RESUMO
Background: N6-methyladenosine (m6A) is the most frequent internal methylation of eukaryotic RNA (ribonucleic acid) transcripts and plays an important function in RNA processing. The current research aimed to investigate the role of m6A-STIM2 axis in cholangiocarcinoma (CCA) progression. Methods: The expression of STIM2 (Stromal Interaction Molecule 2) in CCA was measured using quantitative polymerase chain reaction (PCR) and immunohistochemistry (IHC). STIM2 was examined in vivo for its effects on the malignant phenotypes of CCA cells. The m6A modification of STIM2 was assessed through MeRIP (methylated RNA Immunoprecipitation)-PCR. Results: Based on the GEPIA (Gene Expression Profiling Interactive Analysis) 2 database findings, a low STIM2 mRNA (messenger RNA) level was related to a poor prognosis in individuals with CCA. Quantitative PCR and IHC assays indicated decreased protein satin in CCA tissues and were associated with extrahepatic metastasis. Vianude mice tail vein injection model indicated that increased STIM2 levels suppressed CCA cell metastasis in vivo, while KRT8 (keratin 8) was detected as the direct downstream target of STIM2-mediated CCA cell metastasis in vivo. Meanwhile, based on SRAMP database and MeRIP assays indicated that m6A alteration resulted in abnormal STIM2 expression in CCA via METTL14 and YTHDC2. Conclusions: Our findings revealed the epi-transcriptomic dysregulation in CCA and metastasis by proposing a complicated STIM2-KRT8 regulatory paradigm based on m6A alteration.
RESUMO
Background: High-throughput transcript sequencing plays an important role in the study of hepatocellular carcinoma (HCC) occurrence and development. High-quality biospecimens, especially high-quality RNA, are the most basic prerequisites for obtaining good transcript sequencing data. Our purpose was to explore the treatment conditions of in vitro ischemic tissue samples that can be used to obtain high-integrity RNA before freezing the samples in liquid nitrogen. Materials and Methods: Postoperative tumor tissues (T) and adjacent normal tissues (AN) from 50 HCC patients were randomly selected from May 5, 2017, to June 15, 2017. Postoperative tissue specimens from each HCC patient were stratified by tissue type (T or AN), ischemia time (minutes), and ischemia temperature (°C) into 16 groups: T-4°C-15 minutes, T-4°C-30 minutes, T-4°C-60 minutes, T-4°C-120 minutes, T-24°C-15 minutes, T-24°C-30 minutes, T-24°C-60 minutes, T-24°C-120 minutes, AN-4°C-15 minutes, AN-4°C-30 minutes, AN-4°C-60 minutes, AN-4°C-120 minutes, AN-24°C-15 minutes, AN-24°C-30 minutes, AN-24°C-60 minutes, and AN-24°C-120 minutes. RNA integrity was detected by RNA integrity number (RIN) and 1% agarose gel electrophoresis. Results: At an ischemia temperature of 4°C and ischemia time of >30 minutes, the RIN of T began to decrease. RIN also gradually decreased in T at an ischemia temperature of 4°C and in both T and AN at an ischemia temperature of 24°C for ischemia times 15, 30, 60, and 120 minutes. For an ischemia time ≤15 minutes and ischemia temperature 4°C or 24°C, the RINs of T and AN were significantly different. Furthermore, at ischemia temperature 4°C and ischemia time 30 and 60 minutes or ischemia temperature 24°C and ischemia time 30 minutes, the RIN of T was higher compared with AN. However, there was no significant difference in RIN between T and AN under other treatment conditions. Conclusions: Tissue quality is adversely affected by ischemia time and ischemia temperature. Therefore, temporary ischemia time (≤15 minutes) before snap freezing is key for maintaining high-integrity RNA in HCC tissues.