SF3B4 Depletion Retards the Growth of A549 Non-Small Cell Lung Cancer Cells via UBE4B-Mediated Regulation of p53/p21 and p27 Expression

Jeonbuk National University,Jeehan Lee,정순영,Hye-Hyeon Yun,고정헌,Jeong-Hwa Lee 한국분자세포생물학회 2022 Molecules and cells Vol.45 No.10

Splicing factor B subunit 4 (SF3B4), a component of the U2-pre-mRNA spliceosomal complex, contributes to tumorigenesis in several types of tumors. However, the oncogenic potential of SF3B4 in lung cancer has not yet been determined. The in vivo expression profiles of SF3B4 in non-small cell lung cancer (NSCLC) from publicly available data revealed a significant increase in SF3B4 expression in tumor tissues compared to that in normal tissues. The impact of SF3B4 deletion on the growth of NSCLC cells was determined using a siRNA strategy in A549 lung adenocarcinoma cells. SF3B4 silencing resulted in marked retardation of the A549 cell proliferation, accompanied by the accumulation of cells at the G0/G1 phase and increased expression of p27, p21, and p53. Double knockdown of SF3B4 and p53 resulted in the restoration of p21 expression and partial recovery of cell proliferation, indicating that the p53/p21 axis is involved, at least in part, in the SF3B4-mediated regulation of A549 cell proliferation. We also provided ubiquitination factor E4B (UBE4B) is essential for p53 accumulation after SF3B4 depletion based on followings. First, co-immunoprecipitation showed that SF3B4 interacts with UBE4B. Furthermore, UBE4B levels were decreased by SF3B4 depletion. UBE4B depletion, in turn, reproduced the outcome of SF3B4 depletion, including reduction of polyubiquitinated p53 levels, subsequent induction of p53/p21 and p27, and proliferation retardation. Collectively, our findings indicate the important role of SF3B4 in the regulation of A549 cell proliferation through the UBE4B/p53/p21 axis and p27, implicating the therapeutic strategies for NSCLC targeting SF3B4 and UBE4B.

DNA 형태 적응을 거쳐 P2sir-관련 도움파지 비효율성을 극복하는 박테리오파지 P4 sid⁺ 유도체 정성 연구

김경진,Kim, Kyoung-Jin 한국미생물학회 2016 미생물학회지 Vol.52 No.1

P2-크기 머리에 packaging 될 특정 DAN 크기(28-29 kb long)와 박테리오파지 P4 유전자 sid의 변이가 "P2 sir-관련 도움파지 비효율성"을 극복할 수 있는 요소로 압축되었다. 유전자 sid의 변이 여부가 필수적인지를 확인하기 위해, 정상적인 sid 유전자를 가지며 $P2_{sir3}$-크기의 큰 머리에 packaging 될 DNA 크기가 28.5 kb되는 P4 delRI::kmr을 사용하여 실험하였다. P4 delRI::kmr이 P2 sir3 용원소에 대해 낮은 EOP를 보이므로, 이를 증가시키기 위해 P2 sir3 용원소를 숙주세포로 하여 파지 stock을 제조하였다. 이 과정에서 P4 delRI::kmr이 P2 sir3 용원소에 대해 적응하는 것을 관찰하고, CsCl 부양 균등밀도 편차실험과 분리된 DNA의 전기영동을 통해 그것이 packaging 될 머리 크기에 따른 DNA 형태 변화에 의한 적응이라는 것을 알아냈다. P2 sir3 용원소에 적응된 P4 delRI::kmr과 적응되지 않은 P4 delRI::kmr stock의 burst size 결정 실험은, sid 유전자 변이에 상관없이 packaging 될 DNA 크기에 의해 "P2 sir-관련 도움파지 비효율성"이 극복된다는 것을 보여주었다. A certain size of DNA (28-29 kb long) to be packaged into P2-size head and the mutation in sid gene of bacteriophage P4 are the major factors to overcome "P2 sir-associated helper inefficiency". To clarify whether the presence of sid mutation is essential to overcome "P2 sir-associated helper inefficiency" or not, we tested the P4 derivative, P4 delRI::kmr, which is $sid^+$ and whose genome size supposed to be 28.5 kb long in the case of being packaged into $P2_{sir3}$-sized large head. As P4 delRI::kmr showed the low EOP with P2 sir3 lysogen, P4 delRI::kmr phage stock was prepared in P2 sir3 lysogen host to increase the EOP with P2 sir3 lysogen. Through this process, P4 delRI::kmr had been adapted for P2 sir3 lysogen. With a CsCl buoyant equilibrium density gradient experiment and gel electrophoresis of the isolated DNA, it was evident that the adaptation of P4 delRI::kmr for P2 sir3 lysogen was caused by the conformational change of DNA to be packaged into large head. The burst size determination experiments with P4 delRI::kmr phage stock adapted for P2 sir3 lysogen and normal P4 delRI::kmr phage stock showed that not the sid mutation but the size of DNA to be packaged (28-29 kb long) was essential to overcome "P2 sir-associated helper inefficiency".

박테리오파아지 P2의 sir 변이를 억제하는 새로운 박테리오파아지 P4 유도체인 P4 ost2의 분리와 동정

김경진,Kim, Gyeong-Jin 한국미생물학회 2003 미생물학회지 Vol.39 No.4

P2의 sir 변이를 억제하는 변이체 박테리오파아지 P4를 얻기 위하여, P4 ash8 sid71 kmr intS를 박테리오파아지 P2 sir3 용원소에 plating하여 플라크를 형성하는 P4 ost2를 분리하였다. P4 ost2는 1단계 생장실험에서 P2의 sir 변이를 억제하는 P4 변이체로 나타났다. 제한효소 절단 반응으로 유전체 DNA의 재배열이 일어난 단편을 찾아 cloning과 sequencing을 거쳐 P4 ost2의 구조를 규명하였다. P4 ost2의 DNA는, 6.9 kb의 결실을 가진 P4 ash8 sid71 kmr intS의 불완전한 trimer로 2개의 cos-cleavage 부위를 가지는 28.8 kb의 유전체로 밝혀졌다. CsCl 부양균등밀도 편차실험으로 P4 ost2의 DNA 크기를 확인하였고 파아지 머리 안에 packaging된 DNA에 대한 기초적인 구조를 파악하였다. P4 ost2의 분리 동정을 통해, P4 유전체 상의 cos-cleavage 부위의 개수가 Sir-type 파아지 머리의 packaging 반응과는 관련이 없다고 추정할 수 있었다. Bacteriophage P4 ost2 which is the P4 mutant suppressing sir mutations of Bacteriophage P2, was isolated as a plaque-former by plating P4 ash8 sid 71 kmr intS on the lawn of P2 sir3 lysogen. P4 ost2 turned out to be the P4 mutant suppressing sir mutations of P2 in one-step growth experiments.

박테리오파아지 P2-P4 시스템을 위한 tetracyclin resistance marker 함유 P4 유도체 벡터 플라스미드 조성

김경진 한국미생물학회 2003 미생물학회지 Vol.39 No.2

바이러스 조립 과정 기작 연구를 위한 좋은 재료인 박테리오파아지 P2-P4 시스템에 이용될 벡터 플라스미드를 개발하기 위하여, P4 ash8 sid71을 출발 물질로 삼아 새로운 P4 유도체 벡터를 조성하였다. 유전자 재조합 기법을 써서 쉽게 선택 가능한 tetracyclin 내성 유전자(tetR)를 도입하고 플라스미드 P4의 크기를 조절하였다. 이를 통해 얻어진 P4 ash8(sid71) tetR은 12.09 kb의 크기를 가지며, 필요할 때 P2로 induction하면 생물학적 활성을 가지는 박테리오파아지로 전환 가능하였다. 전환된 파아지의 burst size를 결정하고, CsCl 부양균등밀도 편차실험을 수행하였다. 균등밀도 실험 분포도에서 P2 크기 파아지 머리의 packaging 상한을 추정할 수 있었다. To develop vector plasmid for the bacteriophage P2-P4 system which is a useful experimental tool for the study of viral capsid assembly, we constructed a new P4-derived vector plasmid starting from P4 ash8 sid71. With recombinant DNA technology, a portion of P4 genome was deleted and tetracyclin resistance gene (tetR) was introduced into P4 genome to give P4 selectivity. Resulting P4 ash8(sid71) tetR was 12.09 kb long and could be converted to a viable bacteriophage with P2 infection. The burst size of induced bacteriophage form of P4 ash8(sid71) tetR was determined. The CsCl buoyant equilibrium density gradient experiment of new P4 derivative suggested the upper limit of packaging capacity in P2-size head.

p16INK4a immunohistochemistry is a promising biomarker to predict the outcome of low grade cervical intraepithelial neoplasia: comparison study with HPV genotyping

Sakiko Nishio,Takuma Fujii,Hiroshi Nishio,Kaori Kameyama,Miyuki Saito,Takashi Iwata,Kaneyuki Kubushiro,Daisuke Aoki 대한부인종양학회 2013 Journal of Gynecologic Oncology Vol.24 No.3

Objective: In cervical intraepithelial neoplasia (CIN), p16INK4a immunohistochemistry has been reported to be a useful diagnostic biomarker. However, limited information is available about the association between the p16INK4a immunohistochemistry and the outcomes of CIN. Here, we report p16INK4a immunohistochemistry as an effective biomarker to predict the outcomes of CIN. Methods: p16INK4a immunohistochemistry was performed in patients with CIN from January 2000 to August 2009. Among these patients, we have performed a retrospective analysis of the medical records to evaluate the outcome of CIN 1-2 and performed statistical analysis to determine the correlation between p16INK4a expression and the outcomes. We also performed HPV genotyping and analyzed the relation between the infecting human papillomavirus (HPV) genotype and the outcomes. Results: A total of 244 patients, including 82 with CIN 1, 60 with CIN 2, and 102 with CIN 3, were examined. The rate of p16INK4a overexpression increased with increasing CIN grade, 20.7% for CIN 1, 80.0% for CIN 2, and 89.2% for CIN 3, with significant differences between CIN 1 and CIN 2-3 group. In the 131 CIN 1-2 patients, the progression rate was significantly higher for the patients showing p16INK4a overexpression than for those not showing p16INK4a overexpression (p=0.005); the regression rate was also found to be significantly lower for the patients showing p16INK4a overexpression (p=0.003). High-risk HPV genotypes were detected in 73 patients (73.7%). Both progression and regression rates were not significantly different between the high-risk HPV-positive and HPV-negative groups (p=0.401 and p=0.381, respectively). Conclusion: p16INK4a overexpression was correlated with the outcome of CIN 1-2, and p16INK4a is considered to be a superior biomarker for predicting the outcome of CIN 1-2 compared with HPV genotyping.

과발현된 p16INK4a 유전자의 pRB 의존적 또는 비의존적 pathway틀 통한 간암세포주의 성장 저해

박택규 ( Taek Kyu Park ),정유진 ( Yu Jin Jung ) 건국대학교 기초과학연구소 1999 理學論集 Vol.24 No.-

p16INK4a는 세포주기의 Gl기에서 cyclin Dl과 cdk4/6의 결합을 방해하는 강력한 inhibitor로 RB 유전자산물의 인산화를 조절한다. 먼저 7종의 사람 간암 (human hepatocellular carcinoma:HCC) 세 포주를 대상으로 하여 p16의 분자상태를 조사하였다. 대상 세포주중에서 SKHepl과 SNU449 세 포주는 pl6INK4a locus의 homozygous 결손을 나타내었고 SNU398은 전사 후 조절단계에서의 비활성화로 인한 기능의 상실을 나타내었다. 이는 p16Ink4a가 간암의 carcinogenesis에서 중요한 역할을 함을 시사한다. 또한 유전자 치료를 위한 P161NK4a의 간암세포성장 억제 효과를 알아보기 위하여 p16 유전자를 포함한 아데노바이러스 벡터를 간암 세 포주에 감염시켰다. Hep3B, SNU398, SNU449에서는 P16INK4a가 세포의 성장을 저해했으나 Chang liver, HepG2, SNU182, SKHepl에서는 세포의 성장 저해를 나타내지 않았다. 특히 SKHepl에서의 성장저해 실패는 재조합 아데노바이러스의 낮은 transduction efficiency 때문으로 밝혀졌다. 또한 pRB가 존재하는 SNU449에서 p16INK4a에 의한 성장 저해 작용 시 세포주기 는 GO/G1기에 머물러 있었으나 pRB가 없는 Hep3B에서는 그렇지 않았다. 이러한 결과는 간암에서 pI6INK4a의 유전자치료 대상으로서의 가능성을 제시하는 것으로 pRB에 의존하거나 또는 비의존적이 거나 간에 p16INK4a가 암세포 성장을 방해할 수 있다는 것을 의미한다. The p16INK4a 15 a major inhibitor of cyclin D-cdk4/6 complex and regulates the phosphorylation of the RB gene product. To evaluate the therapeutic efficacy of p16INK4a on human hepatocellular carcinoma cell lines (HCC), we analyzed the inactivation and growth inhibitory effect of p16lNK4a in seven HCCs. Among seven HCC cell lines tested, SKHepI and SNU449 cells showed homozygous deletion of p16lNK4a locus, and SNU398 cell might be inactivated at posttranscription level, indicating the importance of p16INK4a in HCC carcinogenesis. Adenoviral transduction of p16INK4a gene inhibited the cell growth in Hep3B, SNU398, and SNU449, but not in Chang liver, HepG2, SNU182, and SKHepl, which shows p16lNK4a gene deletion. Failure of growth inhibition in SKHepl is due to low transduction efficiency of recombinant adenovirus. While p16lNK4a-mediated growth inhibtion of pRB-positive SNU449 was arrested at the GO/Gl phase of cell cycle, but that of pRB-negative Hep3B was not. These results suggest that therapeutic efficacy of p16lNK4a gene might be considered on the ability of adenoviral transduction and the cytotoxicity of adenoviral vector itself in HCC. Moreover, growth inhibitory effect of p16INK4a could be exerted through either pRB- dependent or-independent pathway.

구강 편평세포암종에서의 p16INK4A와 p15INK4B 발현과 메틸화의 관계

최철영,추정엽,류숙경,조남표 대한구강악안면병리학회 2003 대한구강악안면병리학회지 Vol.27 No.3

p16INK4A and p15INK4B tumor suppressor genes are frequently altered in various human tumors. Hypermethylation of the promoter region of p16INK4A and p15INK4B seem to be the major mechanism of inactivation. To determine whether the change in p16INK4A and p15INK4B methylation status occur in oral squamous cell carcinomas (OSCCs) and benign oral epithelial hyperplasias, we analyzed 46 OSCCs and 20 benign oral epithelial hyperplasias by methylation-specific PCR. We also analyzed a subset of the samples for p16INK4A and p15INK4B protein expression by immunohistochemistry. The promoter region of p15INK4B was hypermethylated in 13 specimens of the 15 finally analyzed OSCCs and three specimens of the five analyzed benign oral epithelial hyperplasias. By immunohistochemical analysis, we confirmed the loss of p15INK4B expression of all hypermethylated specimens. The promoter region of p16INK4A was amplified by both an unmethylated- and a methylated-specific primers in just one OSCCs. The remaining specimens including 11 OSCCs and four benign oral epithelial hyperplasias were normally methylated. By immunohistochemistry, we analyzed the loss of p16INK4A expression in seven specimens of the 12 OSCCs and two specimens of the four benign oral epithelial hyperplasias. Except for one OSCC, however, all specimens showing loss of expression were normally methylated. These results suggest that loss of p16INK4A and p15INK4B protein expression play an important role in the development of both OSCCs and benign oral epithelial hyperplasias.

Construction and characterization of the bacteriophage P4 derivatives whose genome size suitable for packaging into a P2_sir3-sized head

김경진,Kim, Kyoung-Jin The Microbiological Society of Korea 2015 미생물학회지 Vol.51 No.1

"P2 sir-관련 도움파지 비효율성"이라는 용어는 P2 sir 변이체가 그들의 위성 박테리오파지인 P4에 대해 도움파지 역할을 충분히 못하는 것을 가리키는 용어이다. 이 연구의 목적은 이러한 P2 sir-관련 도움파지 비효율성을 극복하는 요인들을 조사하는 것이다. 우선 P2의 cos 영역을 함유하는 P4 sid71 cosP2가 P2 sir3의 도움파지 비효율성을 극복하는지를 조사하였다. 그 결과 P4 sid71 cosP2는 P2 sir3의 도움파지 비효율성을 극복하지 못하였다. P2의 cos 영역 대신에 $P2_{sir}$-sized head에 packaging되는 DNA 크기가 도움파지 비효율성을 극복하는 중요한 요인으로 나타났다. 이 연구에서는, DNA 조작을 통해 packaging 되는 DNA 크기가 P4 ost1과 P4 ost2 사이가 되는 세 종류의 P4 유도체를 조성하였다. 그 중 하나인 P4 sid71 delRI::apr의 CsCl 균등밀도차실험을 거쳐 packaging되는 DNA의 크기를 확인하였다. P4 유도체들의 후손방출량 실험에 따르면 그들은 모두 P2sir3-관련 도움파지 비효율성을 극복하였다. $P2_{sir3}$-sized head에 잘 packaging되는 P4 유도체의 크기는 28-29 kb로 나타났다. The term "P2 sir-associated helper inefficiency" has been used to define the inefficient helper capability of P2 sir mutants for their satellite bacteriophage P4. The aim of this study was to investigate the factors overcoming P2 sir-associated helper inefficiency. At first, we verified whether the P2 cos region containing P4 sid71 cosP2 could overcome P2 sir-associated helper inefficiency with P2 sir3. The result was that P4 sid71 cosP2 could not overcome P2 sir-associated helper inefficiency with P2 sir3. Instead of cos region of P2, the size of the DNA packaged into a $P2_{sir}$-sized head seems to be important for overcoming P2 sir-associated helper inefficiency. In the present work, three kinds of P4 derivatives with packaged DNA sizes between those of P4 ost1 and P4 ost2, were constructed through DNA manipulation. In one P4 derivative, P4 sid71 delRI::apr, the size of the packaged DNA was identified with a CsCl buoyant equilibrium density gradient experiment. According to the burst sizes of the P4 derivatives, they could overcome P2 sir3-associated helper inefficiency. The size of the P4 derivative DNA suitable for packaging into a $P2_{sir3}$-sized head was 28-29 kb.

LiFePO₄와 Li₄P₂O₇의 ⁷Li MAS NMR 특성 연구

한덕영,박남신,이상혁,이학만,김창삼,Han, Doug-Young,Park, Nam-Sin,Lee, Sang-Hyuk,Lee, Hak-Man,Kim, Chang-Sam 한국결정성장학회 2011 한국결정성장학회지 Vol.21 No.1

[ $^7Li$ ]Magic Angle Spinning(MAS) NMR Spectroscopy를 활용하여 $Li_4P_2O_7$와 $LiFePO_4$ 물질에서 $^7Li$ 핵의 NMR 특성 및 화합물 분자내의 국부적 구조 연구를 수행하였다. $Li_4P_2O_7$와 $LiFePO_4$ 물질 연구는 리튬이온전지에서 고체-전해질 경계상(SEI, solid-electrolyte interphase) 물질 연구를 위한 것이다. $Li_4P_2O_7$와 $LiFePO_4$ 분말은 고상합성법으로 제조하였다.$^7Li$MAS NMR 실험은 $27^{\circ}C$에서 $97^{\circ}C$의 영역에서 변온 실험을 수행하였으며 이는 주변 온도 변화 환경에서 $Li_4P_2O_7$ 물질 내의 Li 핵의 구조 변화를 관찰하기 위한 것이다. $^7Li$ MAS NMR 측정 결과 시료 온도가 $27^{\circ}C$에서 $97^{\circ}C$의 온도 분포 영역에서는 $Li_4P_2O_7$ 물질 내부의 Li 핵은 구조적으로 변화하지 않는 것이 확인되었다. 금번 실험을 통하여 $LiFePO_4$ 분말에 5.0 wt%이내로 포함되어있는 $Li_4P_2O_7$ 물질의 $^7Li$ MAS NMR 신호를 측정할 수 있는 측정 조건을 알았다. [ $^7Li$ ]Magic Angle Spinning (MAS) NMR spectroscopy has been used to study the lithium local environments in $Li_4P_2O_7$ and$LiFePO_4$ materials. The purpose of this study was to know the structure of the solid electrolyte interphase (SEI) in lithium ion cells composed of $LiFePO_4$ as cathode material. $Li_4P_2O_7$ and $LiFePO_4$ were prepared by a solid-state reaction. The $^7Li$ MAS NMR experiments were carried out at variable temperatures in order to observe the local structure changes at the temperatures in $Li_4P_2O_7$ system. The $^7Li$ MAS NMR spectra of in $Li_4P_2O_7$ indicate that the lithium local environments in $Li_4P_2O_7$ were not changed in the temperature range between $27^{\circ}C$ and $97^{\circ}C$ Through this work, we confirmed that the small amount of $Li_4P_2O_7$ less than 5.0 wt% in $LiFePO_4$ could be clearly measured by the $^7Li$ MAS NMR spectroscopy at high spinning rate over than 11 kHz.

PRPF4 is a novel therapeutic target for the treatment of breast cancer by influencing growth, migration, invasion, and apoptosis of breast cancer cells via p38 MAPK signaling pathway

Park, Song,Han, Se-Hyeon,Kim, Hyeon-Gyeom,Jeong, Jain,Choi, Minjee,Kim, Hee-Yeon,Kim, Min-Gi,Park, Jin-Kyu,Han, Jee Eun,Cho, Gil-Jae,Kim, Myoung Ok,Ryoo, Zae Young,Choi, Seong-Kyoon Elsevier 2019 Molecular and cellular probes Vol.47 No.-

<P><B>Abstract</B></P> <P>Pre-mRNA processing factor 4 (PRPF4), a core protein in U4/U6 snRNP, maintains snRNP structures by interacting with PRPF3 and cyclophilin H. Expression of the <I>PRPF4</I> gene affects cell survival as well as apoptosis and is responsible for retinitis pigmentosa (RP). Proteomics analysis shows that <I>PRPF4</I> may be a therapeutic target in human cancers. Nevertheless, the exact function and role of the <I>PRPF4</I> gene are unclear. In this study, we assessed the expression of <I>PRPF4</I> gene in human breast cancer cells. First, we confirmed that the <I>PRPF4</I> gene was overexpressed in various breast cancer cell lines. Next, using breast cancer cell lines MCF7 and MDA-MB-468, we established stable cell lines with <I>PRPF4</I> gene knockdown. We also performed microarray analysis to investigate molecular mechanisms underlying <I>PRPF4</I> activity. All cell lines with <I>PRPF4</I> gene knockdown exhibited reduced cell proliferation, remarkable reduction in anchorage-independent colony formation capacity, and reduction of PCNA protein, which is a marker cell of proliferation. Reduced expression of the <I>PRPF4</I> gene induced apoptosis and changes in the expression of associated apoptotic markers in breast cancer cell lines. Knockdown of the <I>PRPF4</I> gene reduced cellular capacity for migration and invasion (the key hallmarks of human cancers) and decreased the expression of genes involved in epithelial-mesenchymal transition (EMT). Microarray results showed that the expression of <I>PPIP5K1</I>, <I>PPIPK2</I>, and <I>YWHAE</I> genes was reduced at the transcriptional level, leading to reduced phosphorylation of p38 MAPK. These findings suggest that knockdown of <I>PRPF4</I> gene slows down breast cancer progression via suppression of p38 MAPK phosphorylation. In conclusion, the <I>PRPF4</I> gene plays an important role in the growth of breast cancer cells and is therefore a potential therapeutic target.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We generated the knockdown of <I>PRPF4 gene</I> in breast cancer cell line for function study. </LI> <LI> Knockdown of <I>PRPF4</I> decreased the proliferation and colony formation of breast cancer cells. </LI> <LI> Knockdown of <I>PRPF4</I> increased apoptosis of breast cancer cells. </LI> <LI> Knockdown of <I>PRPF4</I> decreased migration, invasion and epithelial mesenchymal transition ability of breast cancer cells. </LI> <LI> Knockdown of <I>PRPF4</I> regulates breast cancer cells progression by decreasing p38 MAPK signaling pathway. </LI> </UL> </P>