18β-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells.

Gastric carcinoma (GC) is a common gastrointestinal malignancy worldwide. Based on the cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. To explore the mechanism by which 18β-glycyrrhetinic acid (18β-GRA) regulates mitochondrial ribosomal protein L35 (MRPL35) related signal proteins to inhibit the proliferation of GC cells. Cell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823. The apoptosis and cell cycle were assessed by flow cytometry. Cell invasion and migration were evaluated by Transwell assay, and cell scratch test was used to detect cell migration. Furthermore, a tumor model was established by hypodermic injection of 2.5 × 106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect MRPL35 expression in the engrafted tumors in mice. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry to screen for differentially expressed proteins (DEPs) extracted from GC cells and control cells after 18β-GRA intervention. A detailed bioinformatics analysis of these DEPs was performed, including Gene Ontology annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and so on. Moreover, STRING database (https://string-db.org/) was used to predict protein-protein interaction (PPI) relationships and Western blot was used to detect the expression of proteins of interest in GC cells. The results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose- and time-dependent manner. It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase. The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose, and the migration and invasiveness of GC cells were inhibited. The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice, and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice. Using TMT technology, 609 DEPs, among which 335 were up-regulated and 274 were down-regulated, were identified in 18β-GRA intervention compared with control. We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways, such as cellular processes, biological regulation, and TP53 signaling pathway. Notably, after the drug intervention, MRPL35 expression was significantly down-regulated (P = 0.000247), TP53 expression was up-regulated (P = 0.02676), and BCL2L1 was down-regulated (P = 0.01699). Combined with the Retrieval of Interacting Genes/Proteins database, we analyzed the relationship between MRPL35, TP53, and BCL2L1 signaling proteins, and we found that COPS5, BAX, and BAD proteins can form a PPI network with MRPL35, TP53, and BCL2L1. Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells, MRPL35, TP53, and BCL2L1 showed dose-dependent up/down-regulation, and the expression of COPS5, BAX, and BAD also increased/decreased with the change of 18β-GRA concentration. 18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35, COPS5, TP53, BCL2L1, BAX, and BAD.

Yuan L ，Yang Y ，Li X ，Zhou X ，Du YH ，Liu WJ ，Zhang L ，Yu L ，Ma TT ，Li JX ，Chen Y ，Nan Y ... -  《-》

18β-glycyrrhetinic acid inhibits proliferation of gastric cancer cells through regulating the miR-345-5p/TGM2 signaling pathway.

Gastric cancer (GC) is a common gastrointestinal malignancy worldwide. Based on cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. To explore the molecular mechanism of 18β-glycyrrhetinic acid (18β-GRA) regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells. CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells. Cell cycle and apoptosis were detected by flow cytometry, cell migration was detected by a wound healing assay, the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated, and the cell autophagy level was determined by MDC staining. TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention, and then the protein-protein interaction was predicted using STRING (https://string-db.org/). MicroRNAs (miRNAs) transcriptome analysis was used to detect the miRNA differential expression profile, and use miRBase (https://www.mirbase/) and TargetScan (https://www.targetscan.org/) to predict the miRNA and complementary binding sites. Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells, and western blot was used to detect the expression of autophagy related proteins. Finally, the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression. 18β-GRA could inhibit GC cells viability, promote cell apoptosis, block cell cycle, reduce cell wound healing ability, and inhibit the GC cells growth in vivo. MDC staining results showed that 18β-GRA could promote autophagy in GC cells. By TMT proteomic analysis and miRNAs transcriptome analysis, it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells. Subsequently, we verified that TGM2 is the target of miR-345-5p, and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2. Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced, and LC3II, ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA. Overexpression of miR-345-5p not only inhibited the expression of TGM2, but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle. 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.

Li X ，Ma XL ，Nan Y ，Du YH ，Yang Y ，Lu DD ，Zhang JF ，Chen Y ，Zhang L ，Niu Y ，Yuan L ... -  《-》

18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3.

Gastric cancer (GC) is one of the most common cancer types worldwide, and its prevention and treatment methods have garnered much attention. As the active ingredient of licorice, 18β-glycyrrhetinic acid (18β-GRA) has a variety of pharmacological effects. The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC, in order to provide effective ideas for the clinical prevention and treatment of GC. To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells. Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs (miRNAs) in GC cells after 18β-GRA intervention. Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability, cell colony formation ability was detected by the clone formation assay, and flow cytometry was used to detect the cell cycle and apoptosis. A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells. Tumor tissue morphology was observed by hematoxylin and eosin staining, and microtubule-associated protein light chain 3 (LC3) expression was detected by immunohistochemistry. TransmiR, STRING, and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3 (STAT3)-related information. Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction (qPCR) and the expression levels of STAT3, phosphorylated STAT3 (p-STAT3), and LC3 were detected by western blot analysis. The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system. AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label. LC3 was labeled and autophagy flow was observed under a confocal laser microscope. The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells (P = 4.51E-06). Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability, arrested the cell cycle in the G0/G1 phase, promoted cell apoptosis, and inhibited the growth of subcutaneous tumors in BALB/c nude mice (P < 0.01). No obvious necrosis was observed in the tumor tissue in the negative control group (no drug intervention or lentivirus transfection) and vector group (the blank vector for lentivirus transfection), and more cells were loose and necrotic in the miR-328-3p group. Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3, and STAT3 was closely related to autophagy markers such as p62. After overexpressing miR-328-3p, the expression level of STAT3 mRNA was significantly decreased (P < 0.01) and p-STAT3 was downregulated (P < 0.05). The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT3 3' untranslated regions of the wild-type reporter vector group was significantly decreased (P < 0.001). Overexpressed miR-328-3p combined with bafilomycin A1 (Baf A1) was used to detect the expression of LC3 II. Compared with the vector group, the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated (P < 0.05), and compared with the Baf A1 group, the expression level of LC3 II in the overexpressed miR-328-3p + Baf A1 group was upregulated (P < 0.01). The expression of LC3 II was detected after intervention of 18β-GRA in GC cells, and the results were consistent with the results of miR-328-3p overexpression (P < 0.05). Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis (P < 0.001). qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention (P < 0.05). Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention (P < 0.05). 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

Yang Y ，Nan Y ，Du YH ，Huang SC ，Lu DD ，Zhang JF ，Li X ，Chen Y ，Zhang L ，Yuan L ... -  《-》

Depletion of MRPL35 inhibits gastric carcinoma cell proliferation by regulating downstream signaling proteins.

Gastric carcinoma (GC) is a digestive system disease with high morbidity and mortality. However, early clinical detection is difficult, and the therapeutic effect for advanced disease is not satisfactory. Thus, finding new tumor markers and therapeutic targets conducive to the treatment of GC is imperative. MRPL35 is a member of the large subunit family of mitochondrial ribosomal protein. MRPL35 shows the characteristic of oncogene in colorectal cancer and esophageal cancer, which promotes the exploration of the correlation between MRPL35 and GC. We proposed that the expression of MRPL35 might be critical in GC. To study the effect of MRPL35 knockdown on GC cell proliferation. The expression of MRPL35 in GC was evaluated based on data from the public tumor database UALCAN (www.ualcan.path.uab.edu). The effect of the expression of MRPL35 on the prognosis was evaluated with KMplot (www.kmplot.com). The expression of MRPL35 was assessed on the tissue microarray by immunohistochemistry and the level of MRPL35 mRNA in 25 pairs of clinical GC tissues and matched adjacent tissues was detected by quantitative reverse transcription-polymerase chain reaction. Celigo cell count assay, colony formation assay, and flow cytometry were used to assess the role of MRPL35 in GC cell proliferation and apoptosis in vitro. Additionally, tumor formation experiment in BALB/c nude mice was utilized to determine the effect of MRPL35 on GC cell proliferation. After knockdown of MRPL35, related proteins were identified by isobaric tags for relative and absolute quantification analysis, and the expression of related proteins was detected by Western blot. The expression of MRPL35 was up-regulated in GC (P = 1.77 × 10-4). The Kaplan-Meier plots of the overall survival indicated that high expression of MRPL35 was associated with a poor survival in GC. Compared with adjacent tissues, the expression of MRPL35 in GC tissues was increased, which was related to age (P = 0.03), lymph node metastasis (P = 0.007), and pathological tumor-node-metastasis stage (P = 0.024). Knockdown of MRPL35 inhibited GC cell proliferation and colony formation and induced apoptosis. Animal experiment results showed that knockdown of MRPL35 inhibited tumor formation in BALB/c nude mice. Western blotting analysis showed that after knockdown of MRPL35, the expression of PICK1 and BCL-XL proteins decreased, and that of AGR2 protein increased. Collectively, our findings demonstrate that knockdown of MRPL35 inhibits GC cell proliferation through related proteins including PICK1, BCL-XL, and AGR2.

Yuan L ，Li JX ，Yang Y ，Chen Y ，Ma TT ，Liang S ，Bu Y ，Yu L ，Nan Y ... -  《-》

Dihydroartemisinin inhibits the tumorigenesis and invasion of gastric cancer by regulating STAT1/KDR/MMP9 and P53/BCL2L1/CASP3/7 pathways.

Dihydroartemisinin (DHA), an effective antimalarial drug, has been widely investigated as an anti-tumor agent. Although previous studies have indicated the potential therapeutic effects of DHA on multiple malignancies, its detailed molecular mechanisms in gastric cancer (GC) are still undocumented. In the present study, we applied network pharmacology and bioinformatics (gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses) to obtain the collective targets of DHA and GC and analyzed their involvement in constructing a protein-protein interaction (PPI) network. The top 10% hub targets in this network were identified, and TCGA database was utilized for the single gene analysis of their correlation with the prognosis of GC. CCK8, EdU, Transwell, and flow cytometry analyses were conducted, and subcutaneous xenograft tumor models were constructed to assess the effects of DHA on the tumorigenesis and invasion of GC. Furthermore, the targets of DHA were verified by molecular docking, quantitative real-time PCR (qPCR) and western blot analyses in GC cells. The results indicated that the common targets of DHA and GC were enriched in multiple cancer-related pathways including KDR, STAT1 and apoptosis signaling pathways, where the core genes included KDR, MMP9, STAT1, TP53, CASP3/7 and BCL2L1. The lowered expression of KDR and increased expression of TP53 and CASP7 harbored a favorable survival for patients with GC patients. CASP7 showed a positive correlation with CASP3 but a negative correlation with KDR and could be regarded as an independent protective factor for overall survival in GC. Moreover, DHA treatment induced cell apoptosis and suppressed the cell proliferation, DNA synthesis, cycle progression and invasive capabilities both in vitro and in vivo. DHA also upregulated p53, CASP3, and cleaved-CASP3 and downregulated BCL2L1, MMP9, KDR, p-KDR, STAT1 and p-STAT1 in GC cell lines. In conclusion, DHA could suppress the tumorigenesis and invasion of GC by regulating STAT1/KDR/MMP9 and p53/BCL2L1/CASP3/7 pathways. Our findings might provide a novel approach for the treatment of GC.

Liang R ，Chen W ，Chen XY ，Fan HN ，Zhang J ，Zhu JS ... -  《-》