The Role of the Exonic lncRNA PRKDC-210 in Transcription Regulation.

In recent years, long noncoding RNAs (lncRNAs) have received increasing attention and have been reported to be associated with various genetic abnormalities. However, the functions of many lncRNAs, including those of long exonic noncoding RNAs (lencRNAs), have not yet been elucidated. Here, we used a novel tethering luciferase assay to analyze the transcriptional regulatory functions of five lencRNAs that are upregulated in cancer. We found that the lencRNA PRKDC-210 interacts with MED12, a component of the CDK8 complex, to regulate the transcription of several genes. The transcriptional activation ability of PRKDC-210 was abolished in siRNA-treated CDK8-depleted cells. We also confirmed the enrichment of PRKDC-210 on RNA polymerase II. RNA-seq analysis of cells in which PRKDC-210 or PRKDC mRNA was knocked down using antisense oligonucleotides revealed that PRKDC-210 can affect the expression levels of genes related to fatty acid metabolism. Finally, we used a ChIRP assay to examine PRKDC-210-enriched sites in the genome. Overall, our findings demonstrate that the lencRNA PRKDC-210 promotes transcription through the CDK8 complex pathway at the transcription initiation site. We propose that PRKDC-210 can affect the transcription of adjacent genes after its transcription and splicing.

The U-to-C RNA editing affects the mRNA stability of nuclear genes in Arabidopsis thaliana.

Cytidine-to-uridine (C-to-U) RNA editing has been generally observed in land plants; however, reverse (U-to-C) RNA editing is a rare phenomenon. In this study, we investigated the U-to-C RNA editing-related genes in Arabidopsis tissues and the effects on mRNA stability, with a special focus on PPR proteins. A previous study showed the extensive occurrence of U-to-C RNA editing in 12-day and 20-dayold Arabidopsis seedlings. Here, we have demonstrated the effects of this "reverse" RNA editing on the mRNA stability for all seven edited genes. We also identified U-to-C RNA editing in the nuclear PPR gene (AT2G19280) in 12-day-old seedlings of Arabidopsis thaliana. The U-to-C RNA editing sites were found in the untranslated region (3' UTR) of the mature mRNA and may affect its secondary structure. We also examined the correlation between U-to-C RNA editing-related genes and their mRNA abundance. Furthermore, we investigated the effects of U-to-C RNA editing in Arabidopsis using the transcription inhibitor actinomycin D (Act D). The addition of Act D to the seedlings of transgenic Arabidopsis generated by Agrobacterium-mediated transformation showed that single nucleotide base conversion adversely affected the mRNA secondary structure and stability.

The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB.

N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-ß-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-ß-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-ß-induced EMT of lung cancer cells through the regulation of JUNB.

Development of a Single Construct System for Site-Directed RNA Editing Using MS2-ADAR.

Site-directed RNA editing (SDRE) technologies have great potential for treating genetic diseases caused by point mutations. Our group and other researchers have developed SDRE methods utilizing adenosine deaminases acting on RNA (ADARs) and guide RNAs recruiting ADARs to target RNAs bearing point mutations. In general, efficient SDRE relies on introducing numerous guide RNAs relative to target genes. However, achieving a large ratio is not possible for gene therapy applications. In order to achieve a realistic ratio, we herein developed a system that can introduce an equal number of genes and guide RNAs into cultured cells using a fusion protein comprising an ADAR fragment and a plasmid vector containing one copy of each gene on a single construct. We transfected the single construct into HEK293T cells and achieved relatively high efficiency (up to 42%). The results demonstrate that efficient SDRE is possible when the copy number is similar for all three factors (target gene, guide RNA, and ADAR enzyme). This method is expected to be capable of highly efficient gene repair in vivo, making it applicable for gene therapy.

Effective RNA Knockdown Using CRISPR-Cas13a and Molecular Targeting of the EML4-ALK Transcript in H3122 Lung Cancer Cells.

RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA-guide RNA complex residing at the 5' of the crRNA. The activity of Cas13a was maximal for guide RNAs 24-30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70-76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.

Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45-55 of the DMD gene, might improve patients' symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45-55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44-56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5' splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

A view of pre-mRNA splicing from RNase R resistant RNAs.

During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs) may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues.

Improvement of C-to-U RNA editing using an artificial MS2-APOBEC system.

RNA cytidine deamination (C-to-U editing) has been achieved using the MS2-apolipoprotein B-editing catalytic polypeptide-like (APOBEC)1 editing system. Here, we fused the cytidine deaminase (CDA) enzymes APOBEC3A and APOBEC3G with the MS2 system and examined their RNA editing efficiencies in transfected HEK 293T cells. Given the single-stranded RNA preferences of APOBEC3A and APOBEC3G, we designed unconventional guide RNAs that induced a loop at the target sequence, allowing the target to form a single-stranded structure. Because APOBEC3A and APOBEC3G have different base preferences (5'-TC and 5'-CC, respectively), we introduced the D317W mutation into APOBEC3G to convert its base preference to that of APOBEC3A. Upon co-transfection with a guide RNA that induced the formation of a 14 nt loop on the target sequence, MS2-fused APOBEC3A and APOBEC3G showed high editing efficiency. While the D317W mutation of APOBEC3G led to a slight improvement in editing efficiency, the difference was not statistically significant. These findings indicate that APOBEC3A and APOBEC3G can induce C-to-U RNA editing when transfected with a loop guide RNA. Moreover, the editing efficiency of APOBEC3G can be enhanced by site-specific mutation to alter the base preference. Overall, our results demonstrate that the MS2 system can fuse and catalyze reactions with different enzymes, suggesting that it holds an even greater potential for RNA editing than is utilized currently.

Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System.

Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA.

MEF2C/p300-mediated epigenetic remodeling promotes the maturation of induced cardiomyocytes.

Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where MEF2C acts as a pioneer factor with GATA4 and TBX5 (GT). However, the generation of functional and mature iCMs is inefficient, and the molecular mechanisms underlying this process remain largely unknown. Here, we found that the overexpression of transcriptionally activated MEF2C via fusion of the powerful MYOD transactivation domain combined with GT increased the generation of beating iCMs by 30-fold. Activated MEF2C with GT generated iCMs that were transcriptionally, structurally, and functionally more mature than those generated by native MEF2C with GT. Mechanistically, activated MEF2C recruited p300 and multiple cardiogenic TFs to cardiac loci to induce chromatin remodeling. In contrast, p300 inhibition suppressed cardiac gene expression, inhibited iCM maturation, and decreased the beating iCM numbers. Splicing isoforms of MEF2C with similar transcriptional activities did not promote functional iCM generation. Thus, MEF2C/p300-mediated epigenetic remodeling promotes iCM maturation.

Functional gene expression analysis of tissue-specific isoforms of Mef2c.

Mef2c contains three alternative exons and generates six Mef2c isoforms in mice. Mef2c α1ß isoforms are expressed in neuronal tissues, α2 isoforms are expressed in muscle, and α1 isoforms are expressed in many other tissues. The γ region inclusion and skipped isoforms are present in equal amounts in many tissues. In this study, differences in the transcriptional activities of each tissue-specific isoform of Mef2c in neuronal cells were examined. Using an MEF2-responsive reporter, exon ß-dependent transactivation was found in neuronal cells, as well as in other cell lines previously described. Microarray analysis was used to examine the transcriptional activities of each Mef2c isoform and to assess differences in endogenous gene expression induced by the different isoforms. The results showed significant gene expression changes due to overexpression of Mef2c isoforms in both an isoforms-dependent and -independent manner.

Alternative splicing produces structural and functional changes in CUGBP2.

BACKGROUND: CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons. RESULTS: The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure. CONCLUSION: Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

Integrated analysis of ALK higher expression in human cancer and downregulation in LUAD using RNA molecular scissors.

PURPOSE: Anaplastic lymphoma kinase (ALK) is an endorsed molecular target in ALK-rearranged carcinomas, including lung adenocarcinoma. However, the clinical advantage of targeting ALK using druggable inhibitors is almost universally restricted by the development of drug resistance. Therefore, a strategy for combating ALK overexpression remains paramount for ALK-driven cancer. METHODS: We systemically analyzed the overexpression pattern of ALK and its clinical consequences, genetic alterations, and their significance in cancer hallmark genes, and correlation using integrated multidimensional approaches. The LwCas13a RNA molecular scissors was used to downregulate ALK-rearrangement by leveraging two target guide RNAs in lung adenocarcinoma (LUAD) cells. Immunocytochemistry, immunoblotting, and MTT assays were conducted to validate the downregulation. RESULTS: We found elevated levels of ALK in several malignancies, including LUAD, than in normal tissues. Higher expression of ALK was significantly associated with worse or shorter survival than patients with lower expression. We identified numerous genetic alterations in ALK, which potentially alter the cancer hallmark genes, including STAT1 and CTSL, in patients with LUAD. Next, we observed that the LwCas13a molecular scissors robustly downregulated both phosphorylated and total ALK chimera protein expression in LUAD cells compared to the control. Furthermore, we found that downregulation of ALK chimera protein substantially inhibited cell viability and induced cell death, including apoptosis. CONCLUSION: Our findings suggest a basis for ALK as a prognostic biomarker and the LwCas13a molecular scissors successfully downregulated the onco-driver ALK-rearrangement protein, which will potentially pave the way toward the development of novel therapeutic strategies for ALK-driven cancer.

Integrated analysis of the clinical consequence and associated gene expression of ALK in ALK-positive human cancers.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is genetically altered in several cancers, including NSCLC, melanoma, lymphoma, and other tumors. Although ALK is associated with various cancers, the relationship between ALK expression and patient prognosis in different cancers is poorly understood. Here, using multidimensional approaches, we revealed the correlation between ALK expression and the clinical outcomes of patients with LUAD, melanoma, OV, DLBC, AML, and BC. We analyzed ALK transcriptional expression, patient survival rate, genetic alteration, protein network, and gene and microRNA (miRNA) co-expression. Compared to that in normal tissues, higher ALK expression was found in LUAD, melanoma, and OV, which are associated with poor patient survival rates. In contrast, lower transcriptional expression was found to decrease the survival rate of patients with DLBC, AML, and BC. A total of 202 missense mutations, 17 truncating mutations, 7 fusions, and 3 in-frame mutations were identified. Further, 17 genes and 19 miRNAs were found to be exclusively co-expressed and echinoderm microtubule-associated protein-like 4 (EML4) was identified as the most positively correlated gene (log odds ratio >3). The gene ontology and signaling pathways of the genes co-expressed with ALK in these six cancers were also identified. Our findings offer a basis for ALK as a prognostic biomarker and therapeutic target in cancers, which will potentially contribute to precision oncology and assist clinicians in identifying suitable treatment options.

C-to-U RNA Editing: A Site Directed RNA Editing Tool for Restoration of Genetic Code.

The restoration of genetic code by editing mutated genes is a potential method for the treatment of genetic diseases/disorders. Genetic disorders are caused by the point mutations of thymine (T) to cytidine (C) or guanosine (G) to adenine (A), for which gene editing (editing of mutated genes) is a promising therapeutic technique. In C-to-Uridine (U) RNA editing, it converts the base C-to-U in RNA molecules and leads to nonsynonymous changes when occurring in coding regions; however, for G-to-A mutations, A-to-I editing occurs. Editing of C-to-U is not as physiologically common as that of A-to-I editing. Although hundreds to thousands of coding sites have been found to be C-to-U edited or editable in humans, the biological significance of this phenomenon remains elusive. In this review, we have tried to provide detailed information on physiological and artificial approaches for C-to-U RNA editing.

Development of Therapeutic RNA Manipulation for Muscular Dystrophy.

Approval of therapeutic RNA molecules, including RNA vaccines, has paved the way for next-generation treatment strategies for various diseases. Oligonucleotide-based therapeutics hold particular promise for treating incurable muscular dystrophies, including Duchenne muscular dystrophy (DMD). DMD is a severe monogenic disease triggered by deletions, duplications, or point mutations in the DMD gene, which encodes a membrane-linked cytoskeletal protein to protect muscle fibers from contraction-induced injury. Patients with DMD inevitably succumb to muscle degeneration and atrophy early in life, leading to premature death from cardiac and respiratory failure. Thus far, the disease has thwarted all curative strategies. Transcriptomic manipulation, employing exon skipping using antisense oligonucleotides (ASO), has made significant progress in the search for DMD therapeutics. Several exon-skipping drugs employing RNA manipulation technology have been approved by regulatory agencies and have shown promise in clinical trials. This review summarizes recent scientific and clinical progress of ASO and other novel RNA manipulations, including RNA-based editing using MS2 coat protein-conjugated adenosine deaminase acting on the RNA (MCP-ADAR) system illustrating the efficacy and limitations of therapies to restore dystrophin. Perhaps lessons from this review will encourage the application of RNA-editing therapy to other neuromuscular disorders.

Alternative splicing of Mef2c promoted by Fox-1 during neural differentiation in P19 cells.

Mef2c protein is one of the MADS-box type transcription factors involved in muscular differentiation and synaptic formation. Previously, it has been reported that the Mef2c gene is responsible for three alternative splicing regulations. Here, we investigated the alternative splicing variants of Mef2c during neural differentiation of P19 cells and during cardio muscular differentiation of P19 clone 6 (P19CL6). We detected that two Mef2c mRNA isoforms, using exon α1 with and without the γ region at exon 10, are mainly produced in immature P19 cells. Remarkably, Mef2c isoforms containing exon ß specifically appeared in the neural cell stage. Because most transcripts contain exon ß in the neural cell stage and in the brain, this suggests that the alternative splicing of exon ß is highly regulated. Among known regulators, Fox-1 was specifically expressed in the neural cell stage in correlation with Mef2c exon ß. Fox-1 promoted exon ß inclusion in transfection experiments using Mef2cß minigene. Moreover, we found that the promotion required RNA-binding activity of Fox-1 and GCAUG sequence located in adjacent intron of exon ß. Taken together, our results suggest that Fox-1, expressed specifically in the neural cell stage, promoted Mef2c exon ß inclusion via the GCAUG.

Double MS2 guided restoration of genetic code in amber (TAG), opal (TGA) and ochre (TAA) stop codon.

The popularity and promise of gene therapy for common genetic diseases are currently increasing. Although effective treatments for genetic disorders are rare, editing of the mutated gene is a possible therapeutic approach for conditions caused by stop codon mutations, including either amber (TAG), opal (TGA) or ochre (TAA) stop codons. Restoration of point-mutated RNAs using artificial RNA editing can be used to modify gene-encoded information and generate functionally distinct proteins from a single gene. By linking the catalytic domain of the RNA editing enzyme, adenosine deaminase acting on RNA (ADAR), to an antisense guide RNA, specific adenosines (A) can be converted to inosine (I), which is recognized as guanosine (G) during translation. In this study, we engineered the deaminase domain of ADAR1 and the MS2 system to target a specific adenosine and restore the G to A mutations. To this end, the ADAR1 deaminase domain was fused with the RNA binding protein, MS2, which binds to MS2 RNA. Guide RNAs of 19 bp were designed to be complementary to target mRNAs, with either 6X stem-loops downstream of the guide RNA and a CMV promoter, or a 1X MS2 stem-loop on either side of the guide RNA and a U6 promoter. The engineered ADAR1 deaminase domain could convert adenosine to inosine at the desired editing site in EGFP, which was edited to contain an amber (TAG), opal (TGA) or ochre (TAA) stop codon. The system could convert the stop codons to a read-through tryptophan codon (TGG) in a cellular system, leading to fluorescence emission, observed using JuLi microscopy. PCR-RFLP and Sanger sequencing of the target transcript were also conducted, revealing an editing efficiency of 20.97 % for the opal stop codon, and 26 % and 17 % for the 5' and 3' A residues, respectively, in the ochre stop codon, using the double MS2. This was a higher editing rate than that achieved using the MS2-6X guide RNA. Observation of restoration of the read-through codon from the three different stop codons over time demonstrated a relatively low percentage of edited codons after 24 h, which increased after 48 h, but decreased again after 72 h. Successful establishment of this system has the potential to represent a new era in the field of gene therapy.

Genome-Wide Identification of U-To-C RNA Editing Events for Nuclear Genes in Arabidopsis thaliana.

Cytosine-to-Uridine (C-to-U) RNA editing involves the deamination phenomenon, which is observed in animal nucleus and plant organelles; however, it has been considered the U-to-C is confined to the organelles of limited non-angiosperm plant species. Although previous RNA-seq-based analysis implied U-to-C RNA editing events in plant nuclear genes, it has not been broadly accepted due to inadequate confirmatory analyses. Here we examined the U-to-C RNA editing in Arabidopsis tissues at different developmental stages of growth. In this study, the high-throughput RNA sequencing (RNA-seq) of 12-day-old and 20-day-old Arabidopsis seedlings was performed, which enabled transcriptome-wide identification of RNA editing sites to analyze differentially expressed genes (DEGs) and nucleotide base conversions. The results showed that DEGs were expressed to higher levels in 12-day-old seedlings than in 20-day-old seedlings. Additionally, pentatricopeptide repeat (PPR) genes were also expressed at higher levels, as indicated by the log2FC values. RNA-seq analysis of 12-day- and 20-day-old Arabidopsis seedlings revealed candidates of U-to-C RNA editing events. Sanger sequencing of both DNA and cDNA for all candidate nucleotide conversions confirmed the seven U-to-C RNA editing sites. This work clearly demonstrated presence of U-to-C RNA editing for nuclear genes in Arabidopsis, which provides the basis to study the mechanism as well as the functions of the unique post-transcriptional modification.

Antiproliferative and antioxidant potentials of bioactive edible vegetable fraction of Achyranthes ferruginea Roxb. in cancer cell line.

In the present study, the aerial parts of Achyranthes ferruginea underwent investigation of their in vitro antioxidant and free radical-scavenging activities in cell-free conditions, their phytoconstituents using gas chromatography-mass spectrometry (GC-MS), and their cytotoxic activity in HeLa cells. A. ferruginea was extracted with 80% methanol and successively fractionated with solvents to yield petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF), and aqueous (AQF) fractions. GC-MS analysis revealed that CHF contained ten phytoconstituents, including different forms of octadecanoic acid methyl esters. The total antioxidant and ferric-reducing antioxidant capacities of the extracts and the standard catechin (CA) were as follows: CA >CHF >PEF >CME (crude methanolic extract) >EAF >AQF, and CA >CHF >EAF >PEF >AQF >CME, respectively. CHF showed the highest DPPH-free radical-scavenging activity, with a median inhibitory concentration of 10.5 ± 0.28 µg/ml, which was slightly higher than that of the standard butylated hydroxytoluene (12.0 ± 0.09 µg/ml). In the hydroxyl radical-scavenging assay, CHF showed identical scavenging activity (9.25 ± 0.73 µg/ml) when compared to CA (10.50 ± 1.06 µg/ml). Moreover, CHF showed strong cytotoxic activity (19.95 ± 1.18 µg/ml) in HeLa cells, which was alike to that of the standards vincristine sulfate and 5-fluorouracil (15.84 ± 1.64 µg/ml and 12.59 ± 1.75 µg/ml, respectively). The in silico study revealed that identified compounds were significantly linked to the targets of various cancer cells and oxidative enzymes. However, online prediction by SwissADME, admetSAR, and PASS showed that it has drug-like, nontoxic, and potential pharmacological actions.