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RASSF10 suppresses hepatocellular carcinoma growth by activating P53 signaling and methylation of RASSF10 is a docetaxel resistant marker.

Jin Y, Cao B, Zhang M, Zhan Q, Herman JG, Yu M, Guo M - Genes Cancer (2015)

Bottom Line: Our previous study found that RASSF10 suppresses colorectal cancer growth by activating P53 signaling.In conclusion, we demonstrated that RASSF10 is frequently methylated in human HCC and its methylation is a potential docetaxel resistant marker.Our data also indicate that RASSF10 suppresses human HCC growth by activating P53 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology & Hepatology, Chinese PLA General Hospital, Beijing, China ; Department of Interventional Radiology, Chinese PLA General Hospital, Beijing, China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common malignances and the second leading cause of cancer related death worldwide. RASSF10 is located on chromosome 11p15.2, a region that shows frequent loss of heterozygosity (LOH) in several cancer types. Our previous study found that RASSF10 suppresses colorectal cancer growth by activating P53 signaling. To explore the epigenetic changes and the mechanism of RASSF10 in human HCC, 69 cases of primary HCC, twenty cases of normal liver tissue samples and 17 HCC cell lines were involved in this study. We found that RASSF10 was methylated in 82.6% (57/69) of human primary HCC and methylation of RASSF10 was significantly associated with tumor size (P < 0.05) and TNM stage (P < 0.05). The expression of RASSF10 was regulated by promoter region methylation. Restoration of RASSF10 expression suppressed cell proliferation, induced apoptosis and G2/M phase arrest, as well as sensitized cells to docetaxel and activated P53 signaling in HepG2 and QGY7703 cells. In conclusion, we demonstrated that RASSF10 is frequently methylated in human HCC and its methylation is a potential docetaxel resistant marker. Our data also indicate that RASSF10 suppresses human HCC growth by activating P53 signaling.

No MeSH data available.


Related in: MedlinePlus

The expression and methylation status of RASSF10 in HCC cells(A) Expression of RASSF10 was detected by semi-quantitative RT-PCR. 5-aza: 5-aza-2′-deoxycytidine; GAPDH: The internal control of RT-PCR. SNU182, SNU387, HBXF344, SNU475, HepG2, BEL7402, LM3, Huh7, PLC/PRF5, QGY7703, Huh1, BEL7402, BEL7405 and QSG7701 are HCC cell lines, DKO is colorectal cancer cell line. (–): absence of 5-aza, ( + ): presence of 5-aza. (B) MSP results of RASSF10 in HCC cell lines and DKO cells. U: unmethylated alleles, M: methylated alleles; IVD: In vitro methylated DNA, methylation control; NL: normal peripheral lymphocytes DNA, unmethylation control.(C) MSP results of RASSF10 after 5-aza treatment in HCC cell lines.
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Figure 1: The expression and methylation status of RASSF10 in HCC cells(A) Expression of RASSF10 was detected by semi-quantitative RT-PCR. 5-aza: 5-aza-2′-deoxycytidine; GAPDH: The internal control of RT-PCR. SNU182, SNU387, HBXF344, SNU475, HepG2, BEL7402, LM3, Huh7, PLC/PRF5, QGY7703, Huh1, BEL7402, BEL7405 and QSG7701 are HCC cell lines, DKO is colorectal cancer cell line. (–): absence of 5-aza, ( + ): presence of 5-aza. (B) MSP results of RASSF10 in HCC cell lines and DKO cells. U: unmethylated alleles, M: methylated alleles; IVD: In vitro methylated DNA, methylation control; NL: normal peripheral lymphocytes DNA, unmethylation control.(C) MSP results of RASSF10 after 5-aza treatment in HCC cell lines.

Mentions: The expression of RASSF10 was detected in HCC cell lines by semi-quantitative RT-PCR. As shown in Figure 1A, loss of RASSF10 expression was found in SNU387, HBXF344, HepG2, PLC/PRF/5, Huh7, BEL7402, LM3, QGY7703, SNU449, SMMC7721, BEL7404, BEL7405, Sk-hep1, QSG7701 and Huh1 cells and reduced expression was found in SNU182 and SNU475 cells. RASSF10 was highly expressed in DKO cells. Promoter region methylation was detected by methylation specific PCR (MSP). Complete methylation was found in SNU387, HBXF344, HepG2, PLC/PRF/5, Huh7, BEL7402, LM3, QGY7703, SNU449, SMMC7721, Sk-hep1, Huh1, BEL7404, BEL7405 and QSG7701 cells, partial methylation was found in SNU182 and SNU475 cells, and unmethylation in DKO cells (Figure 1B). These results demonstrated that loss or reduced expression of RASSF10 correlated with promoter region methylation. Treatment with the DNA methylation transferase inhibitor 5-aza-2′-deoxycytidine (5-aza) restored RASSF10 expression in SNU387, HBXF344, HepG2, PLC/PRF/5, Huh7, BEL7402, LM3, QGY7703 SNU449, SMMC7721, Sk-hep1, Huh1, BEL7404, BEL7405 and QSG7701 cells and increased RASSF10 expression in SNU182 and SNU475 cells (Figure 1A). The above results suggest that RASSF10 expression is regulated by promoter region methylation. To further validate the efficiency of the MSP primers and the methylation density in the promoter region, sodium bisulfite sequencing (BSSQ) was performed in SNU182, SHU475, HepG2, QGY7703 and DKO cells. The BSSQ results were consistent with the MSP results (Figure 2A). As shown in Figure 1C and 2B, the direct effect of 5-aza induction of RASSF10 expression was validated by MSP and BSSQ after 5-aza treatment in SNU182, SHU475, HepG2 and QGY7703 cells.


RASSF10 suppresses hepatocellular carcinoma growth by activating P53 signaling and methylation of RASSF10 is a docetaxel resistant marker.

Jin Y, Cao B, Zhang M, Zhan Q, Herman JG, Yu M, Guo M - Genes Cancer (2015)

The expression and methylation status of RASSF10 in HCC cells(A) Expression of RASSF10 was detected by semi-quantitative RT-PCR. 5-aza: 5-aza-2′-deoxycytidine; GAPDH: The internal control of RT-PCR. SNU182, SNU387, HBXF344, SNU475, HepG2, BEL7402, LM3, Huh7, PLC/PRF5, QGY7703, Huh1, BEL7402, BEL7405 and QSG7701 are HCC cell lines, DKO is colorectal cancer cell line. (–): absence of 5-aza, ( + ): presence of 5-aza. (B) MSP results of RASSF10 in HCC cell lines and DKO cells. U: unmethylated alleles, M: methylated alleles; IVD: In vitro methylated DNA, methylation control; NL: normal peripheral lymphocytes DNA, unmethylation control.(C) MSP results of RASSF10 after 5-aza treatment in HCC cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482244&req=5

Figure 1: The expression and methylation status of RASSF10 in HCC cells(A) Expression of RASSF10 was detected by semi-quantitative RT-PCR. 5-aza: 5-aza-2′-deoxycytidine; GAPDH: The internal control of RT-PCR. SNU182, SNU387, HBXF344, SNU475, HepG2, BEL7402, LM3, Huh7, PLC/PRF5, QGY7703, Huh1, BEL7402, BEL7405 and QSG7701 are HCC cell lines, DKO is colorectal cancer cell line. (–): absence of 5-aza, ( + ): presence of 5-aza. (B) MSP results of RASSF10 in HCC cell lines and DKO cells. U: unmethylated alleles, M: methylated alleles; IVD: In vitro methylated DNA, methylation control; NL: normal peripheral lymphocytes DNA, unmethylation control.(C) MSP results of RASSF10 after 5-aza treatment in HCC cell lines.
Mentions: The expression of RASSF10 was detected in HCC cell lines by semi-quantitative RT-PCR. As shown in Figure 1A, loss of RASSF10 expression was found in SNU387, HBXF344, HepG2, PLC/PRF/5, Huh7, BEL7402, LM3, QGY7703, SNU449, SMMC7721, BEL7404, BEL7405, Sk-hep1, QSG7701 and Huh1 cells and reduced expression was found in SNU182 and SNU475 cells. RASSF10 was highly expressed in DKO cells. Promoter region methylation was detected by methylation specific PCR (MSP). Complete methylation was found in SNU387, HBXF344, HepG2, PLC/PRF/5, Huh7, BEL7402, LM3, QGY7703, SNU449, SMMC7721, Sk-hep1, Huh1, BEL7404, BEL7405 and QSG7701 cells, partial methylation was found in SNU182 and SNU475 cells, and unmethylation in DKO cells (Figure 1B). These results demonstrated that loss or reduced expression of RASSF10 correlated with promoter region methylation. Treatment with the DNA methylation transferase inhibitor 5-aza-2′-deoxycytidine (5-aza) restored RASSF10 expression in SNU387, HBXF344, HepG2, PLC/PRF/5, Huh7, BEL7402, LM3, QGY7703 SNU449, SMMC7721, Sk-hep1, Huh1, BEL7404, BEL7405 and QSG7701 cells and increased RASSF10 expression in SNU182 and SNU475 cells (Figure 1A). The above results suggest that RASSF10 expression is regulated by promoter region methylation. To further validate the efficiency of the MSP primers and the methylation density in the promoter region, sodium bisulfite sequencing (BSSQ) was performed in SNU182, SHU475, HepG2, QGY7703 and DKO cells. The BSSQ results were consistent with the MSP results (Figure 2A). As shown in Figure 1C and 2B, the direct effect of 5-aza induction of RASSF10 expression was validated by MSP and BSSQ after 5-aza treatment in SNU182, SHU475, HepG2 and QGY7703 cells.

Bottom Line: Our previous study found that RASSF10 suppresses colorectal cancer growth by activating P53 signaling.In conclusion, we demonstrated that RASSF10 is frequently methylated in human HCC and its methylation is a potential docetaxel resistant marker.Our data also indicate that RASSF10 suppresses human HCC growth by activating P53 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology & Hepatology, Chinese PLA General Hospital, Beijing, China ; Department of Interventional Radiology, Chinese PLA General Hospital, Beijing, China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common malignances and the second leading cause of cancer related death worldwide. RASSF10 is located on chromosome 11p15.2, a region that shows frequent loss of heterozygosity (LOH) in several cancer types. Our previous study found that RASSF10 suppresses colorectal cancer growth by activating P53 signaling. To explore the epigenetic changes and the mechanism of RASSF10 in human HCC, 69 cases of primary HCC, twenty cases of normal liver tissue samples and 17 HCC cell lines were involved in this study. We found that RASSF10 was methylated in 82.6% (57/69) of human primary HCC and methylation of RASSF10 was significantly associated with tumor size (P < 0.05) and TNM stage (P < 0.05). The expression of RASSF10 was regulated by promoter region methylation. Restoration of RASSF10 expression suppressed cell proliferation, induced apoptosis and G2/M phase arrest, as well as sensitized cells to docetaxel and activated P53 signaling in HepG2 and QGY7703 cells. In conclusion, we demonstrated that RASSF10 is frequently methylated in human HCC and its methylation is a potential docetaxel resistant marker. Our data also indicate that RASSF10 suppresses human HCC growth by activating P53 signaling.

No MeSH data available.


Related in: MedlinePlus