O6-methylguanine-DNA methyl transferase (MGMT) methylation in glioblastoma is an important prognostic and predictive factor that requires an invasive surgical procedure for identification. In several recent studies, MGMT methylation prediction models were developed using MR images, and good diagnostic performance was achieved, which seems to indicate a promising future for radiogenomics. However, the diagnostic performance was not reproducible for numerous research teams when using a larger dataset in the RSNA-MICCAI Brain Tumor Radiogenomic Classification 2021 challenge. To our knowledge, there has been no study regarding the external validation of MGMT prediction models using large-scale multicenter datasets. We tested recent CNN architectures via extensive experiments to investigate whether MGMT methylation in gliomas can be predicted using MRI. With unexpected negative results, approximately 80% of the developed models showed no significant difference with the chance level of 50% in terms of external validation accuracy. In conclusion, MGMT methylation status of gliomas may not be predictable with preoperative MRI, even using deep learning.n = 985); (2) SNUH (n = 400); and (3) BraTS datasets (n = 585). A total of 420 training and validation experiments were performed on combinations of datasets, convolutional neural network (CNN) architectures, MRI sequences, and random seed numbers. The first-place solution of the RSNA-MICCAI radiogenomic challenge was also validated using the external test set (SNUH). For model evaluation, the area under the receiver operating characteristic curve (AUROC), accuracy, precision, and recall were obtained. With unexpected negative results, 80.2% (337/420) and 60.0% (252/420) of the 420 developed models showed no significant difference with a chance level of 50% in terms of test accuracy and test AUROC, respectively. The test AUROC and accuracy of the first-place solution of the BraTS 2021 challenge were 56.2% and 54.8%, respectively, as validated on the SNUH dataset. In conclusion, MGMT methylation status of gliomas may not be predictable with preoperative MR images even using deep learning.O6-methylguanine-DNA methyl transferase (MGMT) methylation prediction models were developed using only small datasets without proper external validation and achieved good diagnostic performance, which seems to indicate a promising future for radiogenomics. However, the diagnostic performance was not reproducible for numerous research teams when using a larger dataset in the RSNA-MICCAI Brain Tumor Radiogenomic Classification 2021 challenge. To our knowledge, there has been no study regarding the external validation of MGMT prediction models using large-scale multicenter datasets. We tested recent CNN architectures via extensive experiments to investigate whether MGMT methylation in gliomas can be predicted using MR images. Specifically, prediction models were developed and validated with different training datasets: (1) the merged (SNUH + BraTS) ( Glioblastoma is the most common primary central nervous system malignancy, with a devastating median survival of 14.6 months even after operation followed by concurrent chemoradiation therapy (CCRT) with adjuvant temozolomide (TMZ) , even win = 31). Finally, the RSNA-MICCAI Brain Tumor Segmentation (BraTS) 2021 Radiogenomic Classification challenge, or task 2 of the BraTS 2021 challenge, was held [n = 247).In some previous works, it was reported that glioblastoma with MGMT methylation showed mass-like edema with nodular enhancement, whereas glioblastoma without MGMT methylation showed infiltrative edema with thick enhancement . Based owas held , and thewas held ,10,11,12n = 985). We performed extensive validation of CNN-based prediction models with large-scale public and independent private datasets.The generalization of models to different datasets is one of the biggest challenges for the application of artificial intelligence techniques in clinical radiology practice . Howevern = 7); (2) suboptimal image quality and consequent poor coregistration (n = 8), and (3) unavailable initial MGMT promoter methylation status (n = 19)  approved this retrospective study with a waiver of informed consent, and this study was carried out in accordance with the Declaration of Helsinki. From November 2014 to January 2019, 434 patients over 18 years old were consecutively and retrospectively enrolled in this study according to the following inclusion criteria: (1) histopathologic diagnostic confirmation of diffuse gliomas  based on 2016 WHO CNS tumor classification at the initial operation; (2) treatment-naïve MRI, and (3) underwent standard treatment, including surgery or stereotactic biopsy. A total of 34 patients were excluded because of the following: (1) missing sequences ((n = 19) . Finallyn = 585), which is a multicentered dataset from the RSNA-MICCAI Brain Tumor Radiogenomic Classification challenge  [To alleviate the small training dataset size, we combined the dataset from our institution, SNUH, with the BraTS 2021 training dataset  . The Braallenge) . For botAll datasets were preprocessed using skull-stripping, coregistration, and intensity rescaling, in an identical manner . For theTo examine the effect of datasets on model performance, a total of three kinds of experiments were performed based on different datasets (d = 3). We (1) trained the model using the SNUH dataset and validated the model using the BraTS 2021 dataset (Experiment 1); (2) trained the model using the BraTS 2021 dataset and validated the model using the SNUH dataset (Experiment 2), and (3) trained and validated the model using the merged (SNUH + BraTS) dataset (Experiment 3). When using the merged dataset, we randomly split it into training, validation and test sets using an 8:1:1 ratio.For each experiment, we validated (1) four representative convolutional neural network (CNN) architecture-based models (m = 4), and (2) seven combinations of sequences (s = 7) as input channels to examine the effect of model architectures and input MRI sequences on the model performance. Thus, a total of 28 combinations were validated in each experiment. Specifically, we tested (1) EfficientNetB0; (2) SEResNet50; (3) SEResNeXt50; and (4) DenseNet121. The model architecture is detailed in the Statistical analysis is detailed in the n = 400; 240 (60%) men, mean age, 52.3 ± 15.2 years old), age was not significantly different between methylation groups (p = 0.655) (n = 63); oligodendroglioma, IDH-mutant, 1p/19q-codeleted (n = 33); and glioblastoma, IDH-wildtype (n = 304). MGMT methylation occurred in (1) 307 out of 585 patients (52.5%) in the BraTS dataset (n = 585); (2) 197 out of 400 patients (49.3%) in the SNUH dataset, and (3) 504 out of 985 patients (51.2%) in the merged dataset. The proportion of methylation was not significantly different between the two datasets (p = 0.319). Progression-free survival (PFS) was significantly longer , 328–526 days)) vs. 974 days ) in the methylated group than in the unmethylated group (p < 0.0001) (p = 0.0001) (p = 0.871); HR, 0.91 . Detailed patient characteristics for the SNUH dataset are summarized in In the SNUH dataset (= 0.655) . Accordi 0.0001) . Patient 0.0001) , and patAccording to seven articles searched from PubMed, the mean dataset size was 155.4 ± 84.6 , and the diagnostic performance was representing by an accuracy of 62–94.7% . Specifip > 0.05), respectively.In our experiments, only 43.8% (184/420) and 39.8% (167/420) of the 420 developed models showed better performance than chance level (50%) in terms of test accuracy and test AUROC, respectively. In the one proportion z-test, 80.2% (337/420) and 60.0% (252/420) of the developed models showed no significant difference with chance level (50%) in terms of test accuracy and test AUROC  46.8% , 55.9% , 56.1% , and 74.2%  on the validation set a; and 2b.In Experiment 2, the best neural network model among 140 (m × s × r) models achieved AUROC, accuracy, precision, and recall of (1) 67.5% , 62.5% , 57.1% , and 100.0%  on the validation set a; and 2b.In Experiment 3, the best neural network model among 140 (m × s × r) models achieved AUROC, accuracy, precision, and recall of (1) 48.8% , 55.1% , 53.8% , and 91.3%  on the validation set a; and 2b.In Experiment 4 or the validation on the BraTS 2021 challenge, the first and second place solutions achieved AUROC, accuracy, precision, and recall of (1) 56.2, 54.8, 53.6, and 59.9% using 3D-ResNet and T1wCE only as the input sequence; and (2) 51.2, 51.3, 51.4, and 18.3% using EfficientNetB0-LSTM and FLAIR-T1wCE-T2w-T1w  as input sequences, respectively, as validated on the SNUH dataset . We coulp > 0.05 in pairwise comparison) (p > 0.05 in pairwise comparison)  . The besparison) . Compariparison) . The besparison) . Full meMGMT methylation is an important predictive factor in glioblastoma, increasing the chemosensitivity of TMZ ,5, playin = 262 patients; because of the rarity of the disease), which increases the risk of overfitting, external validation has rarely been performed. Moreover, because the prediction models have variable validation dataset sizes, their predictive performance are highly dependent on the variable validation method used 3, 4 [n < 1000) [In conventional MRI findings, nodular enhancement, ill-defined enhancing tumor margins, and mass-like edema have been reported to be associated with MGMT methylation. In contrast, infiltrative edema and thick enhancement have been reported to be associated with unmethylated MGMT status ,18,22. Bion)3, 4 ,24. Thusion)3, 4 , are req < 1000) .n = 985) of nearly one thousand patients, and meticulously tested various combinations of models, input sequences, and datasets. The seed number for randomness was also changed and compared because changing the seed number should not significantly affect model performance. As a result, in more than 80% and 60% of the developed models, we did not see a significant difference in terms of test AUROC and accuracy (p > 0.05), respectively, compared with the chance level of 50% in the one proportion z-test for the prediction of MGMT methylation (p > 0.05). In summary, comparing the test AUROC among different trained datasets, different CNN architectures, and different combinations of MRI sequences, there were no significant differences in diagnostic performances in the test sets . Interestingly, the best models in Experiment 1  showed high mean test recall  using EfficientNet-B0 and all the MRI sequences. However, they also showed large variance in test recall, ranging from 25.9% to 100.0%, which indicates unstable performance of the prediction models. Finally, the validation metrics were generally better than the test metrics  with multiparametric inputs, none of the participants among 1555 worldwide teams, including the first place team , could discover reliable MR imaging features that correlate with MGMT methylation in gliomas. Further analysis of the BraTS 2021 challenge is detailed in the n = 985) dataset in total, the best model among the 420 developed models showed an AUROC and accuracy of 64.5%  and 55.6% , respectively, on the test set using the merged dataset. In our study, it is not yet clear what causes the poor performance. This may be caused by (1) overfitting due to the small dataset size, and (2) different data distributions from different hospitals. However, considering that the prediction of IDH mutation in gliomas has shown better performance, achieving 87–92% accuracy in distinguishing IDH mutant gliomas from IDH wild-type tumors using only conventional MRI [In the BraTS 2021 challenge, given the large amount of data  to a relatively small dataset using modern “heavy” neural network architectures with a large number  of parameters, which can be used to extract good latent features compared with previous models. (3) The prediction model did not consider IDH status because the BraTS dataset did not have labels of IDH status for training. This warrants further investigation because IDH status is crucial in the new WHO CNS tumor classification 2021. For future studies, we can incorporate the IDH status to the prediction model for the MGMT methylation in diffuse gliomas because IDH-wildtype and IDH-mutant group shows different tumor biology, although they are classified as adult-type diffuse gliomas, which might lead to different diagnostic performance of MGMT methylation.There are several limitations to this study. (1) We cannot conclude that MGMT prediction is “impossible” using conventional mpMRI. However, it was not achievable with a dataset size of nearly 1000 via thorough experiments, including the external validation of the first place solution of the BraTS 2021 challenge. (2) The risk of overfitting may be alleviated by increasing the number of datasets (In conclusion, contrary to expectations, MGMT methylation cannot be predicted using only conventional structural MRI, even in a deep learning approach with a large-scale multicenter dataset. Although radiogenomics has the capacity to alter how patients with brain tumors are managed in the future, additional tumor characteristics from advanced MRI, such as cellularity and angiogenesis, should be pursued to improve the noninvasive diagnostic performance of MGMT methylation.


These populations are not able to express Lewis antigen and CA 19-9; therefore, CA 19-9 investigation cannot be used for diagnostic and therapeutic measures. In patients highly suspicious of pancreatic cancer where CA 19-9 levels are low, alternative tumor markers such as CA 125 and carcinoembryonic antigen or a combination of various tumor markers can be used to increase sensitivity and specificity in diagnosing pancreatic cancer. A 70-year-old man presented with a complaint of worsening abdominal pain for the last two days. The patient had dark-yellow urine and pale stool. Abdominal ultrasonography and computed tomography scan showed a mass on the head of the pancreas, which was highly suspicious of pancreatic cancer.Pancreatic cancer is the seventh leading cause of cancer-related mortality worldwide and the eleventh most common cause of cancer-related death in Indonesia. In pancreatic cancer, rapid and early diagnosis is crucial. Carbohydrate antigen 19-9 (CA 19-9), the most sensitive and specific tumor marker for pancreatic cancer, may help in diagnosing and determining prognosis; however, some populations do not express CA 19-9. Cases of low CA 19-9 may occur in populations with Lewis The pancreas is an organ that functions as an exocrine and endocrine gland. In 2020, 5,781 cases of pancreatic cancer were reported. Pancreatic cancer is the seventh leading cause of cancer-related mortality globally .The etiology of pancreatic cancer is multifactorial. Non-modifiable risk factors include age over 55 years, especially individuals 70-80 years old; male gender; blood types A, B, and AB; genetics; family history of pancreatic cancer; and type 2 diabetes, especially when the duration is over 10 years. Modifiable risk factors include gastrointestinal and intrapancreatic microflora; smoking or tobacco consumption; occupational exposure to metals or pesticides; alcohol consumption; high red-meat diet, processed meats, cholesterols, and nitrosamine; chronic pancreatitis; acute recurrent pancreatitis; and obesity ,4.Early-stage pancreatic cancer is usually asymptomatic. The non-specific symptoms gradually appear, including abdominal pain, jaundice, pruritus, dark-yellow urine, and pale stool due to biliary system obstruction. In addition, anorexia, significant weight loss, early satiety, dyspepsia, nausea, and malabsorption with steatorrhea may also present . AlthougTumor markers that can help confirm the diagnosis and establish prognosis include carbohydrate antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA), and CA 125 . FurtherA 70-year-old man presented to the Emergency Room (ER) of Wangaya Regional Hospital with a complaint of worsening abdominal pain for the last two days which was mainly localized to the left upper quadrant of the abdomen, with a Visual Analog Scale (VAS) score of 4-5 which worsened after eating. The pain was persistent with nausea, vomiting, and weight loss. In the last year, the patient had experienced several episodes of similar abdominal pain, as well as fatigue and jaundice. Moreover, his urine became dark-yellow similar to tea, and, occasionally, he experienced pale watery stools; his weight decreased by about 10-15 kg. He denied any hematemesis and dark-colored stools. Physical examination showed anemic conjunctiva, icteric sclera, and a palpable mass on the epigastric region of the abdomen with tenderness and normal bowel movement. Before he came to Wangaya Regional Hospital, he admitted that he had sought several medical advice at a local public health center, and was diagnosed with dyspepsia.Blood workup showed normocytic normochromic anemia with hemoglobin of 6.9 g/dL, mean corpuscular volume of 85.4 fL, and mean corpuscular hemoglobin of 30.5 pg, along with increased bilirubin levels  and elevated liver enzymes . Further, blood urea nitrogen level was 22 mg/dL, creatinine 0.7 mg/dL, sodium 141 mEq/L, potassium 3.7 mEq/L, and chloride 100 mEq/L  with normal CA 19-9 levels (≤36 U/mL) at the start of chemotherapy had a longer mean survival rate (30.5 months) compared to most cases with increased CA 19-9  . HoweverA study by Luo et al. ,20 also The low CA 19-9 levels associated with better prognosis might be explained by a hypothesis that CA 19-9 supports any cancer cell metastasis by binding to E-selectin, an adhesion receptor found on the surface of endothelial cells. In addition, according to another hypothesis, CA 19-9 specifically supports the metastasis of pancreatic cancer cells . A studyAn interesting finding of the study was that the CA 125 levels in negative Lewis patients were higher (251.9 ± 642 U/mL) than in positive Lewis patients  . TherefoCA 125, also known as mucin 16 (MUC16), has been recommended by the Chinese Study Group for Pancreatic Cancer for diagnosis, especially in conditions where the CA 19-9 levels are negative and can be used as an additional workup for CA 19-9, especially in hyperbilirubinemia conditions . CA 125 A combination of several tumor markers may increase the sensitivity and specificity. Combined CA 125 and CA 19-9 increases specificity and sensitivity for pancreatic cancer that has not been previously diagnosed [In general, CA 19-9 levels are elevated in pancreatic cancer and have diagnostic, prognostic, therapeutic, and recurrence monitoring benefits. However, there are some cases of pancreatic cancer in which the CA 19-9 test is not reliable, as reported in this case. This phenomenon occurs in negative Lewis antigen populations, with contradicting prognostic implications. In such cases, alternative tumor markers such as CA 125 and CEA or a combination of various tumor markers can be used to increase sensitivity and specificity in diagnosing pancreatic cancer.


Last, FOXQ1 was coexpressed with 47 immune-related genes in pan-cancer (p < 0.05). In conclusion, FOXQ1 expression is closely related to prognosis, clinicopathological parameters, cancer-related pathway activity, the tumor mutational burden, MSI, the tumor microenvironment, immune cell infiltration, and immune-related genes and has the potential to be a diagnostic and prognostic biomarker as well as an immunotherapy target for tumors. Our findings provide important clues for further mechanistic research into FOXQ1.Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family involved in the occurrence and development of different tumors. However, the specific expression patterns and functions of FOXQ1 in pan-cancer remain unclear. Therefore, we collected the expression, mutation, and clinical information data of 33 tumors from The Cancer Genome Atlas database. Via public pan-cancer transcriptome data analysis, we found that FOXQ1 is differentially expressed in various tumors at tissue and cell levels, such as liver hepatocellular carcinoma, colon adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, thyroid carcinoma, and kidney renal clear cell carcinoma. Kaplan–Meier and Cox analyses suggested that FOXQ1 expression was associated with poor overall survival of cutaneous melanoma and thymoma. Its expression was also associated with good disease-specific survival (DSS) in prostate adenocarcinoma but poor DSS in liver hepatocellular carcinoma. In addition, FOXQ1 expression was associated with poor disease-free survival of pancreatic adenocarcinoma. Moreover, FOXQ1 expression was closely related to the tumor mutational burden in 14 tumor types and microsatellite instability (MSI) in 8 tumor types. With an increase in stromal and immune cells, FOXQ1 expression was increased in breast invasive carcinoma, pancreatic adenocarcinoma, thyroid carcinoma, lung adenocarcinoma, and ovarian serous cystadenocarcinoma, while its expression was decreased in pancreatic adenocarcinoma, bladder urothelial carcinoma, and stomach adenocarcinoma. We also found that FOXQ1 expression was related to the infiltration of 22 immune cell types in different tumors ( Cancer is a major public health problem and the leading cause of death worldwide . BecauseStudies have found that tumors occurring in different organs but of the same histological type have strong molecular similarities, such as head and neck, lung, esophageal, bladder, and cervical cancers . There aForkhead box Q1 (FOXQ1), also called HNF-3/fkh homolog-1, is a member of the forkhead box protein family. As a transcription factor, FOXQ1 encodes multiple functional amino acid proteins, activates T cells and autoimmunity, promotes epithelial differentiation, inhibits smooth muscle cell differentiation, and regulates mucous protein expression and particle concentration in gastric surface mucous cells . In addiFOXQ1 can regulate the immune response and influence tumor progression . The expIn this study, The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Oncomine databases were used to analyze the expression profile of FOXQ1 at different levels and the correlation of its expression with prognosis, clinicopathological parameters, cancer-related pathways, tumor mutational burden (TMB), microsatellite instability (MSI), the TME, immune cell infiltration, and immune-related genes to comprehensively unravel the expression profile of FOXQ1 in pan-cancer; understand any changes in expression-related characteristics; and illustrate potential pathogenic mechanisms. Our goal was to provide a reliable basis for identifying potential diagnostic and prognostic biomarkers as well as anticancer immunotherapy targets.https://xenabrowser.net/) was used to download expression, mutation, and clinical information data on 33 tumors. The expression data were normalized by fragments per kilobase of exon model per million mapped fragments. The differential expression of FOXQ1 was identified by a Wilcoxon rank-sum test (p < 0.05). All analyses and visualizations were performed with RStudio 3.6.1. A box diagram was further designed using the “ggpubr” R package. The Oncomine database (https://www.oncomine.org/) was used to validate any differential results with TCGA, and the cutoff criteria were considered to be |log2FC|≥2, p < 0.05, and top 10% gene rank. The online website CCLE  was used to detect FOXQ1 expression in 1,057 cell lines from 36 tumors. Tumors were named as follows: ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B-cell; ESCA: esophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LAML: acute myeloid leukemia; LGG: brain lower-grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; MESO: mesothelioma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumors; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; and UCS: uterine carcinosarcoma; UVM: uveal melanoma.UCSC Xena (p < 0.05), and a survival curve was delineated using the “survminer” and “survival” R packages (p < 0.05). The calculation and visualization were performed using the “Survival” and “forestplot” R packages. The sample size for the prognosis analysis is listed in Survival data in TCGA were used to evaluate the association of FOXQ1 expression with prognosis in different tumor patients. For survival curve analysis, all tumor patients were divided into two groups according to the median FOXQ1 expression. The Kaplan–Meier method and log-rank test were used to analyze the influence of FOXQ1 expression on overall survival (OS), disease-specific survival (DSS), disease-free survival (DFS), and progression-free survival (PFS)  using the “cor.test” command. The visualization was performed with the “fmsb” R package.Mutation data in TCGA were used to assess the TMB and MSI of tumors. TMB was defined as the number of bases mutated per million bases. The MSI score was derived from TCGA data. Next, Spearman correlation analysis was used to assess the association of FOXQ1 expression with the TMB and MSI (p < 0.05) using the “ggplot2,” “ggpubr” and “ggExtra” R packages. In addition, TIMER was used to analyze the association of FOXQ1 with immune cell infiltration (http://timer.comp-genomics.org/).The ESTIMATE algorithm in the “estimate” and “limma” R packages was used to analyze TCGA expression data and thereby calculate the immune score and stromal score. CIBERSORT was used to calculate the immune cell infiltration score. Next, the correlation of FOXQ1 expression with the TME and immune cell infiltration was performed with Spearman correlation analysis (p < 0.05). The “reshape2” and “RColorBrewer” packages were used to perform visualization.In total, 47 immune-related genes were summarized after the literature review, and then TCGA expression data were used for the coexpression of FOXQ1 and immune-related genes. The coexpression profile of FOXQ1 with immune-related genes was evaluated using the “limma” package , thyroid carcinoma (n = 11), bladder urothelial carcinoma (n = 10), and lung adenocarcinoma (n = 10)  ; other r(n = 10) . Its exp(n = 10) . In addip < 0.05), such as CD44, CD86, CD274, TNFRSF9, TIGIT, TNFSF15, TNFRSF18, TNFRSF4, VSIR, and TNFRSF25 (To explore the correlation of FOXQ1 expression with immune-related genes in pan-cancer, coexpression analysis was performed. This work revealed that FOXQ1 was coexpressed with 47 immune-related genes in different cancers (TNFRSF25 . The finTNFRSF25 .This joint analysis of FOXQ1 at different levels is the first to be conducted using pan-cancer expression, survival, and mutation data from TCGA, Oncomine, and CCLE databases. Our results comprehensively summarize the FOXQ1 expression profile at tissue and cell levels and the association of its expression with prognosis, clinicopathological characteristics, cancer-related pathways, TMB, MSI, TME, immune cell infiltration, and immune-related genes. These results deepen our understanding of the FOXQ1 profile in pan-cancer, provide important clues for building immune therapy regimens targeting FOXQ1, and significantly help to guide the exploration of FOXQ1 pathogenic mechanisms in pan-cancer.At the tissue level, TCGA and Oncomine databases showed that FOXQ1 was upregulated in colon adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, thyroid carcinoma, and liver hepatocellular carcinoma and downregulated in kidney cancer and prostate adenocarcinoma. At the cell level, the CCLE database suggested that FOXQ1 was expressed in 1,057 cell lines from 36 tumors, with the background expression level high in pancreatic adenocarcinoma, liver hepatocellular carcinoma, colon adenocarcinoma, lung cancer, and thyroid carcinoma. FOXQ1 has been reported to be highly significantly expressed in colon adenocarcinoma and rectum adenocarcinoma . FOXQ1 iWe also analyzed the correlation of FOXQ1 expression with prognostic and clinicopathological parameters in 33 tumor types. Kaplan–Meier and Cox regression analyses indicated that FOXQ1 expression decreased the OS of skin cutaneous melanoma and thymoma patients. Moreover, its expression influenced the DSS of prostate adenocarcinoma and liver hepatocellular carcinoma. FOXQ1 expression was also associated with poor DFS in pancreatic adenocarcinoma. In addition, FOXQ1 expression affected the tumor progression of bladder urothelial carcinoma, skin cutaneous melanoma, colon adenocarcinoma, thyroid carcinoma, and testicular germ cell tumors and regulated the differentiation degree of tumor cells in bladder urothelial carcinoma, stomach adenocarcinoma, uterine corpus endometrial carcinoma, brain lower-grade glioma, and liver hepatocellular carcinoma. A meta-analysis determined that FOXQ1 expression was related to poor prognosis in malignant solid tumors . FOXQ1 eThe correlation of FOXQ1 expression with cancer-related pathway activity was explored to determine the potential mechanism of FOXQ1 in pan-cancer. The results indicated that FOXQ1 expression was involved in the activation and inactivation of 33 pathways in 12 types of tumors, including the IL6/JAK/STAT3 signaling pathway, allograft rejection pathway, apical junction pathway, epithelial–mesenchymal transition pathway, early estrogen response pathway, interferon–alpha response pathway, RAS signaling pathway, and apoptosis pathway. It has been reported that an activity change in the IL6/JAK/STAT3 signaling pathway can regulate liver hepatocellular carcinoma, chronic myeloid leukemia, and glioma . The epiSpearman correlation analysis was used to identify the relationship of FOXQ1 expression with the TMB and MSI. The results indicated that FOXQ1 expression was associated with the TMB in 14 tumor types, such as cholangiocarcinoma, colon adenocarcinoma, and esophageal carcinoma, and with MSI in 8 tumor types, such as head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, and thyroid carcinoma. A significant correlation has been found between the TMB with the tumor immunotherapy response and the objective response rate of tumor patients . The PFSThe signal transduction pathways involved in FOXQ1 expression might regulate the immune response, which explains the association of FOXQ1 expression with the TME, immune cell infiltration, and immune-related genes. The results showed that with an increase in stromal and immune cells, FOXQ1 expression was increased in breast invasive carcinoma, pancreatic adenocarcinoma, thyroid carcinoma, lung adenocarcinoma, and ovarian serous cystadenocarcinoma, whereas its expression was decreased in pancreatic adenocarcinoma, bladder urothelial carcinoma, and stomach adenocarcinoma. FOXQ1 expression was closely related to the infiltration of 22 immune cell types in 31 tumors, including resting memory CD4 T cells, T follicular helper cells, memory B cells, activated NK cells, plasma cells, naive B cells, M0 macrophages, T regs, resting dendritic cells, and resting mast cells. FOXQ1 was coexpressed with 47 immune-related genes in different cancers, such as CD44, CD86, CD274, TNFRSF9, TIGIT, TNFSF15, TNFRSF18, TNFRSF4, VSIR, and TNFRSF25. The role of FOXQ1 in immunology has been preliminarily confirmed, with previous research showing that cancer-related macrophages induce epithelial–mesenchymal transition by regulating the JAK2/STAT3/miR-506-3p/FOXQ1 axis, thereby enhancing the invasion and migration of colorectal cancer cells . FOXQ1 mIn conclusion, this study is the first to prove that FOXQ1 expression is closely associated with prognosis, clinicopathological parameters, cancer-related pathways, TMB, MSI, TME, immune cell infiltration, and immune-related genes via TCGA, Oncomine, and CCLE databases. We have explored in depth the expression characteristics and possible pathogenic mechanisms of FOXQ1 in tumors, which provide new ideas for the development of novel immunotherapeutic targets.


Group I introns are mobile genetic elements encoding self-splicing ribozymes. Group I introns in nuclear genes are restricted to ribosomal DNA of eukaryotic microorganisms. For example, the myxomycetes, which represent a distinct protist phylum with a unique life strategy, are rich in nucleolar group I introns. We analyzed and compared 75 group I introns at position 516 in the small subunit ribosomal DNA from diverse and distantly related myxomycete taxa. A consensus secondary structure revealed a conserved group IC1 ribozyme core, but with a surprising RNA sequence complexity in the peripheral regions. Five S516 group I introns possess a twintron organization, where a His-Cys homing endonuclease gene insertion was interrupted by a small spliceosomal intron. Eleven S516 introns contained direct repeat arrays with varying lengths of the repeated motif, a varying copy number, and different structural organizations. Phylogenetic analyses of S516 introns and the corresponding host genes revealed a complex inheritance pattern, with both vertical and horizontal transfers. Finally, we reconstructed the evolutionary history of S516 nucleolar group I introns from insertion of mobile-type introns at unoccupied cognate sites, through homing endonuclease gene degradation and loss, and finally to the complete loss of introns. We conclude that myxomycete S516 introns represent a family of genetic elements with surprisingly dynamic structures despite a common function in RNA self-splicing. E. coli rRNA gene numbering system [Group I introns are self-splicing and mobile elements sporadically distributed within genomes of bacteria, bacteriophages, chloroplasts, mitochondria, eukaryotic viruses, and the nuclei of eukaryotes ,2. Nucleg system .Group I introns catalyze their own splicing due to the intron-encoded ribozyme. The splicing mechanism is well described and involves two succeeding transesterification reactions initiated by an attack of an exogenous guanosine (exoG) cofactor ,13. The Nuclear group I introns may also carry additional genes and sequences. Most common are homing endonuclease genes (HEGs), which are found in approximately 5–10% of known nuclear group I introns and code for site-specific endonucleases involved in intron mobility ,20,21,22Mucilago crustacea that contained 34 copies of the intron internal guide sequence [Several examples of direct repeat (DR) arrays within nuclear group I introns have been reported ,11,32,33Naegleria species and in Allovahlkamfia and harbored complex insertions in the P6 segment corresponding to HEGs and lariat-capping ribozymes [In a previous study, we reported self-splicing and structural organization of nuclear S516 group I introns  and haveibozymes ,38,39.In the original S516 intron study [Didymium alpinum isolate Fr-K12, were retrieved from the NCBI database . Spores were germinated and approximately 108 amoeba cells were harvested from DS/2 agar plates, total DNA extracted, and rDNA sequenced as described previously [All rDNA sequences included in this work, except that of ® 2022.0.1 . Multiple sequence alignments were generated using MAFFT version 7.450 [® software program.SSU rDNA sequences (with introns removed) were assembled and aligned in the software program Geneious primeon 7.450 ,41 with on 7.450 ,43, exclon 7.450 ,8,11 usiThe tree-building methods of neighbor joining (NJ), maximum likelihood (ML), maximum parsimony (MP), and minimal evolution (ME), interpreted in MEGA X , were usD. alpinum (Fr-K12) identified a 534-bp group I intron  at position S516 in the nuclear SSU rRNA gene. An RNA secondary structure diagram is presented in Tetrahymena intron [Ribosomal DNA sequence analysis of the myxomycete a intron ,46,47, wD. alpinum Fr-K12) was provided in this study. To assess structural variations among the S516 introns, 143 nucleotide positions in the ribozyme core region common to all 75 introns were strictly aligned according to secondary structure features  tree  is in geL. columbinum isolates cluster together in the SSU rDNA analysis to a well-defined clade (blue box), the corresponding introns are found scattered on the tree, e.g., the L. columbinum (HQ687198) and L. columbinum (HQ687197) introns are distantly related to the other L. columbinum introns. (ii) SSU rDNA analysis strongly support that M. aggregatum and M. cribrariodes are two closely related taxa from the same genus (green box), whereas intron analysis of the core region indicates that the S516 introns are distantly related. This is further supported by structural comparisons , but their corresponding S516 introns suggest a distant relationship. In summary, our phylogenetic analyses suggest that myxomycete S516 introns share a complex evolutionary history, with several clear examples of both vertical and horizontal transfers.Most introns, however, showed a relationship that is not consistent with stable vertical inheritance. To better resolve the evolutionary relationship between S516 introns, we analyzed a smaller dataset consisting of 14 selected taxa , and froparisons , where PL. pseudomaculatum (JQ031985)  (T. varia (KM494993/4/5)  lacking a HEG insertion  , 190 amiY842033) , and 210993/4/5) . Furthernsertion . Interesnsertion A. A nuclnsertion B.Porphyra  [Allovahlkampfia (amoeba-flagellate) [Naegleria (amoeba-flagellate) [Ppo I from the myxomycete Physarum polycephalum [A hallmark of nuclear homing endonucleases is a His-Cys box motif consisting of two zinc coordinating structures (Zn-I and Zn-II) and two catalytic amino acid residues. d algae) , Allovahgellate) , and Naegellate) , as wellcephalum ,52.Elaeomyxa, Comatricha, Colloderma, Lamproderma, Licea, and Trichia), and all except two (Licea and Trichia) classified to the myxomycete order Stemonitales  harbors the largest motif (ca. 220 nt), which is located in P2 and repeated twice. (ii) C. nigricapillitia (AY643824) harbors the smallest motif (only 4 nt) in P2 and is repeated four times. (iii) C. pseudoalpina (DQ903673) harbors DRs in two different extension regions (P1 and P5). While the P1-DR motif is large in size (ca. 110 nt) and repeated four times, the P5-DR is composed of an approximately 60-nt motif and repeated twice. (iv) L. columbinum (HQ687204) also harbors DRs in two extension regions, but in P2 and P9. Two DR motifs are located in P2 (ca. 20 nt and ca. 40 nt), in 5 and 3 copies, respectively, and the DR motif in P9 is 28 nt in size and repeated twice. (v) E. cerifera (JQ031967) harbors three different DR motifs in P6 of 11 nt, 10 nt, and ca. 80 nt in sizes, repeated 4, 6, and 2 times, respectively. (vi) One each of two closely related T. varia introns contains  or lacks  a HEG. Intriguingly, the shorter variant contains a DR, including a HEG remnant sequence, at the proposed HEG deletion site (L. parasitica (JX481297) harbors a complex array in P2. Specifically, the intron folds into a typical group IC1 intron structure (Some isolates showed unique and characteristic DR features : (i) L. ion site . (vii) Ftructure A, and thtructure B. Seven tructure C.D. alpinum Fr-K12 intron. Extension sequences such as HEGs or DR arrays are all located in peripheral segments that do not interfere with the catalytic ribozyme core. HEGs located as insertions into segment P2 in five taxa are interrupted by small spliceosomal introns. Moreover, DR arrays were found in 11 taxa at six peripheral segments, and the L. parasitica intron was found to harbor a highly structured DR motif in 25 copies. Intron evolution analyses indicate a complex inheritance pattern that apparently includes both vertical and horizontal transfers.We report analyses of structural organization and molecular evolution of nucleolar group I intron at position S516 in myxomycetes. All 75 introns belong to the group IC1 subtype and fold at the RNA level into a highly conserved catalytic core, exemplified by the Porphyra red algae [Allovahlkampfia and Naegleria amoeba-flagellates [Naegleria homing endonucleases have been studied in more detail and found to recognize a 19 bp partially symmetrical sequence at the intron-less allele close to the S516 insertion site and to generate a pentanucleotide 3′ overhang at the DNA cleavage site [All HEGs in myxomycete S516 introns encode His-Cys homing endonucleases with a characteristic set of conserved amino acid residues involved in zinc coordination and catalysis. This His-Cys box was found to be highly conserved among S516 HEGs, also outside the myxomycete phylum. These include the ed algae  and the gellates ,39, whicage site ,53. The age site ,29,30,31age site .C. nigricapillitia to 23-nt motif in 25 copies in L. parasitica. A common feature to most arrays was non-identical sequence repeat motifs, which argues against slippage-like mechanisms during replication [Fuligo septica L1949 [M. crustacea S1389 [L. parasitica DR array may suggest a selective structural feature at the RNA level, but no function has currently been assigned.Extension sequences containing DR arrays were noted in six peripheral segments, including P2. The arrays varied from small insertions (4-nt motif in 4 copies) in lication ,55 as a lication ,9,11,33.ca L1949  and M. cea S1389 . The higLamproderma, Echinostelium, and Trichia, but also from Naegleria [Allovhalkampfia [Porphyra [Naegleria martinezi [Bangia fuscopurpurea [Acanthamoeba sp. Isolate [D. alpinum, as well as 93% of the myxomycete S516 introns. HEG deletion was further supported in one of the Trichia S516 introns and suggests a link between deletion of HEG and generation of DR. (4) The complete intron became lost, probably due to homologous recombination with an intron-less allele resulting in SSU rDNA lacking an S516 intron, as noted in the approximately 80% of myxomycetes assessed.The S516 group IC1 intron in myxomycetes and other eukaryotic microorganisms constitute a phylogenetic distinct family with a common origin . While iaegleria , Allovhalkampfia , and PorPorphyra . (2) To artinezi , Bangia purpurea , and an  Isolate . (3) TheThe myxomycete protists represent a unique model system for studying the evolutionary history of self-splicing introns due to an exceptionally high content of nucleolar group I introns. We analyzed 75 group I introns at position 516 in the SSU rDNA of myxomycetes representing five distantly related taxonomic orders. The result reveals a conserved group IC1 ribozyme core, but highly variable peripheral RNA domains containing direct repeat arrays and homing endonuclease genes in 20% of the taxa. All HEGs were interrupted by small spliceosomal introns, which probably facilitate the expression of protein-coding genes embedded in nuclear rDNA. Phylogenetic analysis identified a complex inheritance pattern that can be explained by repeatedly intron gain and loss during the evolution. The current study expands our understanding of the structural organization of group IC1 introns and their evolution.


Scutellaria baicalensis Georgi is an annual herb from the Scutellaria genus that has been extensively used as a traditional medicine for over 2000 years in China. Baicalin and other flavonoids have been identified as the principal bioactive ingredients. The biosynthetic pathway of baicalin in S. baicalensis has been elucidated; however, the specific functions of R2R3-MYB TF, which regulates baicalin synthesis, has not been well characterized in S. baicalensis to date. Here, a S20 R2R3-MYB TF (SbMYB12), which encodes 263 amino acids with a length of 792 bp, was expressed in all tested tissues (mainly in leaves) and responded to exogenous hormone methyl jasmonate (MeJA) treatment. The overexpression of SbMYB12 significantly promoted the accumulation of flavonoids such as baicalin and wogonoside in S. baicalensis hairy roots. Furthermore, biochemical experiments revealed that SbMYB12 is a nuclear-localized transcription activator that binds to the SbCCL7-4, SbCHI-2, and SbF6H-1 promoters to activate their expression. These results illustrate that SbMYB12 positively regulates the generation of baicalin and wogonoside. In summary, this work revealed a novel S20 R2R3-MYB regulator and enhances our understanding of the transcriptional and regulatory mechanisms of baicalin biosynthesis, as well as sheds new light on metabolic engineering in S. baicalensis. R2R3-MYB gene family in a variety of plants  play a critical role in plant development, growth, and stress responses through the self-regulation and control of the expression of target genes . A classthaliana , Populuschocarpa , Brassicica napu , Zea mayZea mays , Ananas  comosus , Oryza sa sativa , Hypericrforatum , etc.). rforatum ,12.MYB (R2R3-MYB) plays a critical role in the positive regulation of flavonoid synthesis, which for Arabidopsis includes S6 (AtMYB11/12/111) and S7 (AtMYB75/90/114) [AtMYB112, which is a member of the stress-resistant S20 family, can positively regulate the accumulation of anthocyanin in Arabidopsis [Flavonoids are ubiquitous in nature and extensively distributed in various plant parts. Studies have revealed that flavonoids are highly conserved in nature, with their synthetic pathway being one of the best-known secondary metabolic pathways in plants today . A parti/90/114) . Studiesbidopsis .Scutellariae Radix is a traditional Chinese remedy derived from the dried roots of S. baicalensis, which is widely used in China. Modern pharmacological studies have shown that baicalin and other flavonoids are the primary pharmacodynamic constituents of S. baicalensis, which have antitumor, antiviral, analgesic, and other beneficial pharmacological effects [S. baicalensis is typically > 10%, which has garnered the attention of many researchers. Recent studies have revealed that baicalin and baicalein can effectively inhibit the replication of the new coronavirus (SARS-CoV-2) [S. baicalensis was observed to increase following water stress, and moderate drought can generally promote the accretion of flavonoids. Suspension cultures of S. baicalensis cells were shown to stimulate the accumulation of flavonoids following light and PEG treatments. The exogenous administration of hormones was observed to stimulate the accumulation of baicalin, which is an active constituent of Baicalaria [S. baicalensis genome, the synthetic pathways of baicalin and other flavonoids were also elucidated. In contrast, very few investigations into the molecular kinetics responsible for the synthesis of flavonoids in S. baicalensis have been undertaken. effects . Thus, iS-CoV-2) ,19. The icalaria . With thAtMYB75, AtMYB90, AtMYB113, and AtMYB114 [MdMYBA, MdMYB1, MdMYB10, and MdMYB110a [SlMYB12 [StAN1 [SmMYB98 [MYB genes at the transcriptome level and found that SbMYB2 and SbMYB7 negatively affected the phenylpropane content in tobacco through the activation of genes involved in flavonoid biosynthesis. However, to date, no studies have investigated the members of R2R3-MYB in S. baicalensis that participate in the regulation of specific flavonoids such as baicalin and wogonoside.R2R3-MYB transcription factors such as AtMYB114 ; MdMYBA,dMYB110a ; and SlM[SlMYB12 , StAN1 [2 [StAN1 ,24, and [SmMYB98  etc., ha[SmMYB98  identifiS. baicalensis, we identified 95 members of the R2R3-MYB family, of which the gene expression levels of SbMYB12 and SbMYB74 were significantly increased following ABA and MeJA treatments [AtMYB112, which belongs to the S20 family in A. thaliana, positively regulated the accumulation of anthocyanins and other flavonoids [R2R3-MYB member (SmMYB98) was reported to positively regulate the synthesis and accumulation of salvianolic acid in the transgenic roots of S. miltiorrhiza. The sequence structure of SbMYB12 is akin to that of AtMYB112 and SmMYB98, which suggests that these proteins may perform similar functions in regulating secondary metabolites.In one of our earlier studies based on the whole-genome data of eatments . AtMYB11avonoids . FurtherR2R3-MYB (SbMYB12) was isolated and characterized from S. baicalensis, which strongly responded to the exogenous MeJA treatment. The regulatory properties of SbMYB12 in overexpressed S. baicalensis hairy root cultures revealed that it enhanced the accumulation of baicalin and wogonoside via the upregulation of a series of key structural genes involved in flavonol biosynthesis. To the best of our knowledge, this is the first report to demonstrate that R2R3-MYB from S. baicalensis has the capability to enhance the accumulation of baicalin and wogonoside.To address this knowledge gap, in this study, a novel member of S20 SbMYB12 was classified in subgroup S20 with AtMYB112, which is responsible for the accumulation of flavonoids and anthocyanins in Arabidopsis [SbMYB12 attracted our attention, as it appeared to play an important role in the regulation of flavonoid biosynthesis in S. baicalensis [SbMYB12 was significantly upregulated in response to MeJA stress, with its expression reaching a peak after 3 h and being upregulated ~10-fold [SbMYB12 was responsive to MeJA.Based on our previous research , SbMYB12bidopsis . Combinecalensis . In part~10-fold . The indSbMYB12, the roots, root pericytes, root phloem, root xylem, stems, leaves, flowers, and calyx of two-year-old S. baicalensis plants were assessed by qRT-PCR. Although the expression of SbMYB12 was verified for all tested tissues, the highest expression occurred in the leaves , Leu (10.3%), Asn (7.6%), Glu (7.2%), Arg (6.5%), and Asp (6.1%) were prominent. There are 35 negatively charged residues (Asp + Glu) and 32 positively charged residues (Arg + Lys). SbMYB12 exhibited an overall average hydropathicity of −0.786, which implies that the protein is hydrophilic. The protein’s instability index (II) was 62.72, meaning that it was unstable. According to the TMHMM and SignalP web software, SbMYB12 lacked signal peptides and transmembrane sites, which suggests that the protein is a non-secretory and/or non-transmembrane protein  revealed that SbMYB12 has higher homology with SiMYB62, SmMYB98, HaMYB62, and NaMYB62  transgenic hairy roots via the A. rhizogenes A4-strain-mediated transformation method   C. OE-1, SbMYB12-OE hairy roots and the CK. As shown in SbMYB12-OE lines was quite different from that of the CK, which may have been caused by different concentrations of secondary metabolites. We quantified the concentrations of five flavonoids  in the SbMYB12-OE lines and the CK  in the promoter regions of SbCLL7-4, SbCHI-2, and SbF6H-1. Y1H assays were performed, which revealed that SbMYB12 interacts with the promoter regions of SbCLL7-4, SbCHI-2, and SbF6H-1  were reported to be primarily involved in stress responses and the regulation of secondary metabolism [AtMYB2 was responsible for ABA-induced salt and drought response genes [AtMYB62 regulated phosphate starvation and gibberellic acid biosynthesis [AtMYB108 was involved in both biotic and abiotic stress responses [AtMYB112 (S20) was engaged with regulating the accumulation of anthocyanins and flavonoids in the phenylpropanoid biosynthetic pathway [S. miltiorrhiza, SmMYB9b-OE (a S20 R2R3-MYB) efficiently increased the tanshinone concentration in S. miltiorrhiza transgenic roots [SmMYB98) positively regulated the synthesis and accumulation of salvianolic acid and tanshinone by activating the transcription of PAL1, GGPPS1, and RAS1 in the transgenic roots of S. miltiorrhiza [SbMYB12 may have functions akin to the homologous genes SmMYB98 and AtMYB112, which are involved in the regulation of secondary metabolites [SbMYB12 responded to different exogenous hormone and abiotic stress treatments, particularly exogenous MeJA, and may play important roles in the regulation of flavonoid biosynthesis in S. baicalensisAs one of the largest TF families in plants, the MYB family was divided into four subfamilies, namely 1R, R2R3, 3R, and 4R-MYBs . Among tubgroups . Among tse genes ; AtMYB62ynthesis ; AtMYB10esponses . Furtheric roots . Subsequic roots  reportediorrhiza . As a meabolites . MoreoveSbMYB12 may be a key regulator of flavonoid biosynthesis, we conducted further investigations. As described earlier, SbMYB12 transcripts were expressed in all tested tissues and preferentially expressed in S. baicalensis leaves  were characterized based on a non-reference transcriptome, and transgenic tobacco plants overexpressing SbMYB2, SbMYB7, or SbMYB8 exhibited a higher phenylpropane content by activating the expression of flavonoid-biosynthesis-related genes [SbMYBs participated in the regulation of S. baicalensis-specific flavonoids such as baicalin and wogonoside. Of particular note was that the SbMYB2, SbMYB7, and SbMYB8 mentioned above were renamed as SbMYB45, SbMYB95, and SbMYB18, respectively, in the latest study based on their sequence similarity [SbMYB12 most likely functions as an activator of some of the S. baicalensis-specific flavonoid biosynthesis in S. baicalensis. However, additional experimental evidence is required to verify this.The flavonoids  were confirmed to be the main active components of ae Radix , which pae Radix ,19,35. Bae Radix . The funed genes ,36. Howemilarity . Thus, bSbMYB12 was localized within the nucleus  and activate their expression . When the lateral buds of S. baicalensis germinated and grew to 2 cm, they were removed and transferred to an MS solid medium  without hormones and cultivated in an incubator to obtain sterile seedlings of S. baicalensis for the transformation experiments.Sterile S. baicalensis plants at the germplasm resource garden of Shaanxi Normal University, respectively, for RNA extraction.Various tissues including roots, pericytes, phloem, xylem, stems, leaves, flowers, and calyx were harvested from two-year-old S. baicalensis plantlets using the TIANGEN RNA Prep Pure Plant kit . TransScript® First-Strand cDNA Synthesis SuperMix  was used for cDNA synthesis. SbMYB12 (evm.model.contig76.33) full-length cDNA was employed as the reference sequence, and the primers ) was employed to predict the primary structure of the SbMYB12 protein, as well as its amino acid composition, molecular weight, theoretical isoelectric point (pI), and stability [https://web.expasy.org/protscale/ (accessed on 20 September 2022)) was used to analyze its hydrophobic properties and charge distributions [https://services.healthtech.dtu.dk/service.php?TMHMM-2.0 (accessed on 20 September 2022)) was used [https://services.healthtech.dtu.dk/service.php?SignalP-5.0 (accessed on 20 September 2022)) was employed to estimate signaling peptides [http://smart.embl-heidelberg.de (accessed on 20 September 2022)) [SbMYB12 were predicted, while its tertiary structure was predicted using SWISS-MODEL (https://swissmodel.expasy.org/ (accessed on 20 September 2022)) [The ExPASy-ProtParam tool ) , the pror 2022)) .SbMYB12 and 17 members from the S20 subgroup R2R3-MYB were used to construct a phylogenetic tree with MEGA11 (https://www.megasoftware.net/dload_mac_beta (accessed on 20 September 2022)) using the neighbor-joining method (NJ) [https://www.lynnon.com/dnaman.html (accessed on 20 September 2022)) was used to perform multiple sequence alignments between SbMYB12 and other MYB TF species. SbMYB12 and other MYB proteins.The hod (NJ) . DNAMAN SbACT7 gene as a reference [SbMYB12 and other corresponding genes were calculated using the 2−ΔΔCt method [Based on previously described procedures , the toteference . SYBR grt method . All priSbMYB12 proteins in cells, SbMYB12-GFP fusion protein expression vectors were developed by amplifying the SbMYB12 coding sequence and fusing it with HBT-GFP-NOS vectors. Arabidopsis leaves were cultured under 12 h light/12 h darkness for four weeks, and mesophyll protoplasts were prepared as previously described [To determine the locations of escribed . The traSbMYB12 was cloned and integrated into the pGBKT7 (BD) vector. Saccharomyces cerevisiae strain AH109  was transformed with recombinant vector BD-SbMYB12 and cultured for 2–3 days at 28 °C on SD/-Trp , then screened on SD/-Trp/-Ade/-His/X-α-gal  media to assay the transactivation activity.Using a Gateway recombinatorial cloning system , the ORFSbMYB12 was cloned into the pK7WG2R (with a dsRed marker gene) to generate the overexpression vector pK7WG2R-SbMYB12 using the Gateway recombinatorial cloning system  [S. baicalensis infected by the Agrobacterium rhizogenes A4 strain  containing the pK7WG2R-SbMYB12 as described previously [SbMYB12-overexpressed hairy roots were confirmed by DsRed protein fluorescence and the presence of rolB, rolC, and SbMYB12 at the genomic DNA level, as described earlier [The full-length CDS of CA, USA) . Transgeeviously . In para earlier . For liqThe flavonoid content was measured using previously described techniques . BrieflySbMYB12 CDS was cloned into the BamH I and Xho I  sites of the pGADT7(AD) vector to generate AD-SbMYB12. The promoter fragments of SbCLL7-4, SbF6H-1, and SbCHI-2 were inserted into the EcoR I and Sac II  sites of the pHIS2 vector to generate pHIS2-pSbCLL7-4, pHIS2-pSbF6H-1, and pHIS2-pSbCHI-2, respectively. The Yeast One-Hybrid (Y1H) assay was performed as described in our previous protocols [The complete rotocols .t-tests. Each bar represents the mean and standard deviation (SD) of three replicates. The significance of a statistical difference was defined as * p ≤ 0.05 and ** p ≤ 0.01, respectively.The SPSS software (version 26.0) was utilized to analyze the differences via one-way ANOVA (followed by Tukey’s comparisons) and Student’s S. baicalensis (SbMYB12) was isolated and functionally characterized. Further, SbMYB12 was found to be a nuclear-localized transcription activator that positively regulates the accumulation of flavonoids, such as baicalin and wogonoside, by directly binding to the promoter regions of the key enzyme genes SbCCL7-4, SbCHI-2, and SbF6H-1. Together, these results enrich the understanding of the function of the R2R3-MYB gene in medicinal plants and illustrate the exploitation of R2R3-MYB in flavonoid biosynthesis, which provides a feasible strategy for enhancing the baicalin and wogonoside concentrations via MYB proteins in S. baicalensis. In addition, SbMYB12 was also the first transcription factor cloned and verified gene function in the hairy root transgenic system of S. baicalensis, which lays the foundation for further research on regulating the functional genes of S. baicalensis.For this study, a novel R2R3-MYB subgroup 20 TF from


Background: Neurofibromatosis type 1 (NF1) is a hereditary cancer syndrome characterized by multiple café-au-lait macules on the skin. Lymphoproliferative malignancies associated with NF1 are limited, although the most common are brain tumors. Case presentation: A 22-year-old woman with NF1 was admitted due to abdominal pain and bloody diarrhea. Her laboratory data exhibited macrocytic anemia and elevated IgA levels. Image studies showed diffuse increased wall thickening in the transverse and descending colon without lymphadenopathy and hepatosplenomegaly. A colonoscopy revealed a hemorrhagic ulcerated mass. Pathological analysis of the tumor tissues confirmed IgA-expressing mucosa-associated lymphoid tissue (MALT) lymphoma with histological transformation. Moreover, whole-exome sequencing in tumor tissues and peripheral blood mononuclear cells identified a somatic frameshift mutation of the A20 gene, which represents the loss of function. The patient responded well to R-CHOP chemotherapy, but the disease relapsed after 1 year, resulting in a lethal outcome. Conclusions: MALT lymphoma in children and young adults is extremely rare and is possibly caused by acquired genetic changes. This case suggests a novel association between hereditary cancer syndrome and early-onset MALT lymphoma. Helicobacter pylori-positive primary gastric MALT lymphoma, the standard treatment is eradication therapy [Mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal marginal zone lymphoma that arises from B cells in the marginal zone of secondary lymphoid tissues ,2,3. It  therapy . The rec therapy ,2,3. App therapy . Histolo therapy ,8,9.Neurofibromatosis type 1 (NF1), previously known as von Recklinghausen disease, is a hereditary cancer syndrome characterized by neurofibromas, multiple café-au-lait macules (CALMs), and optic gliomas, with a frequency of 1/3000–4000 persons ,11,12,13A20 gene , a key regulator of nuclear factor kappa-light-chain-enhancer of activated B cells, in tumor tissues, and peripheral blood mononuclear cells (PBMCs) [In the present study, we report the first case of a young adult patient with NF1 who developed MALT lymphoma with histological transformation to DLBCL. Whole-exome sequencing (WES) identified a somatic mutation in the  (PBMCs) .A 22-year-old Japanese woman was referred to our hospital due to unbearable abdominal pain and continuous bloody diarrhea, which had initially presented as recurrent abdominal pain and watery diarrhea 3 months previously. She was diagnosed with NF1 in infancy because of multiple CALMs (more than six) and axillary freckling. In addition, she had a history of epilepsy, mental retardation, and Addison’s disease, and had been taking two antiepileptic drugs  and low-dose oral corticosteroids for a long time. There was no family history of consanguineous marriage or genetic disorders.Physical examination revealed short stature (−3SD), low body weight (−2SD), pale conjunctivae, multiple CALMs on her limbs and trunk , axillarThe patient was admitted to our hospital urgently due to severe symptoms. A colonoscopy performed the day after admission revealed mucosal redness, edema, and a hemorrhagic ulcerated mass extending from the splenic flexure to the descending colon d,e. A biA20, TNIP1, and FAS genes) and three non-synonymous mutations  in the tumor tissues. These were de novo somatic mutations associated with the development of B-cell lymphoma. Moreover, these frameshift mutations were also identified in the patient’s PBMCs. Sanger sequencing identified a frameshift insertion in the A20 gene, indicating the loss of function in the tumor tissues and PBMCs of our patient  following the Declaration of Helsinki. Briefly, genomic DNA was extracted from the PBMCs of our patient and her parents, and from the resected colonic tissues of the patient, without fixation ,20. WES  patient . HoweverConsidering the risk of secondary malignancies, we selected at least six courses of R-CHOP  as standard therapy for DLBCL, but not followed by radiation therapy or upfront autologous transplantation. After starting one cycle of R-CHOP, the patient had no grade 3/4 non-hematological toxicities, and her symptoms improved dramatically. After six cycles of R-CHOP, there were no visible massive lesions on CT and the colonoscopy showed normal findings; however, the follow-up biopsy specimen revealed residual MALT lymphoma. One year after therapy completion, the disease relapsed and the patient’s condition rapidly deteriorated to multiple organ failure, and eventually, a lethal outcome.We retrospectively reviewed the patient’s medical records to estimate the onset of the lymphoma. IgA levels were initially elevated at the age of 8 years and gradually increased from the age of 16 years, whereas the IgG levels simultaneously decreased a. A CT sNF1 gene encodes neurofibromin, a negative regulator of the RAS proto-oncogene, which affects the RAS/MAPK pathway to promote cell proliferation and survival [NF1 gene have been largely linked to some cancers including melanoma, lung cancer, and breast cancer [NF1 gene in lymphoma are limited [NF1 genetic mutations, our patient met the revised 2021 diagnostic criteria for NF1 [The survival ,11,12,13survival ,15,21. Ft cancer . Among tt cancer . As for t cancer . However limited ,17. Gene limited ,25. Our  for NF1 ,25.MLH1, MSH2, MSH6, and PMS2 [PMS2 gene, but not a biallelic mutation. In this report, we could not establish a definitive diagnosis of CMMRD syndrome in our patient, who had already been diagnosed with NF1. Treatment of cancers in CMMRD is commonly challenging because of the resistance to chemotherapeutics [Multiple CALMs were associated with several inherited genetic syndromes such as NF1 and the McCune–Albright, Noonan, Watson, Legius, and constitutional mismatch repair deficiency (CMMRD) syndromes . CMMRD iand PMS2 . Most paand PMS2 ,28. Notaand PMS2 ,30. The apeutics ,28,29,30apeutics ,28,29,30Helicobacter pylori infection is the major risk factor associated with the development of gastric MALT lymphoma; however, the pathogenesis of MALT lymphoma of the colon remains unclear [A20 gene, which represents the functional loss of the A20 protein. In particular, the A20 genetic mutation allele frequency in PBMCs was identified as 12–15%, supporting the somatic mosaic mutation. Mutations and deletions in the A20 gene are involved in the pathogenesis of MALT lymphoma [A20 gene caused chronic inflammatory or autoimmune conditions in the colon, leading to the accumulation of multiple mutations and the development of MALT lymphoma. Furthermore, mutations in other genes including TP53, BTG1, and BCL7A were also confirmed in the tumor tissues, possibly causing the development of DLBCL. Our patient with suspected CMMRD syndrome may have been predisposed to acquire the A20 genetic mutation. Further analysis of the A20 somatic mutation identified in our patient may clarify the mechanisms of pathogenesis associated with early-onset non-Hodgkin lymphoma, particularly MALT lymphoma.MALT lymphomas are often associated with underlying autoimmune disease and chronic viral infection ,2,3. The unclear . The WESlymphoma . Moreovelymphoma . AlthougAlthough monoclonal gammopathy was found in 15–20% of patients with low-grade and aggressive B-cell lymphoma, the frequency of IgA-expressing lymphomas was very low . The preImmunoproliferative small intestinal disease (IPSID) is a rare variant of MALT lymphoma with villous atrophy in the small intestine, most commonly described as Mediterranean lymphoma  ,38,39. IA20 gene, possibly involved in the development of MALT lymphoma, in tumor tissues, and PBMCs. This case supports that hereditary cancer syndrome should be considered as a differential diagnosis in early-onset MALT lymphoma. Furthermore, identification of the underlying genetic changes in patients with NF1 will enable the pathogenesis and long-term management of secondary malignancies.We present the first case of a young adult patient with NF1 who developed intestinal MALT lymphoma with histological transformation. In addition, we identified a somatic mutation in the


This study is designed to investigate the regulatory effect of MNQ on the key cancer genes in mitogen-activated protein kinase, phosphoinositide 3-kinase, and nuclear factor кB signaling pathways. The expression levels of the genes were compared at different time point using polymerase chain reaction arrays and Ingenuity Pathway Analysis was performed to identify gene networks that are most significant to key cancer genes. A total of 43 differentially expressed genes were identified with 21 up-regulated and 22 down-regulated genes. Up-regulated genes were involved in apoptosis, cell cycle and act as tumor suppressor while down-regulated genes were involved in anti-apoptosis, angiogenesis, cell cycle and act as transcription factor as well as proto-oncogenes. MNQ exhibited multiple regulatory effects on the cancer key genes that targeting at cell proliferation, cell differentiation, cell transformation, apoptosis, reduce inflammatory responses, inhibits angiogenesis and metastasis. The process of carcinogenesis is relay on over-expression of cancer genes through gene amplification, inappropriate expression of normal genes, or mutations in genes ,2. AccumThe complication of carcinogenesis involves several cancer hallmarks. These underlying principles are describing how normal cells transform to malignant or tumor cells. Hallmarks of cancer refers to behaviors of cancer cells that sustained to proliferative signals, resisted to cell death signals, evading from growth suppressors, enabling replicative immortality, genome instability and mutation, continues tumor-promoting inflammation, deregulating cellular energetic, avoiding immune destruction, inducing angiogenesis, and activating invasion and metastasis . MultiplCancer cells are known to have alterations in multiple cellular signaling pathways involving the affected genes . AberranMore than 10 cancer signaling pathways have been identified . In a feImpatiens balsamina, Linn from the family of Balsaminaceae, is an ornamental plant that has been used to treat various skin diseases locally. 2-Methoxy-1,4-naphthoquinone (MNQ), isolated from pericarps of Impatiens balsamina Linn. has been shown to cause cytotoxic towards various cancer cell lines [ll lines , show anll lines ,16, trigll lines , inhibitll lines . Furtherll lines , alteredll lines .PKC is one of the protein kinases that play a role in carcinogenesis and maintenance of malignant phenotype. Moreover, it is also the core upper stream kinase involved in mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and nuclear factor кB (NF-κB) pathways that regulate many down-stream proteins implicated in these cancer signaling pathways. In our previous study, MNQ has been demonstrated to inhibit PKC βI, δ, and ζ ; therefon-butyrate (SnB) were obtained from Sigma. MNQ was isolated from the pericarps of Impatiens balsamina, Linn. The compound was prepared in dimethyl sulfoxide and stored at 4ºC. The reagents obtained from Qiagen  were RT2 First Strand Kit , RT2 STBR Green ROX FAST mastermix .Phorbol 12-myristate 13-acetate (PMA) and sodium 2 incubator .Raji cells obtained from Riken Cell Bank, Japan were maintained in commercial Roswell Park Memorial Institute Media (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) . The cells were incubated in a humidified atmosphere at 37°C with 5% COThe high-performance liquid chromatography (HPLC) chromatogram of the isolated MNQ was first identified by comparing the HPLC chromatogram of MNQ HPLC chromatogram obtained by Teng  in our p5 cells/mL) were incubated in 1 mL of RPMI 1640 medium (supplemented with 10 % FBS) containing 0.05 µM PMA, 3 mM SnB, and MNQ in a 24-well plate and then incubated at 37°C for 6, 12, 24, and 48 h in a CO2 incubator. PMA and SnB was used as inducer and enhancer respectively to cause Epstein-Barr virus activation in Raji cells and directly transform Raji cells into promotion stage.Raji cells  following the manufacturer’s instructions. The genomic DNA elimination mix was prepared accordingly to the manual given by Qiagen.RNA of the treated Raji cells was extracted according to the manufacturer’s instruction using RNeasy Mini Kit (Qiagen). RT2 SYBR Green ROX FAST Mastermix according to manufacturer’s protocol. RT2 Profiler PCR Array for the detection of key cancer genes regulation in MAPK, PI3K, and NF-κB pathways were tested in three independent experiments. The genes expression results were calculated using results generated by the RT cycler and the ∆∆CT method. The CT values for all wells were exported to PCR Array Data Analysis Template Excel sheet  and uploaded to web-based software at www.sabiosciences.com/pcrarraydataanalysis.php. This software helps in calculating CT values collected and shows p-values and fold change for each gene. The connection between genes was analyzed using web-based resources Gene Network Central Pro at http://gncpro.sabiosciences.com/gncpro/gncpro.php.The cDNA was added with RTThe identified key cancer genes were first uploaded onto Qiagen’s Ingenuity Pathway Analysis (IPA) system for core analysis and then overlaid with the global molecular network in the ingenuity pathway knowledge base. IPA was performed to identify canonical pathways, diseases and functions, and gene networks that are most significant to key cancer genes and to categorize differentially expressed genes in specific molecular and cellular functions. Thresholds of two-fold or greater in changes in expression and a p-value of 0.05 or less for significance were used to filter the findings from the analysis with IPA software.−ΔΔCt) was set at greater than 5.0 or less than −4.0.The results were obtained from three separate experiments. In terms of the sensitivity and accuracy, the cutoff fold change . The fold changes of all statistically significant genes involved in these pathways are shown in BAD, EGR1, FOXO1, FOXO3, ITGB1, LTA, TNF, TNFRSF1A, TNFRSF10B, TRADD, YWHAH), tumor suppressor genes  and cell cycle regulation genes . The confirmed 22 down-regulated genes were anti-apoptotic genes , angiogenesis genes , cell cycle regulation genes , transcription factors (FOS and JUN), proto-oncogenes (BCL3).From the 43 genes identified, MNQ up-regulated 21 genes and down-regulated 22 genes. The identified 21 up-regulated genes were apoptotic genes  are involved in MAPK pathway  are involved in PI3K pathway  are involved in NF-κB pathway  are involved in all three pathways  from the total 218 cancer key genes studied in these three signaling pathways ‒3. TheseLAMTOR3, TP53, RB1, CDKN1A, CDKN1B, CDKN2A and CDKN2D and were up-regulated across all time points  in MNQ-treated Raji could possibly explain that Raji cells tried to sustain their survival in the presence of the abovementioned genes in inducing cell cycle arrest and apoptosis. This phenomenon can be observed from LAMTOR3 expression were gradually increased in amount from 6 to 48 h treatment. In addition, EGRF, epidermal growth factor receptor involved in cell cycle was down-regulated by MNQ. Furthermore, proto-oncogene genes that are involved in cell growth and differentiation such as ARAF, BRAF, KRAS, HRAS and NRAS were also down-regulated across all time points. Overall, MNQ mainly caused cell cycle arrest, supressed cell proliferation and induced apoptosis in Raji cells via MAPK pathway.In this study, MNQ activated the apoptosis, tumor suppressor and cell cycle regulatory activities in MAPK pathway, where the genes of e points , Fig. 1.PTEN, FOXO1, FOXO3, YWHAH, MTOR, ITGB1, BAD, and APC genes in PI3K pathway (APC) was found to be the most highest expressed gene across all time points. APC is a tumor suppressor that indirectly regulate a number of key genes involved in cell proliferation. Besides, it also plays a role in cell migration [PTEN is another tumour suppressor gene up-regulated by MNQ that generally involved in the regulation of cell cycle, and it is known to be the target of many anticancer drugs. FOXO1 and FOXO3 have been reported to trigger apoptosis via regulating genes that responsible for cell death such as BIM and PUMA [BAD is a known pro-apoptotic gene that initiate apoptosis. YWHAH encodes for 14-3-3 protein eta has been reported to regulate a wide variety of signaling pathways, and mainly mediated these signaling pathways by binding to phosphoserine-containing proteins. MTOR that encode for mTOR complex 1 and 2 proteins has been reported to regulate cell growth, cell proliferation, cell motility and cell survival [ITGB1 gene encodes Integrin beta-1 surface receptor that are involved in cell proliferation, cell adhesion and recognition as well as metastatic diffusion of tumor cells [PDGFRA, AKT2, AKT3, RPS6KA1, IGF1, and IGF1R, genes. PDGFRA gene has been reported to instruct the making of platelet-derived growth factor receptor alpha (PDGFRA). PDGFRA proteins activate signaling pathways to control many important cellular processes such as cell growth, division and survival. Mutation on PDGFRA has been reported in gastrointestinal stromal tumors and in chronic myeloid leukemia. Besides, highly phosphorylated PDGFRA has been observed in non‒small cell lung cancer and rhabdomyosarcoma [AKT2 and AKT3 are the serine/threonine kinases that regulate cellular metabolism, cell proliferation, cell survival, cell growth and angiogenesis. These genes are highly activated in many cancer cells. The AKT protein kinase has been indicated to transduce growth factors to oncogenes and target the proteins to induce tumor development. Thus, inhibition of the AKT kinase is workable in cancer prevention as down-regulation of AKT signaling is a strategy to prevent cancer formation [RPS6KA1 encodes ribosomal protein S6 kinase alpha-1 kinase that phosphorylate various substrates of the MAPK singling pathway, which is implicated in controlling cell growth and differentiation. RPS6KA1 is also known as RSK1 that present in cytoplasm. RSK1 is an ERK effector that is involved mainly in nuclear signaling, cell growth, survival, and cell proliferation. It has been reported to regulate SRF, c-Fos, and Nur77 transcription factors. RSK1 stimulated cell growth by regulating c-Fos and promotes cyclin D1 expression. Besides, RSK1 has been demonstrated to inhibit neuronal NO synthase in response to mitogenic signaling. RSK1 is highly expressed in prostate and breast cancers. RSK1 triggered survival signals via the Ras/ERK signaling pathway and protected cancer cells from apoptosis. RSK1 is also involved in cell cycle regulation by phosphorylating CDKN1B and preventing CDKN1B translocation into nucleus and thus inhibited G1 progression. RSK1 also promoted cell survival by suppressing the functions of pro-apoptotic proteins BAD and DAPK1. MNQ down-regulated insulin-like growth factor 1 (IGF1) and insulin-like growth factor 1 receptor (IGF1R) significantly. IGF family is a humoral mediator of growth hormone (GH) [In this study, MNQ activated tumor suppressor, apoptosis and cell cycle regulation through upregulating  pathway , Fig. 2,igration . PTEN isand PUMA . BAD is survival . ITGB1 gor cells . In our or cells , MNQ wasosarcoma , AKT2 anormation . Akt kinone (GH) . The IGFone (GH) . IGF hasone (GH) . IGF1R ione (GH) .TRADD, LTA, TNFRSF1A, TNFRSF10B, and TNF genes, and down-regulate NFKB2, NFKB1E, IL6, TIMP1, and BCL3 genes. LTA is a cytokine produced by lymphocytes that belong to tumor necrosis factor family. LTA bound to TNFRSF1A/TNFR1, TNFRSF1B/TNFBR, TNFRSF14 and induced apoptosis. TNFRSF1A is expressed in transformed cells to trigger apoptosis or inflammation [TNFRSF1A has been studied to display resistance towards apoptosis [TNFRSF1A signals apoptosis through caspase-8 [TNFα) has been indicated to promote apoptosis in human endothelial cells through TNFRSF1A and trigger caspase-2 and p53 activation. It took part in variety cellular responses, such as survival, differentiation, proliferation, apoptosis, and migration [TNFRSF1A has been shown to promote the recruitment of TNFR-associated factors (TRAFs), FAS-associated via death domains (FADDs), and TNFR-associated via death domains (TRADDs). TNFRs interact with TRAFs, FADDs and TRADDS to control apoptosis. TNFα has been reported to bind with TNFRSF1A to induce apoptosis by activating caspase-8 [TRADD. NFKB1, NFKB2, NFKB1A, and NFKB1E are the genes encode for protein members of NF-κB family. Two angiogenesis genes of IL6 and TIMP1 were down-regulated by MNQ. IL6 is an inflammatory cytokine that modulate growth and differentiation in tumor cells through STAT3 signaling pathway. Overexpression of IL6 has been reported to be associated with tumor progression through inhibition of cancer cell apoptosis, stimulation of angiogenesis, and drug resistance. Clinical studies have been reported IL6 protein as a regulator that associate with many types of cancers such as multiple myeloma, non‒small cell lung adenocarcinoma, prostate cancer, colorectal cancer, renal cell carcinoma, breast cancer, and ovarian cancer [BCL3 was the only down-regulated proto-oncogene by MNQ. BCL3 is a proto-oncogene deregulated in solid tumors. BCL3 has been revealed to induce proliferation and inhibit apoptosis. BCL3 activated STAT3, an aggressive oncogene in human cancer and promoted metastasis [For NF-κB pathway, MNQ was found to up-regulate ammation . Mutatiopoptosis . TNFRSF1aspase-8 . Tumor nigration . TNFα isigration . TNFRSF1aspase-8 . Tumor naspase-8 , caspaseaspase-8 . STAT1, n cancer ,41. Tissn cancer ,43. BCL3tastasis ,45. In ltastasis -54. Thestastasis  and STATtastasis . In additastasis .BAD, EGR1, FOXO1, FOXO3, ITGB1, LTA, TNF, TNFRSF1A, TNFRSF10B, TRADD, and YWHAH), tumor suppressor genes , and cell cycle genes . On the contrary, MNQ down-regulated anti-apoptotic genes , angiogenesis genes (IL6 and TIMP1), cell cycle genes , transcription factors (FOS and JUN), and proto-oncogenes (BCL3).In a nutshell, MNQ up-regulated apoptotic genes (The findings of this study have revealed the capability of MNQ in regulating the key cancer genes in MAPK, PI3K, and NF-κB signaling pathways. The regulatory effects exerted by MNQ are targeting at cell proliferation, cell differentiation, cell transformation, induce apoptosis, reduce inflammatory responses, inhibits angiogenesis and metastasis. These results underline the need to further investigate the mechanism of actions of MNQ at each molecular level of these genes.