BACKGROUND: Growing evidence suggests that long non-coding RNAs (lncRNAs) exert pivotal roles in fostering chemoresistance across diverse tumors. Nevertheless, the precise involvement of lncRNAs in modulating chemoresistance within the context of gallbladder cancer (GBC) remains obscure. This study aimed to uncover how lncRNAs regulate chemoresistance in gallbladder cancer, offering potential targets to overcome drug resistance. METHODS: To elucidate the relationship between gemcitabine sensitivity and small nucleolar RNA host gene 1 ( SNHG1 ) expression, we utilized publicly available GBC databases, GBC tissues from Renji Hospital collected between January 2017 and December 2019, as well as GBC cell lines. The assessment of SNHG1, miR-23b-3p, and phosphatase and tensin homolog (PTEN) expression was performed using in situ  hybridization, quantitative real-time polymerase chain reaction, and western blotting. The cell counting kit-8 (CCK-8) assay was used to quantify the cell viability. Furthermore, a GBC xenograft model was employed to evaluate the impact of SNHG1 on the therapeutic efficacy of gemcitabine. Receiver operating characteristic (ROC) curve analyses were executed to assess the specificity and sensitivity of SNHG1. RESULTS: Our analyses revealed an inverse correlation between the lncRNA SNHG1 and gemcitabine resistance across genomics of drug sensitivity in cancer (GDSC) and Gene Expression Omnibus (GEO) datasets, GBC cell lines, and patients. Gain-of-function investigations underscored that SNHG1 heightened the gemcitabine sensitivity of GBC cells in both in vitro and in vivo  settings. Mechanistic explorations illuminated that SNHG1 could activate PTEN -a commonly suppressed tumor suppressor gene in cancers-thereby curbing the development of gemcitabine resistance in GBC cells. Notably, microRNA (miRNA) target prediction algorithms unveiled the presence of miR-23b-3p binding sites within SNHG1 and the 3'-untranslated region (UTR) of PTEN . Moreover, SNHG1 acted as a sponge for miR-23b-3p, competitively binding to the 3'-UTR of PTEN , thereby amplifying PTEN expression and heightening the susceptibility of GBC cells to gemcitabine. CONCLUSION The SNHG1/miR-23b-3p/PTEN axis emerges as a pivotal regulator of gemcitabine sensitivity in GBC cells, holding potential as a promising therapeutic target for managing GBC patients.
Humans ; Deoxycytidine/pharmacology* ; PTEN Phosphohydrolase/genetics* ; Gemcitabine ; RNA, Long Noncoding/metabolism* ; MicroRNAs/genetics* ; Gallbladder Neoplasms/genetics* ; Cell Line, Tumor ; Animals ; Mice ; Drug Resistance, Neoplasm/genetics* ; Mice, Nude ; Antimetabolites, Antineoplastic ; Gene Expression Regulation, Neoplastic

This study aimed to investigate the effects of Zhiwei Fuwei Pills on mitochondrial apoptosis in the rat model of precancerous lesions of gastric cancer(PLGC) based on the microRNA-21(miRNA-21)/B-cell lymphoma-2(Bcl-2) signaling pathway. Eighty-five 5-week-old male SPF-grade SD rats were selected, of which 75 were fed with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) for multifactorial modeling, and the PLGC model was established after 26 weeks. The rats were randomly grouped as follows: model, folic acid(0.002 g·kg~(-1)), low-dose(0.42 g·kg~(-1)) Zhiwei Fuwei Pills, medium-dose(0.84 g·kg~(-1)) Zhiwei Fuwei Pills, and high-dose(1.67 g·kg~(-1)) Zhiwei Fuwei Pills, with 15 rats in each group. Additionally, 10 rats were assigned to a blank group and administrated with an equivalent volume of normal saline by gavage. After four weeks of continuous drug administration, the gastric mucosal tissue was collected. Hematoxylin-eosin(HE) staining was performed to reveal the pathological changes in the gastric mucosa. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) was employed to detect apoptosis in gastric mucosal epithelial cells. RT-PCR was adopted to determine the mRNA levels of miRNA-21, phosphatase and tensin homolog(PTEN), Bcl-2, Bcl-2-associated X protein(Bax), and cysteinyl aspartate-specific protease 3(caspase-3). Western blot was employed to determine the protein levels of PTEN, Bcl-2, Bax, and caspase-3. Immunohistochemistry(IHC) was used to detect the positive expression of PTEN, Bcl-2, and Bax in the gastric mucosal tissue. Transmission electron microscopy(TEM) was employed to observe the morphological and structural changes in mitochondria. The results showed that compared with model group, the drug administration groups showed alleviated pathological changes, with increased apoptotic cells, down-regulated mRNA levels of miRNA-21 and Bcl-2, up-regulated mRNA and protein levels of PTEN, Bax, and caspase-3, and down-regulated protein level of Bcl-2. In addition, the drug administration groups exhibited mitochondrial swelling and rupture and reduction of cristae, which indicated mitochondrial apoptosis. These findings suggest that Zhiwei Fuwei Pills can effectively improve mitochondrial apoptosis in PLGC cells by regulating the miRNA-21/Bcl-2 signaling pathway.
Animals ; MicroRNAs/metabolism* ; Male ; Apoptosis/drug effects* ; Stomach Neoplasms/physiopathology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Rats ; Rats, Sprague-Dawley ; Drugs, Chinese Herbal/administration & dosage* ; Mitochondria/genetics* ; Signal Transduction/drug effects* ; Precancerous Conditions/drug therapy* ; Humans ; PTEN Phosphohydrolase/genetics*

OBJECTIVE: By analyzing the differential miRNA in seminal plasma between individuals with normal and abnormal sperm DNA fragmentation index（DFI）, we aim to identify miRNA that may impact sperm DNA integrity and target genes, and attempt to analyze their potential mechanisms of action. METHODS: A total of 161 study subjects were collected and divided into normal control group, DFI-medium group and DFI-abnormal group based on the DFI detection values. Differential miRNA were identified through miRNA chip analysis. Through bioinformatics analysis and target gene prediction, miRNA related to DFI and specific target genes were identified. The relative expression levels of differential miRNA and target genes in each group were compared to explore the impact of their differential expression on DFI. RESULTS: Through miRNA chip analysis, a total of 11 differential miRNA were detected. Bioinformatics analysis suggested that miR-26a-5p may be associated with reduced sperm DNA integrity. And gene prediction indicated that PTEN was a specific target gene of miR-26a-5p. Compared to the normal control group, the relative expression levels of miR-26a-5p in both the DFI-medium group and the DFI-abnormal group showed a decrease, while the relative expression levels of PTEN showed an increase. The relative expression levels of miR-26a-5p in all groups were negatively correlated with DFI values, while the relative expression levels of PTEN showed a positive correlation with DFI values in the DFI-medium group and the DFI-abnormal group. The AUC of miR-26a-5p in the DFI-medium group was 0.740 (P<0.05), with a sensitivity of 73.6% and a specificity of 71.5%; the AUC of PTEN was 0.797 (P<0.05), with a sensitivity of 76.5% and a specificity of 78.4%. In the DFI-abnormal group, the AUC of miR-26a-5p was 0.848 (P<0.05), with a sensitivity of 81.3% and a specificity of 78.1%. While the AUC of PTEN was 0.763 (P<0.05), with a sensitivity of 77.2% and a specificity of 80.2%. CONCLUSION miR-26a-5p affects the integrity of sperm DNA by regulating the expression of PTEN negatively. The relative expression levels of seminal plasma miR-26a-5p and PTEN have good diagnostic value for sperm DNA integrity damage, which can help in the etiological diagnosis and prognosis analysis of abnormal DFI. This provides a diagnostic and treatment approach for the study and diagnosis of DFI abnormalities without clear etiology.
Male ; Humans ; MicroRNAs/genetics* ; PTEN Phosphohydrolase/genetics* ; Spermatozoa ; Semen/metabolism* ; DNA Fragmentation

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.
Female ; Animals ; Mice ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Choristoma ; Endometriosis/genetics* ; TOR Serine-Threonine Kinases/genetics* ; RNA, Messenger ; Signal Transduction ; Body Weight ; Mammals ; PTEN Phosphohydrolase/genetics*

OBJECTIVE: To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM. METHODS: HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected. RESULTS: CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance. CONCLUSION Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.
Humans ; Atorvastatin/pharmacology* ; PTEN Phosphohydrolase/pharmacology* ; Sincalide/metabolism* ; Drug Resistance, Neoplasm/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Leukemia, Promyelocytic, Acute/drug therapy* ; Doxorubicin/pharmacology* ; Apoptosis ; RNA, Small Interfering/pharmacology* ; Glycolysis ; Glucose/therapeutic use* ; Cell Proliferation

The purpose of our study is to investigate the prognostic value of phosphatase and tensin homolog on chromosome 10 (PTEN) expression in patients with de novo metastatic castration naïve prostate cancer (mCNPC). A total of 205 patients with mCNPC at Fudan University Shanghai Cancer Center (Shanghai, China) were retrospectively examined. Immunohistochemical staining of PTEN was performed on prostate biopsy samples of these patients. Associations among clinicopathological features, patient survival and PTEN protein expression were analyzed. PTEN loss occurred in 58 of 205 (28.3%) patients. Loss of PTEN was significantly correlated with high metastatic volume (P = 0.017). No association between PTEN expression and Gleason score was observed. Patients with PTEN loss had significantly shorter progression-free survival (PFS, P < 0.001) and overall survival (OS, P < 0.001) compared with patients with intact PTEN expression. Multivariate analysis showed that elevated alkaline phosphatase, high metastatic volume and PTEN loss were independent poor prognostic factors for PFS. The Eastern Cooperative Oncology Group performance status (ECOG PS)#8805; 2 and PTEN loss were independent poor prognostic factors for OS. The adjusted hazard ratio of PTEN loss for PFS and OS was 1.67 (95% confidence interval [CI]: 1.14-2.43, P = 0.008) and 1.95 (95% CI: 1.23-3.10, P = 0.005), respectively. PTEN loss was also significantly associated with shorter PFS (P = 0.025) and OS (P < 0.001) in patients with low-volume metastatic disease. Our data showed that PTEN loss is an independent predictor for shorter PFS and OS in patients with de novo mCNPC.
China/epidemiology* ; Humans ; Male ; PTEN Phosphohydrolase/genetics* ; Prognosis ; Prostatic Neoplasms ; Retrospective Studies

Objective: To explore the effect of growth arrest-specific5 (GAS5) inhibition on the proliferation, colony formation, invasion, migration andepithelial-mesenchymal transition(EMT), cancer cell stem of HCT-116 and its mechanism. Methods: The colorectal carcinoma (CRC) cell HCT116 was divided into blank control, negative control (NC), si-GAS5 and si-GAS5+ miR-21 inhibitor groups. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expressions of miR-21 and GAS5 at 48 h after transfection. The binding site of GAS5 and miR-21 was determined by luciferase reporter array. Cell proliferation ability was detected by CCK-8 assay. Cell colony ability was detected by colony formation assay. Cell invasion and migration abilities were detected by Transwell assay. Cell cycle and apoptosis were examined by flow cytometer (FCM). The protein levels of EMT associated factors including Snail, N-cadherin, vimentin, E-cadherin, stem cell related factors including CD44, SOX2, Oct2, and PTEN/Akt signal pathway associated factors were examined by western blotting. Results: The expression levels of miR-21 in blank, NC, si-GAS5 group were 1.00±0.10, 1.00±0.10, 1.80±0.20, the absorbance values were 0.51±0.02, 0.50±0.01 and 0.65±0.01, the cell clones were 90±4, 91±5, 200±8, the invaded cells were 118±3, 119±3, 150±4, the migrated cells were 110±2, 108±2, 127±2, the cell ratios in G(1) phase were (49.3±2.1)%, (50.1±2.0)% and (42.2±1.1)%, the cell ratios in S phase were (19.2±1.2)%, (20.2±1.1)% and (28.3±2.2)%, the cell apoptotic ratios were (14.4±2.2)%, (14.5±2.1)% and (7.2±1.3)%. These results indicated that inhibition of GAS5 up regulated the expression level of miR-21, promoted cell proliferation, invasion and migration, decreased G(1)-phase cells and increased S-phase cells, and suppressed cell apoptosis (P<0.05). Moreover, inhibition of GAS5 up regulated the expressions of Snail, N-cadherin, vimentin, Sox2, CD44, Oct2 and p-Akt in HCT-116 cells (P<0.05), while down regulated the expressions of E-cadherin and PTEN (P<0.05). Inhibition of miR-21 reversed the impact of GAS5 knockdown on PTEN/Akt signaling pathway (P<0.05). Conclusion: GAS5 can act as a competing endogenous RNA for miR-21, and down regulation of GAS5 can promote the development of CRC by activating the miR-21/PTEN/Akt signaling pathway and promoting the acquisition of EMT and tumor cell stemness.
Humans ; Cadherins/metabolism* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Colorectal Neoplasms/pathology* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; PTEN Phosphohydrolase/metabolism* ; Vimentin/metabolism*

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.
Adenoviridae/metabolism* ; Animals ; Carrier Proteins ; Cell Proliferation ; Cortactin ; Filamins/genetics* ; Hepatic Stellate Cells/metabolism* ; PTEN Phosphohydrolase/metabolism* ; RNA, Small Interfering/genetics* ; Rats ; Vinculin/genetics*

Choriocarcinoma/pathology* ; Female ; Gene Deletion ; Humans ; PTEN Phosphohydrolase/genetics* ; Pregnancy ; Sequence Deletion ; Stomach Neoplasms/surgery*

This study explored the effects of resveratrol(Res) on the expression of phosphatase and tensin homolog deleted on chromosome ten(PTEN) and the fibrosis of rat renal tubular epithelial cells in a high-glucose environment and the possible mechanism underlying the fibrosis reduction. After the pretreatment of rat renal tubular epithelial cells(NRK-52 E) cultured in a high-glucose condition with Res or PTEN inhibitor SF1670, they were divided into several groups, i.e., normal glucose(NG), normal glucose + SF1670(NS), high glucose(HG), high glucose + SF1670(HS), high glucose + Res at different concentrations(5, 10, 25 μmol·L~(-1)). The expression and distribution of E-cadherin and α-SMA in renal tubular epithelial cells were observed by immunofluorescence cytochemistry. The protein expression levels of PTEN, E-cadherin, α-SMA, p-Akt~((Thr308)) and collagen Ⅳ were determined by Western blot. Real-time PCR was employed to detect the expression of PTEN mRNA. Compared with the NG group, the HG group witnessed the reduced expression of PTEN mRNA, PTEN protein and E-cadherin protein, but saw the increased expression of α-SMA, p-Akt~((Thr308)) and collagen Ⅳ proteins. Besides, with the increase in Res concentration, the expression levels of PTEN mRNA, PTEN protein and E-cadherin protein gradually increased, while those of α-SMA, collagen Ⅳ, p-Akt~((Thr308)) proteins gradually decreased in the Res groups, showing a dose-effect dependence, compared with the HG group. No distinct difference was found between the NS group and the NG group. The expression level of E-cadherin was even lower and those of α-SMA, p-Akt~((Thr308)), and collagen Ⅳ were higher in the HS group than in the HG group, with no marked difference shown in the two groups in terms of PTEN mRNA and protein. Although the PTEN inhibitor did not affect PTEN, the expression changes of the other proteins were opposite to the results after Res treatment and the fibrosis was aggravated, which suggested that SF1670 promoted the fibrosis by inhibiting PTEN, activating Akt and increasing the synthesis of collagen Ⅳ and other extracellular matrix. The results show that Res can antagonize the high glucose-mediated fibrosis of renal tubular epithelial cells. This may be achieved via the up-regulation of PTEN and the inhibition of PI3 K/Akt signaling pathway.
Animals ; Epithelial Cells ; Fibrosis ; Glucose ; PTEN Phosphohydrolase/genetics* ; Rats ; Resveratrol/pharmacology*