DNA methylation polymerase chain reaction (PCR) array of apoptosis-related genes in pleomorphic adenomas of the salivary glands

Pereira, Núbia Braga; Carmo, Ana Carolina de Melo do; Campos, Kelma; Costa, Sara Ferreira dos Santos; Diniz, Marina Gonçalves; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri

doi:10.1016/j.oooo.2017.08.011

Cited by 2 publications

(3 citation statements)

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“…A change in methylation of > 20% was considered as significant, according to the instructions of the manufacturer and comparable to other studies using the EpiTect Methyl II PCR Array System [12,13]. Here, DNA methylation was compared between apoptosis-related genes in pleomorphic adenomas of the salivary glands [13], respectively in genes involved in inflammation and autoimmunity in inflammatory bowel disease [12]. mRNA expression was done in genes with significant methylation changes, according to references [12,13], and analyzed with an unpaired t-test.…”

Section: Discussionmentioning

confidence: 94%

“…Methylation analysis was done according to the manufacturer's instructions. A change in methylation of > 20% was considered as significant, according to the instructions of the manufacturer and comparable to other studies using the EpiTect Methyl II PCR Array System [12,13]. Here, DNA methylation was compared between apoptosis-related genes in pleomorphic adenomas of the salivary glands [13], respectively in genes involved in inflammation and autoimmunity in inflammatory bowel disease [12].…”

Section: Discussionmentioning

confidence: 95%

“…Here, DNA methylation was compared between apoptosis-related genes in pleomorphic adenomas of the salivary glands [13], respectively in genes involved in inflammation and autoimmunity in inflammatory bowel disease [12]. mRNA expression was done in genes with significant methylation changes, according to references [12,13], and analyzed with an unpaired t-test. This An a priori alpha error p of less than 0.05 was considered to indicate statistical significance.…”

Section: Discussionmentioning

confidence: 99%

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Methylation in HT22 cells and primary hippocampal neurons with and without isoflurane exposure

et al. 2020

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Background: Epigenetic modulation may play a role in anesthesia related phenotypes, such as cognitive impairment or memory loss, especially with exposure to anesthetics in the vulnerable phase of brain development. While isoflurane anesthesia can evoke neuroinflammation and neuroapoptosis in young animals, we investigated in a permanent hippocampal cell line (HT22) and in primary hippocampal neurons in an a priori in vitro analysis, whether isoflurane exposure 1) evokes DNA methylation changes in genes involved in apoptosis and inflammation, and 2) results observed in a permanent hippocampal cell line are comparable to primary hippocampal neurons. In case of methylation changes in specific genes, (3) mRNA analysis was performed to assess possible effects on gene expression. Methods: HT22 cells and primary mouse hippocampal neurons were exposed to 3% isoflurane for 4 h and DNA (each 6 single experiments) and RNA (3 single independent experiments) were extracted. Methylation analysis (EpiTect Methyl II PCR Array Systems, Qiagen) included the methylation status of 66 genes involved in apoptosis, cytokine production, inflammatory response, and autoimmunity. Quantitative Real-Time PCR was performed using the Quantitect SYBR Green Kit on a Step One Plus. Results: Methylation status was markedly different between immortalized HT22 cells and cultured primary hippocampal neurons without isoflurane exposure. Of 66 genes investigated, 29 were methylated to a significantly greater degree in HT22 cells compared to primary hippocampal neurons. In cultured primary hippocampal neurons, in contrast, there was a greater methylation in several genes involved in inflammation, accompanied with significant downregulation of C-X-C motif chemokine 12 with isoflurane exposure (p = 0.023). Conclusions: We demonstrate marked differences in gene methylation between HT22 cells and cultured primary hippocampal neurons without isoflurane exposure, with a greater methylation of several genes involved in inflammation upon isoflurane exposure and significant downregulation of Cxcl12 mRNA expression in primary hippocampal neurons. Accordingly, further investigations of anesthesia related DNA methylation should be performed with special consideration being given to the choice of cells targeted for such investigations.

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