Abstract
The expression of human SLFN11 was reported to sensitize cancer cells to DNA damaging agents. This study is to explore the epigenetic change and the function of SLFN11 in human colorectal cancer (CRC). Six CRC cell lines and 128 primary CRC samples were used. SLFN11 was methylated in 55.47% (71/128) of primary CRC. The expression of SLFN11 was regulated by promoter region methylation. Methylation of SLFN11 was significantly associated with age, poor 5-year overall survival and 5-year relapse-free survival (all p < 0.05). SLFN11 suppressed CRC cell growth both in vitro and in vivo and sensitized CRC cells to cisplatin. SLFN11 is frequently methylated in human CRC, and the expression of SLFN11 is regulated by promoter region methylation. Methylation of SLFN11 reduced the sensitivity of CRC cells to cisplatin.
Highlights
Antisense primerRNA isolation & semiquantitative reverse transcription PCR Total RNA was extracted using Trizol reagent; cDNA was synthesized according to the manufacturer’s instructions (Invitrogen, CA, USA) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as control
The expression of SLFN11 is regulated by promoter region methylation in colorectal cancer (CRC) The expression levels of SLFN11 were examined by semiquantitative reverse transcription PCR (RTPCR) to explore the regulation of SLFN11 in CRC
Complete methylation was observed in RKO, DLD1, SW620 and LOVO cells; partial methylation was detected in Ls180; and unmethylation was found in DKO cells (Figure 1B)
Summary
RNA isolation & semiquantitative reverse transcription PCR Total RNA was extracted using Trizol reagent; cDNA was synthesized according to the manufacturer’s instructions (Invitrogen, CA, USA) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as control. The SLFN11 PCR primer sequences were as follows: 5′-AACGCCCGATAACCTTCACA-3′ (forward) and 5′-CTAAGGGGAGGCCCACTAGA-3′ (reverse). The GAPDH PCR primer sequences were as follows: 5′-GACCACAGTCCATGCCATCAC-3′ (forward) and 5′-GTCCACCACCCTGTTGCTGTA-3′ (reverse). KRAS & BRAF mutation detection, bisulfite modification, methylation-specific PCR & bisulfite sequencing Genomic DNA from colorectal cell lines and CRC tissue samples were prepared by the proteinase-K method. SLFN11 methylation-specfic PCR (MSP) and bisulfite sequencing (BSSQ) were performed as described previously [18]. Transfection was performed at a ratio of 1:3 (DNA mass:lipo mass) following the manufacturer’s instructions for Lipofectamine 3000 Reagent (Invitrogen, CA, USA). Transfection was performed according to the manufacturer’s instructions by using Lipofectamine 2000 in SW620 cell (Invitrogen, CA, USA). The sequences were as follows: siRNA-1726 duplex
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