Vector-transfected Cells Research Articles

Let-7 miRNA silencing promotes Kaposi's sarcoma-associated herpesvirus lytic replication via activating mitogen-activated protein kinase kinase kinase kinase 4 and its downstream factors

To explore the effect of let-7 miRNA silencing on Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and the underling mechanism. The pEGFP-C2-let-7 sponge vector was transfected into BCBL-1 and 293T cells with Lipofectamine 2000 to silence the expression of let-7 miRNAs. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of let-7 miRNAs, the transcriptional levels of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), cyclooxygenase-2 (COX-2) and matrix metalloproteinase 13 (MMP-13), and the DNA copy numbers of KSHV open reading frame 50 (ORF50) and open reading frame 72 (ORF72). Western blot was used to detect the total and phosphorylated protein levels of MAP4K4, COX-2, extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK. The expression of let-7 miRNAs was dramatically decreased in the let-7 sponge transfected BCBL-1 and 293T cells compared with that in the vector-transfected cells (P<0.05 for all). The gene copy number and mRNA transcriptional level of KSHV ORF50 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with that in the vector-transfected cells (1.00±0.10 vs. 2.33±0.18 and 1.08±0.48 vs 3.22±0.27, respectively,P<0.001 for both). The gene copy number and mRNA transcriptional level of KSHV ORF72 were also significantly increased in let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells(1.07±0.49 vs 1.67±0.45 and 1.01±0.19 vs 1.54±0.11, respectively,P<0.05 for both). Furthermore, the mRNA transcriptional levels of MAP4K4, COX-2 and MMP-13 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells (1.00±0.05 vs 5.73±0.96, 1.00±0.05 vs 2.68±0.19, 1.00±0.02 vs 2.69±0.25, respectively,P<0.001 for all). Let-7 miRNAs silencing also increased the protein expression levels of MAP4K4, COX-2 and phospho-ERK1/2, while the phospho-JNK and phospho-p38 were not changed in the BCBL-1 and 293T cells. Let-7 silencing may activate the replication of KSHV, possibly through up-regulating MAP4K4 and its downstream molecules COX-2, MMP-13, and phosphorylation of ERK1/2, finally results in the progression of Kaposi sarcoma.

CACNA1H(M1549V) Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil.

We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1H(M1549V), causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1H(M1549V) leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production. We heterologously expressed empty vector, CACNA1H(WT) and CACNA1H(M1549V) in the aldosterone-producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, expression levels, and subcellular distribution of the channel were similar between CACNA1H(WT) and CACNA1H(M1549V). We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1H(WT) led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1H(M1549V) caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1H(WT)- and CACNA1H(M1549V)-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1H(WT) and CACNA1H(M1549V) on CYP11B2 expression. These results directly link CACNA1H(M1549V) to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.

Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil

In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively.

The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met.

The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.

Vector-Based let-7a miRNAs Function as Suppressors of Multiple Oncogenes in SK-N-MC and SHEP Neuroblastoma Cells

Objective: This study was designed to determine the anti-tumor effect of vector-based let-7a miRNA in neuroblastoma cell lines. Methods: A tetracycline (tet)-inducible let-7a expression vector was constructed and used to stably transfect SKN- MC and SHEP neuroblastoma cells. The effects of let-7a overexpression induced by tetracycline on cell proliferation and adhesion were analyzed in vitro by MTT assays and adhesion assays. The mRNA expression of the let-7 target oncogenes NRAS, KRAS, c-myc, and HMGA2 was examined by quantitative real-time reverse transcription polymerase chain reaction. Protein expression of N-RAS, K-RAS, c-Myc, HMGA2, NeuN, and β3- tubulin was analyzed using western blot and immunocytochemistry. Morphology of SK-N-MC and SHEP cells was also observed. Additionally, the let-7a-inducible vector-transfected SHEP cells were subcutaneously injected in nude mice, and tumor growth in vivo was also evaluated. Results: Let-7a expression was significantly induced in neuroblastoma cells and inhibited cell proliferation and adhesion in both SK-N-MC and SHEP cells. Let-7a up-regulated β3-tubulin and NeuN expression in SK-N-MC and SHEP cells. However, short neurite-like outgrowth was observed only in SK-N-MC cells, but not in SHEP cells. Let-7a overexpression decreased the expression of N-RAS and HMGA2 proteins in both SK-N-MC and SHEP cells. c-Myc expression also decreased in SK-N-MC cells. The tumor sizes of SHEP xenografts in the tet-inducible group were smaller than those in the mice without tetracycline induction. Conclusion: Taken together, our data demonstrate the anti-tumor effects of vector-based let-7a miRNA overexpression in SK-N-MC and SHEP neuroblastoma cells against multioncogenes. Let-7a miRNA is a potential therapeutic molecule for the treatment of neuroblastoma.

Eel green fluorescent protein is associated with resistance to oxidative stress

Green fluorescent protein (GFP) from eel (Anguilla japonica) muscle (eelGFP) is unique in the vertebrates and requires bilirubin as a ligand to emit fluorescence. This study was performed to clarify the physiological function of the unique GFP. Investigation of susceptibility to oxidative stress was carried out using three types of cell lines including jellyfish (Aequorea coerulescens) GFP (jfGFP)-, or eel GFP (eelGFP)-expressing HEK293 cells, and control vector-transfected HEK293 cells. Binding of eelGFP to bilirubin was confirmed by the observation of green fluorescence in HEK293-eelGFP cells. The growth rate was compared with the three types of cells in the presence or absence of phenol red which possessed antioxidant activity. The growth rates of HEK293-CV and HEK293-jfGFP under phenol red-free conditions were reduced to 52 and 31% of those under phenol red. Under the phenol red-free condition, HEK293-eelGFP had a growth rate of approximately 70% of the phenol red-containing condition. The eelGFP-expressing cells were approximately 2-fold resistant to oxidative stress such as H2O2 exposure. The fluorescence intensity partially decreased or disappeared after exposure to H2O2, and heterogeneous intensity of fluorescence was also observed in isolated eel skeletal muscle cells. These results suggested eelGFP, but not jfGFP, coupled with bilirubin provided the antioxidant activity to the cells as compared to non-bound free bilirubin.

Targeting TRPM2 Channels Impairs Radiation-Induced Cell Cycle Arrest and Fosters Cell Death of T Cell Leukemia Cells in a Bcl-2-Dependent Manner.

Messenger RNA data of lymphohematopoietic cancer lines suggest a correlation between expression of the cation channel TRPM2 and the antiapoptotic protein Bcl-2. The latter is overexpressed in various tumor entities and mediates therapy resistance. Here, we analyzed the crosstalk between Bcl-2 and TRPM2 channels in T cell leukemia cells during oxidative stress as conferred by ionizing radiation (IR). To this end, the effects of TRPM2 inhibition or knock-down on plasma membrane currents, Ca2+ signaling, mitochondrial superoxide anion formation, and cell cycle progression were compared between irradiated (0–10 Gy) Bcl-2-overexpressing and empty vector-transfected Jurkat cells. As a result, IR stimulated a TRPM2-mediated Ca2+-entry, which was higher in Bcl-2-overexpressing than in control cells and which contributed to IR-induced G2/M cell cycle arrest. TRPM2 inhibition induced a release from G2/M arrest resulting in cell death. Collectively, this data suggests a pivotal function of TRPM2 in the DNA damage response of T cell leukemia cells. Apoptosis-resistant Bcl-2-overexpressing cells even can afford higher TRPM2 activity without risking a hazardous Ca2+-overload-induced mitochondrial superoxide anion formation.

NDRG1 promotes the multidrug resistance of neuroblastoma cells with upregulated expression of drug resistant proteins.

Resistance to chemotherapeutic drugs and recurrence are two major causes of poor prognosis in many tumors including neuroblastoma. This study aimed to investigate the effect of the elevated intracellular NDRG1 expression on drug resistance of human neuroblastoma cells to chemotherapy, for exploring novel approaches for biotherapy of neuroblastoma. Human neuroblastoma KP-N-Ns cell lines were transfected with the lentivirus vector containing human NDRG1 cDNA, with empty vector-transfected or blank cells as controls. Transfection status was confirmed under fluorescence microscope, while PCR assay and western blot analysis were performed to determine the expression changes. MTT and TUNEL assays were used to detect the resistance of the cells to anticancer drugs, including vincristine, cyclophosphamide and so on. Additionally, the expression of drug resistant proteins was detected. Stable lentiviral transfection cell line was successfully established with significantly upregulated NDRG1 expression. MTT assay revealed greater cell growth under NDRG1 overexpression with drugs stimulation, as compared to controls. TUNEL assay also showed less apoptosis of NDRG1 overexpressing cells than those of controls when exposed to these drugs, suggesting the increasing drug resistance through NDRG1 overexpression. Besides, the expression of MDR, LRP-1 and MRP-1 was also increased in NDRG1 overexpressing cells, implying NDRG1-mediated pathways in multidrug resistance of neuroblastoma. NDRG1 could increase the resistance of neuroblastoma cells to chemotherapeutic drugs, with its positive regulation on drug resistant proteins. This study provided new insights for exploring the mechanism of the resistance to chemotherapeutic drugs and also novel approach for biotherapy in neuroblastoma.

Atenolol Renal Secretion Is Mediated by Human Organic Cation Transporter 2 and Multidrug and Toxin Extrusion Proteins.

Atenolol is a β-blocker widely used in the treatment of hypertension. Atenolol is cleared predominantly by the kidney by both glomerular filtration and active secretion, but the molecular mechanisms involved in its renal secretion are unclear. Using a panel of human embryonic kidney cell lines stably expressing human organic cation transporter (hOCT) 1-3, human organic anion transporter (hOAT) 1, hOAT3, human multidrug and toxin extrusion protein (hMATE) 1, and hMATE2-K, we found that atenolol interacted with both organic cation and anion transporters. However, it is transported by hOCT1, hOCT2, hMATE1, and hMATE2-K, but not by hOCT3, hOAT1, and hOAT3. A detailed kinetic analysis coupled with absolute quantification of membrane transporter proteins by liquid chromatography-tandem mass spectrometry revealed that atenolol is an excellent substrate for the renal transporters hOCT2, hMATE1, and hMATE2-K. The Km values for hOCT2, hMATE1, and hMATE2-K are 280 ± 4, 32 ± 5, and 76 ± 14 μM, respectively, and the calculated turnover numbers are 2.76, 0.41, and 2.20 s(-1), respectively. To demonstrate unidirectional transepithelial transport of atenolol, we developed and functionally validated a hOCT2/hMATE1 double-transfected Madin-Darby canine kidney cell culture model. Transwell studies showed that atenolol transport in the basal (B)-to-apical (A) direction is 27-fold higher than in the A-to-B direction, whereas its B-to-A/A-to-B transport ratio was only 2 in the vector-transfected control cells. The overall permeability of atenolol in the B-to-A direction in hOCT2/hMATE1 cells was 44-fold higher than in control cells. Together, our data support that atenolol tubular secretion is mediated through the hOCT2/hMATEs secretion pathway and suggest a significant role of organic cation transporters in the disposition of an important antihypertensive drug.

Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1.

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484]

Abstract 1436: The SxxSS motif of T-cell Factor-4 isoforms regulates Wnt5a expression and EMT in human liver cancer cells

Abstract Background: The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. Previously we identified 14 TCF-4 isoforms derived from human HCC cell lines (Exp Cell Res 2011). The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012). However, K cells often showed morphological alteration, reminiscent of epithelial-mesenchymal transition (EMT), which is involved in non-canonical Wnt signaling (BMC Cancer 2013). The finding suggested that the SxxSS motif had potential to regulate EMT through the non-canonical Wnt signaling pathway. Thus, the AIM of this study was to investigate whether the SxxSS motif modulated expression levels of EMT regulators and Wnt5a, a representative non-canonical Wnt ligand. Methods: The human HCC cell line HAK-1A (Hepatology 1993) was used. TCF-4K mutants (269A, 272A, and 273A) were prepared with conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (269A, 272A, and 273A cells, respectively) were established. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Sh-RNA was used to knockdown wnt5a gene expression. Results: The 269A-mutant cells robustly expressed Wnt5a in both protein and mRNA levels, while empty vector-transfected cells (control), J cells, or K cells did not. Of note, Wnt5a expression was coupled with SLUG expression and EMT-like cellular morphological change. Snail was hardly expressed in the cells examined in this study. When the Wnt5a expression was specifically silenced by using sh-RNA, the expression level of SLUG was clearly decreased. Conclusion: The findings in this study suggest that serine 269 (the first serine) of the SxxSS motif of TCF-4 is a major switch to control Wnt5a transcription, thereby modulating the expression level of the EMT-regulator SLUG in a human HCC cell line. Citation Format: Hironori Koga, Toru Nakamura, Mitsuhiko Abe, Yu Ikezono, Fumitaka Wada, Hirohisa Yano, Takuji Torimura. The SxxSS motif of T-cell Factor-4 isoforms regulates Wnt5a expression and EMT in human liver cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1436. doi:10.1158/1538-7445.AM2015-1436

Role of osteoclasts in heterotopic ossification enhanced by fibrodysplasia ossificans progressiva-related activin-like kinase 2 mutation in mice.

Fibrodysplasia ossificans progressiva (FOP) is a disorder of skeletal malformations and progressive heterotopic ossification. The constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2), is responsible for the pathogenesis of FOP. Although transfection of the causal mutation of FOP into myoblasts enhances osteoclast formation by transforming growth factor-β (TGF-β), the role of osteoclasts in heterotopic ossification is unknown. We therefore examined the effects of alendronate, SB431542 and SB203580 on heterotopic ossification induced by the causal mutation of FOP. Total bone mineral content as well as numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated and alkaline phosphatase (ALP)-positive cells in heterotopic bone were significantly higher in muscle tissues implanted with ALK2 (R206H)-transfected mouse myoblastic C2C12 cells than in the tissues implanted with empty vector-transfected cells in nude mice. Alendronate, an aminobisphosphonate, did not affect total mineral content or numbers of TRAP-positive multinucleated and ALP-positive cells in heterotopic bone, which were enhanced by the implantation of ALK2 (R206H)-transfected C2C12 cells, although it significantly decreased serum levels of cross-linked C-telopeptide of type I collagen, a bone resorption index. Moreover, neither SB431542, an inhibitor of TGF-β receptor type I kinase, nor SB203580, an inhibitor of p38 mitogen-activated protein kinase, affected the increase in heterotopic ossification due to the implantation of ALK2 (R206H)-transfected C2C12 cells. In conclusion, the present study indicates that osteoclast inhibition does not affect heterotopic ossification enhanced by FOP-related mutation.

A study of radiosensitizing effect of PKM2 silencing in lung adenocarcinoma cells and xenografts

Objective To investigate the impacts of pyruvate kinase M2 isoform(PKM2)silencing on the radiosensitivity of lung adenocarcinoma cell line(A549 cells)and the radiation synergy of xenografts, and to explore their mechanisms. Methods Plasmid pshRNA-PKM2 for interference with PKM2 expression was transfected into A549 cells, and empty vector-transfected cells and untransfected cells were set as controls. The silencing efficiency of pshRNA-PKM2 and the expression level of microtubule-associated protein 1 light chain 3(LC3)were measured by Western blot assay. The radiosensitizing effects in A549 cells and xenografts after PKM2 silencing were determined by colony-forming assay and xenografts growth curves. Autophagy formation in A549 cells and xenografts was analyzed by transmission electron microscopy, and the expression level of PKM2 in xenografts was measured by immunohistochemistry. Comparison between groups was made by Student's t-test, and the body weights of nude mice and xenograft volumes were subjected to analysis of variance for continuous variables. Results Stable A549 cell lines transfected with pshRNA-PKM2 were successfully produced. Transfection with pshRNA-PKM2 significantly down-regulated PKM2 expression in A549 cells and xenografts(P=0. 001 ; P=0. 000). The sensitizer enhancement ratios for A549 cells and xenografts were 1. 47 and 2. 00, respectively. Interference with PKM2 expression enhanced radiation-in duced autophagy formation and significantly increased the ratio of LC 3 - Ⅱ / I(P= 0.000 1). Conclusions Silencing of PKM2 expression may enhance the radiosensitivity of A549 cells and xenografts by regulation of autophagy, which holds promise for becoming an effective radiosensitizing target for non-small cell lung cancer, but still needs to be confirmed by further studies. Key words: Pyruvate kinase M2; Irradiation; Autophagy; Cell line, lung adenocarcinom

Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3

Okadaic acid is known as a diarrheal shellfish poison. It is thought that there is no specific target organ for okadaic acid after it has been absorbed into the body. However, the details of its pharmacokinetics are still unknown.In this study, we demonstrated that okadaic acid was more toxic to the hepatocyte-specific uptake transporter OATP1B1- or OATP1B3-expressing cells than control vector-transfected cells. In addition, PP2A activity, which is a target molecule of okadaic acid, was more potently inhibited by okadaic acid in OATP1B1- or OATP1B3-expressing cells compared with control vector-transfected cells. The cytotoxicity of okadaic acid in OATP1B1- or OATP1B3-expressing cells was attenuated by known substrates of OATP1B1- and OATP1B3, but not in control vector-transfected cells. Furthermore, after uptake inhibition study using OATP1B3-expressing cells, Dixon plot showed that okadaic acid inhibited the uptake of hepatotoxin microcystin-LR, which is a substrate for OATP1B1 and OATP1B3, in a competitive manner. These results strongly suggested that okadaic acid is a substrate for OATP1B3 and probably for OATP1B1, and could be involved in unknown caused liver failure and liver cancer. Since okadaic acid possesses cytotoxicity and cell proliferative activity by virtue of its known phosphatase inhibition activity.

Effect of semaphorin4D gene silencing on malignant behavior of human ovarian cancer SKOV3 cells

To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude mice. Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blot were used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SKOV3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0.401 ± 0.051, 0.120 ± 0.035 and 0.014 ± 0.015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ± 0.019, 0.536 ± 0.040,respectively, P < 0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0.196 ± 0.023, 0.074 ± 0.015 and 0.040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.275 ± 0.009, 0.282 ± 0.015, respectively, P < 0.05). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR (inhibitory rate) of pshRNASema 4D-B group was (61.0 ± 3.3)% (P < 0.05). Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.

Glutaredoxin 1 (Grx1) Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation.

Glutaredoxin 1 (Grx1) belongs to the oxidoreductase family and is a component of the endogenous antioxidant defense system. However, its physiological function remains largely unknown. In this study, we investigated whether and how Grx1 overexpression protects the retinal pigment epithelial (RPE) cells against H2O2-induced apoptosis. Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Primary human RPE cells were transfected with Grx1 small interfering RNA (siRNA) or scrambled siRNA. Cell viability was measured with the WST8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Protein kinase B (AKT) activation was examined by Western blot. Protein kinase B glutathionylation was detected by immunoprecipitation followed by immunoblotting with anti-PSSG antibody. Glutaredoxin 1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Conversely, Grx1 gene knockdown sensitized primary human RPE cells to H2O2. Assessment of apoptosis indicated that cells transfected with the Grx1-containing plasmid were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to empty vector-transfected cells. Hydrogen peroxide-induced PSSG accumulation was also attenuated by Grx1 enrichment. Furthermore, Grx1 overexpression prevented H2O2-induced AKT glutathionylation, resulting in a sustained phospho-AKT elevation in RPE cells. Glutaredoxin 1 can protect RPE cells against oxidative stress-induced apoptosis. The mechanism of this protection is associated with its ability to stimulate the phosphorylation of AKT by preventing AKT glutathionylation. Considering Grx1's protective abilities in RPE cells, Grx1 could be a potential pharmacological target for retinal degenerative diseases.

High expression of RELM-α correlates with poor prognosis and promotes angiogenesis in gastric cancer.

Accumulating evidence indicates that resistin-like molecule-α (RELM-α) is involved in angiogenesis, while the clinical significance and the exact role of RELM-α in gastric cancer remain obscure. The aim of the present study was to evaluate the clinical significance of RELM-α in gastric cancer, and to investigate its effective mechanisms in order to identify a potential therapeutic target. The expression levels of RELM-α in 92 gastric cancer and adjacent normal tissues were investigated and the relationship between RELM-α expression and the clinicopathological characteristics was explored. To investigate the potential role of RELM-α in gastric cancer cell biological behavior, the cell proliferation, migration and invasion assays were conducted using two gastric cancer cell lines (SGC7901 and MKN45). We also assessed whether RELM-α gene silencing modulates angiogenesis using small interference RNA in cancer cell lines, and investigated its effect on nuclear factor (NF)-κB activation and vascular endothelial growth factor (VEGF) and MMP-9 expression. Contrasting sharply with the strong RELM-α-positive tumors, adjacent normal tissues and cell lines exhibited negative or weakly positive expression (P<0.01). High expression level of RELM-α was associated with advanced stage and tumor size (P<0.01). The silencing of RELM-α expression by Ad5/F35-siRNA treatment significantly inhibited cell migratory and invasive ability in SGC7901 and MKN45 gastric cancer cells compared with the control and Ad5/F35 vector-transfected cell lines (P<0.01). However, the silencing of RELM-α expression also significantly blocked NF-κB activation and attenuated VEGF and MMP-9 expression. The data demonstrated that RELM-α is a promising novel biomarker of angiogenesis in patients with gastric cancer. The study identified that the silencing of RELM-α expression may regulate the proliferation, invasion and migration of gastric cancer cells by targeting VEGF/MMP-9, and the mechanism involved tissue angiogenesis via the NF-κB signaling pathway.

Regulation of the Brain Organic Cation Transporter Plasma Membrane Monoamine Transporter (PMAT) by Alternative Splicing

PMAT is a polyspecific organic cation transporter highly expressed at the blood‐cerebrospinal fluid (CSF) barrier and facilitates the removal of monoamine neurotransmitters, organic cation drugs and toxicants from the CSF. We found that human SHSY5Y neuronal cells co‐express the full‐length wild type (wt) PMAT mRNA with two alternatively spliced mRNA species. One spliced variant (PMAT_v1) introduced a premature stop codon before transmembrane domain (TMD) 3 and the other (PMAT_v2) had a frame shift resulting in the deletion of TMD 3‐4. The goal of this study was to determine 1) the function and expression of the spliced variants in normal human tissues; 2) if the spliced variants exhibit a regulatory role on wt PMAT. Variant‐specific PCR indicated the spliced variants were co‐expressed with wt PMAT in all tested human tissues (whole brain, choroid plexus, small intestine and colon) at levels 37‐84% of the wt PMAT. When expressed in HEK293 cells, wt PMAT‐expressing cells showed up to 20‐fold increase in [3H]MPP+ uptake as compared to vector‐transfected cells. In contrast, there was no significant increase in uptake in cells‐expressing PMAT_v1 or PMAT_v2. PMAT_v1 protein was not detected by immunocytochemistry in HEK cells while PMAT_v2 was exclusively expressed in intracellular organelles. Co‐expression of PMAT_v1 or PMAT_v2 with wt PMAT in HEK293 cells did not significantly interfere with the normal activity of wt PMAT. Together our data showed PMAT pre‐mRNA is alternatively spliced in normal human tissues; and the overall PMAT activity in certain human tissues and cells may be regulated by altering the ratio between the functional and the non‐functional mRNA species.This study was supported by NIH Grants R01GM066233 and T32GM07750.

Human amniotic epithelial cells: Proliferation and apoptosis during their hepatic differentiation

Human amniotic epithelial cells: Proliferation and apoptosis during their hepatic differentiation

Overexpression of microRNA-30a inhibits hepatitis B virus X protein-induced autophagosome formation in hepatic cells.

Hepatitis B virus (HBV) enters the host and survives by using several mechanisms. One of the ways that HBV survives and replicates in the host cells is by inducing autophagy. Previous reports have shown that microRNA (miRNA)-30a inhibits autophagosome formation in cancer cells. Hence, we hypothesized that overexpression of miRNA-30a could inhibit HBV-induced autophagosome formation in hepatic cells. To study this, both HepG2 cells and HepG2.2.1.5 cells (HBV-expressing stable cell line) were transfected with miRNA-30a, and the cells were collected either for RNA isolation or protein isolation after 72h of transfection. Beclin-1 expression was significantly higher in untransfected HepG2.2.1.5 cells than in HepG2 cells. Western blots showed that miRNA-30a overexpression resulted in a significant decrease in beclin-1 expression (eight-fold and four-fold in HepG2 and HepG2.2.1.5 cells, respectively) and c-myc expression, whereas the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were increased. In contrast, overexpression of HBV X protein (HBx) in HepG2 cells resulted in the enhancement of beclin-1 (six-fold increase as compared with the empty vector-transfected cells) and c-myc expression, whereas the numbers of TUNEL-positive cells were reduced. To confirm these findings, HBx and miRNA-30a were coexpressed in HepG2 cells, and the results showed significant inhibition of autophagosome formation and beclin-1 and c-myc expression, whereas apoptosis increased. These data demonstrate that HBx induces autophagosome formation via beclin-1 expression, whereas miRNA-30a overexpression could successfully inhibit the beclin-1 expression induced by HBx, thereby modulating autophagosome formation in hepatic cells.