Patients with oral squamous cell carcinoma (OSCC) generally have an elevated expression of homeobox C6 (HOXC6) gene. We found that HOXC6 was the significantly upregulated gene in hypopharyngeal squamous cell carcinoma (FaDu) cells using RNA-seq analysis. However, it remains unclear whether HOXC6 plays a role in tumor process mechanism. Our study aimed to explore the potential oncogenic role and the detailed molecular mechanism of HOXC6 in FaDu cells. In this study, Sirt1 was validated to be overexpressed in FaDu cells and associated with HOXC6 expression. Overexpression of HOXC6 promoted the cell colony formation, whereas inhibition of Sirt1 by Sirt1 inhibitor EX527 reduced cell proliferation/colony formation and migration, and induced apoptosis in HOXC6 overexpressed FaDu cells. Interestingly, mechanistic study showed that EX527 mediated Sirt1 suppression led to decreased HOXC6 expression and upregulation of Sirt1 significantly increased the expression of HOXC6. HOXC6 was shown to cooperate with Sirt1 to enhance cell survival. We propose that HOXC6 promotes cell growth/colony formation, and that the HOXC6 may be a progression of hypopharyngeal carcinoma by activating Sirt1 pathways.

Sirtuin 1 (SIRT1), the most conserved nicotinamide adenine dinucleotide-dependent protein deacetylase, is involved in the regulation of energy metabolism, genomic stability, and development. SIRT1 knockout (SIRT1) mice exhibit decreased energy expenditure and hypersensitivity to a high-fat diet (HFD). SIRT1 deficiency in the testis has also been shown to cause male infertility in animal models. Therefore, the present study was conducted to examine the alteration of the testicular function of SIRT1 mice on HFD. Six-week-old mice were fed ad libitum as wild type (WT) and SIRT1 male mice with either a control diet or with HFD for 32 weeks and then were sacrificed. The levels of biomarkers for hepatotoxicity, sex hormones, and cytokines were analyzed in the serum and blood-testis barrier, and the sperm morphology was examined in the testis and epididymal spermatozoa. Interestingly, an enlargement of seminal vesicles was observed in the SIRT1 mice fed with HFD. A significantly higher level of hepatotoxicity was also seen in these mice. The concentration of serum testosterone increased in HFD-fed SIRT1 mice compared to the controls. The levels of interleukin-1β and TNF-α increased in both HFD-fed WT and SIRT1 mice. In RT-PCR, the m RNA expression of tight junction protein 2 and claudin 3 significantly decreased in HFD-fed SIRT1 compared to those of the controls. Degenerative spermatocytes and spermatids were detected in the HFD-fed SIRT1 mice testicular section. Sperm motility decreased in WT and SIRT1 with HFD feeding, and sperm concentration decreased significantly in WT-HFD and SIRT1 mice with or without HFD feeding. Taken together, HFD can alter energy and steroid metabolism in SIRT1-deficient mice, which can lead to imbalances in motility and production of sperm and testosterone that can result in male reproductive disorders.

Sirtuin proteins are evolutionary conserved Sir2-related NAD+-dependent deacetylases and regulate many of cellular processes such as metabolism, inflammation, transcription, and aging. Sirtuin contains activity of either ADP-ribosyl-transferase or deacetyltranfease and their activity is dependent on the localization in cells. However, the expression pattern of Sirtuins has not been well studied. To examine the expression levels of Sirtuins, RT-PCR was performed using total RNAs from various tissues including liver, small intestine, heart, brain, kidney, lung, spleen, stomach, uterus, ovary, and testis. Sirtuins were highly expressed in most of tissues including the testis. Immunostaining assay showed that Sirt1 and Sirt6 were mainly located in the nucleus of germ cells, spermatocytes, and spermatids in the seminiferous tubules, whereas Sirt2 and Sirt5 were exclusively present in the cytoplasm of germ cells and sperma-tocytes. Our results indicate that Sirtuins may function as regulators of spermatogenesis and their activities might be dependent on their location in the seminiferous tubules.

Selenoprotein S (SelS) is widely expressed in diverse tissues where it localizes in the plasma membrane and endoplasmic reticulum. We studied the potential function of SelS in erythrocyte differentiation using K562 cells stably over-expressing SelS wild-type (WT) or one of two SelS point mutants, U188S or U188C. We found that in the K562 cells treated with 1μM Ara-C, SelS gradually declined over five days of treatment. On day 4, intracellular ROS levels were higher in cells expressing SelS-WT than in those expressing a SelS mutant. Moreover, the cell cycle patterns in cells expressing SelS-WT or U188C were similar to the controls. The expression and activation of SIRT1 were also reduced during K562 differentiation. Cells expressing SelS-WT showed elevated SIRT1 expression and activation (phosphorylation), as well as higher levels of FoxO3a expression. SIRT1 activation was diminished slightly in cells expressing SelS-WT after treatment with the ROS scavenger NAC (12 mM), but not in those expressing a SelS mutant. After four days of Ara-C treatment, SelS-WT-expressing cells showed elevated transcription of β-globin, y-globin, ε-globin, GATA-1 and zfpm-1, whereas cells expressing a SelS mutant did not. These results suggest that the suppression of SelS acts as a trigger for proerythrocyte differentiation via the ROS-mediated downregulation of SIRT1.

Sirtuin-1(Sirt1)은 NAD+ dependent deacetylase로써 유전자 침묵, 포도당과 지질대사, 세포자살 등 다양한 cellular process에 연관되어 있다. 최근 이뤄진 여러 연구에서 Sirt1 Knockout mouse가 정자형성 장애와 불임증상을 가진다는 것이 보고되었다. Egr1(Early Growth Response 1)은 Egr family에 속하는 전사인자로서 mitogenesis와 differentiation에 필요한 유전자들의 전사를 활성화시킨다. 또한 이는 Sirt1의 전사를 조절하는 것으로 알려져 있다. 본 연구에서는 생쥐 정소 내에서 Egr1의 유무에 의한 Sirt1의 발현 수준 차이를 밝히고자 Egr1 knockout mouse와 wild type mouse를 이용하여 Sirt1의 발현패턴을 분석하였다. Sirt1의 발현을 분석하기 위하여 Quantitative RT-PCR과 conventional RT-PCR을 이용하여 각각 ICR strain adult male mice와 Egr1 Knockout adult male mice의 testicular cDNA에서 mRNA 수준에서 Sirt1의 발현수준을 분석하였다. 또한 Immunohistochemistry를 이용하여 Sirt1단백질의 localization을 분석하였다. 분석한 결과, Wild type과 Egr1 Knockout mouse의 정소 내에서 Sirt1 mRNA의 발현 수준은 큰 차이를 보이지 않았으나, Egr1 Knockout mouse의 정소 내에서 Sirt1 단백질의 발현수준은 wild type과 비교하여 현저히 떨어졌으며, 이 변화는 특히 pachytene spermatocyte에서 큰 차이를 보였다. 반면, Sertoli cell에서는 Pachytene spermatocyte에서만큼 뚜렷한 차이를 관찰할 수 없었다. 본 연구에서 얻은 결과를 바탕으로 Egr1이 pachytene spermatocyte에서 Sirt1 단백질의 발현에 중요한 역할을 할 것이라는 결론을 얻을 수 있었다. Sirt1 Knockout mouse가 불임 증상을 보이며, 정자형성에 장애가 있는 반면, Egr1 KO mouse는 정상적인 생식능력을 가짐으로 보아 premeiotic 또는 meiotic stage에서의 Sirt1 결핍이 이러한 infertility의 원인이 될 것으로 추측된다.

Sirt1 belongs to class III histone/protein deacetylase. Sirt1 KO mice have spermatogenic defects. In this study, expression of Sirt1 and acetylated histone 3k9 (H3k9ac) was examined in developing mouse testes. Among two splicing variants of sirt1 mRNA long form was dominant in developing testis. Testicular sirt1 mRNA levels were low at birth, increased until 14 days post partum (pp) and remained constant thereafter. Sirt1 immunoreactivity was weak to negligible in gonocytes, moderate in spermatogonia, absent in preleptotene spermatocytes, moderate in zygotene spermatocytes, strong in pachytene spermatocytes, and weak in diplotene spermatocytes. Round and elongating spermatids were negative for Sirt1. Acetylated histone 3k9 (H3k9ac) immunoreactivity was moderate in spermatogonia and weak to negligible in preleptotene to pachytene spermatocytes but strong in round and elongating spermatids. In Sertoli cells, nuclear Sirt1 immunoreactivity was absent at birth, increased at 14 days pp and markedly increased at 28 days pp onwards. In immature Sertoli cells culture, FSH and testosterone increased sirt1 mRNA, suggesting that Sirt1 participates in protein deacetylation events during the differentiation of Sertoli cells by gonadotropin. In the Leydig cells, nuclear Sirt1 immunoreactivity was weak until 2 weeks pp and decreased in 4 weeks pp onward. suggesting the protein deacetylation by Sirt1 in Leydig cell precursor/progenitor cells. Mutually exclusive expression between Sirt1 and H3k9ac in pachytene spermatocytes in testis suggests that deacetylation of H3K9ac by Sirt1 participates in the gene silencing and/or chromosome behavior in pachytene sspermatocytes.