scholarly journals Effects of PKM2 Gene Silencing on the Proliferation and Apoptosis of Colorectal Cancer LS-147T and SW620 Cells

2017 ◽  
Vol 42 (5) ◽  
pp. 1769-1778 ◽  
Author(s):  
Ran Ao ◽  
Lin Guan ◽  
Ying Wang ◽  
Jia-Ni Wang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Gene Silencing ◽  
Cell Counting ◽  
Qrt Pcr ◽  
Empty Plasmid ◽  
Proliferation And Apoptosis ◽  
Mrna And Protein Expression ◽  
Sw620 Cells

Background/Aims: This paper aims to explore the effects of pyruvate kinase (PK) M2 gene silencing on the proliferation and apoptosis of colorectal cancer (CRC) LS-147T and SW620 cells. Methods: CRC LS-147T and SW620 cells highly expressing PKM2 were randomly selected by quantitative real-time polymerase chain reaction (qRT-PCR) and then assigned into the blank (no transfection), PKM2-shRNA (transfection with shRNA) and empty plasmid (transfection with empty plasmid) groups. Immunofluorescence was applied to detect PKM2 protein expression. qRT-PCR and Western blotting were conducted to assess mRNA and protein expression of PKM2, p53 and p21. The cell counting kit-8 (CCK-8) assay was used to assess cell proliferation. Flow cytometry was used to assess the cell cycle and apoptosis rate, and a senescence-associated β-galactosidase staining kit was used to assess cell senescence. Results: PKM2 exhibited high mRNA expression among CRC LS-147T and SW620 cells with remarkable protein expression noted in the cytoplasm and nucleus. The PKM2-shRNA group exhibited reduced PKM2 mRNA and protein expression, whereas p53 and p21 expression was increased compared with the blank and empty plasmid groups. Cell proliferation in PKM2-shRNA cells decreased significantly compared with the blank group and empty plasmid groups. The PKM2-shRNA group exhibited more cells in the G1 phase and fewer cells in the G2/M phase compared with the blank and empty plasmid groups. In addition, the PKM2-shRNA group exhibited significantly increased apoptosis rates and β-galactosidase activity compared with the blank and empty plasmid groups. Conclusion: Our study demonstrates that PKM2 gene silencing suppresses proliferation and promotes apoptosis in LS-147T and SW620 cells.

129 microRNA-183~96~182 CLUSTER PROMOTE BOVINE GRANULOSA CELL PROLIFERATION THROUGH COORDINATED REGULATION OF FOXO1

2016 ◽  
Vol 28 (2) ◽  
pp. 194
Author(s):  
S. Gebremedhn ◽  
D. Salilew-Wondim ◽  
M. Hoelker ◽  
F. Rings ◽  
C. Neuhoff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Statistical Significance ◽  
Flow Cytometric Analysis ◽  
Cell Counting ◽  
G1 Arrest ◽  
Mirna Cluster ◽  
Mrna And Protein Expression ◽  
Coordinated Regulation

Among other microRNA clusters, we previously showed that the miR-183~96~182 cluster (miR-183, miR-96, and miR-182) is abundantly expressed in bovine granulosa cells (bGC) of preovulatory dominant follicles obtained at the follicular phase of the bovine oestrous cycle. Moreover, this miRNA cluster are validated to coordinately target the Fork head O1 (FOXO1), a subfamily of transcription factors that regulate genes involved in cell proliferation, apoptosis, cell cycle arrest, and metabolism. However, the functional involvement of miR-183~96~182 cluster in bGC function by regulation of FOXO1 is not yet determined. Here, we aimed to investigate the function of miR-183~96~182 cluster in bGC using in vitro cell culture model. For this, bGC were aspirated from ovarian follicles (Ø 3–5 mm) obtained from local abattoir. Cells were plated in 24-well plate (2.5 × 105 cells well–1) in DMEM/F-12 (Sigma, Germany) supplemented with 10% FBS (GIBCO, Grand Island, NY) and 1% penicillin/streptomycin (GIBCO) and incubated at 37°C in 5% CO2. Transfection of bGC with miRNA mimics, inhibitors, FOXO1-siRNA, and appropriate controls (Exiqon, Vedbæk, Denmark) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cell proliferation was determined using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technology, Kumamoto, Japan). Cell cycle distribution was determined with flow cytometric analysis. Total RNA was isolated using miRNeasy mini kit (Qiagen, Hilden, Germany), quantification of target gene was performed using qPCR, and data were analysed using ΔΔCT method. Differences in the mean expression values between treatments were analysed with two-tailed Student’s t-test and statistical significance was defined at P ≤ 0.05. Results showed that a sponge effect was observed upon inhibition in individual miRNA of the cluster, which could be attributed to the partial sequence similarity among cluster members. Both FOXO1 mRNA and protein expression were significantly reduced upon transfection of bGC with miR-183~96~182 cluster mimics, while miR-183~96~182 cluster inhibition increased both FOXO1 mRNA and protein expression. Transfection of bGC with miR-183~96~182 mimics promoted cell proliferation, while inhibition tends to slow down proliferation. Furthermore, the proportion of bGC under G0/G1 arrest markedly declined (P < 0.05), while the S and G2/M phases increased in response to miR-183~96~182 mimicking. Selective knockdown of FOXO1 with FOXO1-siRNA significantly reduced FOXO1 mRNA and protein expression. Interestingly, knockdown of FOXO1 showed similar phenotypic effects such as that of miR-183~96~182 mimics transfection, which resulted in elevated bGC proliferation and reduction in the proportion of cells under G0/G1 arrest. In conclusion, overexpression of miR-183~96~182 cluster promote bGC proliferation and G0/G1 to S and G2/M cell cycle transition through coordinated regulation of genes in the FOXO1 signaling axis.

Long non-coding RNA Linc00525 as a prognostic factor to regulate proliferation and apoptosis in colorectal cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e15085-e15085 ◽  
Author(s):  
Peng Peng ◽  
Qin Zheng
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Colorectal Cancer Cell ◽  
Qrt Pcr ◽  
Proliferation And Apoptosis ◽  
Colorectal Cancer Cell Lines ◽  
Cancer Tissues

e15085 Background: Colorectal cancer (CRC) is one of the most common digestive system tumors and poses a serious threat to human health. More and more studies have shown that long noncoding RNAs (lncRNAs) play an important role in the occurrence and development of various tumors. They regulate a variety of cancer biology, such as proliferation, apoptosis, invasion and metastasis. Abnormally expressed lncRNAs are closely related to colorectal cancer. The purpose of this study was to find lncRNAs associated with the development of colorectal cancer and to explore its function and mechanism. Methods: (1) By analyzing the expression profile of lncRNAs in colorectal cancer-normal tissues in GEO databases, the abnormal expression of lncRNA LINC00525 was screened. Colorectal cancer tissues, adjacent normal colon tissues and colorectal cancer cell lines (HCT116, DLD1, HT-29, SW480) were detected by qRT-PCR. We analyzed the relationship between expression of LINC00525 and clinical features. (2) The biological functions of LINC00525 in HCT116, DLD1 and SW480 were peformed in vitro by MTT, clone formation assay, EDU and flow cytometry. (3) The effect of LINC00525 on tumorigenesis in vivo was evaluated by nude mice model. (4) The expression of lncRNA LINC00525 was knocked down in colorectal cancer cell lines (HCT116, SW480), and the mRNA expression levels of P15, P21, P27 and KLF2 were detected by qRT-PCR. Results: (1) Microarray data and qRT-PCR verification showed that the expression of lncRNA LINC00525 in colorectal cancer tissues and colorectal cancer cell lines was significantly upregulated. The overexpression of LINC00525 was positively correlated with clinical stage and tumor size. (2) Knockdown of LINC00525 in colorectal cancer cell lines could inhibit cancer cell proliferation and induce apoptosis. In SW480 and HCT116 cell lines, cells were arrested in G0/G1 phase after knocking down LINC00525. (3) Subcutaneous xenograft experiments in nude mice further confirmed that knockdown of LINC00525 could inhibit the tumor growth. (4) After knocking down the expression of LINC00525 in HCT116 and SW480 cell lines, the expression of mRNAs of tumor suppressor genes P15, P21, P27 and KLF2 increased. Conclusions: Our studies suggested that LINC00525 was significantly upregulated in colorectal cancer tissues. Mechanism studies had shown that LINC00525 could regulate the expression of KLF2, P15, P21 and P27 in CRC cell lines, and then affect cell proliferation and apoptosis.

scholarly journals MicroRNA-124 (MiR-124) Inhibits Cell Proliferation, Metastasis and Invasion in Colorectal Cancer by Downregulating Rho-Associated Protein Kinase 1(ROCK1)

2016 ◽  
Vol 38 (5) ◽  
pp. 1785-1795 ◽  
Author(s):  
Liqing Zhou ◽  
Ziran Xu ◽  
Xiaoqiang Ren ◽  
Kaixuan Chen ◽  
Shiyong Xin
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Qrt Pcr ◽  
Normal Controls ◽  
In Cells

Background/Aims: MiR-124 inhibits neoplastic transformation, cell proliferation, and metastasis and downregulates Rho-associated protein kinase (ROCK1) in Colorectal Cancer (CRC). The aim of this study was to further investigate the roles and interactions of ROCK1 and miR-124 and the effects of knockdown of ROCK1and MiR-124 in human Colorectal Cancer (CRC). Methods: Three Colorectal cancer cell lines (HCT116, HT29 and SW620) and one Human Colonic Mucosa Epithelial cell line (NCM460) were studied. The protein expression of ROCK1 was examined by Western-blot and qRT-PCR were performed to examine the expression levels of ROCK1 mRNA and miR-124. Furthermore, We performed transfection of cancer cell line (SW620) with pre-miR-124(mimics), anti-miR-124(inhibitor), ROCK1 siRNA and the control, then observed the affects of ROCK1 protein expression by westen-blot, cell proliferation by EDU (5-ethynyl-2'deoxyuridine assay) and expression levels of ROCK1mRNA by qRT-PCR . A soft agar formation assay, Migration and invasion assays were used to determine the effect of regulation of miR-124 and ROCK1, and survivin on the transformation and invasion capability of colorectal cancer cell. Results: MiR-124 expression was significantly downregulated in CRC cell lines compare to normal (P < 0.05). In contrast, ROCK1 protein expression was significantly increased in CRC cell lines compared to the normal (P < 0.05), whereas the gene (ROCK1mRNA) expression remained unaltered (P > 0.05). ROCK1 mRNA was unaltered in cells transfected with miR-124 mimic and miR-124 inhibitor, compared to normal controls. There was a significant reduction in ROCK1 protein in cells transfected with miR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P < 0.05). Cell proliferation, transformation and invasion of cells transfected with miR-124 inhibitor were significantly increased compared to those in normal controls (P<0.05). However, cell proliferation, transformation and invasion of cells transfected with ROCK1 siRNA were significantly decreased compared to control (P < 0.05). Conclusions: In conclusion, our results demonstrated that miR-124 not only promoted cancer cell hyperplasia and significantly associated with CRC metastasis and progression, but also downregulated ROCK1 protein expression. More importantly, increased ROCK1 expression or inhibited miR-124 expression may constitute effective new therapeutic strategies for the treatment of renal cancer in the future.

scholarly journals COPB2 gene silencing inhibits colorectal cancer cell proliferation and induces apoptosis via the JNK/c-Jun signaling pathway

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0240106
Author(s):  
Yan Wang ◽  
Guangmei Xie ◽  
Min Li ◽  
Juan Du ◽  
Min Wang
Keyword(s):  
Colorectal Cancer ◽  
Gene Silencing ◽  
Signaling Pathway ◽  
Caspase 3 ◽  
Cell Counting ◽  
Apoptosis Pathway ◽  
Proliferation And Apoptosis ◽  
Related Proteins ◽  
Hct116 Cells

Objectives Colorectal cancer (CRC) is one of the most common malignant human tumors. It is associated with high morbidity and mortality rates. In recent years, tumor gene therapy has emerged as a promising new approach for colorectal cancer therapy. Herein, we identify and analyze the role of COPB2 (coatomer protein complex, subunit beta 2) in proliferation and apoptosis of CRC cells. Methods To investigate the role of COPB2 in the proliferation and apoptosis of CRC cells, a shCOPB2 vector and a shCtrl vector were constructed for transfection into RKO and HCT116 cells. Cells proliferation was subsequently measured via cell counting kit-8 (CCK8) assay and Celigo cell counting assay. Apoptosis was measured via flow cytometry. The activity level of Caspase 3/7 was measured. Finally, the level of several JNK/c-Jun apoptosis pathway-related proteins were measured to characterize the mechanism of apoptosis. Results Our results showed that the proliferation rate was decreased and the apoptosis rate was increased in shCOPB2-treated RKO and HCT116 cells compared to those in controls. After the silencing of COPB2, JNK/c-Jun signal pathway activation was increased, the expression levels of apoptosis pathway-related proteins, such as Bad, p53 and Caspase 3, were also increased. Conclusion COPB2 gene silencing can inhibit RKO and HCT116 cells proliferation and induce apoptosis via the JNK/c-Jun signaling pathway.

scholarly journals Resistin is a survival factor for porcine ovarian follicular cells

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 343-355 ◽  
Author(s):  
Agnieszka Rak ◽  
Eliza Drwal ◽  
Anna Wróbel ◽  
Ewa Łucja Gregoraszczuk
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Dna Fragmentation ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Inhibitory Effect ◽  
Ovarian Cell ◽  
Proliferation And Apoptosis ◽  
Mrna And Protein Expression

Previously, we demonstrated the expression of resistin in the porcine ovary, the regulation of its expression and its direct effect on ovarian steroidogenesis. The objective of this study was to examine the effect of resistin on cell proliferation and apoptosis in a co-culture model of porcine granulosa and theca cells. First, we analysed the effect of resistin at 1 and 10 ng/ml alone or in combination with FSH- and IGF1 on ovarian cell proliferation with an alamarBlue assay and protein expression of cyclins A and B using western blot. Next, the mRNA and protein expression of selected pro-apoptotic and pro-survival regulators of cell apoptosis, caspase-9, -8 and -3 activity and DNA fragmentation using real time PCR, western blot, fluorescent assay and an ELISA kit, respectively, were analysed after resistin treatment. Furthermore, we determined the effect of resistin on the protein expression of ERK1/2, Stat and Akt kinase. Using specific inhibitors of these kinases, we also checked caspase-3 activity and protein expression. We found that resistin, at both doses, has no effect on cell proliferation. The results showed that resistin decreased pro-apoptotic genes, which was confirmed on protein expression of selected factors. We demonstrate an inhibitory effect of resistin on caspase activity and DNA fragmentation. Finally, resistin stimulated phosphorylation of the ERK1/2, Stat and Akt and kinases inhibitors reversed resistin action on caspase-3 activity and protein expression to control. All of these results showed that resistin has an inhibitory effect on porcine ovarian cell apoptosis by activation of the MAPK/ERK, JAK/Stat and Akt/PI3 kinase signalling pathways.

scholarly journals FOXO3a-ROS Pathway is Involved in Androgen-Induced Proliferation of Prostate Cancer Cell

2021 ◽  
Author(s):  
Yan Tao ◽  
Jianzhong Lu ◽  
Shengjun Fu ◽  
Lanlan Li ◽  
Shanhui Liu ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Catalase Activity ◽  
Lncap Cells ◽  
Over Expression ◽  
Qrt Pcr ◽  
Mrna And Protein Expression ◽  
Antioxidant Enzyme Catalase

Abstract Background: Although FOXO3a can inhibit the cell proliferation of prostate cancer, its relationship with reactive oxygen species (ROS) in prostate cancer(PCa) has not been reported. Methods: We analyzed the correlation between the expression of FOXO3a and the antioxidant enzyme catalase in prostate cancer through the UALCAN and GEPIA databases. We also constructed a PPI network of FOXO3a via the STRING database. The mRNA and protein expression of FOXO3a and catalase in LNCaP cells after DHT treatment were detected by qRT-PCR and western blot analysis. The effects of FOXO3a on catalase expression were tested by over-expression and siRNA interference respectively. At the same time, the catalase activity and ROS level in LNCaP cells after DHT treatment were detected. The changes of cell proliferation and ROS in LNCaP by antioxidant were also analyzed.Results: We found that the catalase expression was down-regulated and has positively correlation with FOXO3a in PCa by public databases. The results of qRT-PCR and western blot showed that the mRNA and protein expression of FOXO3a and catalase were significantly reduced after DHT treatment in the LNCaP cells. Over-expression and knockdown of FOXO3a can also induce the change of catalase expression. DHT treatment can inhibit catalase activity and increase ROS level. We found that antioxidant treatment reduced DHT-induced proliferation and ROS production.Conclusions: Our data show that the mechanisms by which DHT promotes PCa cell proliferation is that FOXO3a suppresses catalase expression and activates ROS signaling.

scholarly journals KIT is involved in melanocyte proliferation, apoptosis and melanogenesis in the Rex Rabbit

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9402
Author(s):  
Shuaishuai Hu ◽  
Yang Chen ◽  
Bohao Zhao ◽  
Naisu Yang ◽  
Shi Chen ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Expression Patterns ◽  
Melanin Content ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Proliferation And Apoptosis ◽  
Mrna And Protein Expression ◽  
Different Color ◽  
Rex Rabbit

Background Melanocytes play an extremely important role in the process of skin and coat colors in mammals which is regulated by melanin-related genes. Previous studies have demonstrated that KIT is implicated in the process of determining the color of the coat in Rex rabbits. However, the effect of KIT on the proliferation and apoptosis of melanocytes and melanogenesis has not been clarified. Methods The mRNA and protein expression levels of KIT were quantified in different coat colored rabbits by qRT-PCR and a Wes assay. To identify whether KIT functions by regulating of melanogenesis, KIT overexpression and knockdown was conducted in melanocytes, and KIT mRNA expression and melanin-related genes TYR, MITF, PMEL and DCT were quantified by qRT-PCR. To further confirm whether KIT influences melanogenesis in melanocytes, melanin content was quantified using NaOH lysis after overexpression and knockdown of KIT. Melanocyte proliferation was estimated using a CCK-8 assay at 0, 24, 48 and 72 h after transfection, and the rate of apoptosis of melanocytes was measured by fluorescence-activated cell sorting. Results KITmRNA and protein expression levels were significantly different in the skin of Rex rabbits with different color coats (P < 0.05), the greatest levels observed in those with black skin. The mRNA expression levels of KIT significantly affected the mRNA expression of the pigmentation-related genes TYR, MITF, PMEL and DCT (P < 0.01). Melanin content was evidently regulated by the change in expression patterns of KIT (P < 0.01). In addition, KIT clearly promoted melanocyte proliferation, but inhibited apoptosis. Conclusions Our results reveal that KIT is a critical gene in the regulation of melanogenesis, controlling proliferation and apoptosis in melanocytes, providing additional evidence for the mechanism of pigmentation of animal fur.

Icariin Stimulates hFOB 1.19 Osteoblast Proliferation and Differentiation via OPG/RANKL Mediated by the Estrogen Receptor

2020 ◽  
Vol 22 (1) ◽  
pp. 168-175 ◽  
Author(s):  
Lin-Jun Sun ◽  
Chong Li ◽  
Xiang-hao Wen ◽  
Lu Guo ◽  
Zi-Fen Guo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Protein Expression ◽  
Chinese Herb ◽  
Human Osteoblast ◽  
Osteoblast Cells ◽  
Expression Ratio ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Mrna And Protein Expression

Background:: Icariin (ICA), one of the main effective components isolated from the traditional Chinese herb Epimedium brevicornu Maxim., has been reported to possess extensive pharmacological actions, including enhanced sexual function, immune regulation, anti-inflammation, and antiosteoporosis. Methods:: Our study was designed to investigate the effect of ICA on cell proliferation and differentiation and the molecular mechanism of OPG/RANKL mediated by the Estrogen Receptor (ER) in hFOB1.19 human osteoblast cells. Results:: The experimental results show that ICA can stimulate cell proliferation and increase the activity of Alkaline Phosphatase (ALP), Osteocalcin (BGP) and I Collagen (Col I) and a number of calcified nodules. Furthermore, the mRNA and protein expression of OPG and RANKL and the OPG/ RANKL mRNA and protein expression ratios were upregulated by ICA. The above-mentioned results indicated that the optimal concentration of ICA for stimulating osteogenesis was 50ng/mL. Subsequent mechanistic studies comparing 50ng/mL ICA with an estrogen receptor antagonist demonstrated that the effect of the upregulated expression is connected with the estrogen receptor. In conclusion, ICA can regulate bone formation by promoting cell proliferation and differentiation and upregulating the OPG/RANKL expression ratio by the ER in hFOB1.19 human osteoblast cells.

Sign in / Sign up

Export Citation Format

Share Document