OBJECTIVE: Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment. METHODS: The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway. RESULTS: According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway. CONCLUSION JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.
Animals ; Mice ; Humans ; Carcinoma, Hepatocellular/genetics* ; beta Catenin/pharmacology* ; Liver Neoplasms/genetics* ; Drugs, Chinese Herbal/therapeutic use* ; RNA, Messenger/therapeutic use* ; Cell Line, Tumor ; Cell Proliferation ; Cell Movement ; Gene Expression Regulation, Neoplastic

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker─vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein. Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis. Then, by adding N-glycosylation processing inhibitor kifunensine into the medium, complex glycan was inhibited to generate. The residual glycan of purified protein was removed by endoglycosidase H. Finally, AP protein was removed by using human rhinovirus protease and size exclusion chromatography. The concentration of purified Necl-4 protein was monitored by measuring the absorbance at 280 nm and analyzed by SDS-PAGE. Result The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector. The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%. Conclusions By using modified AP mammalian protein expression system, we can easily screen the high productive stable cell lines by using AP activity assay. By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H, we can obtain deglycosylated Necl-4 protein in milligram quantities. Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.

OBJECTIVETo explore the functions of miR-9 and miR-9(*) in SAMP8 mice during the aging and their possible mechanisms.

METHODSSAMP8 mice(4-,8-,12-month old,respectively)were selected,three age-matched SAMR1 mice were used as the control group with three mice in each group. The brains were collected and then sectioned for in situ hybridization of miR-9 and miR-9(*). Mimics or inhibitors of miR-9 and miR-9(*) were transfected into N2a cells,and the effects of overexpression or knockdown of the microRNAs on the cell cycle were detected by flow cytometry. Target genes were predicted by bioinformatic analysis and confirmed by dual luciferase assay.

RESULTSExpressions of miR-9 and miR-9(*) in hippocampus of SAMP8 mice were lower than those of SAMR1 mice. Knockdown of miR-9 and miR-9(*) induced a prolonged G1 phase and a shortened S phase in N2a cells;in contrast,miR-9 and miR-9(*) overexpression showed opposite effects. The predicted target genes of miR-9 were PSEN1,SCN2B,MAP3K3,and BACE1,and that of miR-9(*) was CDKn1c. Dual luciferase reporter gene assay showed that miR-9 targeted MAP3K3 while miR-9(*) targeted CDKn1c.

CONCLUSIONmiR-9 and miR-9(*) play an important role during aging via the target genes MAP3K3 and CDKn1c in the SAMP8 mice.

Aging ; Animals ; Brain ; Cyclin-Dependent Kinase Inhibitor p57 ; Mice ; MicroRNAs

OBJECTIVETo screen the proteins associated with four-and-a-half LIM domains 3 (FHL3) 3' untranslated region (3'UTR) in glioma cells.

METHODSWestern blot was adopted to detect the regulatory effect of poly(C)-binding protein 2 (PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immuno- precipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1 (PTBP1), a binding protein identified by LC-MS/MS.

RESULTSPCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein.

CONCLUSIONSPCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.

3' Untranslated Regions ; Base Sequence ; Blotting, Western ; Brain Neoplasms ; genetics ; metabolism ; Cell Line, Tumor ; Chromatography, Liquid ; DNA Primers ; Glioma ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; LIM Domain Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Tandem Mass Spectrometry

Objective To screen the proteins associated with four-and-a-half LIM domains 3 (FHL3) 3’ untranslated region (3’UTR) in glioma cells.
Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2 (PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3’UTR. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immuno-precipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1 (PTBP1), a binding protein identified by LC-MS/MS.
Results PCBP2 could bind to FHL3 mRNA 3’UTR-A and inhibited the expression of FHL3 in T98G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3’UTR and interacted with PCBP2 protein.
Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3’UTR through forming a protein complex.

OBJECTIVETo screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1).

METHODSWestern blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1.

RESULTSThe expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (> 38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites.

CONCLUSIONSThe high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.

Arginine ; analogs & derivatives ; analysis ; Cell Line, Tumor ; Chromatography, Liquid ; Glioma ; chemistry ; Humans ; Immunoprecipitation ; Protein-Arginine N-Methyltransferases ; analysis ; physiology ; Repressor Proteins ; analysis ; physiology ; Substrate Specificity ; Tandem Mass Spectrometry

OBJECTIVETo screen and identify the possible existence of natural antisense transcript (NAT) within the mouse neocortex.

METHODSSixty-three cerebral cortex layer-specific genes were screened by bioinformatics prediction in mice, among which 31 mice with potential NATs were screened. NAT was identified using reverse transcription polymerase chain reaction (RT-PCR) and then cloned in pGEM-T Vector System for sequencing.

RESULTSAmong 31 genes predicted using bioinformatics, 8 were proved to be NAT positive by RT-PCR.

CONCLUSIONSNATs exist in the mouse neocortex tissue during the development of cerebral cortex. NATs may influence mouse cortical development by regulating the related coding genes.

Animals ; Cell Line ; Cerebral Cortex ; Mice ; Molecular Sequence Data ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics

OBJECTIVETo investigate the molecular mechanism of nectin-like molecule 1 (NECL1) inhibiting the migration and invasion of U251 glioma cells.

METHODSWe infected U251 glioma cells with adeno-nectin-like molecule 1 (Ad-NECL1) or empty adenovirus (Ad). Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells. DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines. The differential expression of osteopontin (OPN), a gene related to migration and invasion, was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry.

RESULTSThe restoration of NECL1 inhibited migration of U251 cells significantly (P<0.05). Altogether 195 genes were found differentially expressed by microarray, in which 175 were up-regulated and 20 down-regulated, including 9 extracellular matrix proteins involved in the migration of cells. Both mRNA and protein expressions of OPN, the most markedly reduced extracellular matrix protein, were found decreased in U251 cells after restoration of NECL1. Immunohistochemical assay also detected an increase of OPN in glioma tissues, related with the progressing of malignant grade.

CONCLUSIONA link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.

Brain Neoplasms ; metabolism ; pathology ; Cell Adhesion Molecules ; physiology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; physiology ; Glioma ; metabolism ; pathology ; Humans ; Nectins ; Neoplasm Invasiveness ; physiopathology ; Neoplasm Metastasis ; physiopathology ; Osteopontin ; genetics

OBJECTIVETo assess the expression level of D-Tyr-tRNA(Tyr) deacylase (DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease (AD).

METHODSAltogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice. Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice. The behavior of the mice was tested by using a Step-through Test System.

RESULTSBoth mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice (P<0.05). In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.

CONCLUSIONSDTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.

Alzheimer Disease ; enzymology ; Aminoacyltransferases ; metabolism ; Animals ; Base Sequence ; DNA Primers ; Disease Models, Animal ; Mice ; Reverse Transcriptase Polymerase Chain Reaction