[Bupleuri Radix-Paeoniae Radix Alba medicated plasma exerts effects on HepG2 hepatoma cells by regulating miR-1297/PTEN signaling axis].

The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2 hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3'-untranslated region(3'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2 hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.

Integrative Bioinformatics Analysis Reveals Potential Target Genes and PTEN Signaling in Breast Cancer and Effect of Zingiber officinale (Ginger) and Allium sativum (Garlic) extract on It.

INTRODUCTION: Breast cancer is the most common type of cancer in women. The construction of a competing gene network is an important step in the identification of the role of hub genes in breast cancers. In the current research, we used a number of bioinformatics tools to construct this network in breast cancer and investigated the combined effect of garlic and ginger on mice model of breast cancer. MATERIALS AND METHODS: We chose female mice weighing 18-20 g that were divided into 7 groups including; the cancer group receiving normal saline, different doses of ginger extract (100 and 500 mg/kg), different doses of garlic (50 and 100 mg/kg), tamoxifen (10 mg/ kg) and simultaneous garlic (100 mg/kg) and ginger (500 mg/kg) for 3 weeks intraperitoneal. Then we anesthetized the mice, isolated the tumor, and determined its size. Glutathione reductase and peroxidase levels and HER2, PTEN, and Cullin3 genes expression were measured. RESULTS: We identified 20 hub genes for breast cancer. In animal phase we found that tumor size in all mice receiving garlic and ginger showed a significant decrease compared to the control. Glutathione reductase showed a significant increase in all groups, especially in ginger 500 and combined groups. Glutathione peroxidase increased almost in all groups, especially in ginger 500. Expression of HER2 decreased in all treated groups. Expression of PTEN increased just in the combined group. CONCLUSION: Taken together, we introduce a number of novel promising diagnostic biomarkers for breast cancer. The use of garlic and ginger in the treatment of cancer can be useful. This action is probably through the antioxidant mechanism, and regulation of the expression of cancer related genes such as PTEN.

Enhanced Chemoprevention of Prostate Cancer by Combining Arctigenin with Green Tea and Quercetin in Prostate-Specific Phosphatase and Tensin Homolog Knockout Mice.

The low bioavailability of most phytochemicals limits their anticancer effects in humans. The present study was designed to test whether combining arctigenin (Arc), a lignan mainly from the seed of Arctium lappa, with green tea (GT) and quercetin (Q) enhances the chemopreventive effect on prostate cancer. We performed in vitro proliferation studies on different cell lines. We observed a strong synergistic anti-proliferative effect of GT+Q+Arc in exposing androgen-sensitive human prostate cancer LNCaP cells. The pre-malignant WPE1-NA22 cell line was more sensitive to this combination. No cytotoxicity was observed in normal prostate epithelial PrEC cells. For an in vivo study, 3-week-old, prostate-specific PTEN (phosphatase and tensin homolog) knockout mice were treated with GT+Q, Arc, GT+Q+Arc, or the control daily until 16 weeks of age. In vivo imaging using prostate-specific membrane antigen (PSMA) probes demonstrated that the prostate tumorigenesis was significantly inhibited by 40% (GT+Q), 60% (Arc at 30 mg/kg bw), and 90% (GT+Q+Arc) compared to the control. A pathological examination showed that all control mice developed invasive prostate adenocarcinoma. In contrast, the primary lesion in the GT+Q and Arc alone groups was high-grade prostatic intraepithelial neoplasia (PIN), with low-grade PIN in the GT+Q+Arc group. The combined effect of GT+Q+Arc was associated with an increased inhibition of the androgen receptor, the PI3K/Akt pathway, Ki67 expression, and angiogenesis. This study demonstrates that combining Arc with GT and Q was highly effective in prostate cancer chemoprevention. These results warrant clinical trials to confirm the efficacy of this combination in humans.

PTEN-induced kinase 1 gene single-nucleotide variants as biomarkers in adjuvant chemotherapy for colorectal cancer: a retrospective study.

BACKGROUND: Fluoropyrimidine-based postoperative adjuvant chemotherapy is globally recommended for high-risk stage II and stage III colon cancer. However, adjuvant chemotherapy is often associated with severe adverse events and is not highly effective in preventing recurrence. Therefore, discovery of novel molecular biomarkers of postoperative adjuvant chemotherapy to identify patients at increased risk of recurrent colorectal cancer is warranted. Autophagy (including mitophagy) is activated under chemotherapy-induced stress and contributes to chemotherapy resistance. Expression of autophagy-related genes and their single-nucleotide polymorphisms are reported to be effective predictors of chemotherapy response in some cancers. Our goal was to evaluate the relationship between single-nucleotide variants of autophagy-related genes and recurrence rates in order to identify novel biomarkers that predict the effect of adjuvant chemotherapy in colorectal cancer. METHODS: We analyzed surgical or biopsy specimens from 84 patients who underwent radical surgery followed by fluoropyrimidine-based adjuvant chemotherapy at Saitama Medical University International Medical Center between January and December 2016. Using targeted enrichment sequencing, we identified single-nucleotide variants and insertions/deletions in 50 genes, including autophagy-related genes, and examined their association with colorectal cancer recurrence rates. RESULTS: We detected 560 single-nucleotide variants and insertions/deletions in the target region. The results of Fisher's exact test indicated that the recurrence rate of colorectal cancer after adjuvant chemotherapy was significantly lower in patients with the single-nucleotide variants (c.1018G > A [p < 0.005] or c.1562A > C [p < 0.01]) of the mitophagy-related gene PTEN-induced kinase 1. CONCLUSIONS: The two single-nucleotide variants of PINK1 gene may be biomarkers of non-recurrence in colorectal cancer patients who received postoperative adjuvant chemotherapy.

Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis.

Lentinan (LNT) isolated from Lentinus edodes is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT on the mouse hepatocellular carcinoma (HCC) cell line Hepa1­6 and its possible mechanism. Mouse HCC apoptosis and its potential associated mechanism were then explored using in vitro and in vivo approaches. For in vitro approaches, the effect of LNT on the proliferation of Hepa1­6 cells was investigated by Cell Counting Kit­8 assay. Annexin V­FITC staining and flow cytometry were applied to explore HCC apoptosis. Western blotting was used to analyze related proteins, such as EGR1, phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (p­Akt), protein kinase B (Akt), B lymphocyte­2 (Bcl­2), Bcl2 family­associated X protein (Bax), etc. Cellular immunofluorescence staining was employed to assess the localization and expression of EGR1 and PTEN in nuclear and cytoplasmic fractions of Hepa1­6 cells. The association between EGR1 and PTEN was explored by EGR1 overexpression in cell lines. For in vivo methods, a mouse model of diethylnitrosamine (DEN)­induced primary liver cancer was established using C57BL/6 mice to investigate the inhibitory effect of LNT on liver cancer. Histopathology of liver tissue from mice was detected by hematoxylin­eosin staining and immunohistochemical assay. In vitro and in vivo results showed that LNT can inhibit the proliferation and promote the apoptosis of mouse HCC cells. Besides, LNT increased the expression of EGR1 in Hepa1­6 cells, which is translocated to the nucleus to function as a transcriptional factor. EGR1 then activates the expression of the tumor suppressor PTEN, thereby inhibiting the activation of the AKT signaling pathway. These data revealed a novel anti­tumor mechanism by which LNT can induce apoptosis to inhibit mouse HCC progression through the EGR1/PTEN/AKT axis. These results provide a scientific basis for the potential use of LNT in drug development and clinical applications associated with primary liver cancer.

[Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway].

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.

Scutellarein regulates the PTEN/AKT/NFκB signaling pathway to reduce pirarubicin related liver inflammation.

The side effects of chemotherapy drugs have been hindering the progress of tumor treatment. The liver is the metabolic site of most drugs, which leads to the frequent occurrence of liver injury. Classical chemotherapy drugs such as pirarubicin (THP) can also cause dose­dependent hepatotoxicity, and the related mechanism is closely related to liver inflammation. Scutellarein (Sc) is a potential Chinese herbal monomer exhibiting liver protection activity, which can effectively alleviate the liver inflammation caused by obesity. In the present study, THP was used to establish a rat model of hepatotoxicity, and Sc was used for treatment. The experimental methods used included measuring body weight, detecting serum biomarkers, observing liver morphology with H&E staining, observing cell apoptosis with TUNEL staining, and detecting the expression of PTEN/AKT/NFκB signaling pathways and inflammatory genes with PCR and western blotting. However, whether Sc can inhibit the liver inflammation induced by THP has not been reported. The experimental results showed that THP led to the upregulation of PTEN and the increase of inflammatory factors in rat liver, while Sc effectively alleviated the aforementioned changes. It was further identified in primary hepatocytes that Sc can effectively inhabited PTEN, regulate AKT/NFκB signaling pathway, inhibit liver inflammation and ultimately protect the liver.

Dingkun Pill modulate ovarian function in chemotherapy-induced premature ovarian insufficiency mice by regulating PTEN/PI3K/AKT/FOXO3a signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Dingkun Pill (DKP) is a traditional Chinese medicine that has been shown to have beneficial effects on reproductive function. However, the specific mechanism underlying its effect on POI is not well understood. AIM OF THE STUDY: To investigate the effect of different doses of Dingkun Pill on ovarian function in cyclophosphamide (CTX)-induced premature ovarian insufficiency (POI) mice and to explore its molecular mechanism through PTEN/PI3K/AKT/FOXO3a signaling pathway. This study will provide valuable insights into the potential clinical application of Dingkun Pill for the treatment of POI. MATERIALS AND METHODS: Fifty female ICR mice were randomly divided into normal control (NC) group, model control (MC) group, and Dingkun Pill low, medium, high dose (DKP-L, M, H) groups. Mice were injected with CTX to construct the POI model. Mice in the DKP-L, M, and H groups were given 0.9 g/kg, 1.8 g/kg, and 3.6 g/kg of Dingkun Pill suspension for 21 days, respectively. Mice in the NC and MC groups were given the same amount of normal saline by gavage. Changes in body weight, estrous cycle and gonadal index were observed in each group of mice. Serum levels of FSH, LH, E2 and AMH were detected by ELISA. Hematoxylin-eosin (HE) staining observed the changes of ovarian pathological morphology and follicle counts at all levels. qRT-PCR was used to measure the levels of the PTEN and FOXO3a genes in ovarian tissue. The expression of PTEN/PI3K/AKT/FOXO3a signaling pathway related proteins were detected by Western-blot and immunohistochemistry (IHC). RESULTS: In POI mice, Dingkun Pill increased body weight, promoted the recovery of estrous cycle, increased ovarian index, and improved pathological morphology of the ovaries. The FSH level decreased in the medium dose group (P < 0.05), the LH level reduced significantly in the medium and high dose groups (P < 0.01), and the E2 level in the high dose group increased (P < 0.05). There was no significant difference in AMH levels across all dose groups. The number of growing follicles improved at all levels in the low and medium dose groups, but declined significantly in the high dose group. However, the number of corpus luteum increased significantly in the high dose group (P < 0.001), and the atretic follicles in the three dose groups decreased. Results from qRT-PCR, Western-blot and IHC showed that the moderate dose of Dingkun Pill suppressed the levels of the p-PI3K and p-AKT proteins by upregulating the expression of PTEN in the ovarian tissues of POI mice, thereby inhibiting the expression of the key protein p-FOXO3a. However, the inhibitory effect of the higher dose may be less than that of the lower and intermediate dose groups. CONCLUSIONS: The Dingkun Pill modulated hormonal levels, promoted follicle growth and induced ovulation in mice with CTX-induced POI, with better results in the low and moderate dose groups. Its mechanism may be related to the regulation of the PTEN/PI3K/AKT/FOXO3a signaling pathway.

miR-363-3p/PTEN is involved in the regulation of lipid metabolism by genistein in HepG2 cells via ERß.

BACKGROUND: Genistein (GEN) is one of the most well-known phytoestrogens identified in various legumes. Although increasing evidence shows GEN has a potential use in phytotherapy to regulate lipid metabolism, its therapeutic mechanisms have not yet been completely elucidated, especially epigenetic alterations of miRNAs to alleviate lipid accumulation in the liver remains unknown. PURPOSE: To clarify how GEN modulates the miRNA profile in HepG2 cells and investigate molecular mechanisms of the modulated miRNA on regulating hepatic lipid metabolism. METHODS: The miRNA microarray was performed to compare the miRNAs expression patterns, followed by determining principal miRNA and its target gene associated with hepatic lipid metabolism modulated by GEN. miR-363-3p mimics (mi) and phosphatase and tensin homolog (PTEN)-siRNA were transfected into HepG2 cells and GEN was further treated with the cells for 24 h RESULTS: GEN induced downregulation of miR-363-3p and upregulation of PTEN, which was a target mRNA of miR-363-3p. The miR-363-3p mi led to an upregulation of sterol-regulatory element-binding protein-1c (SREBP-1c) and its downstream lipid synthesis-related factors in HepG2 cells. In addition, the inhibition of PTEN led to an increase of lipogenesis, which was associated with the AKT/mTOR signal regulation. However, GEN treatment could abrogate the lipogenic effects of miR-363-3p mi or PTEN siRNA. The modulation was associated with estrogen receptor ß (ERß). CONCLUSION: We discerned a new mechanism that GEN regulated hepatic lipid metabolism by inhibiting miR-363-3p, which could be mediated via ERß and by targeting PTEN in HepG2 cells. Additionally, GEN reduced hepatic lipid accumulation by regulating PTEN-AKT/mTOR signal. It implicated a protective role of GEN by elucidating its epigenetic modification of the miRNA modulated by ERß on improving hepatic lipid metabolism and provided novel evidence of the mechanism on targeting miR-363-3p/PTEN in treating hepatic lipid disorders.

Rheum officinale and Salvia miltiorrhiza inhibit renal fibrosis via miR-21/PTEN/Akt signaling pathway in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of the main components of many famous Chinese herbal formulas, Rheum palmatum L. and Salvia miltiorhiza Bunge (RS) are extensively used to treat chronic kidney disease (CKD). RS has been proved to improve renal function and relieve renal fibrosis (RF), but the potential mechanism remains a mystery. AIM OF THE STUDY: The purpose of this study is to determine whether microRNA-21 (miR-21) is associated with RF progression, as well as whether RS protects against RF through miR-21/PTEN/AKT signaling. MATERIALS AND METHODS: (1) The rat model of RF was established using unilateral ureteral obstruction (UUO). After UUO surgery, miR-21 levels in plasma were detected by RT-PCR and RF scores were assessed by Masson's trichrome stain at days 3, 7, 14 and 21. The correlation analysis of the above two indexes was carried out by Spearman correlation analysis. (2) Human proximal tubular epithelial cells (HK-2) was transfected with miR-21 mimic and inhibitor, and then the levels of phosphatase and tensin homolog (PTEN) protein and mRNA were measured with Western blotting and RT-PCR, respectively. (3) TGF-ß (10 ng/mL) was added into HK-2 cells to induce fibrosis, followed by the intervention of RS-containing rat serum. PTEN and protein kinase-B (Akt) phosphorylation, as well as the expression of PTEN protein in HK-2 cells, were assessed by RT-PCR, Western blotting and immunofluorescence. (4) The rat models of RF were prepared by UUO and treated with RS. Serum creatinine and urea nitrogen levels were measured. RF score was determined by Masson's trichrome stain. RT-PCR was used to determine the expression of miR-21, PTEN, and Akt mRNA. Western blotting was used to determine the expression of PTEN and Akt proteins. RESULTS: A positive correlation was found between plasma miR-21 levels and RF scores of rats after UUO surgery at Days 3, 7, 14 and 21. It was confirmed that miR-21 targeted PTEN. RS drug-containing serum could rise the expression of PTEN and reduce Akt phosphorylation of HK-2 cells induced by TGF-ß. Moreover, RS drug-containing serum could increase PTEN expression and reduce Akt phosphorylation induced by miR-21 mimic in HK-2 cells. The rats treated with RS had significantly decreased serum creatinine and urea nitrogen levels and a lower RF score. RS also decreased miR-21 and Akt expressions, increased PTEN expression of UUO rats. CONCLUSION: There was a positive correlation between plasma miR-21 levels and RF scores. The inhibitory effect of RS on RF might be mediated by miR-21/PTEN/AKT signaling.

Benign goiters requiring thyroidectomy as the signal for PTEN hamartoma tumor syndrome diagnosis.

PTEN hamartoma tumor syndrome (PHTS) is a rare genetic cancer and tumor predisposition syndrome. Due to the wide spectrum of clinical manifestations and variable age at onset, the pathways leading to a PHTS diagnosis are difficult and highly variable. Many patients were found to have PHTS after a cancer diagnosis, missing the opportunity of prevention or enhanced cancer screening. This retrospective study evaluated a PHTS cohort followed in a high-risk surveillance clinic in a comprehensive cancer institution. A significant portion of the patients (60.9%, 14/23) had at least one cancer diagnosis (average age 34.6 years at diagnosis). A significant portion (78.3%, 18/23) were affected with clinically significant goiters (age 27.9 years), and many (60.9%, 14/23) had partial or total thyroidectomy (age 27.1 years). The average age at goiter diagnosis or thyroidectomy is younger than a cancer diagnosis. In 12 individuals who were affected with clinically significant goiter and cancer, all cancers were diagnosed after the thyroid disease (6.3 years). As clinically significant thyroid nodules in childhood or early young adulthood are common in PHTS, but uncommon for general population, these early onset thyroid nodules may alert the clinician to initiate PHTS-targeted evaluation and genetic testing.

BAT26 Only Microsatellite Instability with High Tumor Mutation Burden-A Rare Entity Associated with PTEN Protein Loss and High PD-L1 Expression.

Detecting microsatellite instability (MSI) in advanced cancers is crucial for clinical decision-making, as it helps in identifying patients with differential treatment responses and prognoses. BAT26 is a highly sensitive MSI marker that defines the mismatch repair (MMR) status with high sensitivity and specificity. However, isolated BAT26-only instability is rare and has not been previously reported. Of the 6476 cases tested using pentaplex MSI polymerase chain reaction, we identified two BAT26-only instability cases (0.03%) in this study. The case #1 patient was diagnosed with endometrial adenocarcinoma without MMR germline mutations. The endometrial tumor showed BAT26-only instability, partial loss of MLH1/PMS2 protein expression, and a high programmed cell death ligand 1 (PD-L1) combined positive score (CPS = 8). The tumor exhibited a somatic phosphatase and tensin homolog (PTEN) R303P missense mutation and loss of the PTEN protein. On a comprehensive cancer panel sequencing with ≥500 genes, the tumor showed an MSI score of 11.38% and high tumor mutation burden (TMB) (19.5 mt/mb). The case #2 patient was diagnosed with colorectal carcinoma with proficient MMR and PTEN protein loss without PTEN alteration, as well as a high PD-L1 CPS (CPS = 10). A pathogenic KRAS A146T mutation was detected with an MSI score of 3.36% and high TMB (13 mt/mb). In conclusion, BAT26-only instability is very rare and associated with PTEN protein loss, high TMB, and a high PD-L1 score. Our results suggest that patients with BAT26-only instability may show good responses to immunotherapy.

MiR-17-5p Inhibits the Proliferation and Metastasis of Gastric Cancer Cells by Targeting PTEN Protein.

Context: Effectively inhibiting gastric-cancer metastasis and decreasing the recurrence of gastric cancer after surgery are urgent problems. In recent years, abnormal microRNA (miRNA) expression has been found in gastric cancer, and miRNA-17-5p has been found to regulate the occurrence and progression of various cancers. It's necessary to analyze miRNA-17-5p's action mechanism in gastric cancer. Objective: The study intended to investigate: (1) miR-17-5p expression in gastric-cancer tissues and adjacent normal tissues in clinical patients, (2) to explore the regulatory effects of miR-17-5p on the biological behavior of gastric cancer at the cellular level, by constructing a gastric-cancer cell model that uses overexpressing miR-17-5p, and (3) to discuss preliminarily its action mechanism. Design: The research team designed a laboratory study. Setting: The study took place at the First Affiliated Hospital of Jinzhou Medical University in Jinzhou, Liaoning Province, China. Participants: Clinical specimens of gastric-cancer tissues and adjacent normal tissues were obtained from 20 patients who had undergone surgical resection for gastric cancer at the hospital. Outcome Measures: Quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) was employed to detect miR-17-5p expression in the gastric-cancer tissues and cells. The si-miR-17-5p was transfected into gastric-cancer cell lines to establish a cell model, with the si-miR-17-5p becoming the intervention group and a negative control group, the si-NC group also being created. The cultured SGC-7901 cells were divided into miR-17-5p mimic group (si-miR-17-5p) and negative control group (si-NC) by transfecting si-miR-17-5p and transfection reagent Lipo2000 for research. Cell proliferation was detected using the cell counting kit-8 (CCK-8) assay; cell invasion and migration were measured using a Transwell assay; and cell migration was also measured using a wound-healing assay. A bioinformatics prediction, luciferase reporter gene assay, and western blot assay were applied to verify the downstream target protein of miR-17-5p. Results: The miR-17-5p levels were significantly higher in gastric-cancer tissues and cell lines than in adjacent normal tissues or gastric epithelial cells. Gastric-cancer cells that were transfected with si-miR-17-5p were found to have significantly inhibited cell proliferation. The si-miR-17-5p could significantly suppress the invasion and metastasis of gastric-cancer cells. A bioinformatics prediction concluded that phosphatase and tensin homolog (PTEN) was the downstream target protein of miR-17-5p, which was confirmed by the Luciferase reporter gene assay and Western blot assay. Conclusions: The miR-17-5p was upregulated in gastric-cancer patients. Simultaneously, si-miR-17-5p dramatically inhibited the proliferation, invasion, and metastasis of gastric-cancer cells, and PTEN protein was its downstream target protein. The study provides a new approach to the treatment of gastric cancer.

Emodin regulates the autophagy via the miR-371a-5p/PTEN axis to inhibit hepatic malignancy.

Emodin has been reported to fulfill an important function in suppressing the vicious outcome of liver cancer. We aimed to elucidate the partial underlying molecular mechanism of emodin in inhibiting liver cancer, and we applied miRNA-sequence analysis and corresponding molecular functional experiments to find that the inhibitory effect of emodin on liver cancer was partly mediated by cellular autophagy through the miR-371a-5p/PTEN axis. The expression level of miR-371a-5p was down-regulated after emodin treatment in liver cancer cell lines (LCCLs). Restoring the expression level of miR-371a-5p attenuated the suppression of emodin on LCCLs. Additionally, we performed the prediction in relevant online databases and found that PTEN might functioned as a downstream target of miR-371a-5p to participate in the regulation on the above process. What's more, the detection of autophagy-related protein markers showed that LC3II was elevated accompanied by the decreased P62. The above results revealed that PTEN functioned as a key target to regulate the autophagy in the process where emodin inhibited the malignant outcome of LCCLs via miR-371a-5p, which further provided a theoretical basis for the application of traditional Chinese medicine (TCM) on clinical tumors.

Fuzheng Kang' ai decoction inhibits cell proliferation, migration and invasion by modulating mir-21-5p/human phosphatase and tensin homology deleted on chromosome ten in lung cancer cells.

OBJECTIVE: To elucidate the potential molecular mechanism by which Fuzheng Kang'ai decoction (, FZKA) inhibits proliferation, migration, and invasion of lung cancer cells. METHODS: Varying FZKA concentrations were used to manage lung cancer cells (A549 and PC9). We employed: cell counting kit-8 (CCK-8) and plate clone for-mation assays to examine the cell viability; flow cytometry (FCM) to analyze the cycle arrest; transwell and wound-healing assays to assess the cell invasion and migration, respectively. Further, a quantitative real-time polymerase chain reaction (qRT-PCR) assay was adopted to evaluate the miR-21-5p expression. For protein expression analysis, we employed the Western blot technique. Recombinant miR-21-5p overexpression adenovirus vector harboring GFP was constructed and transfected into A549 and PC9, after which we explored the effect of FZKA on miR-21-5p overexpression. RESULTS: Notably, treatment with FZKA inhibited viability, clone-formation ability, invasion, and migration of lung cancer cells. Mechanistically, FZKA markedly suppressed miR-21-5p expression but elevated the human phosphatase and tensin homology deleted on chromosome ten (PTEN) protein level in both A549 and PC9 cells. Over-expression of miR-21-5p lowered PTEN protein expression. Besides, overexpressed miR-21-5p levels with adenovirus antagonized FZKA-upregulated PTEN protein expression. CONCLUSION: The present study demonstrates how FZKA modulates cell biological behaviors, for instance, it impedes the proliferation by upregulating PTEN expression with miR-21-5p as the target. These findings unveil the potential novel molecular mechanisms from the microRNA aspect by which FZKA suppresses the growth of human lung cancer cells.

Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway.

OBJECTIVE: To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/ reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2 (PINK1/PARKIN) signaling pathway. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells; the hypoxia/ reoxygenation (H/R) model was established in tri-gas incubator; 2', 7'-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species (ROS); the changes of mitochondrial membrane potential was determined by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining; the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits; flow cytometry was used to detect the ratio of apoptotic cells; transmission electron microscope was used to observe the ultrastructure of H9C2 cells; Western blot was used to detect the protein changes of mitochondrial 20 kDa outer membrane protein (TOM20), translocase of inner mitochondrial membrane 23 (TIM23), presenilins associated rhomboid-like protein (PARL), PINK1, PARKIN and mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa (P62), microtubule-associated protein 1 light chain 3 beta (LC3B); the mRNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction; immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin. RESULTS: Resveratrol could inhibit the proliferation of H9C2 cells in a time- and concentration- dependent manner; however, pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels, alleviate the loss of mitochondrial membrane potential induced by H/R, inhibit H/R-induced apoptosis of H9C2 cells, and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage. Resveratrol could further increase the levels of p62, PINK1, PARKIN protein, the expression of PINK1, PARKIN mRNA and the ratio of LC3BⅡ/LC3BⅠin H/R-induced H9C2 cells, inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells, and further reduce the expression of TOM20,TIM23, PARL, Mfn1 and Mfn2 protein in H/R-induced H9C2 cells. The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells. CONCLUSIONS: Resveratrol can protect H9C2 cells from H/R injury, which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.

Leonurus japonicus Houttuyn induces reactive oxygen species-mediated apoptosis via regulation of miR-19a-3p/PTEN/PI3K/AKT in U937 and THP-1 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houttuyn is a medicinal ingredient in more than 300 prescriptions in traditional Korean medicine. It is especially important for women's health and blood-related diseases. Recent research revealed that Leonurus japonicus Houttuyn extracts have antioxidative, anticancer, analgesic, anti-inflammatory, and neuroprotective properties. AIM OF THE STUDY: However, its underlying anti-cancerous mechanisms remain unclear. This study elucidated the anticancer mechanism of Leonurus japonicus Houttuyn in U937 and THP-1 cancer cells. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC) was used for detecting main compound of Leonurus japonicus Houttuyn, rutin. EZ-Cytox cell viability assay, Western blot analysis, live and dead cell assay, 2', 7' dichlorofluorescin diacetate (DCFDA) assay, quantitative real-time PCR (qRT-PCR) analysis, and microRNA (miR) mimic transfection assay were applied to further investigate anti-cancer efficacies and underlying mechanism in U937 and THP-1 cells. RESULTS: The main compound of Leonurus japonicus Houttuyn, rutin was detected using HPLC. The cytotoxic effect of Leonurus japonicus Houttuyn was exerted in U937 and THP-1 cancer cells but not in MDBK and IEC-6 normal cells. Leonurus japonicus Houttuyn decreased mitochondria membrane potential (ΔΨm). Consistently, Leonurus japonicus Houttuyn reduced the expression of survivin and cleaved caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). Cell death was increased in Leonurus japonicus Houttuyn treated groups. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and CCAAT-enhancer-binding protein homologous protein (CHOP) was increased and phosphatidylinositol-3-kinase (PI3K) and Protein kinase B (AKT) were decreased by Leonurus japonicus Houttuyn. Reactive oxygen speices generation was elevated by Leonurus japonicus Houttuyn and its cytotoxicity was reversed by N-acetyl-l-cysteine (NAC) pretreatment. Moreover, onco-microRNA (miR), miR-19a-3p was suppressed by Leonurus japonicus Houttuyn and transfection of miR-19a-3p mimic reversed the regulated PTEN, p-AKT, CHOP expression, attenuating Leonurus japonicus Houttuyn induced apoptosis. CONCLUSIONS: These findings indicated that Leonurus japonicus Houttuyn has anti-cancer effects by regulation of PTEN/PI3K/AKT signal pathway and ROS-related ER stress-induced apoptosis via regulation of miR-19a-3p. Leonurus japonicus Houttuyn may be an effective candidate for triggering PTEN-dependent apoptosis of cancer cells related to acute myeloid leukemia.

Buyang Huanwu Decoction promotes angiogenesis in myocardial infarction through suppression of PTEN and activation of the PI3K/Akt signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Myocardial infarction (MI) is the most severe subtype of coronary artery disease. Recent studies have demonstrated that the repair process and prognosis of MI are closely related to microcirculatory function in myocardial tissue. Buyang Huanwu Decoction (BYHWD) has shown great potential in the treatment of MI. However, the effects and mechanisms of BYHWD on angiogenesis post-MI remain unclear. AIM OF THE STUDY: The study aimed to explore the promotion of angiogenesis by BYHWD post-MI and the potential mechanisms in vivo and in vitro. MATERIALS AND METHODS: MI in mice was induced by permanent ligature of the coronary artery. The sample was divided into sham, model, and BYHWD treatment groups. After four weeks, the effects of BYHWD treatment on cardiac function were evaluated by echocardiography and HE and Masson staining. Angiogenesis was detected by CD 31 immunofluorescence staining in vivo. Then, various databases were searched to identify the corresponding targets of BYHWD in order to explore the molecular mechanisms underlying its effects in MI. Moreover, Western blot and immunohistochemistry were employed to measure the PTEN/PI3K/Akt/GSK3ß signalling pathway and VEGFA expression in MI mice. Finally, the effects of BYHWD on cell angiogenesis and the activation of the PTEN/PI3K/Akt/GSK3ß pathway in primary HUVECs were investigated. Overexpression of PTEN was achieved by an adenovirus vector encoding PTEN. RESULTS: BYHWD significantly promoted angiogenesis and improved cardiac function in MI mice. Target prediction analysis suggested that BYHWD ameliorates MI via the PI3K/Akt pathway. BYHWD promoted angiogenesis post-MI by suppressing PTEN and activating the PI3K/Akt/GSK3ß signalling pathway in vivo and in vitro. Moreover, the effects of BYHWD on HUVEC angiogenesis and the expression of PI3K/Akt/GSK3ß signalling pathway-associated proteins were partially abrogated by the overexpression of PTEN. CONCLUSION: Collectively, this study demonstrates that BYHWD exerts cardioprotective effects against MI by targeting angiogenesis. These effects are related to suppressing PTEN and activating the PI3K/Akt/GSK3ß signalling pathway by BYHWD.

The Role of PTEN/PI3K/AKT Signaling Pathway in Apoptosis of Liver Cells in Cocks with Manganese Toxicity.

PTEN/PI3K/AKT signaling pathway is an important pathway for cell proliferation and apoptosis. Exposure to excess manganese (Mn) can cause damage in organisms. However, whether Mn toxicity can cause apoptosis is still not clear. In order to explore the mechanism of PTEN/PI3K/AKT signaling pathway responsible for Mn-induced apoptotic injury, 160 Hyline cocks were divided into four groups; there were the control group (Con group), the low-dose Mn group (L group), the medium-dose Mn group (M group), and the high-dose Mn group (H group). The cocks in Con group, L group, M group, and H group were fed with MnCl2 diet containing 100, 600, 900, and 1800 mg/kg, respectively. The growth status of cocks in each group was observed on days 30, 60, and 90. Thirty cocks were randomly selected from each group and sacrificed on day 90 for optical microscope observation and fluorescence microscopic observation, as well as for transcription-level expression of apoptosis-related genes and heat shock proteins (HSPs) in the liver. The results showed that the growth status of cocks was gradually depressed with the extension of feeding time and with the increase of Mn dose. On day 90, the results of optical microscope observation and fluorescence microscope observation showed that damage and apoptosis appeared in the cock liver cells under Mn exposure groups. The results of transcription-level detection of apoptosis-related genes and HSPs indicated that Mn exposure upregulated eleven pro-apoptotic genes (including RIP1, RIP3, MLKL, Bax, Caspase-3, FADD, Cyt-C, ERK, JNK, Caspase-8, and P38) and downregulated one anti-apoptotic gene Bcl-2, further meaning that exposure to Mn-induced apoptosis in cock liver cells and PTEN/PI3K/AKT signaling pathway took part in molecular mechanism of apoptosis caused by excess Mn. Moreover, in our experiment, the increase of four HSPs (including HSP27, HSP40, HSP60, and HSP70) was found after Mn treatment for 90 days, which indicated that Mn stress triggered HSPs and HSPs were involved in molecular mechanism of Mn poisoning in cock livers. In addition, we also found there was upregulated dose-dependent manner in fifteen detected genes and there was downregulated dose-dependent manner in Bcl-2, indicating that the apoptosis caused by Mn poisoning in cock liver cells was dose-dependent.

Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.