Role Of SIRT6 In Gastric Cancer And Underlying Mechanism

Gastric cancer, one of the most common gastrointestinal malignancies worldwide, is responsible for approximately 8% of cancer death. The prevalence of gastric cancer in China is high and 5-years survival rate of gastric carcinoma patients is less than 20%. Although novel therapies such as interventional therapy and molecular targeted treatment have undoubtedly improved prognosis and outcome for gastric cancer, a high mortality rate is still the recurrent problem during clinical follow-up. It is believed that tumorigenesis can be perceived as dysfunction of signal transduction network and the disturbance of signaling molecules is a major fact of cancer development and progression. Thus, discovery of new biomarkers and the great insights into the molecular mechanisms involved in gastric cancer occurrence and development are required.The SIRT family (SIRT1-7) is a class of NAD+-dependent acetylases, deacetylases and ADP-ribosyltransferases, which play diverse parts in cancer by affecting the response to genomic instability, regulating cancer-associated metabolism and modifying the tumour microenvironment. SIRT6 is nuclear proteins with deacetylase activity against several targets. Analysis of The Cancer Genome database reveals that SIRT6 is deleted or downregulated in 20% of all kinds of cancer, including colon cancers and rectal adenocarcinoma. However, in certain other types of cancer, high levels of SIRT6 are associated with worse outcomes, such as breast cancer and pancreatic carcinoma. The role of SIRT6 in tumor development is controversial and only limited data are available."Warburg effect" is the universal property of cancers cells, which is characterized as increased uptake of glucose and the conversion of glucose to lactate in cancer cells, rather than catabolizing glucose via the TCA cycle under adequate oxygen tension. Oncogenic alterations and overexpression of transcriptional factors are involved in the reprogramming of metabolism pathway. Consistent with it having a metabolism regulator role, deletion of SIRT6 in mice causes increased glycolysis. Studies proved that SIRT6 regulates glucose homoeostasis via suppressing the expression of multiple glycolytic genes. HIF-1α the critical regulator of metabolic shift to glycolysis and glutaminolysis. SIRT6 represses the expression of multiple glycolytic genes that are known to be HIF-1α targets, including Pdkl, ATP-dependent 6-phosphofructokinase, muscle type (Pfkm; also known as Pfk1) and glucose transporter 1, suggesting that SIRT6-mediated repression of HIF-la may suppress "Warburg effect" in tumors, In this study, immunohistochemical analysis was used to investigate HIF-1a and SIRT6 protein expression in gastric cancer, and the correlation of HIF-1a and SIRT6 expression with clinicopathological factors were discussed. Glycolysis related enzymes were analyzed by RT-PCR method and correlation of HK2, PDK1, LDH5, Glutl mRNA expression with HIF-1a and SIRT6 were investigated by Pearson correlation analysis.ObjectiveTo investigate HIF-1a and SIRT6 expression in gastric carcinoma tissue and analyze the correlation with clinicopathological factors; to study glycolysis related enzymes mRNA expression in gastric carcinoma tissue and analyze the correlation with HIF-1a and SIRT6; to observe the effects of SIRT6 on SGC7901 cell and explore the molecule mechanism.Methods1. The matched fresh tissue samples of primary gastric cancer and adjacent normal tissues from the same patient used for immunohistochemistry, western blot and Real-Time PCR were collected from 40 GC patients. The association between SIRT6 and several clinicopathological factors, including age, gender, tumor infiltration, tumor differentiation, lymph node involvement and tumor grade were analyzed by two-sided chi-squared test.2. mRNA expression of HK2, PDK1, LDH5, Glut1 in primary gastric cancer and adjacent normal tissues were examined by RT-PCR methods. The association between SIRT6 and glycolysis related enzymes were analyzed by Pearson correlation.3. pcDNA3.1 (+) SIRT6 plasmid were transfected into SGC7901 cell. Cell viability assay were detected by CCK-8 kit. Cell apoptosis was analyzed by TUNEL method. Concentration of glucose and lactic acid were examined by commercial kit. mRNA expression of HK2, PDK1, LDH5, Glutl were also discussed.Results1. SIRT6 expression in 40 paired paraffin-embedded GC tissues and adjacent non-tumoral tissues were investigated by immunohistochemistry. Positive SIRT6 expression was detected in 25 of 40 GC patients. The average SIRT6 protein levels were significantly decreased in tumor tissues, indicating that SIRT6 was frequently repressed in GC. SIRT6 mRNA levels were also compared in these samples by RT-PCR. SIRT6 mRNA levels were significantly downregulated in GC tissues compared with non-tumoral tissues.2. HIF-la expression in 40 paired paraffin-embedded GC tissues and adjacent non-tumoral tissues were investigated by immunohistochemistry. Positive HIF-la expression was detected in 30 of 40 GC patients. The average HIF-laprotein levels were significantly increased in tumor tissues, indicating that HIF-lawas frequently overexpressed in GC. HIF-la mRNA levels were also compared in these samples by RT-PCR. HIF-la mRNA levels were significantly upregulated in GC tissues compared with non-tumoral tissues, suggesting that HIF-la overexpression in GC is regulated in a transcription-dependent manner.3. Correlative analysis of SIRT6 protein levels with clinicopathologic features suggested significant association between decreased SIRT6 expression and Lesions infiltration depth, tumor grade, TNM stage and lymphatic metastasis.4. Correlative analysis of HIF-la protein levels with clinicopathologic features suggested significant association between enhensive HIF-la expression and Lesions infiltration depth, tumor grade, TNM stage, lymphatic metastasis and tumor size.5. HK2, PDK1, LDH5, Glutl mRNA expression in 40 paired paraffin-embedded GC tissues and adjacent non-tumoral tissues were investigated by RT-PCR. Up-regulated HK2, PDK1, LDH5, Glutl mRNA levels were observed in GC tissues. Positive expression rate of HK2, PDK1, LDH5, Glutl in GC tissues were 67.5%,62.5%, 72.5% and 72.5% respectively.6. Pearson correlation analysis of HIF-1a levels with HK2, PDK1, LDH5 and Glutl suggested significant association between HIF-la expression and Up-regulated mRNA expression of HK2, PDK1, LDH5 and Glutl.7. Pearson correlation analysis of SIRT6 levels with HK2, PDK1, LDH5 and Glutl suggested significant association between SIRT6 expression and Up-regulated mRNA expression of HK2, PDK1, LDH5 and Glutl.8. To further elucidate the tumour suppressor role of SIRT6 in GC tumorigenesis, pcDNA3.1 (+) SIRT6 plasmid were transfected into SGC7901 cell line. Overexpressing wild-type SIRT6 in SGC7901 cells exhibited decreased growth rates and increased apoptosis.9. wild-type SIRT6 overexpression in SGC7901 cells inhibited HK2, LDH5 and Glutl mRNA expression. Moreover, decrease glucose uptake and lactic acid production were also observed.Conclusion1. Compared with adjacent non-tumoral tissues, positive SIRT6 expression was significantly decreased in GC tumor tissues. Decreased SIRT6 expression were significant association with Lesions infiltration depth, tumor grade, TNM stage and lymphatic metastasis. Positive HIF-la expression was significantly increased in GC tumor tissues. Up-regulated HIF-la expression was significant association with Lesions infiltration depth, tumor grade, TNM stage, lymphatic metastasis and tumor size.2. Up-regulated HK2, PDK1, LDH5, Glutl mRNA expression was observed in GC tissues. HIF-la and SIRT6 expression were significant association with HK2, PDK1, LDH5, Glutl mRNA expression.3. SIRT6 was demonstrated to act as an tumor suppressive in GC development by inhibiting glycolysis activity via blocking HIF-la expression.