Chong Guo

UConn Health

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Changchun Liu

UConn Health

Yixun Li

Education University of Hong Kong

Xin‐Yuan Guan

Chinese University of Hong Kong

Shenju Cheng

First Affiliated Hospital of Kunming Medical University

Jiongyu Zhang

UConn Health

Lori Avery

UConn Health

Ziyue Li

UConn Health

Zijie Liu

Chinese Academy of Medical Sciences & Peking Union Medical College

Bo Nie

First Affiliated Hospital of Kunming Medical University

Chengyu Hou

Guangzhou University of Chinese Medicine

Cooperative Institutions

First Affiliated Hospital of Kunming Medical University

Kunming Medical University

UConn Health

University of Connecticut

Innovation Team (China)

Chinese Academy of Medical Sciences & Peking Union Medical College

Sichuan University

Peking University

Shanghai Jiao Tong University

Soochow University

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Integrated analysis of DNA methylation profiling and gene expression profiling identifies novel markers in lung cancer in Xuanwei, China

PLoS ONE (2018)

Juan Wang Yong Duan Qinghe Meng Rong Gong Chong Guo

Background Aberrant DNA methylation occurs frequently in cancer. The aim of this study was to identify novel methylation markers in lung cancer in Xuanwei, China, through integrated genome-wide DNA methylation and gene expression studies. Methods Differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were detected on 10 paired lung cancer tissues and noncancerous lung tissues by methylated DNA immunoprecipitation combined with microarray (MeDIP-chip) and gene expression microarray analyses, respectively. Integrated analysis of DMRs and DEGs was performed to screen out candidate methylation-related genes. Both methylation and expression changes of the candidate genes were further validated and analyzed. Results Compared with normal lung tissues, lung cancer tissues expressed a total of 6,899 DMRs, including 5,788 hypermethylated regions and 1,111 hypomethylated regions. Integrated analysis of DMRs and DEGs identified 45 tumor-specific candidate genes: 38 genes whose DMRs were hypermethylated and expression was downregulated, and 7 genes whose DMRs were hypomethylated and expression was upregulated. The methylation and expression validation results identified 4 candidate genes (STXBP6, BCL6B, FZD10, and HSPB6) that were significantly hypermethylated and downregulated in most of the tumor tissues compared with the noncancerous lung tissues. Conclusions This integrated analysis of genome-wide DNA methylation and gene expression in lung cancer in Xuanwei revealed several genes regulated by promoter methylation that have not been described in lung cancer before. These results provide new insight into the carcinogenesis of lung cancer in Xuanwei and represent promising new diagnostic and therapeutic targets.

Differentially methylated regions

Methylated DNA immunoprecipitation

10.1371/journal.pone.0203155

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Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

Angewandte Chemie (2024)

Jiongyu Zhang Ziyue Li Chong Guo Xin‐Yuan Guan Lori Avery

Abstract The CRISPR‐Cas12a system has emerged as a powerful tool for next‐generation nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐ cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full‐size RNA targets, although LbCas12a exhibits weaker trans‐ cleavage activity than AsCas12a on both single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the “ U niversal N uclease for I dentification of V irus E mpowered by R NA‐ Se nsing” (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double‐stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry.

10.1002/ange.202403123

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[Silencing DNMT1 Attenuates the Effect of WIF-1 Gene Promoter Methylation on the Biological Behavior of Chronic Myeloid Leukemia K562 Cells].

PubMed (2021)

Kun Wu Zhen Jin Yi-Xun Li Xin Li Shenju Cheng

To investigate the effect of silencing DNA methyltransferase 1(DNMT1) to the methylation of the promoter of the tumor suppressor gene wnt-1 (WIF-1) in human chronic myeloid leukemia (CML) cells.DNMT1 siRNAi plasmid was constructed and DNMT1 siRNAi was transfected into CML K562 cells. RT-PCR and Western blot were used to detect the expression of DNMT1 gene and related protein, and methylation PCR was used to detect WIF-1 gene promoter methylation level. The trypan blue exclusion and MTT assay were used to detect the cell proliferation, flow cytometry were used to detect the cell apoptosis rate, colony formation assay was used to detect cell colony formation ability. Expression of Wnt/β- catenin and its downstream signaling pathway proteins were detected by Western blot after DNMT1 gene was silenced.The expression level of DNMT1 mRNA and its related protein in the experimental group were significantly lower than those in the control group and negative control group (P<0.05). After 72 hours of successful transfection, the WIF-1 gene in the control group and negative control group were completely methylated, while in the experimental group, the methylation level significantly decreased. The results of MSP showed that the PCR product amplified by the unmethylated WIF-1 primer in the experimental group increased significantly,while by the methylated WIF-1 primer decreased significantly. After 48 h of transfection, the OD value, viable cell number and colony formation of the cells in experimental group were significantly lower than those in the negative control group and the control group (P<0.05). The apoptosis rate of the cells in experimental group was significantly higher than those in the negative control group and control group (P<0.05). The expression levels of β- actin, myc, cyclin D1 and TCF-1 in K562 cells in the experimental group were significantly lower than those in the negative control group and control group (P<0.05).Silencing DNMT1 gene can inhibit the proliferation and promote the apoptosis of K562 cells. The mechanism may be related to reverse the hypermethylation level of the WIF-1 gene promoter, thereby inhibit the activity of the Wnt/β- catenin signaling pathway.沉默DNMT1减弱WIF-1基因启动子甲基化对慢性髓系白血病K562细胞生物学行为的影响.探讨沉默DNA甲基转移酶（DNMT1）对人慢性髓系白血病细胞内抑癌基因wnt抑制因子-1（WIF-1）基因启动子甲基化的影响。.构建DNMT1 siRNAi质粒，将DNMT1 siRNAi转染至慢性髓系白血病K562细胞中，采用RT-PCR和Western blot法检测DNMT1基因和相关蛋白的表达，并利用甲基化PCR检测WIF-1基因启动子甲基化水平，台盼蓝拒染法和MTT 法检测细胞的增殖能力，流式细胞术检测细胞的凋亡率，集落形成实验检测细胞的集落形成能力，Western blot法检测沉默DNMT1基因后 Wnt/β-catenin及其下游信号通路蛋白的表达情况。.实验组DNMT1 mRNA和相关蛋白表达水平显著低于对照组和阴性对照组（P<0.05）；成功转染72 h后，对照组和阴性对照组WIF-1基因处于完全甲基化状态，而实验组WIF-1基因启动子区域甲基化水平显著降低。MSP检测结果显示，实验组未甲基化的WIF-1引物扩增出的PCR产物含量显著增加，而甲基化的WIF-1引物扩增出的PCR产物含量显著降低。转染成功48 h后，实验组的OD值、活细胞数及集落形成数均显著低于阴性对照组和对照组（P<0.05）。实验组的细胞凋亡率显著高于阴性对照组和对照组（P<0.05）。实验组K562细胞中β-actin、myc、cyclin D1和TCF-1蛋白表达水平均显著低于阴性对照组和对照组（P<0.05。.沉默DNMT1基因能够抑制 K562 细胞增殖，促进其凋亡，其作用机制可能与逆转WIF-1基因启动子高甲基化水平，从而抑制Wnt/β-catenin信号通路的活性有关。.

DNMT1

K562 cells

10.19746/j.cnki.issn.1009-2137.2021.06.013

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MEF2A is a transcription factor for circPVT1 and contributes to the malignancy of acute myeloid leukemia

International Journal of Oncology (2024)

Kun Wu Yuntao Li Bo Nie Chong Guo Xiaobo Ma

Acute myeloid leukemia (AML) is a hematological malignancy with a high relapse rate and a poor survival rate. The circular RNA circPVT1 and myocyte enhancer factor 2A (MEF2A) have unique functions in the progression of AML; however, the underlying mechanisms and clinical significance remain to be clarified. Bioinformatics and database analyses were used to assess the transcription factors and target genes of circPVT1. Dual‑luciferase reporter gene and argonaute 2‑RNA immunoprecipitation assays were used to verify the targeted relationships. The expression levels of related genes and proteins were detected by reverse transcription‑quantitative PCR and western blotting. Cell viability and apoptosis were detected by Cell Counting Kit‑8 assay and flow cytometry, respectively. The results revealed that circPVT1 was highly expressed in AML samples and cell lines, and that MEF2A regulated the expression of circPVT1. MEF2A overexpression promoted cell viability and epithelial‑mesenchymal transition (EMT), and inhibited cell apoptosis. In addition, circPVT1 was revealed to target the regulation of microRNA (miR)‑455‑3p, and miR‑455‑3p targeted the regulation of MCL1 expression, thus indicating that circPVT1 promoted MCL1 expression through its interaction with miR‑455‑3p. Furthermore, cells were transfected with the small interfering RNA‑(si)‑circPVT1, miR‑455‑3p inhibitor or si‑MCL1, and si‑circPVT1 and si‑MCL1 inhibited the viability and EMT of NB4 and HL‑60 cells. However, the miR‑455‑3p inhibitor had the opposite effect on cells. In conclusion, MEF2A may act as a transcription factor of circPVT1 to promote the malignant process of AML, and knockdown of circPVT1 could inhibit the viability and EMT of AML cells through the miR‑455‑3p/MCL1 axis.

10.3892/ijo.2024.5699

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MicroRNA‑18a‑5p regulates the Warburg effect by targeting hypoxia‑inducible factor 1α in the K562/ADM cell line

Experimental and Therapeutic Medicine (2021)

Kun Wu Chong Guo Yixun Li Jinrong Yang Qiang Zhou

The Warburg effect is involved in drug resistance and recurrence of cancer, and poses a challenge for the treatment of chronic myelogenous leukemia (CML). Hypoxia‑inducible factor 1α (HIF‑1α) plays a key role in the Warburg effect. microRNAs (miRs) targeting HIF‑1α have potential of regulating such aberrant metabolic process. The present study demonstrated that miR‑18a‑5p was expressed at a low level in K562/ADM cells via reverse transcription‑quantitative PCR (RT‑qPCR). The results of the luciferase reporter assay indicated that miR‑18a‑5p could specifically bind the 3'‑untranslated region of HIF‑1α. Through RT‑qPCR and western blotting, it was revealed that miR‑18a‑5p downregulated the expression of HIF‑1α. By inhibiting HIF‑1α, miR‑18a‑5p suppressed aerobic glycolysis in K562/ADM cells, according to the results produced by glucose uptake, lactate production, pyruvate level and ATP synthesis measurement, along with the results obtained from extracellular acidification rate and oxygen consumption rate assays. These results provided new evidence that miR‑18a‑5p may suppress the Warburg effect by targeting HIF‑1α. Furthermore, via CCK‑8 and flow cytometry assays, cells transfected with miR‑18a‑5p mimics were more sensitive to Adriamycin (AMD) compared with AMD group. Reversing the Warburg effect by miR‑30a‑5p might provide a potential therapeutic strategy for CML.

Warburg Effect

K562 cells

Anaerobic glycolysis

10.3892/etm.2021.10503

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Distinguishing Four HCV Genotypes by Loop Mediated Isothermal Amplification Assay

Clinical Laboratory (2019)

Chuanqi Yang Xiaofei Li Huan Li Chong Guo Yixun Li

Genotyping of hepatitis C virus (HCV) is of great significance for clinical diagnosis and treatment. The object of this work was to establish a loop-mediated isothermal amplification (LAMP) method for four subtypes of HCV prevalent in China.Gene sequences of HCV-1b, 2a, 3, and 6a were downloaded from Genebank. Primer sets of the four HCV subtypes for LAMP method were designed by Primer Explorer V.4. LAMP reactions were performed at 65°C for 60 minutes. Sera of HCV-1b, 2a, 3, and 6a, HBV-A, HBV-B, HIV-1, HIV-2 and HAV, synthesized nucleic acid of HCV-4a, and 5a were used for evaluation of LAMP performance. For specificity evaluation, each primer set for HCV-1b, 2a, 3, and 6a was tested with all kinds of sera. Each serum of the above 4 HCV subtypes was diluted and aliquoted into 10 parts. RNA was subsequently extracted, which was detected by LAMP and Real-time PCR to verify limit of detection (LOD).The LAMP assay established in this study could detect and distinguish HCV-1b, 2a, 3, and 6a. Consistent with gene sequencing methods, specificity of HCV-1b, 2a, 3, and 6a was 100%, 85%, 90%, and 100% detected by LAMP, respectively. The LOD of HCV-1b, 3, and 6a was 1.5 x 103 IU/mL. The LOD of HCV2a was 1.0 x 103 IU/mL with the LAMP method.The LAMP method established in this study is feasible for distinguishing HCV-1b, 2a, 3, and 6a specifically and sensitively.

Primer (cosmetics)

SYBR Green I

10.7754/clin.lab.2019.190142

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Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR‐Cas12a

Advanced Science (2024)

Xin‐Yuan Guan Rui Yang Jiongyu Zhang Jeong Moon Chengyu Hou

Abstract CRISPR‐Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR‐Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR‐Cas12a enzyme, a dual‐mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high‐risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high‐risk pooled HPV subtypes. To enable low‐cost point‐of‐care testing, the assay is incorporated into a paper‐based microfluidic chip. Furthermore, the clinical performance of the paper‐based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.

Nucleic acid detection

10.1002/advs.202411021

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Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

Angewandte Chemie International Edition (2024)

Jiongyu Zhang Ziyue Li Chong Guo Xin‐Yuan Guan Lori Avery

Transcription

10.1002/anie.202403123

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Interpreting CRISPR-Cas12a enzyme kinetics through free energy change of nucleic acids

Nucleic Acids Research (2024)

Jiongyu Zhang Xin‐Yuan Guan Jeong Moon Shuo Zhang Zhengyang Jia

Abstract While CRISPR has revolutionized biotechnology, predicting CRISPR-Cas nuclease activity remains a challenge. Herein, through the trans-cleavage feature of CRISPR-Cas12a, we investigate the correlation between CRISPR enzyme kinetics and the free energy change of crRNA and DNA targets from their initial thermodynamic states to a presumed transition state before hybridization. By subjecting computationally designed CRISPR RNAs (crRNAs), we unravel a linear correlation between the trans-cleavage kinetics of Cas12a and the energy barrier for crRNA spacer and single-stranded DNA target unwinding. This correlation shifts to a parabolic relationship with the energy consumption required for double-stranded DNA target separation. We further validate these correlations using ∼100 randomly selected crRNA/DNA pairs from viral genomes. Through machine learning methods, we reveal the synergistic effect of free energy change of crRNA and DNA on categorizing Cas12a activity on a two-dimensional map. Furthermore, by examining other potential factors, we find that the free energy change is the predominant factor governing Cas12a kinetics. This study will not only empower sequence design for numerous applications of CRISPR-Cas12a systems, but can also extend to activity prediction for a variety of enzymatic reactions driven by nucleic acid dynamics.

Trans-activating crRNA

Nuclease

10.1093/nar/gkae1124

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DNMT1 inhibition improves the activity of memory-like natural killer cells by enhancing the level of autophagy

Molecular Biology Reports (2024)

Yixun Li Chong Guo Fujia Zhang Shenju Cheng Yanhong Li

Acute myeloid leukemia (AML) is a common hematological tumor, but it is difficult to treat. DNMT1 is a DNA methyltransferase whose main function is to maintain stable DNA methylation during the DNA replication process. DNMT1 also plays an important role in AML, but its function in cytokine-induced memory-like natural killer (CIML NK) cell activity remains unclear. In this study, we isolated primary NK cells from the peripheral blood of healthy volunteers and AML patients and treated them with 10 ng/mL IL-12, 50 ng/mL IL-15 and 50 ng/mL IL-18 to promote their differentiation into CIML NK cells. The activity of CIML NK cells was evaluated by RT‒qPCR, western blotting, ELISAs, and flow cytometry. DNMT1 was highly expressed in NK cells from AML patients. Knocking down DNMT1 significantly increased the expression of CD25, CD137, CD107a, IFN-γ, and TNF-α and increased the activity of CIML NK cells. Mechanistically, knocking down DNMT1 promoted autophagy by activating the AMPK/mTOR signaling pathway, thereby enhancing the activity of CIML NK cells and alleviating the progression of AML. Our study revealed that the downregulation of DNMT expression may be a new target for the treatment of AML.

DNMT1

10.1007/s11033-024-10181-9

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