A pan-cancer analysis unveiling the function of NR4A family genes in tumor immune microenvironment, prognosis, and drug response

Park Seong-Woo,Han Mi-Ryung 한국유전학회 2024 Genes & Genomics Vol.46 No.8

Background NR4A family genes play crucial roles in cancers. However, the role of NR4A family genes in cancers remains paradoxical as they promote or suppress tumorigenesis. Objective We aimed to conduct comprehensive analyses of the association between the expression of NR4A family genes and tumor microenvironment (TME) based on bioinformatics methods. Methods We collected RNA-seq data from 33 cancer types and 20 normal tissue sites from the TCGA and GTEx databases. Expression patterns of NR4A family genes and their associations with DNA methylation, miRNA, overall survival, drug responses, and tumor microenvironment were investigated. Results Significant downregulation of all NR4A family genes was observed in 15 cancer types. DNA promoter methylation and expression of NR4A family genes were negatively correlated in five cancers. The expression of 10 miRNAs targeting NR4A family genes was negatively correlated with the expression of NR4A family genes. High expression of all NR4A family genes was associated with poor prognosis in stomach adenocarcinoma and increased expressions of NR4A2 and NR4A3 were associated with poor prognosis in adrenocortical carcinoma. In addition, we found an elevated expression of NR4A2, which enhances the response to various chemotherapeutic drugs, whereas NR4A3 decreases drug sensitivity. Interestingly, in breast cancer, NR4A3 was significantly associated with C2 (IFN-γ dominant), C3 (inflammatory), and C6 (TGF-β dominant) immune subtypes and infiltrated immune cell types, implying both oncogenic and tumor-suppressive functions of NR4A3 in breast cancer. Conclusion The NR4A family genes have the potential to serve as a diagnostic, prognostic, and immunological marker of human cancers. Background NR4A family genes play crucial roles in cancers. However, the role of NR4A family genes in cancers remains paradoxical as they promote or suppress tumorigenesis. Objective We aimed to conduct comprehensive analyses of the association between the expression of NR4A family genes and tumor microenvironment (TME) based on bioinformatics methods. Methods We collected RNA-seq data from 33 cancer types and 20 normal tissue sites from the TCGA and GTEx databases. Expression patterns of NR4A family genes and their associations with DNA methylation, miRNA, overall survival, drug responses, and tumor microenvironment were investigated. Results Significant downregulation of all NR4A family genes was observed in 15 cancer types. DNA promoter methylation and expression of NR4A family genes were negatively correlated in five cancers. The expression of 10 miRNAs targeting NR4A family genes was negatively correlated with the expression of NR4A family genes. High expression of all NR4A family genes was associated with poor prognosis in stomach adenocarcinoma and increased expressions of NR4A2 and NR4A3 were associated with poor prognosis in adrenocortical carcinoma. In addition, we found an elevated expression of NR4A2, which enhances the response to various chemotherapeutic drugs, whereas NR4A3 decreases drug sensitivity. Interestingly, in breast cancer, NR4A3 was significantly associated with C2 (IFN-γ dominant), C3 (inflammatory), and C6 (TGF-β dominant) immune subtypes and infiltrated immune cell types, implying both oncogenic and tumor-suppressive functions of NR4A3 in breast cancer. Conclusion The NR4A family genes have the potential to serve as a diagnostic, prognostic, and immunological marker of human cancers.

연어 뇌에서 N-Methyl-D-Aspartate 수용체 아단위 NR2A와 NR2B의 분포

진덕희,문일수 한국생명과학회 1999 생명과학회지 Vol.9 No.6

본 연구는 연어 뇌 연접의 단백질구성에 대한 기초연구로서, 기억형성에 중요한 역활을 하는 NMDA 수용체의 분포와 PTK에 의한 인산화에 대하여 조사하였다. 본 실험에 사용한 연어 뇌의 PSD 분획에서는 Coomassie로 염색할 경우 20여개 분명한 단백질띠를 확인할 수 있었으며, 소량으로 존재하여 전체적으로 도말되어 보이는 펩타이드의 수는 알 수 없었다. 이들 중 180kD 크기의 단백질은 phosphotyrosine 특이성 항체에 상대적으로 잘 인식이 되었다. 또한 이 180 kDa의 단백질들은 쥐에 있어서의 NR2A 및 NR2B의 위치인 분자량 약 180kD의 위치와 동일한 것으로 보아 연어에 있어서도 후각기능에 관계하는 NR2A 및 NR2B가 존재함을 추정할 수 있다. We carried out immunoblot analyses to study expression and subcellular distribution of the N-methyl-D-aspartate receptor(NR) subunits in salmon (Chum Salmon, Oncorhynchus keta). We prepared subcellular fractions such as brain homogenates, synaptosomes, and postsynaptic density (PSD) from salmon brains, and analyzed protein compositions by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In a Coomassie-stained 6% SDS-gel, about 20 distinct major protein bands could be identified in the PSD fraction. Immunoblot analyses using antibodies against rat NR subunit 2A and 2B antigens (NR2A and NR2B, respectively) showed weak but evident signals at the 180 kDa positions in the salmon PSD fractions. However, in contrast to rat NRs, the salmon NR2A and NR2B are not recognized by a phosphotyrosine-specific antibody suggesting that the salmon NRs are regulated differently from those of the rat by protein tyrosine kinases. Our results indicate that NR2A and NR2B subunits are expressed in the salmon PSD fraction but not regulated by tyrosine phosphorylation.

저산소증과 산소재공급 후 흰쥐 해마 CAI 신경세포들의 변화에 따른 NMDA수용체아단위 NR2B와 c-fos mRNA 발현의 변화

정용욱,고복현 대한해부학회 1999 Anatomy & Cell Biology Vol.32 No.5

저산소증 흰쥐 대뇌피질에서 NMDA수용체 아단위 조절자 NR2B의 증가에 따른 세 포골격단백질의 분해

정용욱,문일수,고복현 대한해부학회 1997 Anatomy & Cell Biology Vol.30 No.5

Changes of NR2B and c-fos mRNA Expression in Rat Hippocampal CA1 Neuronal Change Following Hypoxia and Reoxygenation

정용욱(Yong Wook Jung),고복현(Bok Hyun Ko) 대한해부학회 1999 Anatomy & Cell Biology Vol.32 No.5

저산소증 이후 나타나는 해마 CA1 지역 신경세포의 변화는 NMDA수용체 아단위 특히 NR2의 농도와 깊은 관련이 있다. NR2 아단위들은 NMDA 수용체의 조절자로서 NR1이 어떠한 NR2와 결합하느냐에 의해 NMDA 수용체의 특성이 결정되어 지는 특징이 있다. NMDA 수용체의 과 자극은 수용체를 통한 칼슘통로를 열게 되고 이로 인한 칼슘의 과다유입은 c-fos의 발현에 중요한 역할을 할 것으로 생각된다. 본 실험은 저산소증 이 후 산소재공급 상태에서 해마 CA1지역의 NMDA 수용체 아단위들의 발현변화가 c-fos의 발현과 신경세포들의 변화에 미치는 영향을 mRNA in situ hybridization과 TUNEL 방법을 이용하여 알아보았다. 실험의 결과에 의하면 NMDA 수용체 아단위 가운데 NR2B 아단위의 발현이 대조군에 비하여 가장 증가 하였으며 시간대별 변화의 정도도 가장 심하였다. 이는 NR2B 아단위가 저산소증으로 초래되는 NMDA 수용체 성상의 변화를 결정하는 주된 아단위 임을 의미한다. 산소재공급 후 0시간에서 6시간까지 TUNEL 양성반응과 c-fos의 발현은 대조군에 비해 의미 있게 증가하였으나 그 이후 TUNEL 양성반응은 산소재공급 6시간대에 비해 감소하였으며 c-fos의 발현은 일시적 감소 후 3일에서 6일까지 다시 증가하였다. 따라서 NR2B의 발현증가와 이로 인해 초래될 칼슘의 과다한 유입은 신경세포의 자연사와 c-fos의 활성화에 중요한 역할을 할 것으로 생각되며 c-fos의 활성화는 이들 유전자의 발현시간에 따라 CA1 신경세포의 자연사에 다른 역할을 수행할 것으로 생각된다. Vulnerability of hippocampal CA1 neurons after brain hypoxia is coupled with high concentration of NMDA receptor subunits especially NR2 subunits. These subunits can be regarded as modulators, since NMDA receptors display different properties depending on which of the NR2 subunits assembles with NR1. Stimulation of NMDA receptors opens a receptor-gated Ca2+ channel; and the Ca2+ influx is believed to play a critical role in c-fos expression that require alterations in gene expression. We examined the effect of the NMDA subunits (NR1, NR2A, NR2B) mRNA level on the c-fos activation and CA1 neuronal change by in situ hybridization and TUNEL method following hypoxia and reoxygenation. Present studies show that high expression and variability of NR2B mRNA in CA1 region, compared with those of NR1 and NR2A, at our experimental time window suggest that NR2B is main functional subunit in the determining of properties of NMDA receptor in hypoxic hippocampal CA1 cells. From 0 hour to 6 hours, TUNEL positive reaction and c-fos mRNA expression were steadily increased. Thereafter, TUNEL positive reaction was decreased compared with that of 6 hours and c-fos mRNA expression was decreased and then steadily re-increased from 3 days to 6 days. Therefore, high expression of NR2B and subsequent Ca2+ influx may play important roles in the hippocampal CA1 neuronal apoptosis and c-fos expression. In addition, c-fos expression may play different roles in neuronal apoptosis in a time-dependent manner.

Expression of Functional Subunits of N-Methy1-D-aspartate Receptor in Hypoxic Neonatial Rat

Lee, Sung Chang,Jung, Yong Wook 東國大學校醫學硏究所 2005 東國醫學 Vol.12 No.1

발생중인 뇌의 특정기간은 허혈이나 저산소증에 매우 민감하다. 특히 해마 신경세포의 사망은 주산기 저산소증으로 인한 뇌 손상의 가장 전형적 형태이며 이는 가끔 다른 지역의 뇌 손상과 동반된다. 발생중인 해마신경세포의 손상에 NMDA수용체의 아단위인 NR2A와 NR2B의 발현변화가 동반되는지를 알아보기 위하여 생후 7일된 흰쥐를 92% N₂와 8% O₂로 구성된 저산소증 상황에 2시간 노출하였다. 해마조직은 저산소증 처리 후 7일과 14일 뒤에 얻었으며 NR2A와 NR2B의 mRNA 양의 변화는 NR2A와 NR2B 아단위에 대한 antisense probe를 이용한 in situ hybridization 방법을 이용하여 알아보았다. 실험결과에 의하면 저산소증 처리 후 14일 된 생후 21일 흰쥐 해마에서 NR2B의 발현은 의미있게 증가하였으며 이 시기에 특히 CA3 지역의 신경세포들의 숫자가 감소되고 이와 반대로 별아교세포의 숫자는 증가하였다. 그러나 이 시기 NR2A의 발현은 대조군에 비해 감소하였다. 저산소증에 대한 신생흰쥐 해마에서의 NR2B의 발현증가는 NMDA 수용체의 기능적 변화를 초래하는 NR2 아단위의 발현변화가 발생 중 흰쥐 해마에서의 특정 신경세포집단의 손상발생에 중요한 역할을 할 수 있을 가능성을 의미한다. In this study, we examined whether transcriptional changes of NR2A and NR2B subunits of the N-methyl-D-aspartate (NMDA) receptors related to the neuronal cell death to hypoxic toxicity are involved in developing neurons in the hippocampus. Fourteen days after hypoxia, the expression of NR2B significantly increased whereas NR2A showed distinct reduction with prominant reduction of actual numbers and accompanied reactive astrocytosis. Alteration of NR2A and NR2B expression to hypoxia insults suggest the possibility that the changes of the NR2B containing NMDA receptors play a crucial role in the occurrence of specific hippocampal neuronal injury on development.

일시적 허혈이 흰쥐 대뇌피질에서 NMDA수용체 아단위 2A, 2B와 신경미세섬유(NF200)에 미치는 영향

정용욱,문일수,고복현 대한해부학회 1998 Anatomy & Cell Biology Vol.31 No.4

Involvement of serine phosphorylation of spinal cord NR-2B subunit of the N-methyl-D-aspartate receptor following electroacupuncture stimulation

Kang Byeol-Rim,Choi Byung-Tae,Yoon Hyun-Min,Min Young-Kwang,Ahn Chang-Beohm 대한침구의학회 2007 대한침구의학회지 Vol.24 No.2

Objective : This study was to examine the low frequency EA is associated with NMDAR and phosphorylation of NMDAR NR-2B subunits in the spinal cordMethods : We investigated that only those rats without overt signs of spinal cord or root damage such as paralysis or lameness were used for experimentation. The NMDA antagonist, D-2-amino-5-phosphonopentanoic acid dissolved in sterile saline and injected intrathecally. Two stainless-steel needles were inserted in each hind leg at those acupoints corresponding to Zusanli(S36) and Sanyinjiao(Sp6) in man and wereconnected to an electric stimulator The EA wit 2 ㎐ stimulation started immediately after AP-5 injection for 30 min. Results : EA analgesia was slightly reduced by intrathecal injection of NMDAR antagonist AP-5, but analgesic effects of EA still showed at 60 minutes after termination of stimulation in all EA-treated group. In the Western blot analysis, the levels of NMDAR NR-2B and phospho-NR-2B were slightly induced by EA stimulation in the spinal cord. These expressions were significantly inhibited by spinal blockage of AP-5. As for regional reaction of NMDAR NR-2B and phospho-NR-2B, immunoreactions were observed throughout all laminae of the dorsal horn of spinal cord and weaker ones showed in the neck region. The mean IOD of phospho-NR-2B in the superficial laminae and nucleus proprius of EA-treated rats were significantly increased compared with normal ones, these expressions were decreased in EA-treated with AP-5 groupsConclusion : It is concluded that EA stimulation may be involved NMDAR activation through phosphorylation of spinal NMDAR NR-2B subunit. 목적 : 저주파에 해당하는 2 Hz 전침 자극이 척수 N-methyl-D-aspartate receptor (NMDAR)의 NR-2B subunit의 발현 및 인산화에 미치는 영향을 조사하였다. 방법 : Sprague-Dawley계 흰쥐를 Størkson 등의 방법에 의해 척수막의 지주막하강에 catheter를 삽입하는 수술을 행한 후 마비 등의 척수 손상을 나타내지 않는 개체를 대상으로 하였다. N-methyl-D-aspartate (NMDA) antagonist인 D-2-amino-5- phosphonopentanoic acid (AP-5)를 투여한 후 족삼리와 삼음교에 해당하는 부위에 30분간 전침 자극하였다. 무통각 여부는 hot plate test를 시행하였으며 NMDAR NR-2 subunit 발현과 인산화 여부는 Western blot과 면역조직화학적으로 살펴보았다. 결과 : 전침 무통각은 전침 자극 후 180분 후까지 지속되었으며 NMDA antagonist인 AP-5를 투여하였을 때 전침 무통각이 저하되었으나 유의성은 나타내지 않았다. Western blot 분석으로 보아 NMDAR NR-2B 및 인산화 NR-2B의 발현은 전침자극에 의해 미약한 증가를 보이나 AP-5투여에 의해 현저한 저해를 보였다. 면역조직화학에 의한 척수배각 구역별 발현을 보면 NMDAR NR-2B 및 인산화 NR-2B는 전 배각에 걸쳐 관찰되나 경부 (층판 Ⅴ-Ⅵ)에서 약한 반응을 보였다. 전침 자극에 의한 각 군별 NR-2B 발현은 유의한 차이를 보여 주지 않았으나 인산화 NR-2B는 천층 (층판 Ⅰ-Ⅱ)및 고유핵 (층판 Ⅲ-Ⅳ)에서 유의성 있는 증가를 보였다. 전침 자극시 AP-5 투여는 유의성은 보이지 않았으나 인산화 NR-2B발현을 저해하였다. 결론 : 저주파 2 Hz 전침에 의한 무통각은 NMDA antagonist인 AP-5 투여에 의해 저해될 뿐아니라 NMDAR NR-2B subunit의 인산화를 저해하는 것으로 보아 전침 무통각의 과정에 NMDAR 및 NMDAR NR-2B의 인산화가 관여함을 알 수 있다.

The Role of Nuclear Receptor Subfamily 1 Group H Member 4 (NR1H4) in Colon Cancer Cell Survival through the Regulation of c-Myc Stability

Lee, Yun Jeong,Lee, Eun-Young,Choi, Bo Hee,Jang, Hyonchol,Myung, Jae-Kyung,You, Hye Jin Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.5

Nuclear receptor subfamily group H member 4 (NR1H4), also known as farnesoid X receptor, has been implicated in several cellular processes in the liver and intestine. Preclinical and clinical studies have suggested a role of NR1H4 in colon cancer development; however, how NR1H4 regulates colon cancer cell growth and survival remains unclear. We generated NR1H4 knockout (KO) colon cancer cells using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (CAS9) technology and explored the effects of NR1H4 KO in colon cancer cell proliferation, survival, and apoptosis. Interestingly, NR1H4 KO cells showed impaired cell proliferation, reduced colony formation, and increased apoptotic cell death compared to control colon cancer cells. We identified MYC as an important mediator of the signaling pathway alterations induced by NR1H4 KO. NR1H4 silencing in colon cancer cells resulted in reduced MYC protein levels, while NR1H4 activation using an NR1H4 ligand, chenodeoxycholic acid, resulted in time- and dose-dependent MYC induction. Moreover, NR1H4 KO enhanced the anti-cancer effects of doxorubicin and cisplatin, supporting the role of MYC in the enhanced apoptosis observed in NR1H4 KO cells. Taken together, our findings suggest that modulating NR1H4 activity in colon cancer cells might be a promising alternative approach to treat cancer using MYC-targeting agents.

Production and Characterization of Nitrate Reductase Deficient Mutants in Petunia parviflora

Lee, Cheol-Hee The Plant Resources Society of Korea 2006 한국자원식물학회지 Vol.19 No.6

Nitrate reductase deficient (NR) mutant lines were selected indirectly by their resistance to 100mM chlorate in cell cultures of P. parviflora. A total of 585 chlorate resistant lines were confirmed by a second passage on a high concentration of chlorate. Frequency of spontaneous mutation was $9.7{\times}10^{-7}$ in 3 month old suspension-cultured cells, and in non-selective media containing amino acids as sole nitrogen source. The frequency of mutation could be increased up to 11-fold by culture for 12 months. Out of 40 randomly selected calli, 22 were fully deficient in NR. The rest of the clones contained a decreased level of NR activity. Further characterization was carried out in 13 mutant lines which were fully deficient in NR and in 5 mutant lines containing residual (0-7.0%) NR activity, as compared to wild-type cells cultured on the same medium. The $NR^-$ mutants were tentatively classified as defective in the NR apoenzyme (nia-type; 11 mutant lines including the 5 with residual NR activity) or in the molybdenum cofactor (cnx-type; 7 mutant lines) by the XDH activity. The cnx-type could be further classified into two groups. In one group (5 mutant lines) of these, the NR activity could be partially restored by nonphysiologically high (1.0mM) molybdate in the culture medium. Both types of $NR^-$ mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They also proved to be extremely sensitive to the standard medium ($MSP_1$) containing nitrate and ammonium. Shoot regeneration was obtained only in the $NR^-$ mutants, which contained residual NR activity, but they so far have failed to grow into plants.