Leilei Zhou

Cisplatin-resistant lung cancer cell–derived exosomes increase cisplatin resistance of recipient cells in exosomal miR-100–5p-dependent manner

Exosomes derived from lung cancer cells confer cisplatin (DDP) resistance to other cancer cells. However, the underlying mechanism is still unknown. A549 resistance to DDP (A549/DDP) was established. Microarray was used to analyze microRNA (miRNA) expression profiles of A549 cells, A549/DDP cells, A549 exosomes, and A549/DDP exosomes. There was a strong correlation of miRNA profiles between exosomes and their maternal cells. A total of 11 miRNAs were significantly upregulated both in A549/DDP cells compared with A549 cells and in exosomes derived from A549/DDP cells in contrast to exosomes from A549 cells. A total of 31 downregulated miRNAs were also observed. miR-100–5p was the most prominent decreased miRNA in DDP-resistant exosomes compared with the corresponding sensitive ones. Downregulated miR-100–5p was proved to be involved in DDP resistance in A549 cells, and mammalian target of rapamycin (mTOR) expression was reverse regulated by miR-100–5p. Exosomes confer recipient cells’ resistance to DDP in an exosomal miR-100–5p-dependent manner with mTOR as its potential target both in vitro and in vivo. Exosomes from DDP-resistant lung cancer cells A549 can alter other lung cancer cells’ sensitivity to DDP in exosomal miR-100–5p-dependent manner. Our study provides new insights into the molecular mechanism of DDP resistance in lung cancer.

Epithelial-to-mesenchymal transition correlates with gefitinib resistance in NSCLC cells and the liver X receptor ligand GW3965 reverses gefitinib resistance through inhibition of vimentin

The role of epithelial-to-mesenchymal transition in cancer drug resistance is increasingly acknowledged. We examined whether epithelial-to-mesenchymal transition affects gefitinib resistance in non-small cell lung cancer (NSCLC) cells. Cell viability was detected by CCK-8 assay, VIM expression levels were determined by quantitative real-time polymerase chain reaction. Western blot and immunocytochemistry were performed to determine the protein expression level of vimentin. We observed morphologic differences between gefitinib-sensitive and -insensitive cells. Compared with the sensitive parental cell line, HCC827, vimentin expression levels were increased in HCC827 cells with acquired gefitinib resistance. Vimentin expression was also markedly upregulated in cells with intrinsic gefitinib resistance, and upregulated vimentin expression was correlated with gefitinib sensitivity. Our previous study demonstrated that coadministration of gefitinib and GW3965 resulted in decreased cell proliferation and induced apoptosis. Therefore, we investigated the relationship among GW3965, vimentin, and gefitinib resistance in NSCLC cells by analysis of the expression of vimentin in cells treated with a combination of gefitinib and GW3965. Gefitinib treatment led to increased levels of intracellular vimentin, while combined treatment with gefitinib and GW3965 resulted in decreased vimentin expression levels through reduction of gefitinib drug resistance in NSCLC cells. Overall, these findings suggest that vimentin expression is associated with sensitivity to gefitinib, and our study highlights the potential usefulness of the drug, GW3965, for reversal of gefitinib resistance through inhibition of vimentin expression.

Liver X receptors agonist GW3965 re-sensitizes gefitinib-resistant human non-small cell lung cancer cell to gefitinib treatment by inhibiting NF-κB in vitro

The recent research shows that the inhibition of the nuclear factor-κB (NF-κB) pathway is a promising therapeutic option for patients who progress after treatment with the novel mutant-selective EGFR-TKIs. For propose to find a nontoxic drug to reverse the acquired gefitinib resistance, we examined whether the Liver X Receptors agonist GW3965 affect gefitinib resistance of HCC827/GR-8-2 cells. Cell viability was measured by CCK-8 assay. Levels of NF-κB, p-AKT and caspases were detected by Western blot analysis. Immunocytochemical analysis was used to detect the expression of NF-κB, p-AKT intracellularly. Induction of apoptosis and cell cycle arrest was measured by Flow cytometry assay. And results revealed that more than 90% of HCC827/GR-8-2 cells lived upon treatment with gefitinib at a dose of 5μM for 48h. However, when under the combine treatment of GW3965 (5μM) & gefitinib(5μM), cell death rate was increased observably. Co-administration of gefitinib & GW3965 induced cell apoptosis and cell cycle arrest. Additionally, we observed a dose-dependent- down-regulation of NF-κB in HCC827/GR-8-2 cells treated with gefitinib & GW3965. GW3965 and gefitinib synergistically decreased cell proliferation and induced apoptosis by inhibiting NF-κB signaling pathway in gefitinib resistant cells. These findings support our hypothesis that GW3965 could act as a useful drug to reverse the gefitinib resistance.

Genome-wide profiling of micro-RNA expression in gefitinib-resistant human lung adenocarcinoma using microarray for the identification of miR-149-5p modulation

To understand the mechanism involved in gefitinib resistance, we established gefitinib-resistant human HCC827/GR-8-1 cell line from the parental HCC827 cell line. We compared the micro-RNA expression profiles of the HCC827 cells HCC827/GR-8-1 using Agilent micro-RNA microarrays. The micro-RNAs, such as the miR-149-5p, were up- or downregulated and associated with acquired gefitinib resistance. Quantitative real-time polymerase chain reaction was then performed to verify the expression patterns of different micro-RNAs. The result showed that miR-149-5p was upregulated in the HCC827/GR-8-1 cell line. To investigate the biological function of miR-149-5p in non–small cell lung cancer cells acquired gefitinib resistance, we examined cell proliferation using a cell counting kit-8 assay. Cell viability was evaluated after the miR-149-5p mimics, inhibitors, and negative control were separately transfected into the non–small cell lung cancer cells. The results showed that the non–small cell lung cancer cells transfected with miR-149-5p mimics exhibited reduced cell motility. The drug-sensitivity assay results revealed that the overexpression of miR-149-5p effectively evaluates the half maximal inhibitory concentration values of the cell in response to gefitinib, and the downregulation of miR-149-5p can attenuate the half maximal inhibitory concentration values of the cell lines in response to gefitinib. Furthermore, the levels of miR-149-5p in the HCC827 and HCC827/GR-8-1 cells were inversely correlated with caspase-3 expression. In conclusion, this study revealed that miR-149-5p is upregulated in the HCC827/GR-8-1 cells and involved in the acquired gefitinib resistance.

High YBX1 expression indicates poor prognosis and promotes cell migration and invasion in nasopharyngeal carcinoma

Abstract Y‐box binding protein‐1 (YBX1) is a multifunctional protein and often acts as an indicator of poor prognosis in cancers. Increasing evidence has shown that the levels of YBX1 protein were closely associated with multidrug resistance, relapse, metastasis and poor prognosis in cancers. However, its role in nasopharyngeal carcinoma (NPC) metastasis remains unknown. In our study, we discovered that the expression of YBX1 was increased in nasopharyngeal carcinoma tissues. YBX1 protein levels positively correlated with T stage and metastasis of NPC patients. Moreover, expression of YBX1 was negatively correlated with membrane E‐cadherin levels and positively correlated with Vimentin expression. In vitro, the expression of YBX1 was closely related to the invasive and migratory ability of nasopharyngeal carcinoma cells. Knockdown of YBX1 inhibited migration and invasion in 5–8 F cells, and over‐expression of YBX1 promoted CNE1 cells migration and invasion. Transforming growth factor‐&bgr;1 (TGF‐&bgr;1) treatment led to epithelial‐to‐mesenchymal transition (EMT) in CNE1 cells accompanied by elevated YBX1 expression. On the contrary, knockdown of YBX1 partially inhibited the TGF‐&bgr;1‐induced CNE1 cell migration, together with changes of EMT‐associated markers. Our study revealed that TGF‐&bgr;1/YBX1 signaling might be one of novel mechanisms mediating EMT in NPC, providing a new target for the treatment of nasopharyngeal carcinoma. HighlightsYBX1 overexpressed in cytoplasmic of nasopharyngeal carcinoma cells.YBX1 expression associated with the EMT process in nasopharyngeal carcinoma.TGF‐&bgr;1 induced YBX1 expression in NPC cell lines.TGF‐&bgr;1/YBX1 signaling mediated the EMT process in NPC cell lines.

PPARγ agonist efatutazone and gefitinib synergistically inhibit the proliferation of EGFR-TKI-resistant lung adenocarcinoma cells via the PPARγ/PTEN/Akt pathway

ABSTRACT Development of acquired resistance to EGFR‐TKI therapy continues to be a serious clinical problem in Lung adenocarcinoma management. Peroxisome proliferator‐activated receptor gamma (PPAR&ggr;) agonists demonstrate anti‐tumor activity likely via transactivating genes that regulate cell proliferation, differentiation and apoptosis. Efatutazone, a novel later generation PPAR&ggr; agonist, selectively activates PPAR&ggr; target genes and has antiproliferative effects in a range of malignancies. However, the exact function and molecular mechanism of PPAR&ggr; agonists efatutazone in EGFR‐TKI gefitinib‐resistance of Lung adenocarcinoma has not been determined. In this study, we studied the development of acquired resistance to an EGFR‐TKI gefitinib in lung adenocarcinoma cells and investigated the antiproliferative effects of efatutazone in the acquired resistant cells. The treatment of gefitinib‐resistant cells with efatutazone reduced the growth of gefitinib‐resistant cells in a dose‐ and time‐dependent manner, and facilitated the anti‐proliferative effects of gefitinib. Mechanistic investigations suggested that efatutazone acted by upregulating protein expression of PPAR&ggr;, phosphatase and tensin homolog (PTEN), inactivating the Akt pathway, followed by dephosphorylation of p21Cip1 at Thr145 without affecting the transcriptional levels. Our results suggested that efatutazone, alone or in combination with gefitinib, might offer therapeutic effects in lung adenocarcinoma. HIGHLIGHTSThe low expression of PPARG predicts poor overall survival for patients with lung adenocarcinoma.Treatment with efatutazone and gefitinib produced a synergistic effect on inhibiting proliferation of EGFR‐TKI‐resistant cells.The PPAR&ggr;/PTEN/Akt/p21 signaling pathway might be involved in above synergistic effect.Efatutazone is a potential therapeutic agent for the NSCLC patients who develop acquired resistance to EGFRT‐KI.

MicroRNA-106b-5p regulates cisplatin chemosensitivity by targeting polycystic kidney disease-2 in non-small-cell lung cancer

Systemic therapy with cytotoxic agents remains one of the main treatment methods for non-small-cell lung cancer (NSCLC). Cisplatin is a commonly used chemotherapeutic agent, that, when combined with other drugs, is an effective treatment for NSCLC. However, effective cancer therapy is hindered by a patient’s resistance to cisplatin. Unfortunately, the potential mechanism underlying such resistance remains unclear. In this study, we explored the mechanism of microRNA-106b-5p (miR-106b-5p), which is involved in the resistance to cisplatin in the A549 cell line of NSCLC. Quantitative real-time PCR was used to test the expression of miR-106-5p in the A549 and the A549/DDP cell line of NSCLC. The cell counting kit-8 assay was used to detect cell viability. Flow cytometry was used to measure cell cycle and cell apoptosis. Luciferase reporter assays and western blot were performed to confirm whether polycystic kidney disease-2 (PKD2) is a direct target gene of miR-106b-5p. Immunohistochemistry was performed to examine the distribution of PKD2 expression in patients who are sensitive and resistant to cisplatin. The experiments indicated that the expression of miR-106b-5p was significantly decreased in A549/DDP compared with that in A549. MiR-106b-5p affected the tolerance of cells to cisplatin by negatively regulating PKD2. Upregulation of miR-106b-5p or downregulation of PKD2 expression can cause A549/DDP cells to become considerably more sensitive to cisplatin. The results showed that miR-106b-5p enhanced the sensitivity of A549/DDP cells to cisplatin by targeting the expression of PKD2. These findings suggest that the use of miR-106b-5p may be a promising clinical strategy in the treatment of NSCLC.

MiRNA signature predicts the response of patients with advanced lung adenocarcinoma to platinum-based treatment

BackgroundAccumulating literature proved that miRNAs can regulate the sensitivity of platinum and act as a promising candidate to predict the response of patients with lung adenocarcinoma to chemotherapy. However, most studies on miRNAs were restricted to in vitro experiments. This study aimed to evaluate whether miRNAs alone or in combination (miRNA signature) can act as predictive biomarkers of platinum-based chemotherapy in patients with lung adenocarcinoma.MethodsEight miRNAs that most probably predict the efficacy of platinum were screened in 111 tumor tissues of lung adenocarcinoma. Univariate and multivariate Cox analyses, Kaplan–Meier survival curve analysis, Chi-square test, and univariate and multivariate logistic regression analyses were employed to determine whether miRNA expression is associated with the response of patients to platinum-based chemotherapy. The maximum significant odds ratio value was acquired by multiple cycles of multivariate logistic regression analysis. The cut-off points of miRNAs were obtained. A miRNA chemo-sensibility index (CI) formula was established, and its prediction performance was confirmed in another independent set (n = 31).ResultsUnderexpression of three miRNAs (miRNA-21, miRNA-125b, and miRNA-224) was independently associated with the chemotherapy sensitivity of patients with lung adenocarcinoma. The miRNA CI formula containing these three miRNAs was calculated as (1.364 × miR-21) + (1.323 × miR-125b) + (1.131 × miR-224). A high CI was related to platinum-based chemotherapy resistance, and its prediction performance was confirmed in the testing set. The MAPK, PI3K-Akt, Ras, and cGMP-PKG signaling pathways were considered to be most probably correlated with platinum resistance.ConclusionOur miRNA CI formula can act as an independent predictor to predict the response of patients with lung adenocarcinoma to platinum-based chemotherapy.

T-cell lymphomas associated gene expression signature: Bioinformatics analysis based on gene expression Omnibus

Due to the low incidence and the heterogeneity of subtypes, the biological process of T‐cell lymphomas is largely unknown. Although many genes have been detected in T‐cell lymphomas, the role of these genes in biological process of T‐cell lymphomas was not further analyzed.

LncRNA HOXA11-AS drives cisplatin resistance of human LUAD cells via modulating miR-454-3p/Stat3

Over the past several years, long non‐coding RNAs (lncRNAs) have attracted more and more attention due to their special functions. They are vital biomarkers in multiple diseases. LncRNA HOMEOBOX A11 (HOXA11) has been found to be aberrantly expressed in some kinds of malignant tumors. In this study, we mainly discuss the oncogenic role of it in promoting malignant progression and chemoresistance in lung adenocarcinoma (LUAD) cells. The expression of HOXA11‐AS was much stronger in cisplatin‐resistant LUAD cells. Based on The Cancer Genome Atlas database, patients with high expression of HOXA11‐AS had shorter survival time. Additionally, knockdown of HOXA11‐AS caused positive changes in cell activities of LUAD. For example, cell proliferation and migration were weakened, the epithelial mesenchymal transition process was reversed, and apoptosis was induced. These changes were more obvious in cells treated with cisplatin. Next, the HOXA11‐AS/miR‐454‐3p/Stat3 (signal transducer and activator of transcription 3) pathway was found to influence the cisplatin resistance of LUAD cells. HOXA11‐AS specifically acted as a competing endogenous RNA (ceRNA) in LUAD cells. The combinations among these three genes were demonstrated. Finally, rescue assays were applied to demonstrate the ceRNA pattern consisting of HOXA11‐AS, miR‐454‐3p and Stat3. In conclusion, lncRNA HOXA11‐AS acted as a ceRNA to promote cisplatin resistance of human LUAD cells via the miR‐454‐3p/Stat3 axis.