Development of Efficient Transformation Protocol for Soybean (Glycine max L.) and Characterization of Transgene Expression after Agrobacterium-mediated Gene Transfer

Kijong Lee,Bu-Young Yi,Kyung-Hwan Kim,Jung-Bong Kim,Seok-Cheol Suh,Hee-Jong Woo,Kong-Sik Shin,Soon-Jong Kweon 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.1

Increasing vitamin E activity in economically important oil crops such as soybean will enhance the nutritional value of these crops. An improved soybean transformation system involving pre-culture of soybean seed on medium supplemented with 1 mg/L 6-benzylaminopurine under dark conditions was established. To improve the nutritional value of soybean by increasing the α-tocopherol content, soybeans were transformed with γ-tocopherol methyltransferase (γ-TMT) gene by Agrobacterium-mediated transformation. Frequency of soybean transformation was significantly increased from 0.5 to 4.3% by this modified system, and 13 lines of transgenic soybean plants containing γ-TMT were obtained. The molecular characterization using polymerase chain reaction (PCR), reverse transcriptase PCR, and Southern blot analysis confirmed insertion and inheritance of the transgene in the transgenic plants and their progeny. Morphologically normal and fertile transgenic plants were analyzed, and the majority of transgenic soybean transmitted herbicide resistance at 3:1 or 15:1 ratios to their progeny. Alpha-tocopherol content in transgenic soybean seeds was determined by high-performance liquid chromatography; over-expression of γ-TMT resulted in a 41-fold increase in α-tocopherol over wild-type soybean seeds.

Heterogeneous Exhaustion Status of Tumor-Infiltrating CD8 T Cells Determines Distinct Subgroups of Hepatocellular Carcinoma Patients

( Hyung-don Kim ),( Gi-won Song ),( Seongyeol Park ),( Min Kyung Jung ),( Kijong Yi ),( Min Hwan Kim ),( Hyo Jeong Kang ),( Chang-hoon Yoo ),( Kyung Hwan Kim ),( Sukyeong Eo ),( Deok-bog Moon ),( Seun 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8⁺ T cells isolated from HCC specimens. Methods: We obtained HCC specimens, along with adjacent non-tumor tissues and blood samples, from 79 patients who underwent surgical resection at Asan Medical Center (in Seoul, Korea) from April 2016 through December 2017. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8⁺ T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA-seq. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8⁺ T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8⁺ T cells were identified using HLA-A^*0201 dextramers. Results: PD1-high, PD1-intermediate, and PD1-negative CD8+ T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and a low proportion was TCF1⁺, TBET^high/ eomesodermin^low, and CD127+. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in proportions of PD1-high CD8⁺ T cells led to the identification of 2 subgroups of HCCs; HCCs with a larger proportion of PD1-high cells were more aggressive than HCCs with a smaller proportion. Incubation of CD8⁺ T cells from HCCs with high proportions of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3. Conclusions: We found HCC specimens to contain CD8⁺ T cells that express different levels of PD1. HCCs with high proportions of PD1-high CD8⁺ T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with high proportions of PD1-high CD8⁺ T cells might be more susceptible to combined immune checkpoint blockade-based therapies.

MicroRNA Expression Signatures Associated With <i>BRAF</i> -Mutated Versus <i>KRAS</i> -Mutated Colorectal Cancers

Choi, Yong Won,Song, Young Soo,Lee, Hyunwoo,Yi, Kijong,Kim, Young-Bae,Suh, Kwang Wook,Lee, Dakeun Williams & Wilkins Co 2016 Medicine Vol.95 No.15

<P><B>Abstract</B></P><P><I>BRAF</I> and <I>KRAS</I> genes are known to play a similar role in the activation of <I>RAS-RAF-MEK-ERK</I> signaling pathway in colorectal tumorigenesis. However, <I>BRAF</I>-mutated colorectal cancers (CRCs) have distinct clinicopathologic characteristics different from those of the <I>KRAS</I> mutated ones as in comparison the <I>BRAF</I>-mutated CRCs are associated with a much worse prognosis for the afflicted patients. This study aimed to determine the different miRNA expression signatures associated with <I>BRAF</I>-mutated CRCs in comparison to <I>KRAS</I>-mutated ones, and to identify the specific miRNAs possibly mediating the aggressive phenotype of the <I>BRAF</I>-mutated CRCs.</P><P>We screened 535 formalin-fixed paraffin-embedded CRC tissue samples for the <I>BRAF V600E</I> mutation, and selected 7 <I>BRAF</I>-mutated and 7 <I>KRAS</I>-mutated CRCs that were tumor size, stage, and microsatellite status-matched. Affymetrix GeneChip® miRNA 4.0 Array was used for detection of miRNA expression differences in the selected samples. We validated the array results by quantitative reverse transcription polymerase chain reaction (qRT-PCR) for selected miRNAs.</P><P>A total of 10 differentially expressed (DE) miRNAs associated with <I>BRAF</I>-mutated CRCs were obtained, including miR-31-5p, miR-877-5p, miR-362-5p, and miR-425-3p. miR-31-5p showed the highest fold change (8.3-fold) among all of the miRNAs analyzed. From the analyses of GO biological processes, the DE-miRNAs were functionally relevant to cellular proliferation such as positive regulation of gene expression (<I>P</I> = 1.26 × 10<SUP>−10</SUP>), transcription (<I>P</I> = 9.70 × 10<SUP>−10</SUP>), and RNA metabolic process (<I>P</I> = 1.97 × 10<SUP>−9</SUP>). Bioinformatics analysis showed that the DE-miRNAs were significantly enriched in cancer-associated pathways including neutrophin signaling (<I>P</I> = 6.84 × 10<SUP>−5</SUP>), pathways in cancer (<I>P</I> = 0.0016), Wnt signaling (<I>P</I> = 0.0027), and MAPK signaling pathway (<I>P</I> = 0.0036).</P><P>Our results suggest that the DE-miRNAs in <I>BRAF</I>-mutated CRCs in comparison to <I>KRAS</I>-mutated CRCs are implicated in the aggressive phenotype of the <I>BRAF</I>-mutated CRCs. Further experimental validation is required to confirm these results.</P>

Association Between Expression Level of PD1 by Tumor-Infiltrating CD8⁺ T Cells and Features of Hepatocellular Carcinoma

Kim, Hyung-Don,Song, Gi-Won,Park, Seongyeol,Jung, Min Kyung,Kim, Min Hwan,Kang, Hyo Jeong,Yoo, Changhoon,Yi, Kijong,Kim, Kyung Hwan,Eo, Sukyeong,Moon, Deok-Bog,Hong, Seung-Mo,Ju, Young Seok,Shin, Eui- Elsevier 2018 Gastroenterology Vol.155 No.6

<P><B>Background & Aims</B></P> <P>T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8<SUP>+</SUP> T cells isolated from HCC specimens.</P> <P><B>Methods</B></P> <P>We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8<SUP>+</SUP> T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8<SUP>+</SUP> T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8<SUP>+</SUP> T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry.</P> <P><B>Results</B></P> <P>PD1-high, PD1-intermediate, and PD1-negative CD8<SUP>+</SUP> T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1<SUP>+</SUP>, TBET<SUP>high</SUP>/eomesodermin<SUP>low</SUP>, and CD127<SUP>+</SUP>. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8<SUP>+</SUP> T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8<SUP>+</SUP> T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3.</P> <P><B>Conclusions</B></P> <P>We found HCC specimens to contain CD8<SUP>+</SUP> T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8<SUP>+</SUP> T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8<SUP>+</SUP> T cells might be more susceptible to combined immune checkpoint blockade–based therapies.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Tracing Oncogene Rearrangements in the Mutational History of Lung Adenocarcinoma

Lee, Jake June-Koo,Park, Seongyeol,Park, Hansol,Kim, Sehui,Lee, Jongkeun,Lee, Junehawk,Youk, Jeonghwan,Yi, Kijong,An, Yohan,Park, In Kyu,Kang, Chang Hyun,Chung, Doo Hyun,Kim, Tae Min,Jeon, Yoon Kyung Elsevier 2019 Cell Vol.177 No.7