Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters

Abstract

Pleomorphic adenoma gene-like 1 gene (PLAGL1) encodes a zinc-finger nuclear transcription factor which promotes apoptosis and cell cycle arrest. Loss or downregulation of its expression has been observed in various human neoplasms. This study compared PLAGL1 expression in colorectal cancer (CRC) tissue and colon mucosa of healthy subjects at the mRNA and protein levels, and estimated its prognostic value. The PLAGL1 mRNA levels were also determined in CRC cell lines. We collected paired tumor tissue and unchanged mucosa of the large intestine from 121 CRC patients as well as 72 colon biopsies of healthy subjects obtained during screening colonoscopy. PLAGL1 mRNA levels were determined by quantitative PCR, while PLAGL1 protein expression was estimated by western blotting and immunohistochemistry. PLAGL1 mRNA level in tumor tissue was ~2-fold lower than in samples of corresponding unchanged tissues and biopsies of healthy colon mucosa. Downregulated expression of PLAGL1 mRNA was also observed in all tested CRC cell lines. Although the average content of PLAGL1 protein did not differ significantly between tumor and unchanged tissues of CRC patients or colon mucosa of healthy individuals, the decreased PLAGL1 protein levels in tumor specimens correlated with lymph node involvement, the presence of metastases and higher TNM disease stage. The PLAGL1 expression level did not correlate significantly with patient overall survival; however, the hazard ratio for patients whose tumor tissues showed reduced PLAGL1 immunohistochemical staining was twice higher than in patients with increased PLAGL1 immunoreactivity. In conclusion, these results suggest that dysregulation of PLAGL1 expression may be involved to some extent in the progression of CRC, but the so far collected patient survival data do not confirm applicability of the PLAGL1 expression level as a prognostic factor in CRC.

Altered Expression of DDR1 in Clear Cell Renal Cell Carcinoma Correlates With miR-199a/b-5p and Patients' Outcome

“…Protein extraction, SDS-PAGE and WB were carried out according to the modified method described previously (21). The levels of protein in homogenates of paired tumor and normal renal tissue specimens were determined using rabbit anti-human antibodies against DDR1 (diluted 1:500; #sc-532; Santa Cruz Biotechnology, Dallas, TX, USA) and actin beta (ACTB; diluted 1:100; #A2066; Sigma-Aldrich) as internal loading control.…”

“…DDR1 mRNA quantification. Total RNA was extracted and reverse transcribed as previously described (21,22). The levels of DDR1 transcripts in homogenates of paired tumor and renal tissue specimens were determined by RT-qPCR and normalized to peptidylprolylisomerase A (PPIA) and TATA box binding protein (TBP) mRNAs content.…”

Expression of the EP300, TP53 and BAX genes in colorectal cancer: Correlations with clinicopathological parameters and survival

“…Total RNA extraction, reverse transcription and real-time quantitative PCR. Total RNA was extracted from all studied tissues and reverse transcribed as previously described (30). Quantification of genes expression was carried out using ABI 7500/7500 Fast Real-Time PCR System (Life Technologies, Applied Biosystems, Foster City, CA, USA).…”

“…Immunoreactivity of the studied proteins was analyzed in sections of 49 tumor and 37 unchanged colorectal tissues of CRC patients. Immunohistochemistry was performed according to previously described methods (30), using rabbit primary antibodies directed against p300 [diluted 1:400 in phosphate-buffered saline (PBS); #ab61217] or BAX (1:400; #ab32503) (both from Abcam, Cambridge, UK). The p53 protein immunostaining was conducted using the Leica ST5010 Autostainer (Leica, Wetzlar, Germany) and ready-to-use antibodies (#IR616; Dako, Glostrup, Denmark) according to the manufacturer's instructions.…”

IKBKB expression in clear cell renal cell carcinoma is associated with tumor grade and patient outcomes

“…Procedures were performed according to the method described previously (22) with some modifications. Briefly, the samples were homogenized in radioimmunoprecipitation lysis buffer supplemented with 1:100 protease inhibitor cocktail, 1:100 phosphatase inhibitor cocktail and 5 mM EDTA (Sigma-Aldrich; Merck, KGaA, Darmstadt, Germany).…”