OBJECTIVES: Oxaliplatin (OXA) and 5-fluorouracil (5-FU) are 2 commonly used chemotherapeutic agents for colorectal cancer (CRC). MicroRNAs (miRNAs, miRs) play crucial roles in the development of chemoresistance in various cancers. However, the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear. This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms. METHODS: CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs between drug-sensitive and resistant groups (OXA or 5-FU) were analyzed, and miR-224-5p was identified as the target miRNA. Chemoresistant cell lines HCT15-OXR, HCT15-5-FU, SW480-OXR, and SW480-5-FU were established. Transient transfections were performed using miR-224-5p mimics, inhibitors, and their respective negative controls (control mimic, control inhibitor) in these cell lines. Cells were treated with different concentrations of OXA or 5-FU post-transfection, and the half-maximal inhibitory concentration (IC₅₀) was determined using the cell counting kit-8 (CCK-8) assay. Cell proliferation was assessed by CCK-8 and colony formation assays. The expression levels of miR-224-5p, LC3, and P62 were measured by real-time polymerase chain reaction (real-time PCR) and/or Western blotting. Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay. TargetScan 8.0, miRTarBase, miRPathDB, and HADb were used to predict B-cell lymphoma-2 (Bcl-2) as a potential miR-244-5p target, which was further validated by dual-luciferase reporter assays. RESULTS: Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression, whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity. Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells, which was reversed by miR-224-5p overexpression. Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p. CONCLUSIONS MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.
Humans ; MicroRNAs/physiology* ; Colorectal Neoplasms/drug therapy* ; Drug Resistance, Neoplasm/genetics* ; Autophagy/drug effects* ; Fluorouracil/pharmacology* ; Oxaliplatin ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Gene Expression Regulation, Neoplastic

BACKGROUND: Enzalutamide, a second-generation androgen receptor (AR) pathway inhibitor, is widely used in the treatment of castration-resistant prostate cancer. However, after a period of enzalutamide treatment, patients inevitably develop drug resistance. In this study, we characterized leucine-rich repeated G-protein-coupled receptor 5 (LGR5) and explored its potential therapeutic value in prostate cancer. METHODS: A total of 142 pairs of tumor and adjacent formalin-fixed paraf-fin-embedded tissue samples from patients with prostate cancer were collected from the Pathology Department at Sun Yat-sen Memorial Hos-pital. LGR5 was screened by sequencing data of enzalutamide-resistant cell lines combined with sequencing data of lesions with different Gleason scores from the same patients. The biological function of LGR5 and its effect on enzalutamide resistance were investigated in vitro and in vivo . Glutathione-S-transferase (GST) pull-down, coimmunoprecipitation, Western blotting, and immunofluorescence assays were used to explore the specific binding mechanism of LGR5 and related pathway changes. RESULTS: LGR5 was significantly upregulated in prostate cancer and negatively correlated with poor patient prognosis. Overexpression of LGR5 promoted the malignant progression of prostate cancer and reduced sensitivity to enzalutamide in vitro and in vivo . LGR5 promoted the phosphorylation of glycogen synthase kinase-3β (GSK-3β) by binding heat shock protein 90,000 alpha B1 (HSP90AB1) and mediated the activation of the Wingless/integrated (WNT)/β-catenin signaling pathway. The increased β-catenin in the cytoplasm entered the nucleus and bound to the nuclear AR, promoting the transcription level of AR, which led to the enhanced tolerance of prostate cancer to enzalutamide. Reducing HSP90AB1 binding to LGR5 significantly enhanced sensitivity to enzalutamide. CONCLUSIONS LGR5 directly binds to HSP90AB1 and mediates GSK-3β phosphorylation, promoting AR expression by regulating the WNT/β-catenin signaling pathway, thereby conferring resistance to enzalutamide treatment in prostate cancer.
Male ; Humans ; Phenylthiohydantoin/pharmacology* ; Benzamides ; Receptors, G-Protein-Coupled/genetics* ; Nitriles ; Cell Line, Tumor ; HSP90 Heat-Shock Proteins/metabolism* ; Drug Resistance, Neoplasm/genetics* ; Prostatic Neoplasms/drug therapy* ; beta Catenin/metabolism* ; Receptors, Androgen/genetics* ; Animals ; Mice ; Wnt Signaling Pathway/physiology*

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer's disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP^751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
Amyloid beta-Protein Precursor/physiology* ; Apoptosis/drug effects* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Fluorouracil/pharmacology* ; Humans ; Liver Neoplasms/drug therapy* ; Mitochondria/physiology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; bcl-X Protein/genetics*

Hepatocellular carcinoma (HCC) is one of the most common types of liver cancer and is the second leading cause of cancer mortality with an estimated 745 500 deaths annually (Jemal et al., 2011). Although new therapeutic modalities including novel chemotherapeutic interventions and targeted therapy have been applied, the prognosis of HCC patients remains unsatisfactory due to the high incidence of intrahepatic and distal metastases (Siegel et al., 2018).
Apoptosis Regulatory Proteins/physiology* ; Biomarkers ; Carcinoma, Hepatocellular/pathology* ; Female ; Genome ; Humans ; Hypoxia ; Liver Neoplasms/pathology* ; Male ; MicroRNAs/analysis* ; Neoplasm Staging ; Prognosis ; Repressor Proteins/physiology*

BACKGROUND: Long noncoding RNAs (lncRNAs) play pivotal roles in various malignant tumors. Epidermal growth factor receptor (EGFR) signaling is associated with the pathogenesis of cutaneous squamous cell carcinoma (cSCC). This study aimed to explore the role of LINC00520 in the development of cSCC via EGFR and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways. METHODS: A microarray analysis was applied to screen differentially expressed lncRNAs in cSCC samples. The A431 cSCC cell line was transfected and assigned different groups. The expression patterns of LINC00520, EGFR, and intermediates in the PI3K/Akt pathway were characterized using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis. Cell proliferation, migration, and invasion were detected using the MTT assay, scratch test, and Transwell assay, respectively. Cell-based experiments and a tumorigenicity assay were conducted to assess the effect of LINC00520 on cSCC progression. This study was ended in September 2017. Comparisons between two groups were analyzed with t-test and comparisons among multiple groups were analyzed using one-way analysis of variance. The nonparametric Wilcoxon rank sum test was used to analyze skewed data. The enumerated data were analyzed using the chi-square test or Fisher exact test. RESULTS: Data from chip GSE66359 revealed depletion of LINC00520 in cSCC. Cells transfected with LINC00520 vector and LINC00520 vector + si-EGFR showed elevated LINC00520 level but decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the si-LINC00520 group showed opposite trends (all P < 0.05). Compared with the LINC00520 vector group, the LINC00520 vector + si-EGFR group showed decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the LINC00520 vector + EGFR vector group showed opposite results (all P < 0.05). CONCLUSION Based on our results, LINC00520-targeted EGFR inhibition might result in the inactivation of the PI3K/Akt pathway, thus inhibiting cSCC development.
Animals ; Carcinoma, Squamous Cell ; pathology ; prevention & control ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Disease Progression ; ErbB Receptors ; antagonists & inhibitors ; Female ; Humans ; Lymphatic Metastasis ; Mice ; Neoplasm Invasiveness ; Phosphatidylinositol 3-Kinases ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; RNA, Long Noncoding ; physiology ; Signal Transduction ; physiology ; Skin Neoplasms ; pathology ; prevention & control

OBJECTIVE: To investigate the effects of tetrandrine (Tet) on proliferation and activation of rat cardiac fibroblasts. METHODS: Firstly, the cell counting kit-8 (cck-8) assay was applied to detect the effects of Tet with different concentrations on proliferation of cardiac fibroblasts. Secondly, transforming growth factor (TGF-β)with a concentration of 5 μg/L was used to induce the cardiac fibroblast activation, and Western blot was performed to measure the expression variation of β-catenin, vimentin (Vm), fibronectin (Fn) and smooth muscle α-actin (SMA). At last, the real-time PCR was conducted to measure the expression change of collagen-1(Col-1) and collagen-3(Col-3). RESULTS: The cck-8 assay showed that the Tet with different concentrations respectively, which were 0.5 μmol/L, 1 μmol/L, 2 μmol/L, 4 μmol/L, and 8 μmol/L, significantly inhibited the proliferation of cardiac fibroblasts. The viability was decreased to 94.4%,84.9%,74.9%,63.8%and 50.3% respectively of the control group when the Tet concentration changed, and the difference was statistically significant, P=0.043, P<0.001, P<0.001, P<0.001, P<0.001 respectively. Western blot revealed that the expressions of β-catenin, Fn, SMA and Vm, were up-regulated by TGF-β(5 μg/L), the result showed that the difference was statistically significant, and the P values were 0.001,0.008,0.010,0.001 respectively. Then, the up-regulation of β-catenin, Fn and SMA was attenuated by pre-treatment of Tet, and the result also displayed that the difference was statistically significant, and the P values were 0.009, 0.005, 0.019,respectively. While there was no significant change in the expression of Vm, according to Western blotting, and P>0.05,at the same time, real-time PCR indicated that the up-regulations of Col-1 and Col-3 which were induced by TGF-β were blocked by pre-treatment of Tet, the result showed that the difference was statistically significant, P<0.001. CONCLUSION According to the experimental results, we can draw the conclusion that: the Tet can significantly inhibit the proliferation of cardiac fibroblasts, meanwhile, it can block the activation of cardiac fibroblasts, which is induced by TGF-β. It is supposed that the Tet may probably have anti myocardial fibrosis, which indicates that it may probably be a medicine which is used to block the cardiac remodeling.
Actins ; Animals ; Benzylisoquinolines/pharmacology* ; Blotting, Western ; Calcium Channel Blockers/pharmacology* ; Cell Proliferation ; Collagen ; Collagen Type I ; Fibroblasts/physiology* ; Fibrosis ; Myocardium/cytology* ; Neoplasm Proteins/metabolism* ; Rats ; Signal Transduction ; Transforming Growth Factor beta/metabolism* ; Transforming Growth Factor beta1

Brain Ischemia ; pathology ; physiopathology ; Humans ; Neoplasm Proteins ; metabolism ; Pyroptosis ; physiology ; Signal Transduction ; physiology

OBJECTIVE: To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation,migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs). METHODS: Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy,nanoparticle tracking analysis and Western blotting.Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized.HucMSCs were divided into control group,PBS intervention group and exosome intervention group,and CCK-8 assay,wound healing assay,Transwell assay,Western blotting and cell karyotype analysis were used to observe the proliferation,migration,invasion,expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs. RESULTS: The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm,and most of them were oval or spherical capsules rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells.Compared with the control cells and PBS-treated cells,hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (<0.05) with accelerated healing rate (<0.05 at 24 h;<0.01 at 48 h),increased invasive cells (<0.01),enhanced protein expressions of MMP2(<0.05 PBS group;<0.01 control group) and MMP9(<0.05),but their karyotype still remained 46XY without any abnormalities. CONCLUSIONS Piwil2-iCSC-derived exosomes can promote the proliferation,migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.
Argonaute Proteins ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Exosomes ; physiology ; Fetal Blood ; cytology ; Humans ; Karyotyping ; Mesenchymal Stem Cells ; pathology ; Neoplasm Invasiveness ; Neoplastic Stem Cells ; Umbilical Cord ; Wound Healing

OBJECTIVE: To explore the role of SIRT1 in the occurrence of epithelial-mesenchymal transition (EMT) in EC-9706 and Eca-109 cells and the possible mechanism. METHODS: Three chemically synthesized siRNA targeting SIRT1 were transfected into EC-9706 and Eca-109 cells with the non-transfected cells and cells transfected with the negative siRNAs as controls. Real-time PCR and Western blotting were used to detect the expressions of SIRT1, E-cadherin, vimentin, Snail, Twist1 and ZEB in the cells. Transwell invasion assay and wounding healing assay were used to examine the changes in the invasion and metastasis abilities of the cells after transfection. RESULTS: EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3 showed significantly decreased mRNA and protein expressions of SIRT1 ( < 0.05). Transwell invasion assay and wounding healing assay showed that transfection with SIRT1 siRNA1 and SIRT1 siRNA3 caused significantly lowered invasion and metastasis abilities in EC-9706 and Eca-109 cells ( < 0.05). In EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3, the expression level of E-cadherin was significantly increased while the expressions of vimentin, Snail and Twist were significantly lowered ( < 0.05). CONCLUSIONS SIRT1 participates in the invasion and metastasis of EC-9706 and Eca- 109 cells probably by inducing EMT via regulating the expression of Snail.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; physiology ; Humans ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Sirtuin 1 ; genetics ; metabolism ; Snail Family Transcription Factors ; metabolism ; Transfection ; Twist-Related Protein 1 ; metabolism ; Vimentin ; metabolism ; Zinc Finger E-box-Binding Homeobox 1 ; metabolism

OBJECTIVETo predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin (L-OHP) in human colorectal cancer cells.

METHODSL-OHP-resistant human colorectal cancer cell line (HCT116/L-OHP) was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control (NC-miRNA) were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPover-expressing miR-145 and HCT116/L-OHP. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98(GPR98) as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPover-expressing GPR98 and HCT116/L-OHP. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHP) were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation (A value) and sensitivity to L-OHP (the lower the IC50, the stronger the sensitivity) in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein (gp), multiple drug-resistance protein 1(MRP1), cancer-inhibition gene PTEN.

RESULTSHCT116/L-OHP cell line was successfully established with ICof (42.34±1.05) mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than (9.81±0.95) mg/L (t=39.784, P=0.000) and lower than 1.00±0.09 (t=13.021, P=0.000) in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPcells were established successfully, with relative miR-145 expression of 10.01±1.05, which was higher than 1.06±0.14 in HCT116/L-OHPand 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHPcells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHP(mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHPcells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHPcells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHPcells was lower as compared to HCT116/L-OHPcells (1.41±0.16 vs. 1.98±0.13, P<0.05).

CONCLUSIONMiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.

ATP Binding Cassette Transporter, Sub-Family B ; drug effects ; ATP-Binding Cassette, Sub-Family B, Member 1 ; drug effects ; Cell Line, Tumor ; drug effects ; physiology ; Colorectal Neoplasms ; physiopathology ; Down-Regulation ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; physiology ; HCT116 Cells ; drug effects ; physiology ; Humans ; In Vitro Techniques ; MicroRNAs ; genetics ; pharmacology ; Multidrug Resistance-Associated Proteins ; drug effects ; Organoplatinum Compounds ; pharmacology ; PTEN Phosphohydrolase ; drug effects ; RNA, Messenger ; Receptors, G-Protein-Coupled ; drug effects ; genetics