BACKGROUND: Lung cancer is a major threat to human health. The molecular mechanisms related to the occurrence and development of lung cancer are complex and poorly known. Exploring molecular markers related to the development of lung cancer is helpful to improve the effect of early diagnosis and treatment. Long non-coding RNA (lncRNA) THAP7-AS1 is known to be highly expressed in gastric cancer, but has been less studied in other cancers. The aim of the study is to explore the role and mechanism of methyltransferase-like 3 (METTL3) mediated up-regulation of N6-methyladenosine (m6A) modified lncRNA THAP7-AS1 expression in promoting the development of lung cancer. METHODS: Samples of 120 lung cancer and corresponding paracancerous tissues were collected. LncRNA microarrays were used to analyze differentially expressed lncRNAs. THAP7-AS1 levels were detected in lung cancer, adjacent normal tissues and lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The diagnostic value of THAP7-AS1 in lung cancer and the relationship between THAP7-AS1 expression and survival rate and clinicopathological parameters were analyzed. Bioinformatics analysis, methylated RNA immunoprecipitation (meRIP), RNA pull-down and RNA-immunoprecipitation (RIP) assay were used to investigate the molecular regulation mechanism of THAP7-AS1. Cell proliferation, migration, invasion and tumorigenesis of SPC-A-1 and NCI-H1299 cells were determined by MTS, colony-formation, scratch, Transwell and xenotransplantation in vivo, respectively. Expression levels of phosphoinositide 3-kinase/protein kenase B (PI3K/AKT) signal pathway related protein were detected by Western blot. RESULTS: Expression levels of THAP7-AS1 were higher in lung cancer tissues and cell lines (P<0.05). THAP7-AS1 has certain diagnostic value in lung cancer [area under the curve (AUC)=0.737], and its expression associated with overall survival rate, tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05). METTL3-mediated m6A modification enhanced THAP7-AS1 expression. The cell proliferation, migration, invasion and the volume and mass of transplanted tumor were all higher in the THAP7-AS1 group compared with the NC group and sh-NC group of SPC-A-1 and NCI-H1299 cells, while the cell proliferation, migration and invasion were lower in the sh-THAP7-AS1 group (P<0.05). THAP7-AS1 binds specifically to Cullin 4B (CUL4B). The cell proliferation, migration, invasion, and expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3 kinase, catalytic subunit delta (PIK3CD), phospho-phosphatidylinositol 3-kinase (p-PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) were higher in the THAP7-AS1 group compared with the Vector group of SPC-A-1 and NCI-H1299 cells (P<0.05). CONCLUSIONS LncRNA THAP7-AS1 is stably expressed through m6A modification mediated by METTL3, and combines with CUL4B to activate PI3K/AKT signal pathway, which promotes the occurrence and development of lung cancer.
Humans ; Lung Neoplasms/pathology* ; RNA, Long Noncoding/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Up-Regulation ; Proto-Oncogene Proteins c-akt/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Methyltransferases/metabolism* ; Cullin Proteins/genetics*

OBJECTIVE: To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. METHODS: HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. RESULTS: HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). CONCLUSION HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; ErbB Receptors/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Cell Proliferation ; RNA, Messenger/genetics* ; Cell Movement ; Cell Line, Tumor ; Chalcone/analogs & derivatives* ; Quinones

We investigated the effects of decursin on the proliferation, apoptosis, and migration of colorectal cancer HT29 and HCT116 cells through the phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway. Decursin(10, 30, 60, and 90 μmol·L~(-1)) was used to treat HT29 and HCT116 cells. The survival, colony formation ability, proliferation, apoptosis, wound hea-ling area, and migration of the HT29 and HCT116 cells exposed to decursin were examined by cell counting kit-8(CCK8), cloning formation experiments, Ki67 immunofluorescence staining, flow cytometry, wound healing assay, and Transwell assay, respectively. Western blot was employed to determine the expression levels of epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), vimentin, B-cell lymphoma/leukemia-2(Bcl-2), Bcl-2-associated X protein(Bax), tumor suppressor protein p53, PI3K, and Akt. Compared with the control group, decursin significantly inhibited the proliferation and colony number and promoted the apoptosis of HT29 and HCT116 cells, and it significantly down-regulated the expression of Bcl-2 and up-regulated the expression of Bax. Decursin inhibited the wound healing and migration of the cells, significantly down-regulated the expression of N-cadherin and vimentin, and up-regulated the expression of E-cadherin. In addition, it significantly down-regulated the expression of PI3K and Akt and up-regulated that of p53. In summary, decursin may regulate epithelial-mesenchymal transition(EMT) via the PI3K/Akt signaling pathway, thereby affecting the proliferation, apoptosis, and migration of colorectal cancer cells.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; bcl-2-Associated X Protein ; Vimentin/metabolism* ; Cell Proliferation ; Signal Transduction ; Apoptosis ; Cell Line, Tumor ; Colorectal Neoplasms/genetics* ; Cadherins/genetics* ; Cell Movement

This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.
Rats ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Caspase 3/genetics* ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Plant Bark ; Anti-Inflammatory Agents/therapeutic use* ; Arthritis, Experimental/chemically induced* ; Inflammation/drug therapy* ; Cytokines/metabolism* ; Proto-Oncogene Proteins c-bcl-2 ; Apoptosis

Objective To investigate the effect of viral myocarditis serum exosomal miR-320 on apoptosis of cardiomyocytes and its mechanism. Methods The model of viral myocarditis mice was established by intraperitoneal injection of Coxsackie virus B3. Serum exosomes were extracted by serum exosome extraction kit and co-cultured with cardiomyocytes. The uptake of exosomes by cardiomyocytes was detected by laser confocal microscopy. Cardiomyocytes were transfected with miR-320 inhibitor or mimic, and the expression level of miR-320 was detected by real-time quantitative PCR. Flow cytometry was used to detect cardiomyocyte apoptosis rate, and the expression levels of B cell lymphoma 2 (Bcl2) and Bcl2-related X protein (BAX) were tested by Western blot analysis. The prediction of miR-320 target genes and GO and KEGG enrichment analysis were tested by online database. The relationship between miR-320 and its target gene phosphoinositide-3-kinase regulatory subunit 1(Pik3r1) was examined by luciferase reporter gene. The effect of miR-320 on AKT/mTOR pathway protein was detected by Western blot analysis. Results Viral myocarditis serum exosomes promoted cardiomyocyte apoptosis, and increased the level of BAX while the level of Bcl2 was decreased. miR-320 was significantly up-regulated in myocardial tissue of viral myocarditis mice, and both pri-miR-320 and mature of miR-320 were up-regulated greatly in cardiomyocytes. The level of miR-320 in cardiomyocytes treated with viral myocarditis serum exosomes was significantly up-regulated, while transfection of miR-320 inhibitor counteracted miR-320 overexpression and reduced apoptosis rate caused by exosomes. Pik3r1 is the target gene of miR-320, and its overexpression reversed cardiomyocyte apoptosis induced by miR-320 up-regulation. The overexpression of miR-320 inhibited AKT/mTOR pathway activation. Conclusion Viral myocarditis serum exosome-derived miR-320 promotes apoptosis of mouse cardiomyocytes by inhibiting AKT/mTOR pathway by targeting Pik3r1.
Mice ; Animals ; Myocytes, Cardiac ; Phosphatidylinositol 3-Kinase/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Myocarditis/pathology* ; Exosomes/metabolism* ; bcl-2-Associated X Protein/metabolism* ; MicroRNAs/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis/genetics*

OBJECTIVE: To investigate the effect of Akt2 inhibitor on macrophage polarization in the periapical tissue in a rat model of periapical inflammation. METHODS: Rat models of periapical inflammation were established in 28 normal SD rats by opening the pulp cavity of the mandibular first molars, followed by injection of normal saline and Akt2 inhibitor into the left and right medullary cavities, respectively. Four rats without any treatment served as the healthy control group. At 7, 14, 21 and 28 days after modeling, 7 rat models and 1 control rat were randomly selected for observation of inflammatory infiltration in the periapical tissues by X-ray and HE staining. Immunohistochemistry was used to detect the expression and localization of Akt2, macrophages and the inflammatory mediators. RT-PCR was performed to detect the mRNA expressions of Akt2, CD86, CD163, inflammatory mediators, miR-155-5p and C/EBPβ to analyze the changes in macrophage polarization. RESULTS: X-ray and HE staining showed that periapical inflammation was the most obvious at 21 days after modeling in the rats. Immunohistochemistry and RT-PCR showed that compared with those in the control rats, the expressions of Akt2, CD86, CD163, miR-155-5p, C/EBPβ, and IL-10 increased significantly in the rat models at 21 days (P < 0.05). Compared with saline treatment, treatment with the Akt2 inhibitor significantly decreased the expression levels of Akt2, CD86, miR-155-5p and IL-6 and the ratio of CD86⁺M1/CD163⁺M2 macrophages (P < 0.05) and increased the expression levels of CD163, C/EBPβ and IL-10 in the rat models (P < 0.05). CONCLUSION Inhibition of Akt2 can delay the progression of periapical inflammation in rats and promote M2 macrophage polarization in the periapical inflammatory microenvironment possibly by reducing miR-155-5p expression and activating the expression of C/EBPβ in the Akt signaling pathway.
Rats ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; MicroRNAs/genetics* ; Interleukin-10 ; Rats, Sprague-Dawley ; Macrophages/metabolism* ; Inflammation/metabolism*

BACKGROUND: There have been many significant advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC). However, the mechanism underlying the progression of NSCLC is still not clear. Plant homodomain finger-like domain-containing protein 5A (PHF5A) plays an important role in processes of chromatin remodeling, morphological development of tissues and organs and maintenance of stem cell pluripotency. This study aims to investigate the role of PHF5A in the proliferation and migration of NSCLC. METHODS: A549 and PC-9 PHF5A overexpression cell lines were constructed. PHF5A expression was decreased in H292 and H1299 cells by using siRNA. Flow cytometry was used to detect the cell cycle. MTT assay and clone formation assay were used to examine the proliferative ability of NSCLC, while migration assay and wound healing assay were performed to evaluate the ability of migration. Western blot analysis was used to measure the expressions of PI3K, p-AKT and the associated downstream factors. RESULTS: Up-regulation of PHF5A in A549 and PC-9 cells increased the proliferation rate, while down-regulation of PHF5A in H292 and H1299 cells inhibited the proliferation rate at 24 h, 48 h and 72 h (P<0.05). The metastatic ability was elevated in the PHF5A-overexpresion groups, while reduced in the PHF5A-down-regulation group (P<0.05). In addition, reduced expression of PHF5A induced cell cycle arrest at G1/S phase (P<0.05). Furthermore, decreased expression of PHF5A reduced the expression levels of PI3K, phosphorylation of AKT, c-Myc (P<0.05) and elevated the expression of p21 (P<0.05). CONCLUSIONS These results demonstrated that PHF5A may play an important role in progression of NSCLC by regulating the PI3K/AKT signaling pathway.
Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic ; Trans-Activators/genetics* ; RNA-Binding Proteins/metabolism*

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.
Humans ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; MicroRNAs/metabolism* ; Glioma/genetics* ; Genes, Tumor Suppressor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor

Treating patients with esophageal squamous cell carcinoma (ESCC) is challenging due to the high chemoresistance. Growth differentiation factor 15 (GDF15) is crucial in the development of various types of tumors and negatively related to the prognosis of ESCC patients according to our previous research. In this study, the link between GDF15 and chemotherapy resistance in ESCC was further explored. The relationship between GDF15 and the chemotherapy response was investigated through in vitro and in vivo studies. ESCC patients with high levels of GDF15 expression showed an inferior chemotherapeutic response. GDF15 improved the tolerance of ESCC cell lines to low-dose cisplatin by regulating AKT phosphorylation via TGFBR2. Through an in vivo study, we further validated that the anti-GDF15 antibody improved the tumor inhibition effect of cisplatin. Metabolomics showed that GDF15 could alter cellular metabolism and enhance the expression of UGT1A. AKT and TGFBR2 inhibition resulted in the reversal of the GDF15-induced expression of UGT1A, indicating that TGFBR2-AKT pathway-dependent metabolic pathways were involved in the resistance of ESCC cells to cisplatin. The present investigation suggests that a high level of GDF15 expression leads to ESCC chemoresistance and that GDF15 can be targeted during chemotherapy, resulting in beneficial therapeutic outcomes.
Humans ; Esophageal Squamous Cell Carcinoma/drug therapy* ; Cisplatin/metabolism* ; Esophageal Neoplasms/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinoma, Squamous Cell/genetics* ; Growth Differentiation Factor 15/therapeutic use* ; Receptor, Transforming Growth Factor-beta Type II/therapeutic use* ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic

OBJECTIVE: Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness. METHODS: A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected. RESULTS: Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin⁺/CD31⁺ ratio, rather than the number of CD31⁺ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group. CONCLUSION Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.
Humans ; Animals ; Mice ; Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Proliferating Cell Nuclear Antigen/therapeutic use* ; Mice, Nude ; Glycogen Synthase Kinase 3 beta/genetics* ; beta Catenin/therapeutic use* ; Liver Neoplasms/drug therapy* ; Desmin/therapeutic use* ; Ki-67 Antigen ; Cell Line, Tumor ; Hypoxia ; RNA, Messenger/therapeutic use* ; Cell Proliferation