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Suppression of wingless-type MMTV integration site family, member 1 expression by small interfering RNA inhibits U251 glioma cell growth in vitro.

ABSTRACT: A Wingless-type MMTV integration site family, member 1 (Wnt-1) RNA interference expression vector was constructed during the present study, which was used to transfect the glioma U251 cell line and investigate its effect on glioma. Two 21-base oligonucleotides complementary to the coding sequence that was flanking the loop sequence were designed to form a DNA hairpin template for the target small interfering RNA (siRNA). The siRNA templates were cloned into the siRNA expression vector, pGPU6/green fluorescent protein (GFP)/Neo and the sequence was confirmed by DNA sequencing. The pGPU6/GFP/Neo-short hairpin RNA (shRNA)-Wnt-1 vector was subsequently transfected into U251 cells, and reverse transcription polymerase chain reaction and western blot analysis were used to evaluate the Wnt-1 gene silencing effect on U251 cell growth by MTT assay and flow cytometry. The Wnt-1 protein expression was significantly reduced following transfection with the recombinant plasmid, as determined by western blot analysis of the transfected U251 cells. This transfection exhibited a significantly higher death rate, as shown by MTT. Thus, the present study demonstrated that the pGPU6/GFP/Neo-shRNA-Wnt-1 vector inhibited Wnt-1 protein expression. However, further investigations regarding the Wnt signaling pathway in glioma pathogenesis are required.

SUBMITTER: Dong L

PROVIDER: S-EPMC4246625 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Suppression of wingless-type MMTV integration site family, member 1 expression by small interfering RNA inhibits U251 glioma cell growth <i>in vitro.</i>

Dong Lun L Duan Xiao-Chun XC Han Chong-Xu CX Zhang Hengzhu H Wu Yongkang Y

Oncology letters 20141030 1

A Wingless-type MMTV integration site family, member 1 (<i>Wnt-1</i>) RNA interference expression vector was constructed during the present study, which was used to transfect the glioma U251 cell line and investigate its effect on glioma. Two 21-base oligonucleotides complementary to the coding sequence that was flanking the loop sequence were designed to form a DNA hairpin template for the target small interfering RNA (siRNA). The siRNA templates were cloned into the siRNA expression vector, pG ...[more]

PMID: 25435937

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Project description:Energy metabolic reprogramming (EMR) allows for the rearrangement of a series of metabolic genes and proteins when tumor cells adapt to their microenvironment. EMR is characterized by changes in the metabolic pattern and metabolic intermediates to meet the needs of tumor cells for their malignant proliferation and infiltrative growth. The present study investigated the role of low-dose paclitaxel (PTX) in changing the expression levels of key genes and proteins during glycolysis in CD133+ U251 glioma cells and explored the relevant regulatory mechanisms of action at the molecular level. CD133 immunomagnetic beads were applied to malignant CD133+ U251 glioma cells, which were then divided into a negative control and an experimental group treated with 1, 2, 4 or 8 µM PTX for 72 h. Cell Counting Kit-8 (CCK-8) was used to measure U251 cell proliferation. RNA and protein were extracted from the malignant glioma cells in all groups to observe changes in the expression levels of key glycolytic enzymes, such as glucose transporter 1 (GLUT1), pyruvate kinase M (PKM) and lactate dehydrogenase A (LDHA), using reverse transcription-quantitative PCR and western blot assays. Transwell migration assays were performed to quantify the effects of PTX solution on U251 cells. CD133+ U251 glioma cells were isolated successfully. CD1133+ cells had a higher rate of proliferation compared with CD1133- cells. In CD1133+ cells treated with PTX, a dose-dependent reduction in the expression levels of the key glycolytic enzymes GLUT1, PKM and LDHA was observed at both the mRNA and protein levels. PTX solution also inhibited cell migration. Differences between the control and experimental groups were statistically significant (P<0.05). Since glycolysis plays an indispensable role in the proliferation and migration of stem cell-like glioma cells, PTX may inhibit tumor cell growth by downregulating the gene and protein expression levels of glycolytic enzymes in CD133+ glioma cells.

Project description:ObjectiveCardiac surgery and the use of cardiopulmonary bypass initiate a systemic inflammatory response. Wingless-related integration site (WNT) signaling is part of the innate immunity and has been attributed a major role in the regulation of inflammation. In preclinical research, WNT-5a may sustain an inflammatory response and cause endothelial dysfunction. Our aim was to investigate WNT signaling after cardiac surgery and its association with postoperative inflammation (Clinicaltrials.gov, NCT04058496).MethodsIn this prospective, single-center, observational study, 64 consecutive patients for coronary artery bypass grafting (CABG) ± valve surgery were assigned into three groups: off-pump CABG (n = 28), on-pump CABG (n = 16) and combined valve-CABG surgery (n = 20). Blood samples were acquired before surgery, at intensive care unit (ICU) admission and 4, 8, and 48 h thereafter. Plasma concentrations of WNT-5a and its antagonists Secreted frizzled-related protein 1 (sFRP-1), Secreted frizzled-related protein 5 (sFRP-5), and WNT inhibitory factor 1 (WIF-1) were determined by enzyme-linked immunosorbent assay. In addition, plasma concentrations of six inflammatory cytokines were measured by multiplex immunoassay. Parameters were analyzed for evolution of plasma concentration over time, interactions, intergroup differences, and association with clinical outcome parameters.ResultsAt baseline, WNT-5a, sFRP-1, and WIF-1 were present in a minimal concentration, while sFRP-5 was elevated. A higher baseline value of WNT-5a, sFRP-5, and WIF-1 resulted in higher subsequent values of the respective parameter. At ICU admission, WNT-5a and sFRP-5 reached their maximum and minimum value, respectively. WIF-1 decreased over time and was lowest 8 h after surgery. sFRP-1 changed minimally over time. While WNT-5a returned to the baseline within 48 h, sFRP-5 and WIF-1 did not reach their baseline value at 48 h. Of the investigated WNT system components, only WIF-1 partially reflected the severity of surgery. WNT-5a and WIF-1 had an impact on postoperative fluid balance and noradrenaline requirement.ConclusionWNT-5a, sFRP-5, and WIF-1 are part of the systemic inflammatory response after cardiac surgery. WNT-5a peaks immediately after cardiac surgery and returns to baseline within 48 h, presumably modulated by its antagonist sFRP-5. Based on this translational study, WNT-5a antagonism may be further investigated to assess potentially beneficial effects in patients with a dysregulated inflammation after cardiac surgery.

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Suppression of wingless-type MMTV integration site family, member 1 expression by small interfering RNA inhibits U251 glioma cell growth in vitro.

Publications

Suppression of wingless-type MMTV integration site family, member 1 expression by small interfering RNA inhibits U251 glioma cell growth <i>in vitro.</i>

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