FERMT1 suppression induces anti-tumor effects and reduces stemness in glioma cancer cells
Zhigang PanChuhan KeHanlin ZhengXiumei GuoWen GaoXinyue HuangChunhui ChenYu XiongShuni ZhengFeng ZhengWeipeng Hu
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Abstract Objective Glioma is a leading cause of mortality worldwide, its recurrence poses a major challenge in achieving effective treatment outcomes. Cancer stem cells (CSCs) have emerged as key contributors to tumor relapse and chemotherapy resistance, making them attractive targets for glioma cancer therapy. This study investigated the potential of FERMT1 as a prognostic biomarker and its role in regulating stemness through cell cycle in glioma. Methods Using data from TCGA-GBM, GSE4290, GSE50161 and GSE147352 for analysis of FERMT1 expression in glioma tissues. Then, the effects of FERMT1 knockdown on cell cycle, proliferation, sphere formation ability, invasion and migration were investigated. The influences of FERMT1 on expression of glycolysis-related proteins and levels of ATP, glucose, lactate and G6PDH were also explored. Furthermore, the effects of FERMT1 knockdown on cellular metabolism were evidenced. Results Significant upregulation of FERMT1 in glioma tissues was observed. Silencing FERMT1 not only affected the cell cycle but also led to a notable reduction in proliferation, invasion and migration. The expression of glycolysis-associated proteins including GLUT1, GLUT3, GLUT4, and SCO2 were reduced by FERMT1 knockdown, resulted in increased ATP and glucose as well as decreased lactic acid and G6PDH levels. FERMT1 knockdown also inhibited cellular metabolism. Moreover, FERMT1 knockdown significantly reduced sphere diameter, along with inhibiting the expression of transcription factors associated with stemness in glioma cells. Conclusion These findings demonstrated that FERMT1 could be an ideal target for the advancement of innovative strategies against glioma treatment via modulating cellular process involved in stemness regulation and metabolism.Purpose The purpose of this study was to detect the expression pattern of SPZ1 in glioma samples and to clarify its biological functions in the malignant progression of glioma. Our results provide a novel molecular target for glioma. Methods SPZ1 levels in 40 pairs of glioma and non-tumoral ones were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The differences in clinical indicators and prognosis between glioma patients expressing high and low levels of SPZ1 were compared. After knockdown of SPZ1 by transfection of sh-SPZ1, migratory and invasive abilities of A172 and U251 cells were examined by transwell migration and invasion assays. The interaction between SPZ1 and its target gene CXXC4 was finally explored by Western blot and dual-luciferase reporter assay. Results SPZ1 was upregulated in glioma tissues than non-tumoral ones, and the difference was statistically significant. Cell function experiments showed that knockdown of SPZ1 weakened the migratory and invasive abilities of A172 and U251 cells. CXXC4 was identified as the target gene binding to SPZ1. Knockdown of CXXC4 abolished the role of SPZ1 knockdown in inhibiting glioma progression. Conclusions SPZ1 stimulates glioma's malignant progression via targeting CXXC4.
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Glioma is a type of malignant intracranial tumor. Extensive research has identified the participation of long noncoding RNAs (lncRNAs) in glioma progression. This study investigated the mechanism of LINC00294 in mitochondrial function and glioma cell apoptosis. Glioma miRNA and mRNA microarray datasets were obtained, and differentially expressed lncRNAs in glioma were screened through various databases. The LINC00294 expression in glioma patients and glioma cells was detected. Glioma cells were treated under hypoxic conditions and transfected with LINC00294 silencing. The apoptosis and mitochondrial function of glioma cells were measured. The expressions of and relations among miR‐21‐5p, CASKIN1, and cAMP in glioma cells were analyzed. Under hypoxic conditions and LINC00294 silencing, the apoptosis and mitochondrial function of glioma cells were detected after inhibiting miR‐21‐5p or overexpressing CASKIN1. Our results indicated that LINC00294 was downregulated in glioma. LINC00294 silencing inhibited glioma cell apoptosis under hypoxia. LINC00294 silencing reversed the inhibition of hypoxia on mitochondrial function under hypoxia. LINC00294 promoted the CASKIN1 expression by sponging miR‐21‐5p and activated the cAMP pathway. Inhibition of miR‐21‐5p or overexpression of CASKIN1 annulled the effects of LINC00294 silencing on mitochondrial function and glioma cell apoptosis under hypoxia. In conclusion, LINC00294 elevated the CASKIN1 expression by sponging miR‐21‐5p and activating the cAMP signaling pathway, thus inhibiting mitochondrial function and facilitating glioma cell apoptosis.
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RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction.Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP.Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.
Knockdown resistance
RNA Silencing
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Small interfering RNA (siRNA) molecules achieve sequence- specific gene silencing through a process known as RNA interference (RNAi). Compared to other nucleic acid-based therapeutics aimed at post-transcriptional gene silencing, such as antisense oligodeoxynucleotides, siRNA molecules achieve greater magnitude and duration of gene silencing at significantly lower doses. While the duration of gene knockdown by siRNA typically lasts around 1 week in rapidly dividing cells, recent reports of knockdown lasting for several weeks in nondividing cells indicate that dilution due to cell division may be a limiting factor in rapidly dividing cells. To determine if cell division directly impacts the duration of gene knockdown by siRNA, we chose to investigate the kinetics of siRNA-mediated gene silencing in luciferase-expressing cell lines with different observed doubling times using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function. In vitro and in vivo, the duration of gene knockdown is inversely proportional to the rate of cell division. Consistent with previous reports, luciferase protein levels recover to pre-treatment values within less than 1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting around 1 week in subcutaneous tumors in A/J mice and 3-4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. Here, we will present our latest results describing the effects of cell doubling time, siRNA stability, and dosing schedule on siRNA- mediated gene silencing. Specifically, we will investigate whether the duration of knockdown using chemically modified siRNA molecules exhibits a similar dependence on cell doubling time. The implications of these findings will be highlighted using model calculations to determine the dosing schedule required to maintain persistent silencing of target proteins and to predict when maximum mRNA or protein knockdown will occur, an especially important factor when trying to observe a therapeutic effect resulting from protein knockdown. The approach of bioluminescent imaging combined with mathematical modeling provides insights into siRNA function that will hopefully be of practical use for both researchers and clinicians alike.
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<p>Chemoresistance properties of MUC16 and effect of cisplatin on apoptosis of MUC16 knockdown cells. A & B, MUC16 knockdown (H1975-shMUC16 seq1 and 2) cells were highly sensitive to cisplatin (A) and gemcitabine (B). C, The percentage of apoptotic cells was significantly higher in MUC16 knockdown cells (H292-shMUC16) treated with 5μM cisplatin. In contrast, no significant change was observed in the untreated scramble (H292-SCR) and MUC16 knockdown cells. D, We performed stable knockdown of Muc16 in K1418, the result shows that Muc16 is significantly decreased as compared to scramble cells. E, The p53 target gene p21 was significantly increased in MUC16 knockdown (H292-shMUC16) cells. *P<0.05, **P<0.01, ***P<0.001, and NS non-significant.</p>
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The purpose of this study was to detect the expression pattern of SPZ1 in glioma samples and to clarify its biological functions in the malignant progression of glioma. Our results provide a novel molecular target for glioma.SPZ1 levels in 40 pairs of glioma and non-tumoral ones were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The differences in clinical indicators and prognosis between glioma patients expressing high and low levels of SPZ1 were compared. After knockdown of SPZ1 by transfection of sh-SPZ1, migratory and invasive abilities of A172 and U251 cells were examined by transwell migration and invasion assays. The interaction between SPZ1 and its target gene CXXC4 was finally explored by Western blot and dual-luciferase reporter assay.SPZ1 was upregulated in glioma tissues than non-tumoral ones, and the difference was statistically significant. Cell function experiments showed that knockdown of SPZ1 weakened the migratory and invasive abilities of A172 and U251 cells. CXXC4 was identified as the target gene binding to SPZ1. Knockdown of CXXC4 abolished the role of SPZ1 knockdown in inhibiting glioma progression.SPZ1 stimulates glioma's malignant progression via targeting CXXC4.
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This study aimed to investigate FAM84B expression in glioma tissues and explore the role of FAM84B in promoting the proliferation of glioma cells and the mechanism of regulating the cell cycle pathways.The TCGA database was adopted to analyze FAM84B expression in glioma tissues. The FAM84B expression was detected by qRT-PCR in patients with glioma, especially that in glioma cells, U251, LN-229, U98, and U87. Two glioma cell lines U87 and T98 were selected for siRNA transfection, which were divided into si-NC si-FAM84B-1 and si-FAM84B-2 groups. The effect of FAM84B on the proliferation of glioma cells was detected with the MTT experiment and that on the glioma cell cycle was detected with the flow cytometry. The signaling pathways potentially regulated by FAM84B in glioma were analyzed through the bioinformatics analysis. The expression of proteins, Cyclin D1, CDK4, Cdk6, and p21, in the cell cycle-related pathways in cells of each group was detected by the Western blot.TCGA database results showed a significantly higher FAM84B expression in glioma tissues than that in paracancerous tissues. According to the detection of qRT-PCR, FAM84B expressed the highest in the glioma cell line U87 (P < 0.05). Compared with the serum of healthy controls, FAM84B mRNA expression significantly increased in patients with gliomas. And compared with the si-NC group, the proliferation ability of U87 and T98 cells decreased and the cell cycle was blocked in the G0/G1 phase in both si-FAM84B transfection groups (P < 0.05). According to the bioinformatics analysis, FAM84B regulated the cell cycle pathways in glioma. FAM84B siRNA inhibited the expression of key proteins, Cyclin D1, CDK2, CDK4, and Cdk6, of the cell cycle pathways in glioma cells and promoted the expression of P53 and P21 proteins.In conclusion, FAM84B may inhibit the proliferation of glioma cells by regulating the cell cycle pathways.
Cyclin-dependent kinase 6
U87
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Objective To develope gene knockdown cell model with artificial microRNA in setting up gene knockdown cell model.Methods We constructed vectors,and prepared siRNA fragments targeting on DJ-1.Then we transciently transfected the artificial miRNA and siRNA into MN9D cells by lipofectamine2000 reagent,the mRNA and protein expression level of DJ-1 gene were detected by RT-PCR and Western blot.Results Compared with control group,DJ-1 expression level was significantly decreased in both artificial miRNA and siRNA groups.DJ-1 was knockdowned and DJ-1 was decreased 90%(P0.05)at mRNA expression level,and decreased 70%~85%(P0.05) at protein level in MN9D cells transfected with the artificial miRNA.While DJ-1 was decreased by 50%~70%(P0.05)at mRNA level,and decreased by 20%~50%(P0.05)at protein level in MN9D cells transfected with siRNA.Comparing with siRNA,miRNA was more effective in silencing DJ-1.Conclusion The artificial miRNA and siRNA are both effective in silencing gene.miRNA has more significant function in knockingdown DJ-1 than siRNA.
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