KR20230128740A - A marker composition for diagnosing tuberculous pulmonary fibrosis comprising miR-148a and a pharmaceutical composition for preventing or treating tuberculous pulmonary fibrosis comprising a mimic of miR-148a - Google Patents
A marker composition for diagnosing tuberculous pulmonary fibrosis comprising miR-148a and a pharmaceutical composition for preventing or treating tuberculous pulmonary fibrosis comprising a mimic of miR-148a Download PDFInfo
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Abstract
서론: 결핵균 침투에 대한 반응으로 일어나는 흉막 중피세포에 의한 세포외 기질 생산은 결핵성 섬유증에 기여한다. NOX4는 결핵성 섬유증의 발병기전에 관여한다. 본 발명에서는 NOX4 유전자를 표적으로 하는 microRNA가 결핵성 흉막 섬유증에 대한 보호 효과를 나타내는지 여부를 평가했다.
방법: TargetScan 예측 소프트웨어를 사용하여 NOX4 유전자의 3' UTRs에 결합하는 후보 microRNA를 식별하고 microRNA-148a(miR-148a)를 가장 가능성 있는 후보 miRNA로 선택했다. MiR-148a와 NOX4 사이의 인과 관계를 조사하기 위해 Met5A 세포에서 억제 및 강제 발현 분석을 수행했다. 결핵성 흉막 섬유증에서 miR-148a의 역할에 대한 연구는 BCG 흉막 감염 마우스 모델을 사용하여 수행하였다.
결과: 열처리 사멸 결핵균 (HKMT)은 NOX4 및 POLDIP2 발현을 유도한다. 본 발명은 NOX4 및 POLDIP2 발현에 대한 miR-148a의 억제 효과를 보여준다. miR-148a의 증가된 발현은 PMC 세포에서 HKMT 유도 콜라겐-1 합성을 억제했다. BCG 흉막염 모델에서 miR-148a는 섬유화 및 상피 중간엽 전이를 유의하게 감소시킨다.
결론: 본 발명은 miR-148a가 NOX4와 POLDIP2를 조절함으로써 결핵성 흉막 섬유증을 예방할 수 있음을 제시한다.Introduction: Extracellular matrix production by pleural mesothelial cells in response to M. tuberculosis infiltration contributes to tuberculous fibrosis. NOX4 is involved in the pathogenesis of tuberculous fibrosis. In the present invention, we evaluated whether a microRNA targeting the NOX4 gene had a protective effect against tuberculous pleural fibrosis.
Methods: TargetScan prediction software was used to identify candidate microRNAs that bind to the 3' UTRs of the NOX4 gene, and microRNA-148a (miR-148a) was selected as the most likely candidate miRNA. To investigate the causal relationship between MiR-148a and NOX4, suppression and forced expression assays were performed in Met5A cells. A study on the role of miR-148a in tuberculous pleural fibrosis was performed using a BCG pleural infection mouse model.
Results: Heat-killed Mycobacterium tuberculosis (HKMT) induces NOX4 and POLDIP2 expression. The present invention shows the inhibitory effect of miR-148a on NOX4 and POLDIP2 expression. Increased expression of miR-148a inhibited HKMT-induced collagen-1 synthesis in PMC cells. In the BCG pleurisy model, miR-148a significantly reduces fibrosis and epithelial-mesenchymal transition.
CONCLUSIONS: The present invention suggests that miR-148a can prevent tuberculous pleural fibrosis by regulating NOX4 and POLDIP2.
Description
본 발명은 miR-148a를 포함하는 결핵성 흉막 섬유증 진단용 마커 조성물 및 miRNA 모방체를 포함하는 결핵성 흉막 섬유증 예방 또는 치료용 약학적 조성물에 관한 것으로서, 더욱 자세하게는 결핵성 흉막 섬유증 모델에서 miR-148a의 발현이 증가하는 것을 이용하여 결핵성 흉막 섬유증을 진단할 수 있는 마커 조성물, 및 miR-148a의 발현을 증가시킴으로써 결핵성 흉막 섬유증을 예방 또는 치료할 수 있는 약학적 조성물에 관한 것이다.The present invention relates to a marker composition for diagnosing tuberculous pleural fibrosis containing miR-148a and a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis containing miRNA mimics, and more particularly, in a tuberculous pleural fibrosis model, the expression of miR-148a is The present invention relates to a marker composition capable of diagnosing tuberculous pleural fibrosis using an increase in miR-148a expression, and a pharmaceutical composition capable of preventing or treating tuberculous pleural fibrosis by increasing the expression of miR-148a.
결핵 (Tuberculosis; TB)은 전 세계적으로 990만 건의 발병 사례를 유발하는 공중 보건 문제이다. 결핵 사망률은 감소하고 있으며 치료 사례의 85% (6600만 건)가 치료 성공으로 생존한다 [1]. 그러나 결핵 생존자의 최대 절반은 미생물학적 치료에도 불구하고 만성 폐기능 장애와 삶의 질 저하를 호소한다 [2-4]. 이것은 결핵성 섬유증에서 세포외 기질 (extracellular matrix; ECM)의 과도한 침착, 경직 및 실질 흉터와 관련이 있다 [5, 6]. 폐 기능 장애를 초래하는 폐 구조의 잔류 왜곡은 ECM에서 분해되는 정도에 달려 있다 [7]. 결핵성 섬유증과 관련된 근본적인 면역 기전을 조사하면 치료 목표를 찾는 데 도움이 될 수 있다.Tuberculosis (TB) is a public health problem causing 9.9 million cases worldwide. Tuberculosis mortality is declining and 85% (66 million cases) of treated cases survive with successful treatment [1]. However, up to half of tuberculosis survivors complain of chronic lung dysfunction and reduced quality of life despite microbiological treatment [2-4]. It is associated with excessive deposition of the extracellular matrix (ECM), stiffness, and parenchymal scarring in tuberculous fibrosis [5, 6]. Residual distortions in lung structure that lead to lung dysfunction depend on the degree of degradation in the ECM [7]. Investigating the underlying immune mechanisms involved in tuberculous fibrosis may help find therapeutic targets.
NADPH 산화효소 4 (NOX4)는 활성산소종 (ROS)의 주요 공급원이며 특발성 폐 섬유증, 블레오마이신 유발 폐 손상 및 폐암을 포함한 여러 폐 질환에서 상향 조절된다 [8-12]. 우리는 최근 NOX4 신호가 ERK-ROS 신호 및 EMT 경로를 조절하여 결핵성 섬유증에 기여한다고 보고했다 [13]. NOX4 발현을 길항하면 결핵성 흉막염에서 손상된 보호성 자가포식이 회복된다.NADPH oxidase 4 (NOX4) is a major source of reactive oxygen species (ROS) and is upregulated in several lung diseases including idiopathic pulmonary fibrosis, bleomycin-induced lung injury and lung cancer [8-12]. We recently reported that NOX4 signaling contributes to tuberculous fibrosis by regulating ERK-ROS signaling and EMT pathways [13]. Antagonizing NOX4 expression restores impaired protective autophagy in tuberculous pleurisy.
MicroRNA는 약 19-24개의 뉴클레오티드로 구성된 짧은 단일 가닥 비암호화 RNA 분자이다 [14]. miRNA는 주로 표적 mRNA의 공통 서열에 결합하여 표적 유전자 발현을 하향 조절하고 전사 인자 보조 작용에 의해 표적 유전자를 비활성화할 수 있다 [14-16]. miRNA의 잠재력은 암, 신경 퇴행성 질환, 심혈관 질환 및 감염병과 같은 다양한 질병의 치료 대안으로 많은 주목을 받았다 [17]. 일부 miRNA가 결핵의 발병기전에 관여하는 것으로 보고되었지만 결핵 섬유증에서 miRNA의 역할을 탐구한 연구는 거의 없다 [18].MicroRNAs are short single-stranded noncoding RNA molecules consisting of about 19–24 nucleotides [14]. miRNAs can down-regulate target gene expression by primarily binding to consensus sequences of target mRNAs and inactivate target genes by transcription factor co-action [14-16]. The potential of miRNAs has attracted much attention as an alternative treatment for various diseases such as cancer, neurodegenerative diseases, cardiovascular diseases and infectious diseases [17]. Although some miRNAs have been reported to be involved in the pathogenesis of tuberculosis, few studies have explored the role of miRNAs in tuberculosis fibrosis [18].
본 발명의 목적은 miR-148a를 포함하는 결핵성 흉막 섬유증 진단용 마커 조성물을 제공하는 것이다.An object of the present invention is to provide a diagnostic marker composition for tuberculous pleural fibrosis containing miR-148a.
본 발명의 다른 목적은 miR-148a의 발현량을 측정하는 제제를 포함하는 결핵성 흉막 섬유증 진단용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for diagnosing tuberculous pleural fibrosis comprising an agent for measuring the expression level of miR-148a.
본 발명의 또 다른 목적은 miR-148a의 발현량을 측정하는 제제를 포함하는 결핵성 흉막 섬유증 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for diagnosing tuberculous pleural fibrosis comprising an agent for measuring the expression level of miR-148a.
본 발명의 또 다른 목적은 피검체 유래의 생물학적 시료로부터 miR-148a의 발현량을 측정하는 측정 단계를 포함하는 결핵성 흉막 섬유증의 진단을 위한 정보제공방법을 제공하는 것이다.Another object of the present invention is to provide an information providing method for diagnosing tuberculous pleural fibrosis, which includes a measurement step of measuring the expression level of miR-148a from a biological sample derived from a subject.
본 발명의 또 다른 목적은 miR-148a을 코딩하는 유전자의 발현제 또는 miR-148a의 활성제를 포함하는 결핵성 흉막 섬유증 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis, comprising a miR-148a-encoding gene expression agent or a miR-148a activator.
본 발명에서 우리는 결핵성 섬유증에서 NOX4를 조절하고 새로운 치료 표적을 식별하는 잠재적인 miRNA를 찾고자 했다.In the present invention, we sought to find potential miRNAs that regulate NOX4 and identify new therapeutic targets in tuberculous fibrosis.
우리는 miR-148a의 과발현이 결핵성 섬유증에서 NOX4와 POLDIP2를 표적으로 하여 섬유화를 감소시켰다고 보고한다. 흥미롭게도 miR-148a와 NOX4는 상호 부정적인 피드백 루프로 구성된다. miR-148a는 NOX4의 전사 인자인 NF-kB의 발현을 감소시켰고, NOX4/POLDIP2는 miR-148a의 전사를 직접적으로 감소시켰다. 또한, 우리는 결핵성 흉막 삼출이 miR-148a의 발현 증가를 나타내므로 진단 마커로서의 잠재적 역할을 뒷받침한다는 것을 보여주었다.We report that overexpression of miR-148a reduced fibrosis by targeting NOX4 and POLDIP2 in tuberculous fibrosis. Interestingly, miR-148a and NOX4 constitute a mutually negative feedback loop. miR-148a reduced the expression of NF-kB, a transcription factor of NOX4, and NOX4/POLDIP2 directly decreased the transcription of miR-148a. Furthermore, we showed that tuberculous pleural effusions showed increased expression of miR-148a, supporting its potential role as a diagnostic marker.
본 발명에서 miR-148a는 결핵성 섬유증과 관련된 새로운 miRNA로 확인되었다.In the present invention, miR-148a was identified as a new miRNA related to tuberculous fibrosis.
이전 연구에 따르면, 우리의 데이터는 NOX4 신호전달이 결핵성 섬유증에 기여하고 POLDIP2와 상호작용이 있음을 보여준다 [13,22]. 본 발명에서 우리는 생물정보학 분석을 사용하여 NOX4가 miR-148a의 잠재적인 표적 유전자임을 확인했다. 우리는 HKMT가 초기에 miR-148a의 발현을 하향 조절하고 장기간 노출 후에 MiR-148a의 발현을 상향 조절했음을 밝혔다. 기능적으로 miR-148a 모방체는 NOX4, POLDIP2 및 콜라겐 1A mRNA의 HKMT 유도 상향 조절을 되돌린다. 마찬가지로, BCG 흉막염의 인 비보 모델에서 miR-148a 모방체는 NOX4를 억제함으로써 친염증성 사이토카인의 생성과 ECM의 침착을 감소시킨다. 이러한 보호 역할은 miR-148a의 발현이 상향 조절된 마우스에서 확인되었다. 우리는 miR-148a가 결핵 감염 후 섬유증을 효과적으로 억제할 수 있다고 추측할 수 있다. 또한, 이 결과는 결핵성 흉수에서 miR-148a 상향 조절이 보호 메커니즘 또는 항상성 반응임을 나타낸다. 임상적으로, 흉수 miR-148a는 결핵성 섬유증의 식별을 위한 잠재적으로 유용한 바이오마커일 수 있다. 최근 연구에 따르면 체액에서 순환하는 miRNA가 다양한 질병의 조기 진단 및 치료 반응 모니터링에 기여할 수 있다는 가능성이 제기되었다 [23, 24]. 또한, miRNA는 단백질 바이오마커에 비해 더 안정적이고 높은 민감도와 특이성을 가지고 있다 [25].According to previous studies, our data show that NOX4 signaling contributes to tuberculous fibrosis and interacts with POLDIP2 [13,22]. In the present invention, we identified NOX4 as a potential target gene for miR-148a using bioinformatics analysis. We revealed that HKMT initially downregulated the expression of miR-148a and upregulated the expression of MiR-148a after prolonged exposure. Functionally, miR-148a mimics reverse HKMT-induced upregulation of NOX4, POLDIP2 and collagen 1A mRNAs. Similarly, in an in vivo model of BCG pleurisy, miR-148a mimics reduce the production of pro-inflammatory cytokines and ECM deposition by inhibiting NOX4. This protective role was confirmed in mice with upregulated expression of miR-148a. We can speculate that miR-148a can effectively inhibit fibrosis after tuberculosis infection. Furthermore, these results indicate that miR-148a upregulation in tuberculous pleural effusion is a protective mechanism or homeostatic response. Clinically, pleural miR-148a may be a potentially useful biomarker for the identification of tuberculous fibrosis. Recent studies have raised the possibility that miRNAs circulating in bodily fluids may contribute to early diagnosis and treatment response monitoring of various diseases [23, 24]. In addition, miRNAs are more stable and have higher sensitivity and specificity than protein biomarkers [25].
일부 miRNA는 만성 섬유성 폐 질환의 ECM 복구 또는 TB의 임상 결과와 관련이 있는 것으로 제안되었다 [18,26]. 폐 섬유성 질환과 miR-148a의 관련성에 대해서는 알려진 바가 거의 없다. miR-148a가 폐암과 간 섬유화를 억제한다는 보고가 있다 [27,28]. MiR-148a는 Wnt1 신호 전달을 표적으로 하여 비소세포 폐암 세포의 이동 및 침습을 억제했다 [29]. miR-148a의 과발현은 간 성상 세포 활성화 및 간 섬유증에 필요한 ERBB3을 하향 조절한다 [28].Some miRNAs have been suggested to be associated with ECM repair in chronic fibrotic lung disease or the clinical outcome of TB [18,26]. Little is known about the association of miR-148a with pulmonary fibrotic disease. It has been reported that miR-148a inhibits lung cancer and liver fibrosis [27,28]. MiR-148a inhibited the migration and invasion of NSCLC cells by targeting Wnt1 signaling [29]. Overexpression of miR-148a downregulates ERBB3, which is required for hepatic stellate cell activation and liver fibrosis [28].
우리는 miR-148a가 NOX4에 직접적으로 결합하는 것을 입증하지 않았지만, 우리의 데이터는 miR-148a가 NOX4에 간접적으로 조절함을 시사한다. NF-kB, SMAD2/3, E2F, HIF1α 및 Nrf2와 같은 여러 전사 인자가 NOX4 프로모터 활성을 조절하는 것으로 보고되었다 [31]. NF-kB는 인간 식세포와 혈관 평활근 세포에서 NADPH 산화효소를 자극한다 [32,33]. Lu 등은 저산소증이 NOX4 프로모터에 대한 NF-kB 소단위, P65의 결합을 증가시켰고 PPARγ가 음성 조절자로서 그 결합을 저하시켰다고 보고했다 [34]. 본 발명에서 miR-148a 모방체는 NOX4의 전사 인자인 NF-kB의 발현을 감소시켜 NOX4 수준을 하향 조절하였다.Although we did not demonstrate direct binding of miR-148a to NOX4, our data suggest that miR-148a indirectly regulates NOX4. Several transcription factors such as NF-kB, SMAD2/3, E2F, HIF1α and Nrf2 have been reported to regulate NOX4 promoter activity [31]. NF-kB stimulates NADPH oxidase in human phagocytes and vascular smooth muscle cells [32,33]. Lu et al reported that hypoxia increased the binding of the NF-kB subunit, P65, to the NOX4 promoter and decreased its binding with PPARγ as a negative regulator [34]. In the present invention, miR-148a mimics down-regulated NOX4 levels by reducing the expression of NF-kB, a transcription factor for NOX4.
또한, 본 발명에서 ChIP 분석은 NOX4/POLDIP2가 miR-148a 유전자의 프로모터에 있는 특정 부위에 결합하고 HKMT 처리에 의해 그 결합이 강화되었음을 보여주었다. 이뿐만 아니라, 우리의 데이터는 마이크로RNA/표적 단백질 네트워크로서 NOX4와 miR-148a 사이의 자동 조절 피드백의 존재를 뒷받침한다. 결핵 섬유증은 miR-148a를 활성화하여 NOX4를 억제하고, 반대로 NOX4는 miR-148a를 억제하여 자기 제어적인 보호 메커니즘을 제공한다 (도 7).In addition, the ChIP analysis in the present invention showed that NOX4/POLDIP2 binds to a specific site in the miR-148a promoter and that the binding is enhanced by HKMT treatment. In addition to this, our data support the existence of an autoregulatory feedback between NOX4 and miR-148a as a microRNA/target protein network. Tuberculous fibrosis activates miR-148a to inhibit NOX4, and conversely, NOX4 inhibits miR-148a to provide an autoregulatory protective mechanism (Fig. 7).
결론적으로, 본 발명은 miR-148a가 전사 인자 NF-kB를 하향 조절함으로써 NOX4의 억제를 통해 결핵성 섬유증을 약화시킬 수 있음을 보여준다. 특히, NOX4는 miR-148a를 하향 조절하여 음의 피드백 루프를 형성한다. 이는 miR-148a가 결핵성 섬유증의 치료 표적이 될 수 있음을 제시한다.In conclusion, the present invention shows that miR-148a can attenuate tuberculous fibrosis through inhibition of NOX4 by downregulating the transcription factor NF-kB. In particular, NOX4 downregulates miR-148a, forming a negative feedback loop. This suggests that miR-148a could be a therapeutic target for tuberculous fibrosis.
본 발명은 miR-148a 또는 miR-148a 모방체를 포함하는 결핵성 흉막 섬유증 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis containing miR-148a or a miR-148a mimic.
또한, 본 발명은 상기 miR-148a 모방체의 일 실시예로서, 5'-AAAGUUCUGAGACACUCCGACU-3'과 같은 서열을 갖는 것인, 결핵성 흉막 섬유증 예방 또는 치료용 약학 조성물에 관한 것이다. miR-148a 모방체는 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 용이하게 제조할 수 있으며, 가능한 miRNA-148a 모방체의 수는 제한이 없으며, 위 miRNA-148a 모방체 서열은 예시적인 기재일 뿐이다. 이는 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 자명함을 밝혀둔다.In addition, the present invention relates to a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis, which has a sequence such as 5'-AAAGUUCUGAGACACUCCGACU-3', as an embodiment of the miR-148a mimic. miR-148a mimics can be easily prepared by those skilled in the art, the number of possible miRNA-148a mimics is not limited, and the above miRNA-148a mimics sequences are exemplary. It's just a description. It is made clear to those skilled in the art to which the present invention belongs.
또한, 본 발명은 상기 약학 조성물이 다른 결핵성 흉막 섬유증 예방 또는 치료용 약학 조성물과 조합한 것을 특징으로 하는, 결핵성 흉막 섬유증 예방 또는 치료용 약학 조성물에 관한 것이다.In addition, the present invention relates to a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis, characterized in that the pharmaceutical composition is combined with another pharmaceutical composition for preventing or treating tuberculous pleural fibrosis.
또한, 본 발명은 miR-148a를 포함하는 결핵성 흉막 섬유증 진단용 마커 조성물에 관한 것이다.In addition, the present invention relates to a marker composition for diagnosis of tuberculous pleural fibrosis containing miR-148a.
또한, 본 발명은 miR-148a의 발현량을 측정하는 제제를 포함하는 결핵성 흉막 섬유증 진단용 조성물에 관한 것이다.In addition, the present invention relates to a composition for diagnosing tuberculous pleural fibrosis comprising an agent for measuring the expression level of miR-148a.
또한, 본 발명은 miR-148a의 발현량을 측정하는 제제가 상기 miRNA에 상보적으로 결합하는 센스 및 안티센스 프라이머, 또는 프로브인 것인, 결핵성 흉막 섬유증 진단용 조성물에 관한 것이다.In addition, the present invention relates to a composition for diagnosing tuberculous pleural fibrosis, wherein the agent for measuring the expression level of miR-148a is a sense and antisense primer or probe that complementarily binds to the miRNA.
또한, 본 발명은 miR-148a의 발현량을 측정하는 제제를 포함하는 결핵성 흉막 섬유증 진단용 키트에 관한 것이다.In addition, the present invention relates to a kit for diagnosing tuberculous pleural fibrosis comprising an agent for measuring the expression level of miR-148a.
또한, 본 발명은 피검체 유래의 생물학적 시료로부터 miR-148a의 발현량을 측정하는 측정 단계를 포함하는 결핵성 흉막 섬유증 진단을 위한 정보제공방법에 관한 것이다.In addition, the present invention relates to a method for providing information for diagnosing tuberculous pleural fibrosis, comprising a measuring step of measuring the expression level of miR-148a from a biological sample derived from a subject.
또한, 본 발명은 miR-148a의 발현량이 중합효소연쇄반응(PCR), 차세대 염기서열 분석(Next generation sequencing; NGS), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR) 및 마이크로어레이(microarray)로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것인, 결핵성 흉막 섬유증 진단을 위한 정보제공방법에 관한 것이다.In addition, the present invention is a polymerase chain reaction (PCR), next generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real- It relates to an information providing method for diagnosing tuberculous pleural fibrosis, which is measured through one or more methods selected from the group consisting of time PCR) and microarray.
또한, 본 발명에서 생물학적 시료는 조직 및 세포로 이루어진 군으로부터 선택되는 1종 이상인 것인, 결핵성 흉막 섬유증 진단을 위한 정보제공방법에 관한 것이다.In addition, the present invention relates to a method for providing information for diagnosing tuberculous pleural fibrosis, wherein the biological sample is at least one selected from the group consisting of tissues and cells.
또한, 본 발명의 정보제공방법은 측정 단계에서 상기 피검체 시료에서의 miR-148a의 발현량이 정상개체 시료에서의 miR-148a의 발현량보다 증가한 경우 결핵성 흉막 섬유증으로 판단하는 것인, 결핵성 흉막 섬유증 진단을 위한 정보제공방법에 관한 것이다.In addition, in the information providing method of the present invention, tuberculous pleural fibrosis is judged as tuberculous pleural fibrosis when the expression level of miR-148a in the subject sample is higher than the expression level of miR-148a in the normal subject sample in the measurement step. It relates to a method of providing information for diagnosis.
본 발명의 miR-148a 또는 miR-148a 모방체를 유효성분으로 함유하는 결핵성 흉막 섬유증 예방 또는 치료용 조성물은 조성물 총 중량에 대하여 상기 유효성분을 0.0001 내지 50 중량%로 포함한다.The composition for preventing or treating tuberculous pleural fibrosis containing miR-148a or a miR-148a mimic as an active ingredient of the present invention includes 0.0001 to 50% by weight of the active ingredient with respect to the total weight of the composition.
본 발명의 조성물은 상기 miR-148a 또는 miR-148a 모방체에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 본 발명의 조성물은 임상 투여시 경구 또는 비경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있다.The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the miR-148a or the miR-148a mimic. The composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.
본 발명의 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 더 나아가 당해 기술분야의 적정한 방법으로 또는 레밍턴의 문헌 (Remington's Pharmaceutical Science (최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. A pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets, and may act specifically on target organs. A target organ-specific antibody or other ligand may be combined with the carrier so as to be used. Furthermore, it can be preferably formulated according to each component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA).
본 발명의 조성물은 특히 등장 멸균 용액 또는 멸균수 또는 적절한 생리 식염수의 첨가에 따라 주사 가능한 용액의 조성을 위해 동결건조 조성물을 포함할 수 있다. 사용되는 유효성분의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하며, 일일 투여량은 약 0.0001 내지 100 mg/kg이고, 바람직하게는 0.001 내지 10 mg/kg이며, 하루 일 회 내지 수 회 나누어 투여될 수 있다. The compositions of the present invention may include lyophilized compositions, particularly for the formulation of isotonic sterile solutions or injectable solutions upon addition of sterile water or appropriate physiological saline. The dose of the active ingredient used varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease, etc., and the daily dose is about 0.0001 to 100 mg. /kg, preferably 0.001 to 10 mg/kg, and may be divided and administered once or several times a day.
본 발명의 조성물은 단독으로, 또는 방사선 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법 등과 병용하여 사용할 수 있다.The composition of the present invention may be used alone or in combination with radiation therapy, chemotherapy, and methods using biological response modifiers.
본 발명은 miR-148a를 포함하는 결핵성 흉막 섬유화 진단용 마커 조성물 및 miR-148a 또는 miR-148a 모방체 (mimic)를 포함하는 결핵성 흉막 섬유화 예방 또는 치료용 약학적 조성물에 관한 것으로서, 결핵성 흉막 섬유화 유도 모델에서 miR-148a가 유의적으로 증가하는 점을 이용하여 결핵성 흉막 섬유화를 진단할 수 있고, miR-148a 또는 miR-148a 모방체를 이용하여 결핵성 흉막 섬유화를 예방 또는 치료할 수 있으므로, 이를 의학적 용도로 이용할 수 있다.The present invention relates to a marker composition for diagnosing tuberculous pleural fibrosis containing miR-148a and a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis containing miR-148a or a miR-148a mimic, which is a model for inducing tuberculous pleural fibrosis. Tuberculosis pleural fibrosis can be diagnosed using the fact that miR-148a is significantly increased in the can
도 1a ~ 도 1d. NOX4 및 POLDIP2는 상호 관계로 HKMT (열 사멸 결핵균, Heat-killed Mycobacterium tuberculosis) 유도 콜라겐-1A 합성을 매개한다.
도 1a: HKMT 처리 후 시간 경과에 따른 Met5A 세포에서의 NOX4, POLDIP2 및 콜라겐-1A mRNA 발현,
도 1b: NOX4를 표적으로 하는 Si RNA (SiNOX4)와 SiCtrl 존재 하에서 HKMT 처리한 Met5A 세포에서의 NOX4, POLDIP2 및 콜라겐-1A mRNA 발현,
도 1c: POLDIP2를 표적으로 하는 SiRNA (SiPOLDIP2) 및 SiCtrl 존재 하에서 HKMT 처리한 Met5A 세포에서 NOX4, POLDIP2 및 콜라겐-1A mRNA 발현,
도 1d: 웨스턴 블롯 및 면역침전법에 의해 확인된 HKMT 처리 후 Met5A 세포에서 NOX4와 POLDIP2 간의 상호 작용.
도 2a~도 2c는 HKMT 처리에 따른 중피 세포에서 miRNA 발현을 나타낸다.
도 2a: 야생형 마우스 및 NOX4 KO 마우스의 mPMC (Primary mouse pleural mesothelial cells)에서의 miRNA 발현 (n=3),
도 2b: HKMT 자극 유무에 따른 야생형 마우스 및 NOX4 KO 마우스의 mPMC에서의 miRNA 발현,
도 2c: HKMT 자극 유무에 따른 SiCtrl 또는 SiNOX4로 형질감염된 인간 Met5A 세포에서의 miRNA 발현.
도 3(a) ~ 도 3(e)는 miR-148a가 HKMT 자극의 초기 단계에서 감소했지만 노출 시간이 증가함에 따라 증가함을 보여준다.
도 3a: 표시된 시간 동안 HKMT (10 ng/mL)를 가하여 사전 배양된 Met5A 세포에서의 NOX4, POLDIP2 mRNA 발현 (n=3),
도 3b: 표시된 시간 동안 HKMT (10 ng/mL)를 가하여 사전 배양된 Met5A 세포에서 miRNA 발현을 qRT-PCR로 분석하였다 (n=3). 결과는 평균 ± 표준편차로 나타내었다.
도 3c: 표시된 시간 동안 HKMT (10 ng/mL)를 가하여 사전 배양된 WT-mPMC에서 NOX4, POLDIP2 mRNA의 발현 (n=3).
도 3d: 표시된 시간 동안 HKMT (10 ng/mL)를 가하여 사전 배양된 WT-mPMC에서 miRNA 발현을 qRT-PCR로 분석하였다 (n=3). 결과는 평균 ± SEM으로 나타내었다.
도 3e: 결핵성 흉막삼출액 및 여출액에서 순환하는 miRNA 수준의 발현.
도 4a ~ 도 4e는 miR-148a가 NOX4를 조절하여 결핵성 콜라겐-1A 발현을 감소시킬 수 있음을 보여준다.
도 4a: HKMT 처리 유무에 따른 miR-148a 모방체 및 miR-148 억제제로 형질감염된 Met5A의 miR-148a 수준,
도 4b: HKMT 처리 유무에 따른 Ctrl 모방체 및 miR-148a로 형질감염된 Met5A의 NOX4 mRNA, POLDIP2 mRNA 및 콜라겐-1A mRNA,
도 4c: HKMT 처리 유무에 따른 Ctrl 억제제 및 miR-148 억제제로 형질감염된 Met5A의 NOX4 mRNA, POLDIP2 mRNA 및 콜라겐-1A mRNA,
도 4d: HKMT 처리 유무에 따른 miR-148a 모방체로 형질감염된 Met5A에서 NF-kB의 발현,
도 4e: miR-148a 프로모터에서 NF-kB 결합의 ChIP 분석. Met5A 세포를 HKMT로 3시간 동안 처리하거나 처리하지 않고 특정 항-NOX4 항체 및 특정 항-POLDIP2 항체로 면역침전하기 위하여 염색질을 수집하였다. 정량 데이터는 평균 ± 표준편차로 표시된다.
도 5a ~ 도 5e는 miR-148a가 마우스에서 폐 섬유증을 예방함을 보여준다.
도 5a: 실험 설계의 개략도,
도 5b: miR-148a 발현,
도 5c: BCG 처리된 마우스를 miR-148a 모방체로 후처리한 폐 조직에서 상피 중간엽 전이(EMT) 마커, NOX4 및 POLDIP2의 감소를 나타내는 면역블롯 결과,
도 5d: 4개군의 흉막 세포의 총 갯수,
도 5e: Giemsa로 염색된 사이토스핀의 현미경 사진,
도 5f: 혈청에서 평가된 사이토카인 수준(IL-6, TNF-α 및 IFN-γ).
도 6a ~ 도 6d는 miR-148a 후처리가 NOX4 발현을 조절하고 마우스 폐에서 콜라겐-1A 침투를 억제함을 보여준다.
도 6a: H&E 염색,
도 6b: NOX4의 면역 염색,
도 6c: 마우스 폐에서 Masson Trichrome 면역 염색,
도 6d: NOX4 및 메조텔린 (중피 세포에 대한 마커)의 면역형광 염색. 연한 노란색 영역은 NOX4 (녹색)와 메조텔린 (빨간색)의 공동 위치를 나타낸다. DAPI: 4',6-디아미디노-2-페닐인돌 디하이드로클로라이드.
도 7은 miR-148a에 의한 결핵성 섬유증의 조절을 나타낸다. 만성 HKMT 노출은 miR-148a 발현을 유도한다. 처음에 결핵균은 miRNA 148a를 하향 조절하지만 나중에는 miR-148a를 상향 조절한다. 중피 세포에서 miR-148a의 과발현은 NOX4 및 POLDIP2 발현을 차단하여 ECM 침착 및 폐 섬유화를 방지한다. NOX4는 또한 음성 피드백 메커니즘을 제공하는 miR-148a의 발현을 억제할 수 있다.
도 8은 마우스 NOX4 유전자의 3' UTR에 결합하는 잠재적인 miRNA에 대한 예측이다. 표적 스캔은 마우스 NOX4 유전자의 3' UTR과 상호작용할 수 있는 miRNA를 검색하는 데 사용되었다. miR-9-5p, miR-148-3p, miR-196-5p, miR-203-3p 및 miR-192-5p/215-5p가 확인되었다.
도 9는 마우스 폐의 miR-148a 수준이 miR-148a 모방체 (20 nmol/kg)를 정맥 주사한 후 2일째 최고조에 달했음을 보여준다.1a to 1d. NOX4 and POLDIP2 reciprocally mediate HKMT (Heat-killed Mycobacterium tuberculosis) induced collagen-1A synthesis.
Figure 1a: NOX4, POLDIP2 and Collagen-1A mRNA expression in Met5A cells over time after HKMT treatment,
Figure 1b: Expression of NOX4, POLDIP2 and collagen-1A mRNA in Met5A cells treated with HKMT in the presence of NOX4-targeting Si RNA (SiNOX4) and SiCtrl,
Figure 1c: Expression of NOX4, POLDIP2 and collagen-1A mRNA in Met5A cells treated with HKMT in the presence of SiRNA targeting POLDIP2 (SiPOLDIP2) and SiCtrl,
Figure 1d: Interaction between NOX4 and POLDIP2 in Met5A cells after HKMT treatment confirmed by western blot and immunoprecipitation.
2a to 2c show miRNA expression in mesothelial cells following HKMT treatment.
Figure 2a: miRNA expression in mPMC (Primary mouse pleural mesothelial cells) of wild-type mice and NOX4 KO mice (n = 3),
Figure 2b: miRNA expression in mPMC of wild-type mice and NOX4 KO mice with and without HKMT stimulation,
Figure 2c: miRNA expression in human Met5A cells transfected with SiCtrl or SiNOX4 with or without HKMT stimulation.
3(a) to 3(e) show that miR-148a decreased in the initial stage of HKMT stimulation but increased with increasing exposure time.
Figure 3a: NOX4, POLDIP2 mRNA expression in Met5A cells pre-incubated with HKMT (10 ng/mL) for the indicated times (n=3),
Figure 3b: miRNA expression was analyzed by qRT-PCR in Met5A cells pre-incubated with HKMT (10 ng/mL) for the indicated times (n=3). Results are expressed as mean ± standard deviation.
Figure 3c: NOX4, POLDIP2 mRNA expression in WT-mPMC pre-incubated with HKMT (10 ng/mL) for the indicated times (n=3).
Figure 3d: miRNA expression was analyzed by qRT-PCR in WT-mPMC pre-incubated with HKMT (10 ng/mL) for the indicated times (n=3). Results are expressed as mean ± SEM.
Figure 3e: Expression of circulating miRNA levels in tuberculous pleural effusions and filtrate.
4A to 4E show that miR-148a can reduce tuberculous collagen-1A expression by regulating NOX4.
Figure 4a: miR-148a levels of Met5A transfected with miR-148a mimics and miR-148 inhibitors with and without HKMT treatment,
Figure 4b: NOX4 mRNA, POLDIP2 mRNA and collagen-1A mRNA of Met5A transfected with Ctrl mimic and miR-148a with or without HKMT treatment,
Figure 4c: NOX4 mRNA, POLDIP2 mRNA and collagen-1A mRNA of Met5A transfected with Ctrl inhibitor and miR-148 inhibitor with or without HKMT treatment,
Figure 4d: Expression of NF-kB in Met5A transfected with miR-148a mimics with and without HKMT treatment,
Figure 4e: ChIP analysis of NF-kB binding at the miR-148a promoter. Met5A cells were treated with or without HKMT for 3 hours and chromatin was collected for immunoprecipitation with a specific anti-NOX4 antibody and a specific anti-POLDIP2 antibody. Quantitative data are presented as mean±standard deviation.
5A-5E show that miR-148a prevents pulmonary fibrosis in mice.
Figure 5a: schematic diagram of the experimental design;
Figure 5b: miR-148a expression,
Figure 5c: Immunoblot results showing reduction of epithelial-mesenchymal transition (EMT) markers, NOX4 and POLDIP2, in lung tissues post-treated with miR-148a mimics from BCG-treated mice.
Figure 5d: Total number of pleural cells in 4 groups,
Figure 5e: Micrograph of cytospins stained with Giemsa,
5F: Cytokine levels assessed in serum (IL-6, TNF-α and IFN-γ).
6A to 6D show that miR-148a post-treatment modulates NOX4 expression and inhibits collagen-1A infiltration in mouse lungs.
Figure 6a: H&E staining,
Figure 6b: Immunostaining of NOX4,
Figure 6c: Masson's Trichrome immunostaining in mouse lungs.
6D: Immunofluorescent staining of NOX4 and mesothelin (markers for mesothelial cells). The light yellow area represents the co-localization of NOX4 (green) and mesothelin (red). DAPI: 4',6-diamidino-2-phenylindole dihydrochloride.
Figure 7 shows the regulation of tuberculous fibrosis by miR-148a. Chronic HKMT exposure induces miR-148a expression. Mycobacterium tuberculosis initially downregulates miRNA 148a but later upregulates miR-148a. Overexpression of miR-148a in mesothelial cells blocks NOX4 and POLDIP2 expression, preventing ECM deposition and lung fibrosis. NOX4 can also suppress the expression of miR-148a providing a negative feedback mechanism.
8 is a prediction of potential miRNAs that bind to the 3' UTR of the mouse NOX4 gene. A target scan was used to search for miRNAs that could interact with the 3' UTR of the mouse NOX4 gene. miR-9-5p, miR-148-3p, miR-196-5p, miR-203-3p and miR-192-5p/215-5p were identified.
Figure 9 shows that the level of miR-148a in the lungs of mice peaked 2 days after intravenous injection of miR-148a mimic (20 nmol/kg).
아래에서는 구체적인 실시예와 실험예를 들어 본 발명의 구성을 좀 더 자세히 설명한다. 그러나, 본 발명의 범위가 실시예 및 실험예의 기재에만 한정된 것이 아님은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 자명하다.In the following, the configuration of the present invention will be described in more detail with specific examples and experimental examples. However, it is apparent to those skilled in the art that the scope of the present invention is not limited only to the description of Examples and Experimental Examples.
세포주와 동물cell lines and animals
인간 중피 세포주 Met5A는 American Type Culture Collection (ATCC, Manassas, VA, USA)에서 구입했다. C57BL/6 계통의 야생형 NOX4 (NOX4-WT) 마우스 및 녹아웃 NOX4 (NOX4-KO) 마우스는 박 교수 (연세대, 서울)로부터 입수하였다. 모든 동물 실험은 한림대학교 동물 연구 및 사용 위원회 (IACUC)의 승인을 받았다 (NO: Hallym 2020-21).The human mesothelial cell line Met5A was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Wild-type NOX4 (NOX4-WT) mice and knockout NOX4 (NOX4-KO) mice of the C57BL/6 strain were obtained from Professor Park (Yonsei University, Seoul). All animal experiments were approved by the Animal Research and Use Committee (IACUC), Hallym University (NO: Hallym 2020-21).
마우스 흉막 중피 세포 (mPMC)의 분리Isolation of mouse pleural mesothelial cells (mPMC)
NOX4-WT 또는 NOX-KO 마우스 (3~4주령)의 흉부 횡격막 표면, 심장 및 폐에서 원발성 마우스 흉막 중피 세포 (Primary mouse pleural mesothelial cells; mPMC)를 얻었다. 각 조직을 분리하고 DPBS 세척 후 0.25% 트립신-EDTA 용액에서 1시간 동안 배양하였다. 1500 rpm에서 3분간 원심분리한 후, 세포 펠렛을 DMEM (Dulbecco's modified Eagle medium) 배지 (15% FBS, 1% 페니실린/스트렙토마이신)에 현탁하고 5% CO2 인큐베이터에서 하루 동안 배양하였다. 다음날, 접시에 붙은 세포를 DPBS로 세척하고 DMEM (15% FBS, 1% penicillin/streptomycin)으로 2~3일 동안 성장시켰다. 부착되지 않은 세포는 플레이트에 다시 부착하여 2~3일 후에 사용하였다.Primary mouse pleural mesothelial cells (mPMC) were obtained from the thoracic diaphragmatic surface, heart and lung of NOX4-WT or NOX-KO mice (3-4 weeks old). Each tissue was separated and cultured for 1 hour in 0.25% trypsin-EDTA solution after washing with DPBS. After centrifugation at 1500 rpm for 3 minutes, the cell pellet was suspended in Dulbecco's modified Eagle medium (DMEM) medium (15% FBS, 1% penicillin/streptomycin) and cultured in a 5% CO 2 incubator for one day. The next day, cells attached to the dish were washed with DPBS and grown in DMEM (15% FBS, 1% penicillin/streptomycin) for 2-3 days. Non-adherent cells were reattached to the plate and used after 2-3 days.
열 사멸 결핵균 처리heat killed tuberculosis treatment
열 사멸 결핵균 (Heat-killed Mycobacterium tuberculosis; HKMT)(InvivoGen, San Diego, CA, USA)을 사용하여 Met5A 및 mPMC에서 결핵을 연구했다. 60-70% 성장한 세포를 표시된 기간 동안 10ng/mL HKMT로 처리하여 형질감염 시키거나 시키지 않았다. 내인성 유전자 발현을 억제하기 위해 siNOX4 (센스 5' CUGUUGUGGACCCAAUUCA 3' 및 안티센스 5' UGAAUUGGGUCCACAACAG 3'), siPoldip2 (센스 5' CUCUUGUUCACUUUACCUU 3' 및 안티센스 5' AAGGUAAAGUGAACAAG 3')를 실험에 사용했다. 스크램블된 siRNA (센스: 5'-GGTCAAGACACTATTAACA-3' 및 안티센스: 5'-GGATTCCTAG TGT ATTTCA-3')를 대조군 (SiCtrl)으로 사용했다. 시험관 내 결핵에 대한 miR-148a의 효과를 확인하기 위해, 200 pmol의 miR-148a 모방체 (5' AAAGUUCUGAGACACUCCGACU 3') 또는 miR-148a 억제제 (5, UCAGUGCACUACAGAACUUUGU 3'), 대조군 모방체 및 대조군 억제제를 이용하여 jet PRIME 형질감염 시약 (PolyPlus Transfection, New York, NY)으로 형질감염시켰다.Heat-killed Mycobacterium tuberculosis (HKMT) (InvivoGen, San Diego, CA, USA) was used to study tuberculosis in Met5A and mPMC. Cells with 60-70% growth were transfected or not treated with 10 ng/mL HKMT for the indicated time period. siNOX4 (sense 5' CUGUUGUGGACCCAAUUCA 3' and antisense 5' UGAAUUGGGUCCACAACAG 3') and siPoldip2 (sense 5' CUCUUGUUCACUUUACCUU 3' and antisense 5' AAGGUAAAGUGAACAAG 3') were used to suppress endogenous gene expression. Scrambled siRNA (sense: 5'-GGTCAAGACACTATTAACA-3' and antisense: 5'-GGATTCCTAG TGT ATTTCA-3') was used as a control (SiCtrl). To determine the effect of miR-148a on tuberculosis in vitro, 200 pmol of miR-148a mimic (5' AAAGUUCUGAGACACUCCGACU 3') or miR-148a inhibitor (5, UCAGUGCACUACAGAACUUUGU 3'), control mimic and control inhibitor were used. were transfected with jet PRIME transfection reagent (PolyPlus Transfection, New York, NY).
잠재적인 miRNA의 식별Identification of potential miRNAs
TargetScan 프로그램은 NOX4의 3' 비번역 영역 (3' UTR) 영역에 직접 결합하여 유전자 발현을 조절하는 것으로 예측되는 추정 miRNA를 식별하는 데 사용되었다 (도 8).The TargetScan program was used to identify putative miRNAs predicted to modulate gene expression by directly binding to the 3' untranslated region (3' UTR) region of NOX4 (Fig. 8).
면역블롯 및 면역침전 분석Immunoblot and immunoprecipitation analysis
면역블롯 분석 및 면역침전 분석은 표준 절차에 따라 수행되었다. 간단히 설명하면, 세포는 RIPA 완충액 (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% 소듐 데옥시콜레이트, 0.1% SDS 및 1% Nonidet-P40)에 용해되었다. 면역블롯을 수행하기 위해 단백질 용해물을 5X 시료 완충액과 혼합하고, 8분 동안 담즙화하고, 50 ㎍의 단백질을 10% SDS-폴리아크릴아미드 겔 전기영동(SDS-PAGE)으로 분리하고, PVDF 막 (Thermo Scientific, Waltham, MA, 미국)으로 옮겼다. NOX4에 대한 일차 항체 (1:2000 희석, Proteintech, 중국 우한), POLDIP2에 대한 일차 항체 ZO-1 (1:1,000 희석, Abcam, Cambridge, UK), Snail에 대한 일차 항체 (1:1,000 희석, Cell Signaling Technology, Danvers, MA, USA)에 이어 HRP-접합 이차 항체 (1:1,000 희석, Thermo Scientific, Waltham, MA, USA)와 함께 배양하였다. 막을 벗겨내고 β-액틴에 대한 1차 항체 (1:1,000 희석; Cell Signaling Technology, Danvers, MA, USA)로 재탐색 (reprobe)했다.Immunoblot analysis and immunoprecipitation analysis were performed according to standard procedures. Briefly, cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS and 1% Nonidet-P40). To perform immunoblots, protein lysates were mixed with 5X sample buffer, biliary lysis for 8 min, and 50 μg of protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and plated on a PVDF membrane. (Thermo Scientific, Waltham, MA, USA). Primary antibody to NOX4 (1:2000 dilution, Proteintech, Wuhan, China), primary antibody to POLDIP2 ZO-1 (1:1,000 dilution, Abcam, Cambridge, UK), primary antibody to Snail (1:1,000 dilution, Cell Signaling Technology, Danvers, MA, USA) followed by incubation with HRP-conjugated secondary antibody (1:1,000 dilution, Thermo Scientific, Waltham, MA, USA). The membrane was peeled off and reprobed with a primary antibody against β-actin (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA).
면역침전을 위해 세포 용해물 (500μg)을 각각의 1차 항체 (2μg)로 4℃에서 밤새 면역침전시켰다. 40㎕의 Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Dallas, TX, USA)를 첨가하고 4℃에서 1시간 동안 배양하였다. 용균 완충액으로 비드를 3번 세척한 후, 2X 시료 완충액을 추가했다. 시료를 8분 동안 끓이고 10% SDS-PAGE를 분석한 후 면역 침전을 수행했다.For immunoprecipitation, cell lysates (500 μg) were immunoprecipitated overnight at 4° C. with each primary antibody (2 μg). 40 μl of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Dallas, TX, USA) was added and incubated at 4° C. for 1 hour. After washing the beads 3 times with lysis buffer, 2X sample buffer was added. Samples were boiled for 8 minutes and analyzed by 10% SDS-PAGE followed by immunoprecipitation.
정량적 실시간 PCR (qRT-PCR)Quantitative real-time PCR (qRT-PCR)
easy-Blue 시약 (iNtRON Biotechnology, Seoul, Korea)을 사용하여 Met5A 및 mPMC에서 총 RNA 샘플을 추출했다. 가닥 cDNA는 Maxime RT PreMix Kit (iNtRON Biotechnology, Seoul, Korea)를 사용하여 합성되었다. miRNeasy Mini Kit (Qiagen, Germantown, MD, USA)를 사용하여 동결된 마우스 폐 조직 및 Met5A 또는 mPMC에서 총 miRNA를 추출한 다음 miScript Transcription Kit (Qiagen, Germantown, MD, USA)를 사용하여 역전사를 수행했다. 정량적 실시간 PCR (qRT-PCR)을 위해 Rotor-Gene Q (Qiagen)에 SYBR Green PCR Kit (Qiagen, Germantown, MD, USA)를 사용했다. qRT-PCR에 대한 프라이머 서열은 표 1 및 표 2에 제시되어 있다.Total RNA samples were extracted from Met5A and mPMC using easy-Blue reagent (iNtRON Biotechnology, Seoul, Korea). Stranded cDNA was synthesized using the Maxime RT PreMix Kit (iNtRON Biotechnology, Seoul, Korea). Total miRNA was extracted from frozen mouse lung tissue and Met5A or mPMC using the miRNeasy Mini Kit (Qiagen, Germantown, MD, USA), followed by reverse transcription using the miScript Transcription Kit (Qiagen, Germantown, MD, USA). For quantitative real-time PCR (qRT-PCR), Rotor-Gene Q (Qiagen) with SYBR Green PCR Kit (Qiagen, Germantown, MD, USA) was used. Primer sequences for qRT-PCR are presented in Tables 1 and 2.
ChIP 분석 (Chromatin Immunoprecipitation Assay)ChIP assay (Chromatin Immunoprecipitation Assay)
ChIP 분석은 배양된 Met5A 세포에 대해 제조업체의 지침(Cell Signaling Technology, Danvers, MA, USA)에 따라 수행되었다. 간단히 말해서, 세포를 37℃에서 10분간 1% 포름알데히드로 가교시키고 SDS 용해 완충액(1% SDS, 10 mM EDTA 및 50 mM Tris-HCl, pH 8.1)에서 용해시켰다. 염색질 추출물을 분리하고 DNA를 전단하기 위해 얼음 위에서 초음파 처리하여 추가로 단편화하였다. 핵 추출물을 항-NOX4 항체 또는 항-POLDIP2 항체 또는 토끼 IgG로 4℃에서 오버나잇으로 면역침전시켰다. 정제된 면역침전 DNA/단백질 복합체를 PCR에 적용하였다. miR-148a 프로모터에 대한 단백질의 결합 부위를 확인하기 위해 miR-148의 프로모터 영역에서 약 150~200bp DNA 단편을 증폭하기 위해 3쌍의 특이적 프라이머를 합성하였다. PCR에 사용된 프라이머 서열은 다음과 같다. miR-148 프로모터-1 서열 [5'-CAGCACGAGGAACTTGACCCA-3' (센스) 및 5'-GCAAGGC ACTGCACACACTAAC-3' (안티센스)], miR-148 프로모터-2 서열 [5'- TGGAGGTTT GGGTTGGTGAG -3' (센스) 및 5'- TACCAAGGGCTTCCCAGAGA -3' (안티센스)], miR-148 프로모터-3 서열 [5'- GCAGTGCCTTGCAGGAATTT-3' (센스) 및 5'- ATCTCCACAGCCCAAAAGCA -3' (안티센스)]. IgG는 음성 대조군으로 사용되었다.ChIP assays were performed on cultured Met5A cells according to the manufacturer's instructions (Cell Signaling Technology, Danvers, MA, USA). Briefly, cells were cross-linked with 1% formaldehyde at 37°C for 10 minutes and lysed in SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl, pH 8.1). Chromatin extracts were isolated and further fragmented by sonication on ice to shear DNA. Nuclear extracts were immunoprecipitated overnight at 4°C with anti-NOX4 antibody or anti-POLDIP2 antibody or rabbit IgG. Purified immunoprecipitated DNA/protein complexes were subjected to PCR. To confirm the binding site of the protein to the miR-148a promoter, three pairs of specific primers were synthesized to amplify a DNA fragment of about 150-200bp in the promoter region of miR-148. Primer sequences used for PCR are as follows. miR-148 promoter-1 sequence [5'-CAGCACGAGGAACTTGACCCA-3' (sense) and 5'-GCAAGGC ACTGCACACACTAAC-3' (antisense)], miR-148 promoter-2 sequence [5'- TGGAGGTTT GGGTTGGTGAG -3' (sense) ) and 5′-TACCAAGGGCTTCCCAGAGA-3′ (antisense)], miR-148 promoter-3 sequence [5′-GCAGTGCCTTGCAGGAATTT-3′ (sense) and 5′-ATCTCCACAGCCCAAAAGCA-3′ (antisense)]. IgG was used as a negative control.
ELISA (Enzyme-linked Immunosorbent Assay)ELISA (Enzyme-linked Immunosorbent Assay)
마우스 혈청에서 IL-6, TNF-α, IFN-γ의 발현은 ELISA Kit (Abbkine Wuhan, China)를 이용하여 측정하였다.The expression of IL-6, TNF-α, and IFN-γ in mouse serum was measured using an ELISA Kit (Abbkine Wuhan, China).
BCG-유도 흉막염 확립과 miRNA 모방체의 인 비보 치료Establishment of BCG-induced pleurisy and in vivo treatment of miRNA mimics
miRNA 148a 모방 치료 전에 0일째에 106 CFU BCG Pasteur가 든 100uL 식염수를 흉막강 내로 주입하여 BCG 유도 흉막염 동물 모델을 제조하였다 [13]. 인 비보 결핵 섬유증에 대한 miR-148a의 효과를 확인하기 위해 대조군 모방체 또는 miR-148a 모방체 (20 nmol/kg)와 인 비보-JetPEI (PolyPlus Transfection, New York, NY) 형질감염 시약 100 ul를 총 14일 동안 0일부터 이틀에 한 번씩 정맥 주사했다. 15일째에 모든 마우스를 희생시키고 폐 조직과 혈청을 수집했다. 흉강은 희생 전에 1ml 2 mmol/L EDTA-PBS로 세척하였다. 흉막 삼출액을 원심분리하고 세포 펠렛을 100uL PBS에서 재구성했다. 총 세포수 및 diff-Quick 염색한 cytospin의 계수는 종래 기술과 같이 수행하였다 [13].An animal model for BCG-induced pleurisy was prepared by injecting 100uL saline containing 10 6 CFU BCG Pasteur into the pleural cavity on day 0 before miRNA 148a mimic treatment [13]. To confirm the effect of miR-148a on in vivo tuberculosis fibrosis, 100 ul of the control mimic or miR-148a mimic (20 nmol/kg) and in vivo-JetPEI (PolyPlus Transfection, New York, NY) transfection reagent were used. It was injected intravenously every other day from day 0 for a total of 14 days. On day 15 all mice were sacrificed and lung tissue and serum collected. The thoracic cavity was washed with 1 ml 2 mmol/L EDTA-PBS before sacrifice. Pleural effusions were centrifuged and cell pellets were reconstituted in 100uL PBS. Total cell counts and diff-Quick-stained cytospin were counted as in the prior art [13].
조직학, 면역조직화학 및 면역형광 염색Histology, immunohistochemistry and immunofluorescence staining
폐 조직의 파라핀 절편은 폐 염증 및 섬유증을 판별하기 위하여 H&E로 염색되었다. 절편을 탈파라핀화한 후, 폐 조직은 NOX4 항체 (Cusabio, Wuhan, China)와 콜라겐을 염색하는 Masson Trichrome Stain Kit (IMEB Inc., Chicago, IL, USA)로 면역염색되었다. 면역형광 염색을 위해 절편을 NOX4에 대한 항체 (1:100 희석, Santa Cruz Biotechnology, Santa Cruz, CA, USA) 및 메조텔린에 대한 항체 (1:100 희석, Santa Cruz Biotechnology, Santa Cruz, CA, USA)와 함께 밤새 4℃로 배양했다. PBS로 세척한 후, 슬라이드를 Alexa Fluor 546-컨쥬게이션된 염소 항-마우스 IgG1, Alexa Fluor 488-컨쥬게이션된 염소 항-토끼 IgG 2차 항체 및 DAPI (Sigma, St Loise, MO, USA)와 함께 암실에서 배양했다. 모든 샘플은 형광 현미경 (Olympus FV500; Olympus, Tokyo, Japan)으로 검사하였다.Paraffin sections of lung tissue were stained with H&E to determine lung inflammation and fibrosis. After deparaffinization of sections, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China) and the Masson Trichrome Stain Kit (IMEB Inc., Chicago, IL, USA) to stain collagen. For immunofluorescence staining, sections were prepared with an antibody to NOX4 (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an antibody to mesothelin (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). ) and incubated overnight at 4°C. After washing with PBS, slides were stained with Alexa Fluor 546-conjugated goat anti-mouse IgG1, Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody and DAPI (Sigma, St Loise, MO, USA). cultured in the dark. All samples were examined under a fluorescence microscope (Olympus FV500; Olympus, Tokyo, Japan).
환자 및 샘플 수집Patient and sample collection
이 연구의 환자 샘플은 춘천성심병원 기관조사위원회 (IRB no.2012-27)에서 승인한 연구 프로토콜에 따라 제공받았다. 모든 연구 참가자는 이 연구에 참여하기 위해 서면 동의서를 제공했다. 데이터에는 환자를 확인할 수 있는 어떤 정보도 포함되지 않았다. 결핵성 흉막삼출액 (Tuberculous pleural effusion; TPE)은 두 가지 기준으로 정의되었다: 흉막액 검체에서 양성 배양 결과를 나타내거나 또는 마이코박테리아 조직학적 특징이 있는 흉막 생검 (n=3). 흉수 삼출액은 Light의 기준으로 정의되었다 (n= 3) [21]. 제조사의 지시에 따라 miRNeasy Serum/Plasma Advanced Kit (Qiagen, Germantown, MD, USA)를 사용하여 흉수 (n=6)에서 순환하는 miRNA를 추출하였으며, 연구대상자의 특성은 표 3과 같다.Patient samples in this study were provided according to the research protocol approved by the Institutional Investigation Committee of Chuncheon Sacred Heart Hospital (IRB no.2012-27). All study participants gave written informed consent to participate in this study. The data did not contain any information that could identify the patient. Tuberculous pleural effusion (TPE) was defined by two criteria: a positive culture result on a pleural fluid specimen or a pleural biopsy with mycobacterial histology (n=3). Pleural fluid effusion was defined by Light's criteria (n = 3) [21]. Circulating miRNAs were extracted from the pleural effusion (n = 6) using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions, and the characteristics of the study subjects are shown in Table 3.
통계 분석statistical analysis
데이터는 평균 ± 표준편차로 표시된다. 통계적 비교는 양방향 분산 분석 이후 GraphPad Prism5로 수행되었다. 그룹 간의 차이의 유의성은 Student's unpaired t-test를 사용하여 결정되었다. p 값 < 0.05는 유의한 것으로 간주되었다.Data are presented as mean±standard deviation. Statistical comparisons were performed with GraphPad Prism5 after two-way analysis of variance. Significance of differences between groups was determined using Student's unpaired t-test. A p value < 0.05 was considered significant.
결과 1: HKMT 처리 후 중피 세포의 NOX4 및 POLDIP2 발현은 상향조절된다.Result 1: Expression of NOX4 and POLDIP2 in mesothelial cells is upregulated after HKMT treatment.
우리의 이전 연구는 HKMT (열 사멸 결핵균) 처리가 중피 세포에서 POLDIP2 및 콜라겐과 함께 NOX4의 발현을 향상시키는 것을 보여주었다 (도 1a). HKMT 처리는 상호 의존적으로 NOX4와 POLDIP2의 발현을 증가시켰다. NOX4의 하향 조절은 POLDIP2의 발현을 감소시킨 반면, POLDIP2의 하향 조절은 NOX4의 발현을 감소시켰다 (도 1b, 1c). 면역 침전은 NOX4와 POLDIP2 사이의 직접적인 상호작용을 입증하였다 (도 1d).Our previous study showed that HKMT (heat killed Mycobacterium tuberculosis) treatment enhanced the expression of NOX4 along with POLDIP2 and collagen in mesothelial cells (Fig. 1a). HKMT treatment increased the expression of NOX4 and POLDIP2 in a mutually dependent manner. Downregulation of NOX4 reduced the expression of POLDIP2, whereas downregulation of POLDIP2 reduced the expression of NOX4 (Fig. 1b, 1c). Immunoprecipitation demonstrated a direct interaction between NOX4 and POLDIP2 (Fig. 1d).
결과 2: miR-148은 결핵균 노출 초기에는 과소발현되지만 이후에는 증가한다.Result 2: miR-148 was under-expressed in the early stage of exposure to Mycobacterium tuberculosis, but increased thereafter.
TargetScan 예측 소프트웨어는 NOX4 mRNA의 번역되지 않은 영역(3' UTR)에 있는 부위에 결합하는 것으로 추정되는, 척추동물 사이에 광범위하게 보존된 여러 miRNA를 밝혔다 (도 8). 우리는 3시간 동안 HKMT를 처리하거나 처리하지 않고 야생형 또는 NOX4 KO 마우스에서 얻은 mPMC에서 후보 miRNA의 발현을 평가했다 (도 2a). 후보 miRNA 중 miR-9, miR-148a 및 miR-196은 야생형 (WT) 마우스보다 NOX4 KO 마우스의 mPMC에서 더 높았다. 3시간 HKMT 처리 후 miR-9 및 miR-148a의 발현은 WT 마우스 PMC와 Met 5A 세포 모두에서 하향 조절되었다 (도 2b, 도 2c). NOX4 녹아웃 또는 SiRNA NOX4 처리는 3시간 HKMT 처리에 의해 유도된 miR-9 및 miR-148의 감소를 회복시키지 않았다.TargetScan prediction software revealed several widely conserved miRNAs among vertebrates presumably binding to sites in the untranslated region (3' UTR) of NOX4 mRNA (Fig. 8). We evaluated the expression of candidate miRNAs in mPMCs obtained from wild-type or NOX4 KO mice with or without HKMT treatment for 3 h (Fig. 2a). Among the candidate miRNAs, miR-9, miR-148a and miR-196 were higher in mPMC of NOX4 KO mice than in wild-type (WT) mice. Expressions of miR-9 and miR-148a were downregulated in both WT mouse PMCs and Met 5A cells after 3 h HKMT treatment (Fig. 2b, Fig. 2c). NOX4 knockout or siRNA NOX4 treatment did not restore the reduction of miR-9 and miR-148 induced by 3 h HKMT treatment.
우리는 인간 Met5A 세포와 마우스 PMC에서 HKMT 처리 후 시간에 따른 miRNA의 변화를 평가했다 (도 3a-d). 특히 miR-148a는 시간이 지남에 따라 뚜렷한 경향을 보인다. miR-148a는 HKMT 처리 후 처음에는 감소했으나 장기간 노출되면 증가하는 경향이 있다. NOX4 와 miR-148a의 시간대별 발현은 차이를 보였다. 마우스 PMC 실험결과에 따르면 NOX4는 HKMT 처치 24시간에 증가하고 72시간은 감소하는 경향을 보였으나, 반면 miR-148a는 24시간에 감소하고 72시간에서 증가하는 경향을 보였다. miR-148a의 이러한 변화를 관찰함으로써 본 발명자들은 miR-148a를 결핵성 섬유증에서 NOX4 조절에 민감한 후보로 선정하였다. miR-148a의 결합 부위는 NOX4 mRNA의 3' UTR에서 컴퓨터를 통해 예측되었다 (표 4).We evaluated changes in miRNAs over time after HKMT treatment in human Met5A cells and mouse PMCs (Fig. 3a–d). In particular, miR-148a shows a distinct trend over time. miR-148a initially decreased after HKMT treatment, but tended to increase with prolonged exposure. The time-dependent expression of NOX4 and miR-148a showed a difference. According to the results of mouse PMC experiments, NOX4 increased at 24 hours and decreased at 72 hours of HKMT treatment, whereas miR-148a decreased at 24 hours and increased at 72 hours. By observing these changes in miR-148a, we selected miR-148a as a candidate sensitive to NOX4 regulation in tuberculous fibrosis. The binding site of miR-148a was predicted by computer in the 3' UTR of NOX4 mRNA (Table 4).
결핵성 흉수 (Tuberculous pleural effusion; TB-PE)는 결핵균 노출 후 만성 상태를 나타낸다. 장기간 HKMT에 노출된 Met5A와 일맥상통하게도, 순환하는 miR-148의 발현은 여출액 (transudate)보다 결핵성 흉막삼출액(TB-PE)에서 더 높았다.Tuberculous pleural effusion (TB-PE) represents a chronic condition following exposure to Mycobacterium tuberculosis. Consistent with Met5A exposed to long-term HKMT, circulating miR-148 expression was higher in tuberculous pleural effusion (TB-PE) than in transudate.
결과 3: MiR-148은 Met5A 세포에서 NOX4/POLDIP2의 발현을 조절한다.Result 3: MiR-148 regulates the expression of NOX4/POLDIP2 in Met5A cells.
결핵성 섬유증에서 NOX4와 miR-148a의 상관관계를 검증하기 위해 miR-148a의 합성 모방체 (mimic) 및 억제제를 사용하여 miR-148a의 발현 수준이 NOX4의 발현 수준에 영향을 미치는지 조사했다. Met5A 세포를 miR-148 모방체 또는 miR-148a 억제제로 형질감염시키고 HKMT로 3시간 동안 처리하였다. 도 4b는 miR-148a 수준이 증가하면 NOX4, POLDIP2 및 콜라겐 1A의 HKMT 유도 전사가 현저히 감소한다는 것을 보여준다. 반대로, miR-148 억제제를 사용한 miR-148a의 녹다운은 NOX4, POLDIP2 및 콜라겐 1A의 수준을 현저히 증가시켰다 (도 4c).To verify the correlation between NOX4 and miR-148a in tuberculous fibrosis, we investigated whether the expression level of miR-148a affects the expression level of NOX4 using synthetic miR-148a mimics and inhibitors. Met5A cells were transfected with miR-148 mimics or miR-148a inhibitors and treated with HKMT for 3 hours. Figure 4b shows that increasing miR-148a levels markedly reduced HKMT-induced transcription of NOX4, POLDIP2 and collagen 1A. Conversely, knockdown of miR-148a using a miR-148 inhibitor markedly increased the levels of NOX4, POLDIP2 and collagen 1A (Fig. 4c).
HKMT 처리는 NOX4의 전사 인자로 알려진 NF-kB를 상향 조절하고 miR-148a 모방체는 HKMT 유도 NF-kB를 하향조절하였다 (도 4d). ChIP 분석은 HKMT가 miR-148a 프로모터-1 서열에 대한 NOX4/POLDIP2의 결합을 증가시킴을 입증했다 (도 4e). 이러한 결과는 NOX4가 유전자 전사를 직접 조절함으로써 결핵성 섬유증에서 miR-148a 하향 조절을 매개할 수 있음을 시사한다. 종합하면 NOX4와 miR-148a 사이에는 상호 억제 관계가 있는 것으로 보인다.HKMT treatment up-regulated NF-kB, a known transcription factor of NOX4, and miR-148a mimic down-regulated HKMT-induced NF-kB (Fig. 4d). ChIP assay demonstrated that HKMT increased the binding of NOX4/POLDIP2 to the miR-148a promoter-1 sequence (Fig. 4e). These results suggest that NOX4 can mediate miR-148a downregulation in tuberculous fibrosis by directly regulating gene transcription. Taken together, it appears that there is a reciprocal inhibitory relationship between NOX4 and miR-148a.
결과 4: MiR-148은 실험적 결핵성 섬유증을 예방한다.Result 4: MiR-148 prevents experimental tuberculous fibrosis.
miR-148a의 증가는 대조군 마우스와 비교하여 miRNA 꼬리 정맥 주사 후 2일째에 관찰되었다 (도 9). 결핵성 섬유증의 발병에서 miR-148a의 잠재적 역할을 결정하기 위해, miR-148a 모방체 또는 대조군 모방체 (20nmol/kg)를 BCG 주사 후 2주 동안 이틀에 한 번 투여했다 (도 5a). 2주 후, 폐 조직에서 miR-148a의 발현은 PBS + 대조군에 비해 BCG + 대조군에서 더 높았고, 4개 그룹 중 BCG + miR-148a 모방체 투여군에서 가장 높았다 (도 5b). miR-148a 모방체 투여는 BCG 유도 NOX4, POLDIP2 및 Snail 단백질의 발현을 유의하게 감소시켰다 (도 5c). BCG 유도 흉막염에서 miR-148a 모방체의 효과를 조사하기 위해 세포 보충 (cell recruitment)과 사이토카인 축적을 측정했다. 감염 후 15일째에 흉막 세포의 총 수가 유의하게 증가했다 (PBS + 대조군 : 21.3 x 104 ±12.74 x 104/mL, BCG + 대조군: 786.7 x 104 ±369.5 x 104/mL, p = 0.023) (도 5d). BCG + miR-148a 모방체 투여군 마우스의 총 세포수는 BCG + 대조군보다 낮았다 (BCG + miR-148a 모방체: 232.8 x 104 ±313.0 x 104/mL, p = 0.084). 흉막 세포의 현미경 분석은 PBS + 대조군 및 BCG + miR-148a 모방체 투여군과 비교하여 BCG + 대조군에서 마우스의 다핵 거대 대식세포 (multinucleated giant macrophages)가 증가함을 보여주었다 (도 5e). 유사하게, 혈청에서 측정된 사이토카인 (IL-6, TNF-α 및 IFN-γ)의 농도는 PBS + 대조군보다 BCG + 대조군에서 더 높았고, miR-148a 모방체 후처리는 BCG에 의해 유도된 사이토카인의 발현을 감소시켰다 (도 5f). 이러한 결과는 miR-148a가 NOX4 수준을 조절하고 BCG 흉막염에서 흉막 염증과 섬유증을 실질적으로 조절한다는 것을 시사한다.An increase in miR-148a was observed 2 days after miRNA tail vein injection compared to control mice (FIG. 9). To determine the potential role of miR-148a in the pathogenesis of tuberculous fibrosis, miR-148a mimics or control mimics (20 nmol/kg) were administered once every other day for 2 weeks after BCG injection (Fig. 5a). After 2 weeks, the expression of miR-148a in lung tissue was higher in the BCG + control group than in the PBS + control group, and was highest in the BCG + miR-148a mimic administration group among the four groups (Fig. 5b). Administration of miR-148a mimics significantly reduced the expression of BCG-induced NOX4, POLDIP2 and Snail proteins (Fig. 5c). To investigate the effects of miR-148a mimics in BCG-induced pleurisy, cell recruitment and cytokine accumulation were measured. On day 15 after infection, the total number of pleural cells was significantly increased (PBS + control: 21.3 x 10 4 ±12.74 x 10 4 /mL, BCG + control: 786.7 x 10 4 ±369.5 x 10 4 /mL, p = 0.023 ) (Fig. 5d). The total number of cells in mice treated with BCG + miR-148a mimic was lower than that in BCG + control group (BCG + miR-148a mimic: 232.8 x 10 4 ±313.0 x 10 4 /mL, p = 0.084). Microscopic analysis of pleural cells showed an increase in multinucleated giant macrophages in mice in the BCG + control group compared to the PBS + control group and the BCG + miR-148a mimic administration group (FIG. 5e). Similarly, concentrations of cytokines (IL-6, TNF-α and IFN-γ) measured in serum were higher in the BCG + control group than in the PBS + control group, and posttreatment with miR-148a mimics induced BCG-induced cytokinesis. decreased the expression of Cain (Fig. 5f). These results suggest that miR-148a regulates NOX4 levels and substantially modulates pleural inflammation and fibrosis in BCG pleurisy.
H&E 염색은 miR-148a 발현이 증가된 마우스에서 BCG 유도 섬유증의 개선을 보여주었다. BCG 감염 15일 후, BCG + 대조군의 모든 마우스는 PBS + 대조군보다 흉막 증식증 및 폐 세포 침윤이 증가했다. BCG + miR-148a 모방체 투여군의 마우스는 BCG + 대조군의 마우스에 비해 중피하, 육아종 및 흉막의 증식이 감소했다 (도 6a). NOX4에 대한 마우스의 폐 조직 면역 염색은 상피 기관지 세포에서 약한 발현을 나타내는 PBS + 대조군과 달리 BCG + 대조군에서 두꺼운 격막이 있는 침윤 부위 주변에 현저하게 강한 발현을 나타냈다. BCG + miR-148a 모방체 투여군의 마우스는 폐 조직에서 감소된 NOX4 발현을 보여주었다 (도 6b). BCG + 대조군의 마우스 흉막 절편에도 콜라겐 침착이 있었지만 BCG + miR-148a 모방체 투여군의 마우스는 BCG + 대조군의 마우스에 비해 콜라겐 침착이 적었다 (도 6c). NOX4에 대한 miR-148a의 생체 내 억제 효과는 면역형광 염색으로 확인되었다 (도 6d). 우리는 PBS + 대조군보다 BCG + 대조군에서 중피 세포의 마커인 메조텔린 (mesothelin)과 같은 위치에 있는 NOX4의 수준이 증가했음을 발견했다. miR-148a 모방체 투여는 BCG 유도 메조텔린과 NOX4 발현을 차단했다.H&E staining showed amelioration of BCG-induced fibrosis in mice with increased miR-148a expression. Fifteen days after BCG infection, all mice in the BCG + control group had increased pleural hyperplasia and lung cell infiltration than the PBS + control group. Mice in the BCG + miR-148a mimic-treated group had reduced submesothelial, granuloma, and pleural proliferation compared to BCG + control mice (Fig. 6a). Mouse lung tissue immunostaining for NOX4 showed significantly stronger expression around infiltrates with thick septa in the BCG + control group, in contrast to the PBS + control group, which showed weak expression in epithelial bronchial cells. Mice in the BCG + miR-148a mimic administration group showed reduced NOX4 expression in lung tissue (FIG. 6b). Although the pleural sections of BCG + control mice also showed collagen deposition, mice in the BCG + miR-148a mimic administration group showed less collagen deposition compared to BCG + control mice (Fig. 6c). The in vivo inhibitory effect of miR-148a on NOX4 was confirmed by immunofluorescence staining (Fig. 6d). We found that the level of NOX4 colocalized with mesothelin, a marker of mesothelial cells, was increased in the BCG + control group than in the PBS + control group. Administration of miR-148a mimics blocked BCG-induced mesothelin and NOX4 expression.
<110> Industry Academic Cooporation Foundation, Hallym University <120> A marker composition for diagnosing tuberculous pulmonary fibrosis comprising miR-148a and a pharmaceutical composition for preventing or treating tuberculous pulmonary fibrosis comprising a mimic of miR-148a <130> HallymU-JYHONG-miR148a <160> 38 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 1 cuguugugga cccaauuca 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 2 ugaauugggu ccacaacag 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 3 cucuuguuca cuuuaccuu 19 <210> 4 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 4 aagguaaagu gaacaag 17 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggtcaagaca ctattaaca 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggattcctag tgtatttca 19 <210> 7 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> a miR-148a mimic <400> 7 aaaguucuga gacacuccga cu 22 <210> 8 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> a miR-148a inhibitor <400> 8 ucagugcacu acagaacuuu gu 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ccggctgcat cagtcttaac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tcggcacagt acaggcacaa 20 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 caaaacagaa tggaaaatat gagaccgg 28 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tgattgatgc tcgtgactgc cca 23 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 actggtgaga cctgcgtgta 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aatccatcgg tcatgctctc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gtgctatccc tgtacgcctc 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggccatctct tgctcgaag 19 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 atgacctatg aattgacaga c 21 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tctttggtta tctagctgta tga 23 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 taggtagttt cctgttgttg gg 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gtgaaatgtt taggaccact ag 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 aaagttctga gacactccga ct 22 <210> 22 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ctcgcttcgg cagcaca 17 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 atgacctatg atttgacaga c 21 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 tctttggtta tctagctgta tga 23 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 taggtagttt cctgttgttg gg 22 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gtgaaatgtt taggaccact ag 22 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tcagtgcact acagaacttt gt 22 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gcttcggcag cacatatact aaaat 25 <210> 29 <211> 22 <212> RNA <213> Homo sapiens <400> 29 aacagcugug cuaugccaaa ga 22 <210> 30 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> miRNA-9 <400> 30 gguuucu 7 <210> 31 <211> 23 <212> RNA <213> Homo Sapiens <400> 31 agcuuauccu uaaaaaccaa aga 23 <210> 32 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miRNA <400> 32 aguaugucga ucuauugguu ucu 23 <210> 33 <211> 23 <212> RNA <213> Homo sapiens <400> 33 ucagaaggaa aauuuacuac cuu 23 <210> 34 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-196b <400> 34 ggguuguugu ccuuugaugg au 22 <210> 35 <211> 23 <212> RNA <213> Homo sapiens <400> 35 cacuaauauu auuuuugcac ugu 23 <210> 36 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-148a <400> 36 uguuucaaga caucacguga cu 22 <210> 37 <211> 23 <212> RNA <213> Homo sapiens <400> 37 acauuucaaa aauucagguc aaa 23 <210> 38 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-215 <400> 38 cagacaguuu aguauccagu a 21 <110> Industry Academic Cooperation Foundation, Hallym University <120> A marker composition for diagnosing tuberculous pulmonary fibrosis comprising miR-148a and a pharmaceutical composition for preventing or treating tuberculous pulmonary fibrosis comprising a mimic of miR-148a <130> HallymU-JYHONG-miR148a <160> 38 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> artificial sequence <220> <223> primer <400> 1 cuguugugga cccaauuca 19 <210> 2 <211> 19 <212> RNA <213> artificial sequence <220> <223> primer <400> 2 ugaauugggu ccacaacag 19 <210> 3 <211> 19 <212> RNA <213> artificial sequence <220> <223> primer <400> 3 cucuuguuca cuuuaccuu 19 <210> 4 <211> 17 <212> RNA <213> artificial sequence <220> <223> primer <400> 4 aagguaaagu gaacaag 17 <210> 5 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 5 ggtcaagaca ctattaaca 19 <210> 6 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 6 ggattcctag tgtatttca 19 <210> 7 <211> 22 <212> RNA <213> artificial sequence <220> <223> a miR-148a mimic <400> 7 aaaguucuga gacacuccga cu 22 <210> 8 <211> 22 <212> RNA <213> artificial sequence <220> <223> a miR-148a inhibitor <400> 8 ucagugcacu acagaacuu gu 22 <210> 9 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 9 ccggctgcat cagtcttaac 20 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 10 tcggcacagt acaggcacaa 20 <210> 11 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 11 caaaacagaa tggaaaatat gagaccgg 28 <210> 12 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 12 tgattgatgc tcgtgactgc cca 23 <210> 13 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 13 actggtgaga cctgcgtgta 20 <210> 14 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 14 aatccatcgg tcatgctctc 20 <210> 15 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 15 gtgctatccc tgtacgcctc 20 <210> 16 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 16 ggccatctct tgctcgaag 19 <210> 17 <211> 21 <212> DNA <213> artificial sequence <220> <223> primer <400> 17 atgacctatg aattgacaga c 21 <210> 18 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 18 tctttggtta tctagctgta tga 23 <210> 19 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 19 taggtagttt cctgttgttg gg 22 <210> 20 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 20 gtgaaatgtt taggaccact ag 22 <210> 21 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 21 aaagttctga gacactccga ct 22 <210> 22 <211> 17 <212> DNA <213> artificial sequence <220> <223> primer <400> 22 ctcgcttcgg cagcaca 17 <210> 23 <211> 21 <212> DNA <213> artificial sequence <220> <223> primer <400> 23 atgacctatg atttgacaga c 21 <210> 24 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 24 tctttggtta tctagctgta tga 23 <210> 25 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 25 taggtagttt cctgttgttg gg 22 <210> 26 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 26 gtgaaatgtt taggaccact ag 22 <210> 27 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 27 tcagtgcact acagaacttt gt 22 <210> 28 <211> 25 <212> DNA <213> artificial sequence <220> <223> primer <400> 28 gcttcggcag cacatatact aaaat 25 <210> 29 <211> 22 <212> RNA <213> Homo sapiens <400> 29 aacagcugug cuaugccaaa ga 22 <210> 30 <211> 7 <212> RNA <213> artificial sequence <220> <223> miRNA-9 <400> 30 gguuucu 7 <210> 31 <211> 23 <212> RNA <213> Homo sapiens <400> 31 agcuuauccu uaaaaaccaa aga 23 <210> 32 <211> 23 <212> RNA <213> artificial sequence <220> <223> miRNAs <400> 32 aguaugucga ucuauugguu ucu 23 <210> 33 <211> 23 <212> RNA <213> Homo sapiens <400> 33 ucagaaggaa aauuuacuac cuu 23 <210> 34 <211> 22 <212> RNA <213> artificial sequence <220> <223> miR-196b <400> 34 ggguuguugu ccuuugaugg au 22 <210> 35 <211> 23 <212> RNA <213> Homo sapiens <400> 35 cacuaauauu auuuuugcac ugu 23 <210> 36 <211> 22 <212> RNA <213> artificial sequence <220> <223> miR-148a <400> 36 uguuucaaga caucacguga cu 22 <210> 37 <211> 23 <212> RNA <213> Homo sapiens <400> 37 acauuucaaa aauucagguc aaa 23 <210> 38 <211> 21 <212> RNA <213> artificial sequence <220> <223> miR-215 <400> 38 cagacaguuu aguauccagu a 21
Claims (11)
A pharmaceutical composition for preventing or treating tuberculous pleural fibrosis comprising miR-148a or a miR-148a mimic.
miR-148a 모방체는 5'-AAAGUUCUGAGACACUCCGACU-3'인, 결핵성 흉막 섬유증 예방 또는 치료용 약학 조성물.
The method of claim 1,
The miR-148a mimic is 5'-AAAGUUCUGAGACACUCCGACU-3', a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis.
상기 약학 조성물은 다른 결핵성 흉막 섬유증 예방 또는 치료용 약학 조성물과 조합한, 결핵성 흉막 섬유증 예방 또는 치료용 약학 조성물.
The method of claim 1,
The pharmaceutical composition is combined with other pharmaceutical compositions for preventing or treating tuberculous pleural fibrosis, a pharmaceutical composition for preventing or treating tuberculous pleural fibrosis.
A marker composition for diagnosis of tuberculous pleural fibrosis comprising miR-148a.
A composition for diagnosing tuberculous pleural fibrosis comprising an agent for measuring the expression level of miR-148a.
miR-148a의 발현량을 측정하는 제제는 상기 miRNA에 상보적으로 결합하는 센스 및 안티센스 프라이머, 또는 프로브인 것인, 결핵성 흉막 섬유증 진단용 조성물.
The method of claim 5,
An agent for measuring the expression level of miR-148a is a sense and antisense primer or probe that binds complementary to the miRNA, a composition for diagnosing tuberculous pleural fibrosis.
A kit for diagnosing tuberculous pleural fibrosis comprising an agent for measuring the expression level of miR-148a.
An information providing method for diagnosing tuberculous pleural fibrosis comprising a measurement step of measuring the expression level of miR-148a from a biological sample derived from a subject.
miR-148a의 발현량은 중합효소연쇄반응(PCR), 차세대 염기서열 분석(Next generation sequencing; NGS), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR) 및 마이크로어레이(microarray)로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것인, 결핵성 흉막 섬유증 진단을 위한 정보제공방법.
The method of claim 8,
The expression level of miR-148a was determined by polymerase chain reaction (PCR), next generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR) and A method for providing information for diagnosing tuberculous pleural fibrosis, which is measured through one or more methods selected from the group consisting of microarray.
생물학적 시료는 조직 및 세포로 이루어진 군으로부터 선택되는 1종 이상인 것인, 결핵성 흉막 섬유증 진단을 위한 정보제공방법.
The method of claim 8,
The biological sample is at least one selected from the group consisting of tissues and cells, an information providing method for diagnosing tuberculous pleural fibrosis.
정보제공방법은 측정 단계에서 상기 피검체 시료에서의 miR-148a의 발현량이 정상개체 시료에서의 miR-148a의 발현량보다 증가한 경우 결핵성 흉막 섬유증으로 판단하는 것인, 결핵성 흉막 섬유증 진단을 위한 정보제공방법.The method of claim 8,
The information provision method provides information for the diagnosis of tuberculous pleural fibrosis, in which the expression level of miR-148a in the subject sample is higher than the expression level of miR-148a in the normal subject sample in the measurement step. method.
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