Shenglin Ma

MiR-1244 sensitizes the resistance of non-small cell lung cancer A549 cell to cisplatin

BackgroundCisplatin (DDP)-based chemotherapy is the mainstay of first-line therapy for lung cancer. However, their efficacy is often limited by the existence or development of chemoresistance. The aim of this study was to find and investigate the function of miRNAs in cisplatin (DDP)-resistant non-small cell lung cancer (NSCLC) A549 cell.MethodsQuantitative real-time PCR assay was employed to compare the differences of miRNA expression in both cisplatin-resistant A549 (A549/DDP) cell and the parental A549 cell. The dysregulated miRNAs were then corrected by transfecting oligonucleotides into A549/DDP cells. The cellular sensitivity to cisplatin, cell apoptosis and migration were conducted by MTT, flow cytometry and cell wound healing assay, respectively.ResultsBoth miR-589 and miR-1244 were significantly down-regulated in A549/DDP cell compared to the parental A549, while the expression of miR-182 and miR-224 were increased in A549/DDP cell (Pxa0<xa00.05). Importantly, transfection of the cisplatin-resistant cells with either miR-589 or miR-1244 resulted in an increased sensitivity to cisplatin, indicating that the dysregulated miRNA may play an important role in chemotherapy resistance in cancer cell. The rescued expression of miRNA also reduced cell invasion and increased apoptosis of A549/DDP cell.ConclusionThe study indicates a crucial role of miR-1244 in the progress of cisplatin resistance of A549. Further understanding of miR-1244-mediated signaling pathways may promote the clinical use of miR-1244 in lung cancer therapy.

Increased MIR31HG lncRNA expression increases gefitinib resistance in non-small cell lung cancer cell lines through the EGFR/PI3K/AKT signaling pathway

The aim of the present study was to gain insight into the molecular mechanism of gefitinib resistance in non-small cell lung cancer (NSCLC), and demonstrate whether long noncoding RNA (lncRNA) expression signatures differ between gefitinib-sensitive PC9 and gefitinib-resistant PC9 (PC9-R) cell lines. PC9 and PC9-R cells were treated with gefitinib and, after 48 h, proliferation and apoptosis were analyzed using a Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Microarray expression profiling of lncRNAs was undertaken in both PC9 and PC9-R cells, and the expression profiles were verified by reverse transcription quantitative-polymerase chain reaction. The EGFR/PI3K/AKT signaling pathway and mitochondrial apoptosis protein expression levels were assessed by western blot analysis. The PC9 cell line treated with gefitinib had a more significant effect on cell viability and apoptosis than the PC9-R cell line (P<0.05). Expression of various lncRNAs differed significantly between the two cell lines, and MIR31HG expression in particular was significantly higher in PC9-R cells. As expected, MIR31HG lncRNA knockdown sensitized PC9-R cells to gefitinib, and further experiments revealed that turning off the EGFR/PI3K/AKT signaling pathway activated expression of p53 in PC9-R cells transfected with si-MIR31HG. Furthermore, PC9-R cells transfected with si-MIR31HG induced cell apoptosis through the mitochondrial apoptosis pathway, and arrested the cell cycle in the G0/G1 phase. The results of the current study suggest that MIR31HG lncRNA levels in PC9-R cells are higher than in PC9 cells. Furthermore, overexpression of MIR31HG lncRNAs may contribute to gefitinib resistance in PC9-R cells through the EGFR/PI3K/AKT pathway, which impacts on cell proliferation, apoptosis and the cell cycle. MIR31HG lncRNA may therefore be a novel candidate biomarker for future therapeutic strategies involving EGFR-TKIs.

Chronic necrotizing pulmonary aspergillosis in an immunocompetent patient: report of a rare case

Chronic necrotizing pulmonary aspergillosis (CNPA) is a relatively uncommon manifestation of infection with Aspergillus spp. which mainly affects immunocompromised or immunostressed individuals with underlying lung diseases. Here, we present a case of mediastinum-involved CNPA in an immunocompetent patient with no symptoms and previous good health.

Comparison of the Amplification Refractory Mutation System, Super Amplification Refractory Mutation System, and Droplet Digital PCR for T790 M Mutation Detection in Non-small Cell Lung Cancer after Failure of Tyrosine Kinase Inhibitor Treatment

Plasma mutation detection has the advantages of non-invasiveness and accessibility. Here, we evaluated three methods, the amplification refractory mutation system (ARMS), second-generation ARMS (SuperARMS), and droplet digital PCR (ddPCR), to assess their concordance and feasibility for the detection of mutations in plasma samples. Non-small lung cancer patients with stage IIIB/IV that were resistant to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment were enrolled. Blood samples were collected within 14xa0days after TKI resistance. Each sample was simultaneously assessed by the three methods. In total, 169 patients were enrolled; 54.4% were female, 72.2% were diagnosed with stage IV disease; and 97.6% had adenocarcinoma. T790xa0M mutations were detected in 42 (24.8%) of the 169 samples using ARMS, one of which carried the T790xa0M alone, 22 that also encoded exon 19 deletions, and 19 with L858R mutations. For the SuperARMS assay, 59 (34.9%) samples exhibited the T790xa0M mutation, and 110 (65.1%) showed no detectable T790xa0M mutation. ddPCR showed that 61 (36.1%) samples contained the T790xa0M mutation, whereas 108 (63.9%) were not positive. T790xa0M abundance ranged from 0.04% to 38.2%. The median T790xa0M abundance was 0.15% for total samples and 2.98% for T790xa0M mutation samples. The overall concordance was 78.7% (133/169) among ARMS, SuperARMS, and ddPCR. Compared with patients with stage III disease, patients with stage IV disease exhibited a higher T790xa0M mutation detection rate (28.7% vs. 14.9% by ARMS; 37.7% vs. 27.7% by SuperARMS; and 41.8% vs. 21.3% by ddPCR). Liquid biopsy showed promise and has the advantages of non-invasiveness and accessibility. T790xa0M detection based on circulating tumor DNA showed high concordance. Compared with non-digital platforms, ddPCR showed higher sensitivity and provided both frequency and abundance information, which might be important for treatment decisions.

Lung cancer suppressor gene GPRC5A mediates p53 activity in non‑small cell lung cancer cells in vitro

Cellular tumor antigen p53 (p53) functions to maintain genomic stability and regulate cell apoptosis, while G protein‑coupled receptor class C group 5 memberxa0A (GPCR5A) is a lung cancer suppressor gene whose expression is induced by retinoids. The present inxa0vitro study assessed the effects of p53 on the regulation of GPRC5A expression and on non‑small cell lung cancer (NSCLC) cells. Human NSCLC H1299 (p53‑null) and A549 (wild‑type p53) cell lines were subjected to p53 cDNA and small interfering (si)RNA transfection, respectively. GPRC5A expression was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blotting, and cell behavior was analyzed using cell viability and apoptosis assays. The results of the present study demonstrated that knockdown of GPRC5A expression markedly upregulated tumor cell viability and reduced tumor cell apoptosis, while p53 overexpression in H1299 cells significantly increased the expression level of GPRC5A. p53 overexpression and GPRC5A induction markedly inhibited tumor cell viability and induced apoptosis, while knockdown of p53 resulted in a decrease in GPRC5A expression, inhibited tumor cell apoptosis and increased tumor cell viability. In serum‑free culture conditions, GPRC5A expression was decreased in the two cell lines; this decrease was less marked in p53 cDNA‑transfected H1299 cells and more marked in p53 siRNA‑transfected A549 cells. The results of the present study indicated that p53 antitumor activity may be mediated by GPRC5A in NSCLC cells.

Identification of miRNAs as non-invasive biomarkers for early diagnosis of lung cancers

Current clinical diagnostic methods lack the specificity in detecting lung cancer patients. The issue is particularly critical for stage I and II patients. Considerable evidence showed microRNA plays a very important role in lung carcinogenesis. Here, we identified a panel of 41 miRNAs significantly elevated in patients with lung cancer, of which eight miRNAs were further validated in an independent sample cohort. Classification analysis using the panel of eight miRNAs generated a discriminatory power of 93.3xa0% sensitivity and 93.8xa0% specificity in separating non-small-cell lung cancer (NSCLC) patients from normal controls, indicating the miRNAs have a potential clinical utility in discriminating NSCLC. Interestingly, miR-1244 was found significantly elevated in the serum samples of lung cancer patients, and the test characteristics of the single miRNA were area under the curve (AUC) of 0.832 in NSCLC vs healthy controls, and 0.861 in NSCLC vs patients with unidentified pulmonary nodules. This is the first study showing serum miR-1244 could be a biomarker to screen lung cancer patients from the high-risk population.

Clinical significance of reduced GPRC5A expression in surgically resected non‑small cell lung cancer

G protein-coupled receptor, family C, group 5 member A (GPRC5A) is a retinoid-inducible protein, which has been characterized as a tumor-suppressor gene in lung cancer. The present study further examined GPRC5A expression in non-small cell lung cancer (NSCLC) for any association with the clinical features and treatment outcomes of patients with NSCLC. A total of 30 paired NSCLC tumor and adjacent normal tissues were analyzed for the detection of GPRC5A mRNA and protein using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. Immunohistochemistry was performed to determine the GPRC5A expression levels in 110 NSCLC and 60 para-tumor tissues. The results confirmed significantly lower expression levels of GPRC5A in NSCLC tumors compared with the corresponding noncancerous tissues (P<0.001). Lost GPRC5A expression was significantly associated with the tumor histological type (P=0.008), poor tumor differentiation (P<0.001) and tumor-node-metastasis (TNM) stage (P<0.001). Kaplan-Meier curve analysis revealed that patients with NSCLC with low GPRC5A expression tumors had a worse prognosis compared with those with high GPRC5A expression tumors (P=0.010). The results of multivariate Cox analysis further suggested that low GPRC5A expression was an independent prognostic factor for patients with NSCLC (P<0.001). The results of this study suggest GPRC5A expression has clinical potential as a prognostic biomarker for patients with NSCLC.

Hyperthermia inhibits the motility of gemcitabine-resistant pancreatic cancer PANC-1 cells through the inhibition of epithelial-mesenchymal transition

Pancreatic cancer (PC) is one of the most common types of malignant tumor and the leading cause of cancer‑associated mortality worldwide. The chemotherapeutic drug gemcitabine (GEM) is used as a first‑line chemotherapeutic agent for advanced PC. However, the acquisition of drug resistance is a major limitation of the clinical effect of GEM and commonly leads to increased metastasis. The occurrence of epithelial‑mesenchymal transition (EMT) has been demonstrated to be the underlying mechanism of acquired resistance. It has been reported that heat treatment is able to inhibit EMT in pancreatic adenocarcinoma cells. In the present study the effect of hyperthermia on the sensitivity of GEM‑resistant PC cells was investigated. First a GEM‑resistant PC cell line PANC‑1 (PAN/GEM) was developed and it was demonstrated that drug resistant PAN/GEM cells exhibited significantly increased migratory and invasive abilities compared with control PANC‑1 cells using a Transwell assay. EMT was induced in resistant PAN/GEM cells, followed by reduced epithelial marker epithelial (E)‑cadherin expression and increased mesenchymal marker Vimentin expression compared with control PANC‑1 cells. Next, the Transwell assay demonstrated that the hyperthermia at 42˚C for 1xa0h combined with GEM significantly attenuated migration and invasion in drug resistant PAN/GEM cells, while GEM alone treatment did not significantly affect the migration and invasion. Additionally, EMT in PAN/GEM cells was reversed by hyperthermia, as demonstrated by the restoration of E‑cadherin and downregulation of mesenchymal markers Vimentin, matrix metalloproteinase (MMP)2 and MMP9. Furthermore, an MMP2 inhibitor tissue inhibitor of metalloproteinases (TIMP)2 and MMP9 inhibitor TIMP1 were used to treat PAN/GEM cells and it was demonstrated that both inhibitors increased the inhibition of hyperthermia treatment combined with GEM on cell invasion, suggesting an association between cell invasion and MMP2, and MMP9. Additionally, proliferation of PAN/GEM cells following hyperthermia was assessed using an MTT assay. The results demonstrated that proliferation in PAN/GEM cells treated with hyperthermia was significantly inhibited by GEM compared with GEM alone treated cells, indicating that hyperthermia enhanced the inhibition of GEM on cell growth and resensitized the drug‑resistant cells to GEM. Overall, the results of the present study suggested that hyperthermia is able to resensitize GEM‑resistant PANC‑1 cells to GEM by reversing EMT via the regulation of EMT‑associated factors, therefore inhibiting cell migration and invasion.

Notch1 suppresses prostate cancer cell invasion via the metastasis‑associated 1‑KiSS‑1 metastasis‑suppressor pathway

Notch1 is a type-1 transmembrane receptor which has been demonstrated to be involved in proliferation in various organisms. A number of studies have proposed that Notch signaling may be aberrantly activated, thus contributing to development, invasion and metastasis in a variety of human cancers. In the present study, the function and mechanism of Notch1 in human prostate cancer (PCa) LNCaP cells in vitro was investigated. Notch1 and cleaved-Notch1 expression were evaluated in human PCa cell lines, including LNCaP, PC-3 and DU 145, and the human prostate epithelial RWPE-1 cell line. LNCaP cells were transfected with Notch1-targeting short hairpin RNAs (shRNAs) and the level of proliferation, the ability to invade and the expression of genes associated with cancer cell invasion were subsequently investigated. Notch1 was highly expressed in LNCaP, PC-3 and DU 145 cells compared with RWPE-1 cells, while cleaved-Notch1 was expressed in LNCaP, PC-3 and DU 145 cells, and only to a minimal extent in RWPE-1 cells. Knockdown of Notch1 by shRNA in LNCaP cells markedly decreased cell invasion through Matrigel and inhibited cell proliferation 48 h following transfection. Reverse transcription-quantitative polymerase chain reaction analysis indicated that Notch1-knockdown resulted in a significant reduction of metastasis-associated 1 (MTA1) and increase of KiSS-1 metastasis-suppressor (KISS-1), mitogen-activated protein kinase 4 (MKK4) and cluster of differentiation 82 (KAI1). The present data demonstrated that expression of Notch1 was significantly associated with the invasion of prostate cancer. Knockdown of Notch1 decreased the invasive ability of LNCaP cells, which may be caused by downregulating MTA1 and upregulating KISS-1, MKK4 and KAI1. These findings indicated that targeting Notch1 may provide a novel method of suppressing or treating metastasis in prostate cancer.