elucidate the detailed role of FAM71D in sperm motility.

Wider Implications Of The Findings: This is the first systematic study to document the expression of endogenous FAM71D and a function for FAM71D in sperm motility. It provides new insights into our understanding of sperm motility regulation and causes of male infertility.

Study Funding/competing Interests: This study was funded by the National Natural Science Foundation of China, Guangdong Natural Science Foundation and the Shenzhen Project of Science and Technology. The authors have no competing interests.
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http://dx.doi.org/10.1093/humrep/dex290DOI Listing
November 2017

GNAQ mutation R183Q as a potential cause of familial Sturge-Weber syndrome: A case report.

Oncol Lett 2017 Apr 1;13(4):2665-2669. Epub 2017 Mar 1.

Department of Neurology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, P.R. China.

Sturge-Weber syndrome (SWS) is a rare neurocutaneous disorder whose etiology remains unclear. To investigate the genetic contribution underlying this disease, the genetic variants of a 4-generation family with a history of SWS was analyzed in the present study. SWS was diagnosed in 3 of the family members (II-1, III-11 and IV-6). Sanger sequencing was performed to identify mutations in G protein subunit αq (GNAQ) and RAS p21 protein activator 1 exons in the 3 patients with SWS and other unaffected family members. Notably, a non-synonymous single-nucleotide variant at codon 183 on exon 4 of the GNAQ gene was identified as the only pathogenic site. This variant generated a substitution of arginine (R) with glutamine and resulted in a change of function of the encoded protein. Evolutionary conservation analysis revealed that the mutated residue 183 (R) of GNAQ is highly conserved across several vertebrate species. Furthermore, an immunofluorescence staining assay demonstrated that the substitution of arginine with glutamine resulted in a change in the sub-cellular localization of the GNAQ recombinant protein . These findings may aid in the development of novel diagnostic markers and/or therapeutic targets for the treatment of patients with familial SWS.
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http://dx.doi.org/10.3892/ol.2017.5791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403265PMC
April 2017

A nonsense mutation in Ccdc62 gene is responsible for spermiogenesis defects and male infertility in repro29/repro29 mice.

Biol Reprod 2017 03;96(3):587-597

Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen Peking University-Hong Kong University of Science and Technology Medical Center, Shenzhen, P.R. China.

Phenotype-driven mutagenesis is an unbiased method to identify novel genes involved in spermatogenesis and other reproductive processes. Male repro29/repro29 mice generated by the Reproductive Genomics Program at the Jackson Laboratory were infertile with deformed sperm and poor motility. Using selected exonic capture and massively parallel sequencing technologies, we identified a nonsense mutation in the exon 6 of coiled-coil domain-containing 62 gene (Ccdc62), which results in a formation of a premature stop codon and a truncated protein. Among the tissues examined, CCDC62 was found to be expressed at the highest level in mouse testis by reverse transcriptase-PCR (RT-PCR) and Western blot analysis. With immunofluorescent staining, we demonstrated that CCDC62 was expressed in the cytoplasm and the developing acrosome in the spematids of mouse testis, and was specifically localized at the acrosome in mature sperm. The complementation analysis by mating repro29/+ mice with Ccdc62 -/- mice (generated by CRISPR-Cas9 strategy) further provided genetic proof that the infertility of repro29/repro29 mice was caused by Ccdc62 mutation. Finally, it was found that intracellular colocalization and interaction of CCDC62 and Golgi-associated PDZ and coiled-coil motif-containing protein may be important for acrosome formation. Taken together, this study identified a nonsense mutation in Ccdc62, which directly results in male infertility in repro29/repro29 mice.
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http://dx.doi.org/10.1095/biolreprod.116.141408DOI Listing
March 2017

Deficiency of SPATA46, a Novel Nuclear Membrane Protein, Causes Subfertility in Male Mice.

Biol Reprod 2016 09 3;95(3):58. Epub 2016 Aug 3.

Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Clinical College of Anhui Medical University, Shenzhen, China

Teratozoospermia is generally associated with clinical infertility. Despite numerous studies, the molecular mechanisms underlying male infertility are still poorly understood. In the present study, we demonstrated that deletion of Spata46, a gene encoding a novel protein of unknown function found in mouse testis, was responsible for male subfertility, and the cause of subfertility was characterized as abnormal sperm head shape and a failure of sperm-egg fusion. We also demonstrated that SPATA46 was expressed predominantly in condensed spermatids, with a highly specific localization restricted to the subacrosomal area; the protein is located at the nuclear membrane due to a transmembrane region in the N-terminus of the protein. At the subcellular level, SPATA46-deficient condensed spermatids displayed structural defects consisting of a discontinuous nuclear envelope and a cavity in the nucleus associated with an abnormal nuclear shape. Additionally, in vitro, we determined that the absence of SPATA46 led to accumulation of sperm around the perivitelline space of eggs, and the same phenomenon was also observed for natural sperm incubated with an anti-SPATA46 antibody, suggesting functional relevance of SPATA46 for sperm-egg fusion. Taken together, these results indicated that SPATA46 is a novel protein involved in reshaping of the sperm head and sperm-egg fusion.
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http://dx.doi.org/10.1095/biolreprod.116.140996DOI Listing
September 2016

A Novel Missense Mutation in USP26 Gene Is Associated With Nonobstructive Azoospermia.

Reprod Sci 2016 10 18;23(10):1434-41. Epub 2016 Apr 18.

Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, PR China

Objective: The aim of this study was to evaluate whether ubiquitin-specific peptidase 26 (USP26) gene variations were associated with nonobstructive azoospermia (NOA).

Methods: Seven hundred and seventy-six patients diagnosed with NOA and 709 proven fertile men were included in this study. Genetic variations of infertility-related genes, including USP26, were identified by selected exonic sequencing. The effects of USP26 mutations on androgen receptor (AR) binding, ubiquitination, and transcriptional activity were detected by immunoprecipitation and luciferase assay in Hela and TM4 cells.

Results: Six novel missense mutations and 1 novel synonymous mutation of USP26 unique to the patients with NOA were identified. Of these missense mutations, USP26 R344W remarkably reduced the binding affinity and deubiquitinating activity of USP26 to AR, thus eliminated the inhibitory effect of USP26 on transcriptional activity of AR in Hela and TM4 cells.

Conclusion: A novel USP26 variant p.R344W is associated with NOA probably through affecting AR function.
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http://dx.doi.org/10.1177/1933719116641758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933177PMC
October 2016

miR‑30a‑5p in the tumorigenesis of renal cell carcinoma: A tumor suppressive microRNA.

Mol Med Rep 2016 May 21;13(5):4085-94. Epub 2016 Mar 21.

Department of Urology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, P.R. China.

Renal cell carcinoma (RCC) is the most common type of malignant tumor of the adult kidney and has a poor prognosis. MicroRNAs (miRs) are important in a wide range of biological and pathological processes, including cell differentiation, migration, growth, proliferation, apoptosis and metabolism. The present study aimed to determine the role exerted by miR‑30a‑5p in the tumorigenesis of RCC. The expression levels of miR‑30a‑5p in RCC tissues and RCC‑derived cells were demonstrated to be significantly downregulated by real‑time quantitative polymerase chain reaction (RT‑qPCR). Wound scratch assay, cell proliferation assay and flow cytometric analysis revealed that the abilities of migration and proliferation of the RCC‑derived cells were suppressed, whereas cell apoptosis was promoted, when miR‑30a‑5p was overexpressed in these cells. N‑acetylgalactosaminyltransferase 7 (GALNT7) was predicted to be one target gene of miR‑30a‑5p by bioinformatics analysis. Luciferase reporter assay, RT‑qPCR and western blotting were performed to confirm that GALNT7 is the direct conserved target of miR‑30a‑5p. These results suggested that miR‑30a‑5p has a tumor‑suppressive role in the tumorigenesis of RCC.
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http://dx.doi.org/10.3892/mmr.2016.5024DOI Listing
May 2016

Identification of long-non coding RNA UCA1 as an oncogene in renal cell carcinoma.

Mol Med Rep 2016 Apr 16;13(4):3326-34. Epub 2016 Feb 16.

Department of Urology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, P.R. China.

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, which is associated with poor prognosis and high recurrence. Long non‑coding RNAs (lncRNAs) have been reported to be dysregulated in cancer and to be important in the regulation of carcinogenesis, thus suggesting that this class of molecules may be used as biomarkers in cancer. The lncRNA urothelial carcinoma associated 1 (UCA1) has been observed to be upregulated and to function as an oncogene in certain types of cancer; however, the role of UCA1 in RCC remains to be elucidated. The present study aimed to determine the expression and function of UCA1 in RCC. Quantitative polymerase chain reaction (qPCR) was used to determine the expression levels of UCA1 in 46 paired RCC and adjacent normal tissue samples. Furthermore, qPCR was used to determine the expression levels of UCA1 in four RCC cell lines compared with the human embryonic kidney 293T cell line. The impact of UCA1 on cell migration, proliferation and apoptosis was investigated by wound scratch assay, MTT and flow cytometry, respectively. The results of the present study demonstrated that UCA1 expression levels were significantly increased in RCC tissues and cells, as compared with the controls. Ectopic expression and gene silencing of UCA1 in RCC cell lines exerted opposite effects on cellular proliferation, migration and apoptosis, and the results suggested that UCA1 may function as an oncogene in RCC. These results indicated that UCA1 may be considered as a promising biomarker for diagnosis, and a therapeutic target in RCC. Further research is required to elucidate the role and target genes of UCA1 in RCC.
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http://dx.doi.org/10.3892/mmr.2016.4894DOI Listing
April 2016

Oncogenic microRNA-142-3p is associated with cellular migration, proliferation and apoptosis in renal cell carcinoma.

Oncol Lett 2016 Feb 10;11(2):1235-1241. Epub 2015 Dec 10.

Department of Urology, Peking University Shenzhen Hospital, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, Guangdong 518036, P.R. China; The Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, Guangdong 518036, P.R. China.

MicroRNAs (miRNAs/miRs) serve an important role in the regulation of carcinogenic pathways. RCC is the most prevalent kidney cancer that occurs in adults. miRNAs have gained increasing attention due to their association with RCC tumorigenesis, serving as biomarkers for early detection and progression monitoring, and as potential targets for molecular therapy. Upregulation of miRNA-142-3p has been previously identified in RCC tissues by microarray profile, however, its expression and function in RCC have not yet been validated. In the present study, quantitative polymerase chain reaction was performed to quantify the relative expression of miR-142-3p in 53 paired RCC and adjacent normal tissues. Furthermore, wound healing, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assays were performed to analyze the impacts of miR-142-3p on cellular migration, proliferation and apoptosis. The results demonstrated that miR-142-3p was significantly upregulated in RCC tissues compared with adjacent normal tissues. Downregulation of miR-142-3p, induced by chemically synthesized miR-142-3p inhibitor, significantly suppressed cell migration and proliferation, and promoted cell apoptosis in 786-O and ACHN cells, supporting the theory that miR-142-3p may function as an oncogene in RCC. The potential clinical significance of miR-142-3p, as a biomarker and therapeutic target, provides rationale for further investigation into the miR-142-3p-mediated molecular pathway and how it is associated with RCC development.
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http://dx.doi.org/10.3892/ol.2015.4021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734216PMC
February 2016

Oncogenic cAMP responsive element binding protein 1 is overexpressed upon loss of tumor suppressive miR-10b-5p and miR-363-3p in renal cancer.

Oncol Rep 2016 Apr 20;35(4):1967-78. Epub 2016 Jan 20.

Department of Urology, Peking University Shenzhen Hospital, Institute of Urology of Shenzhen PKU‑HKUST Medical Center, Shenzhen, Guangdong 518036, P.R. China.

Renal cell carcinoma (RCC) is the most common kidney cancer in adults and has a poor prognosis. cAMP responsive element binding protein 1 (CREB1) is a proto‑oncogenic transcription factor involved in malignancies of various organs. However, its functional role(s) have not yet been elucidated in RCC. We investigated the expression pattern, function and regulation of CREB1 in RCC. CREB1 was overexpressed in the RCC tissues and cell lines. Downregulation of CREB1 inhibited RCC tumorigenesis by affecting cell proliferation, migration and apoptosis. Multiple computational algorithms predicted that the 3'‑untranslated region (3'‑UTR) of human CREB1 mRNA is a target for miR‑10b‑5p and miR‑363‑3p. Luciferase reporter assay, qPCR and western blot analysis confirmed that miR‑10b‑5p and miR‑363‑3p bind directly to the 3'‑UTR of CREB1 mRNA and inhibit mRNA and protein expression of CREB1. qPCR data also revealed a significantly lower expression of miR‑10b‑5p and miR‑363‑3p in RCC tissues. Introduction of miR‑10b‑5p and miR‑363‑3p mimics led to suppressed expression of CREB1 and inhibited cell proliferation, migration and apoptosis reduction. Taken together, we propose that CREB1 is an oncogene in RCC and that upregulation of CREB1 by loss of tumor suppressive miR‑10b‑5p and miR‑363‑3p plays an important role in the tumorigenesis of RCC.
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http://dx.doi.org/10.3892/or.2016.4579DOI Listing
April 2016

Identification of miR‑130b as an oncogene in renal cell carcinoma.

Mol Med Rep 2016 Feb 30;13(2):1902-8. Epub 2015 Dec 30.

Department of Urology, Peking University Shenzhen Hospital, Shenzhen PKU‑HKUST Medical Center, Shenzhen, Guangdong 518036, P.R. China.

Renal cell carcinoma (RCC) is the most common type of renal tumor, which has a poor prognosis. Improvements in understanding the underlying molecular biology of RCC has led to systemic treatments, which have markedly improved patient outcomes. Therefore, it is necessary and worthwhile to identify novel biomarkers for RCC. MicroRNAs (miRNAs) have been found to be important in a wide range of biological and pathological processes, including cell differentiation, migration, growth, proliferation, apoptosis and metabolism. Aberrant expression of miRNA‑130b has previously been reported in tumors, however, its role in RCC remains to be elucidated. In the present study, the upregulation of miR‑130b was observed in RCC tissues and cell lines using reverse transcription‑quantitative polymerase chain reaction analysis, which was consistent with previous microRNA profiling in RCC. Furthermore, the effects of miR‑130b on cell migration, proliferation and apoptosis were examined using a wound scratch assay, an MTT assay and flow cytometric analysis, respectively. The results demonstrated that the downregulation of miR‑130b by a synthesized inhibitor inhibited cell migration, suppressed cell proliferation and induced RCC cell apoptosis. The present study was the first, to the best of our knowledge, to suggest that miR‑130b may be a promising biomarker for diagnosis and a therapeutic target for the treatment of RCC. Further investigations are required to examine the roles and target genes of miR‑130b in RCC.
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http://dx.doi.org/10.3892/mmr.2015.4744DOI Listing
February 2016

MicroRNA-20b-5p functions as a tumor suppressor in renal cell carcinoma by regulating cellular proliferation, migration and apoptosis.

Mol Med Rep 2016 Feb 17;13(2):1895-901. Epub 2015 Dec 17.

Department of Urology, Peking University Shenzhen Hospital, Institute of Urology of Shenzhen PKU‑HKUST Medical Center, Shenzhen, Guangdong 518036, P.R. China.

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and is associated with a poor prognosis due to a lack of early‑warning signs, protean clinical manifestations, and resistance to radiotherapy and chemotherapy. Recently, increasing evidence has suggested that microRNAs (miRNAs) are involved in the proliferation, invasion and apoptosis of various types of human cancer cells. In a previous study, miRNA expression profiles from renal cell carcinoma (RCC) revealed that expression of miR‑20b‑5p was significantly downregulated in RCC tissues. The aim of this study was to investigate the expression and functional significance of miR‑20b‑5p in RCC. The expression of miR‑20b‑5p was quantified in 48 paired RCC tissues and cell lines, and compared with adjacent normal tissues and the 293T cell line by reverse transcription‑quantitative polymerase chain reaction. The functional impact of miR‑20b‑5p on cell proliferation, cell migration and apoptosis in the 786‑O and ACHN RCC cell lines, was determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a scratch assay and flow cytometry. To the best of our knowledge, the present study was the first to reveal that miR‑20b‑5p was downregulated in RCC tissues and cell lines. It also demonstrated that upregulation of miR‑20b‑5p inhibited cellular migration and proliferation, and promoted cellular apoptosis, suggesting that miR‑20b‑5p functioned as a potential tumor suppressor. However, further studies are required to fully determine the effects of miR‑20b‑5p and the miR‑20b‑5p‑mediated molecular pathway in RCC and other types of cancer. In conclusion, these results imply that miR‑20b‑5p may be a biomarker for early detection and prognosis prediction, as well as a therapeutic target for RCC.
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http://dx.doi.org/10.3892/mmr.2015.4692DOI Listing
February 2016

Protein Arginine Methyltransferase 6 Involved in Germ Cell Viability during Spermatogenesis and Down-Regulated by the Androgen Receptor.

Int J Mol Sci 2015 Dec 10;16(12):29467-81. Epub 2015 Dec 10.

Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen 518036, China.

Androgens and the androgen receptor (AR) are of great importance to spermatogenesis and male fertility. AR knockout (ARKO) mice display a complete insensitivity to androgens and male infertility; however, the exact molecular mechanism for this effect remains unclear. In this study, we found that the expression levels of Prmt6 mRNA and protein were significantly up-regulated in the testes of ARKO mice compared to wild type (WT) mice. PRMT6 was principally localized to the nucleus of spermatogonia and spermatocytes by immunofluorescence staining. Furthermore, luciferase assay data showed that AR together with testosterone treatment suppressed Prmt6 transcription via binding to the androgen-responsive element (ARE) of the Prmt6 promoter. Moreover, knockdown of Prmt6 suppressed germ cells migration and promoted apoptosis. In addition, both of these cellular activities could not be enhanced by testosterone treatment. Taken together, these data indicate that PRMT6, which was down-regulated by AR and influenced cell migration and apoptosis of germ cells, could play a potentially important role in spermatogenesis.
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http://dx.doi.org/10.3390/ijms161226186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691129PMC
December 2015

MicroRNA-106b functions as an oncogene in renal cell carcinoma by affecting cell proliferation, migration and apoptosis.

Mol Med Rep 2016 Feb 8;13(2):1420-6. Epub 2015 Dec 8.

Department of Urology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, P.R. China.

Kidney cancer is the 14th most common cancer in the world and its prognosis remains poor due to difficult early detection and treatment. Therefore, the identification of biomarkers for early-stage renal cell carcinoma (RCC) is important. MicroRNA-106b (miR-106b) has been described as an oncogene in several types of human cancer. Previous microarray studies have suggested that miR-106b was significantly upregulated in RCC tissues compared with paired normal kidney tissues and may be a promising biomarker for the prediction of early metastasis following nephrectomy. The present study aimed to determine the expression and function of miR-106b in RCC. The expression of miR-106b in RCC tissues and cells, and in paired normal tissues and cells was determined by reverse transcription quantitative polymerase chain reaction, based on the previous sequencing results of miRNAs. Furthermore, a wound scratch assay, MTT assay and flow cytometry were performed to examine the functions of miR-106b on cell migration, proliferation and apoptosis. The results demonstrated that miR-106b was upregulated in RCC tissues and cell lines compared with control normal tissues and cell lines. Downregulation of miR-106b with a synthesized inhibitor suppressed cell migration and proliferation and induced renal cancer cell apoptosis, suggesting that miR-106b can be characterized as an oncogene in RCC. To the best of our knowledge, the present study was the first to reveal that miR-106b is upregulated and affects cellular migration, proliferation and apoptosis in RCC. Further studies are required to examine the role and target genes of miR-106b in RCC.
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http://dx.doi.org/10.3892/mmr.2015.4656DOI Listing
February 2016

miR-362-3p targets nemo-like kinase and functions as a tumor suppressor in renal cancer cells.

Mol Med Rep 2016 Jan 30;13(1):994-1002. Epub 2015 Nov 30.

National‑Regional Key Technology Engineering Laboratory for Clinical Application of Cancer Genomics, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China.

MicroRNAs (miRNAs) have been demonstrated to exhibit abnormal expression patterns in various types of human cancer. The aim of the present study was to identify a novel tumor suppressor microRNA (miR) and investigate its physiological function and mechanism in renal cell carcinoma (RCC). The expression levels of miRNA (miR)‑362‑3p expres were measured in 47 pairs of RCC and adjacent normal tissue samples, using reverse transcription-quantitative polymerase chain reaction analysis. In addition, miR‑362‑3p was transfected into renal cancer cells to investigate its role in the regulation of cell proliferation, migration, invasion, apoptosis and cell cycle. Identification of the target gene of miR‑362‑3p was performed using luciferase reporter assays and western blot analyses. The results demonstrated that the expression levels of miR‑362‑3p were downregulated in the RCC tissue samples, compared with the adjacent normal tissue samples. The upregulation of miR‑362‑3p using a synthesized mimic suppressed the proliferation, migration and invasion of the renal cancer cells, and induced cell apoptosis and G1 phase arrest. Further experiments demonstrated that the overexpression of miR‑362‑3p resulted in decrease expression levels of nemo-like kinase. These results suggested that miR-362-3p functions as a tumor suppressor in RCC, and may serve as a potential molecular target in the treatment of RCC.
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http://dx.doi.org/10.3892/mmr.2015.4632DOI Listing
January 2016

Tumor suppressive microRNA‑429 regulates cellular function by targeting VEGF in clear cell renal cell carcinoma.

Mol Med Rep 2016 Feb 7;13(2):1361-6. Epub 2015 Dec 7.

Department of Urology, Peking University Shenzhen Hospital, Institute of Urology of Shenzhen PKU‑HKUST Medical Center, Shenzhen, Guangdong 518036, P.R. China.

Clear cell renal cell carcinoma (ccRCC) is the predominant and most aggressive type of kidney malignancy, however, the mechanism underlying its carcinogenesis remains to be elucidated. The present study aimed to determine the expression and function of microRNA (miR)‑429 in ccRCC carcinogenesis. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to detect the expression of miR‑429 in ccRCC specimens. Following transfection of miR‑429 synthetic mimics, the expression of miR‑429 was examined and cell proliferation, cell migration, apoptosis and luciferase assays were conducted in ccRCC cell lines. The results demonstrated that expression of miR‑429 was decreased in ccRCC cells. In addition, upregulation of miR‑429 by transfection of mimics reduced cellular proliferation and migration, and induced apoptosis in ACHN and 786‑0 cell lines. Furthermore, miR‑429 decreased the 3'UTR luciferase activity of vascular endothelial growth factor (VEGF) and c‑MYC, and RT‑qPCR analysis demonstrated that the cancer cells transfected with miR‑429 mimics exhibited decreased expression of VEGF, but not c‑MYC. To the best of our knowledge, the present study was the first to reveal that downregulated miR‑429 functioned as a tumor suppressor by restraining cellular proliferation and migration, and inducing apoptosis, as well as targeting VEGF in ccRCC cells.
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http://dx.doi.org/10.3892/mmr.2015.4653DOI Listing
February 2016

Androgen receptor binding to an androgen-responsive element in the promoter of the Srsf4 gene inhibits its expression in mouse Sertoli cells.

Mol Reprod Dev 2015 Dec 3;82(12):976-85. Epub 2015 Sep 3.

Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, P.R. China.

The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y)) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells.
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http://dx.doi.org/10.1002/mrd.22576DOI Listing
December 2015

FAM170B, a novel acrosomal protein involved in fertilization in mice.

Mol Reprod Dev 2015 Oct 30;82(10):787-96. Epub 2015 Sep 30.

Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, P.R. China.

The acrosome is a specialized organelle that covers the anterior region of the sperm nucleus, and plays an essential role in mammalian fertilization. Although acrosome biogenesis is an important aspect of spermiogenesis, the molecular mechanism that regulates this event remains unknown. In the present study, we identified a novel gene, Fam170b (family with sequence similarity 170, member B), exclusively expressed in mouse testes. Fam170b expression first started at postnatal week 3, and increased in an age-dependent manner until plateauing in adulthood. Immunofluorescence staining revealed its enrichment in round spermatids, and redistribution to a perinuclear spot adjacent to the Golgi and the acrosome of elongating spermatids and spermatozoa; this localization was shared between mouse and human spermatozoa. Anti-FAM170B antibody was remarkably found to inhibit murine in vitro fertilization, specifically blocking the acrosome reaction. We further determined that FAM170B interacts with GOPC (Golgi-associated PDZ and coiled-coil motif containing protein) during acrosome formation, as verified by immunofluorescence and co-immunoprecipitation assays. Thus, we document the expression and function for the endogenous acrosomal protein FAM170B during spermiogenesis and fertilization.
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http://dx.doi.org/10.1002/mrd.22523DOI Listing
October 2015

Downregulated microRNA-510-5p acts as a tumor suppressor in renal cell carcinoma.

Mol Med Rep 2015 Aug 29;12(2):3061-6. Epub 2015 Apr 29.

Department of Urology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, P.R. China.

MicroRNA (miR)-510-5p has been demonstrated to be involved in a number of types of malignancy; however, the function of miR-510-5p in renal cancer remains unclear. The present study aimed to determine the expression of miR-510-5p in renal cell carcinoma (RCC) specimens and analyzed the impact of miR-510-5p on renal cancer by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound scratch and apoptosis assays. The results showed that miR-510-5p was significantly downregulated in RCC specimens compared with normal renal specimens. Overexpression of miR-510-5p by synthetic mature mimics reduced cell proliferation and migration and induced an increase in cell apoptosis, indicating that miR-510-5p may act as a tumor suppressor in RCC. The present study firstly revealed that downregulated miR-510-5p functioned as a tumor suppressor by reducing cellular proliferation and migration, and inducing apoptosis in RCC. Further research is required to define target genes of miR-510-5p to determine the cellular mechanism of miR-510-5p in the carcinogenesis of RCC.
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http://dx.doi.org/10.3892/mmr.2015.3704DOI Listing
August 2015

Upregulated microRNA-16 as an oncogene in renal cell carcinoma.

Mol Med Rep 2015 Jul 17;12(1):1399-404. Epub 2015 Mar 17.

Department of Urology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, P.R. China.

MicroRNAs (miRs) are small, endogenous noncoding RNAs that serve a significant function in various biologic processes, including those involved in cancer. The present study aimed to determine the expression and function of miR-16 in renal cell carcinoma (RCC). Quantitative polymerase chain reaction was used to quantify the expression of miR-16 in 48 paired RCC tissues and adjacent normal tissues. The impact of miR-16 on cell proliferation, migration and apoptosis was analyzed by transfecting miR-16 mature molecules into the renal cancer cell lines 786-O and ACHN. The results indicated that miR-16 was significantly upregulated in RCC tissues (P<0.05). Downregulation of miR-16 resulted in reduced cell proliferation and migration and increased levels of apoptosis, while overexpression of miR-16 resulted in accelerated cellular proliferation and migration, suggesting that miR-16 may function as an oncogene in RCC. The present study demonstrated for the first time, to the best of our knowledge, that miR-16 is upregulated in RCC and acts as an oncogene by inducing cellular proliferation, migration and reducing apoptosis. Further study of miR-16 in RCC may clarify the molecular mechanisms of RCC carcinogenesis and aid in the development of novel biomarkers and therapeutic options.
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http://dx.doi.org/10.3892/mmr.2015.3496DOI Listing
July 2015

Identification of miR‑125a‑5p as a tumor suppressor of renal cell carcinoma, regulating cellular proliferation, migration and apoptosis.

Mol Med Rep 2015 Feb 3;11(2):1278-83. Epub 2014 Nov 3.

Department of Urology, Peking University Shenzhen Hospital, Institute of Urology of Shenzhen PKU‑HKUST Medical Center, Shenzhen, Guangdong 518036, P.R. China.

miR‑125a‑5p has been previously described as a tumor suppressor in numerous malignancies, however the expression and function of miR‑125a‑5p in renal cell carcinoma (RCC) remains to be elucidated. In the present study, to explore the potential role of miR‑125a‑5p in RCC, quantitative polymerase chain reaction was used to determine the expression levels of miR‑125a‑5p in renal cancer tissues. The influence of miR‑125a‑5p on cell proliferation, migration and apoptosis was also determined, using an MTT assay, a wound scratch assay and flow cytometry, respectively. The expression of miR‑125a‑5p was shown to be decreased in RCC and the restoration of miR‑125a‑5p by synthetic mimics was shown to suppress cell proliferation and migration, and induce apoptosis. The present results indicate that miR‑125a‑5p may function as a tumor suppressor in RCC. The present study is, to the best of our knowledge, the first to demonstrate the downregulation of miR‑125a‑5p in RCC, and to show the role it has in affecting cellular proliferation, migration and apoptosis. Further research is needed to define the target genes of miR‑125a‑5p and explore the potential of miR‑125a‑5p as a diagnostic or a prognostic biomarker for RCC.
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http://dx.doi.org/10.3892/mmr.2014.2848DOI Listing
February 2015

Hsa-miR-1 downregulates long non-coding RNA urothelial cancer associated 1 in bladder cancer.

Tumour Biol 2014 Oct;35(10):10075-84

MicroRNAs (miRNAs) are known to mainly target protein-coding genes at post-transcriptional level, resulting in mRNA destabilization and/or translational repression. Long non-coding RNAs (lncRNAs) are emerging as a novel set of targets for miRNAs. Here, we report that downregulated hsa-miR-1 and upregulated lncRNA urothelial cancer associated 1 (UCA1) were inversely expressed in bladder cancer. Hsa-miR-1 decreased the expression of UCA1 in bladder cancer cells in an Ago2-slicer-dependent manner. The binding site between UCA1 and hsa-miR-1 was confirmed. Overexpression of hsa-miR-1 inhibited bladder cancer cell growth, induced apoptosis, and decreased cell motility. Knockdown of UCA1 expression phenocopied the effects of upregulation of hsa-miR-1. Transfection of UCA1 expression vector partly reversed the changes caused by transfection of pre-miR-1 plasmids. This study provides evidence for hsa-miR-1 to play tumor suppressive roles via downregulating lncRNA UCA1 in bladder cancer, which may have potential therapeutic significance.
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http://dx.doi.org/10.1007/s13277-014-2321-2DOI Listing
October 2014

Superoxide dismutase attenuates hyperoxia-induced interleukin-8 induction via AP-1.

Free Radic Biol Med 2008 Oct 22;45(8):1143-9. Epub 2008 Jul 22.

CardioPulmonary Research Institute, Department of Medicine, Winthrop University Hospital, State University of New York Stony Brook School of Medicine, Mineola, NY 11501, USA.

Exposure of lung epithelial cells to hyperoxia results in the generation of excess reactive oxygen species (ROS), cell damage, and production of proinflammatory cytokines (interleukin-8; IL-8). Although activation of the NF-kappaB and c-Jun N-terminal kinase (JNK)/activator protein (AP)-1 transcription pathways occurs in hyperoxia, it is unclear whether activation of the AP-1 pathway has a direct impact on IL-8 production and whether overexpression of superoxide dismutase (SOD) can mitigate these proinflammatory processes. A549 cells were exposed to 95% O(2), and ROS production, AP-1 activation, and IL-8 levels were determined. Experimental groups included cells transduced with a recombinant adenovirus encoding CuZnSOD or MnSOD (two- to threefold increased activity) or transfected with a JNK1 small interfering RNA (RNAi). Hyperoxia resulted in significant increases in ROS generation, AP-1 activation, and IL-8 production, which were significantly attenuated by overexpression of either MnSOD or CuZnSOD. JNK1 RNAi also moderated IL-8 induction. The data indicate that activation of JNK1/AP-1 and subsequent IL-8 induction in hyperoxia are mediated by intracellular ROS, with SOD having significant protective effects.
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http://dx.doi.org/10.1016/j.freeradbiomed.2008.07.006DOI Listing
October 2008

HIV antiretroviral drug combination induces endothelial mitochondrial dysfunction and reactive oxygen species production, but not apoptosis.

Toxicol Appl Pharmacol 2007 Oct 30;224(1):60-71. Epub 2007 Jun 30.

Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71103, USA.

Numerous reports now indicate that HIV patients administered long-term antiretroviral therapy (ART) are at a greater risk for developing cardiovascular diseases. Endothelial dysfunction is an initiating event in atherogenesis and may contribute to HIV-associated atherosclerosis. We previously reported that ART induces direct endothelial dysfunction in rodents. In vitro treatment of human umbilical vein endothelial cells (HUVEC) with ART indicated endothelial mitochondrial dysfunction and a significant increase in the production of reactive oxygen species (ROS). In this study, we determined whether ART-induced endothelial dysfunction is mediated via mitochondria-derived ROS and whether this mitochondrial injury culminates in endothelial cell apoptosis. Two major components of ART combination therapy, a nucleoside reverse transcriptase inhibitor and a protease inhibitor, were tested, using AZT and indinavir as representatives for each. Microscopy utilizing fluorescent indicators of ROS and mitochondria demonstrated the mitochondrial localization of ART-induced ROS. MnTBAP, a cell-permeable metalloporphyrin antioxidant, abolished ART-induced ROS production. As a final step in confirming the mitochondrial origin of the ART-induced ROS, HUVEC were transduced with a cytosolic- compared to a mitochondria-targeted catalase. Transduction with the mitochondria-targeted catalase was more effective than cytoplasmic catalase in inhibiting the ROS and 8-isoprostane (8-iso-PGF2alpha) produced after treatment with either AZT or indinavir. However, both mitochondrial and cytoplasmic catalase attenuated ROS and 8-iso-PGF2alpha production induced by the combination treatment, suggesting that in this case, the formation of cytoplasmic ROS may also occur, and thus, that the mechanism of toxicity in the combination treatment group may be different compared to treatment with AZT or indinavir alone. Finally, to determine whether ART-induced mitochondrial dysfunction and ROS production culminate in apoptosis, we performed the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), annexin V and 4',6-diamidino-2-phenylindole (DAPI) staining, and caspase-3 activity assays. However, none of these assays showed appreciable levels of ART-induced apoptosis. Our studies thus suggest that in endothelial cells, ART induces mitochondrial dysfunction with a concomitant increase in mitochondria-derived ROS. This compromised mitochondrial function may be one important factor culminating in endothelial dysfunction, without inducing an increase in apoptosis.
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http://dx.doi.org/10.1016/j.taap.2007.06.010DOI Listing
October 2007

Antioxidants improve antibacterial function in hyperoxia-exposed macrophages.

Free Radic Biol Med 2007 May 15;42(10):1517-23. Epub 2007 Feb 15.

CardioPulmonary Research Institute, Winthrop University Hospital, State University of New York at Stony Brook School of Medicine, Mineola, NY 11501, USA.

Hyperoxia and pulmonary infections are well known to increase the risk of acute and chronic lung injury in newborn infants, but it is not clear whether hyperoxia directly increases the risk of pneumonia. The purpose of this study was to examine: (1) the effects of hyperoxia and antioxidant enzymes on inflammation and bacterial clearance in mononuclear cells and (2) developmental differences between adult and neonatal mononuclear cells in response to hyperoxia. Mouse macrophages were exposed to either room air or 95% O2 for 24 h and then incubated with Pseudomonas aeruginosa. After 1 h, bacterial adherence, phagocytosis, and macrophage inflammatory protein (MIP)-1alpha production were analyzed. Bacterial adherence increased 5.8-fold (p < 0.0001), phagocytosis decreased 60% (p < 0.05), and MIP-1alpha production increased 49% (p < 0.05) in response to hyperoxia. Overexpression of MnSOD or catalase significantly decreased bacterial adherence by 30.5%, but only MnSOD significantly improved bacterial phagocytosis and attenuated MIP-1alpha production. When monocytes from newborns and adults were exposed to hyperoxia, phagocytosis was impaired in both groups. However, adult monocytes were significantly more impaired than neonatal monocytes. Data indicate that hyperoxia significantly increases bacterial adherence while impairing function of mononuclear cells, with adult cells being more impaired than neonatal cells. MnSOD reduces bacterial adherence and inflammation and improves bacterial phagocytosis in mononuclear cells in response to hyperoxia, which should minimize the development of oxidant-induced lung injury as well as reducing nosocomial infections.
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http://dx.doi.org/10.1016/j.freeradbiomed.2007.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1963462PMC
May 2007

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells.

Am J Physiol Lung Cell Mol Physiol 2006 May 30;290(5):L978-86. Epub 2005 Dec 30.

Department of Pediatrics, CardioPulmonary Research Institute, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Suite 505, 222 Station Plaza North, Mineola, NY 11501, USA.

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.
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http://dx.doi.org/10.1152/ajplung.00296.2005DOI Listing
May 2006

Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia.

Am J Physiol Lung Cell Mol Physiol 2005 Apr 3;288(4):L718-26. Epub 2004 Dec 3.

CardioPulmonary Research Institute, Winthrop University Hospital, 222 Station Plaza North, Suite 604, Mineola, NY 11501, USA.

Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50-300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O(2) for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.
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http://dx.doi.org/10.1152/ajplung.00456.2003DOI Listing
April 2005

Superoxide dismutase moderates basal and induced bacterial adherence and interleukin-8 expression in airway epithelial cells.

Am J Physiol Lung Cell Mol Physiol 2004 Dec 30;287(6):L1199-206. Epub 2004 Jul 30.

CardioPulmonary Research Institute, Department of Medicine, Division of Pulmonary and Critical Care Medicine, Winthrop-University Hospital, SUNY Stony Brook School of Medicine, Mineola, NY 11501, USA.

Bacterial infection of the tracheobronchial tree is a frequent, serious complication in patients receiving treatment with oxygen and mechanical ventilation, resulting in increased morbidity and mortality. Using human airway epithelial cell culture models, we examined the effect of hyperoxia on bacterial adherence and the expression of interleukin-8 (IL-8), an important mediator involved in the inflammatory process. A 24-h exposure to 95% O(2) increased Pseudomonas aeruginosa (PA) adherence 57% in A549 cells (P < 0.01) and 115% in 16HBE cells (P < 0.01) but had little effect on Staphylococcus aureus (SA) adherence. Exposure to hyperoxia, followed by a 1-h incubation with SA, further enhanced PA adherence (P < 0.01), suggesting that hyperoxia and SA colonization may enhance the susceptibility of lung epithelial cells to gram-negative infections. IL-8 expression was also increased in cells exposed to both hyperoxia and PA. Stable or transient overexpression of manganese superoxide dismutase reduced both basal and stimulated levels of PA adherence and IL-8 levels in response to exposure to either hyperoxia or PA. These data indicate that hyperoxia increases susceptibility to infection and that the pathways are mediated by reactive oxygen species. Therapeutic intervention strategies designed to prevent accumulation of intracellular reactive oxygen species may reduce opportunistic pulmonary infections.
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http://dx.doi.org/10.1152/ajplung.00457.2003DOI Listing
December 2004

Delivering antioxidants by zip code.

Am J Physiol Lung Cell Mol Physiol 2003 Aug;285(2):L281-2

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http://dx.doi.org/10.1152/ajplung.00092.2003DOI Listing
August 2003

Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury.

Am J Respir Cell Mol Biol 2003 Dec 3;29(6):779-83. Epub 2003 Jul 3.

The CardioPulmonary Research Institute, Winthrop-University Hospital, Suite 505, 222 Station Plaza N, Mineola, NY 11501, USA.

Oxidant insults can lead to apoptotic and nonapoptotic cell death. Lung epithelial cells exposed to high levels of oxygen do not die via apoptosis, but through a much slower, morphologically distinct process involving cell and nuclear swelling. In contrast, H2O2 induces a rapid apoptotic cell death. We first assessed the effect of oxidant exposure on activator protein-1 (c-Jun and Fos) and c-Jun N-terminal kinase (JNK) regulation in MLE12 cells. Both oxidants induced c-Jun and Fos expression, albeit with a different pattern of regulation-hyperoxia (95% O2) induced a biphasic response, whereas H2O2 (500 microM) induced a sustained response. We then examined the role of JNK by Western blot, JNK activity assay, and a pull-down assay and observed an identical pattern of regulation. To assess whether JNK functions in a pro-death or pro-survival capacity, we generated stable cell lines that constitutively express a dominant-negative mutation of JNK resulting in significant inhibition of JNK activity. Inhibition of the JNK pathway in this manner prevented hyperoxic and H2O2-induced cell death. These results demonstrate that hyperoxic cell death is pathway-driven and that both modes of death involve the JNK signaling pathway.
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http://dx.doi.org/10.1165/rcmb.2003-0087RCDOI Listing
December 2003