Combined RNAi targeting human Stat3 and ADAM9 as gene therapy for non-small cell lung cancer.

[This retracts the article DOI: 10.3892/ol.2015.4018.].

Zafirlukast promotes mitochondrial respiration by stimulating mitochondrial biogenesis in human bronchial epithelial cells.

Lung diseases, including asthma, pose a serious global health issue. Loss of mitochondrial function and decreased mitochondrial biogenesis play pivotal roles in the initiation and progression of chronic lung diseases. Thus, maintaining mitochondrial function and homeostasis is an important treatment goal. Zafirlukast is a CysLTR1 antagonist that is widely used as an adjuvant treatment for asthma. In the present study, we investigated the effects of zafirlukast in vitro using human bronchial epithelial cells (BECs). We performed measurements of oxygen consumption and bioenergetics and found that zafirlukast increased mitochondrial respiration and biogenesis in human BECs as evidenced by increased mitochondrial mass and mtDNA/nDNA. Through real-time PCR and western blot analysis, we found that zafirlukast significantly increased the expression of PGC-1α, NRF1, and TFAM at both the mRNA and protein levels. Finally, we determined that these effects are mediated through CREB signaling and that inhibition of CREB with its specific inhibitor H89 abolished the effects of zafirlukast described above. Thus, zafirlukast might have potential in enhancing mitochondrial function by promoting mitochondrial biogenesis in human bronchial epithelial cells through upregulating the expression of PGC-1α and activating the CREB pathway.

Long non-coding RNA CASC9 promotes the progression of retinoblastoma via interacting with miR-145-5p.

Abnormal expression of long non-coding RNA cancer susceptibility candidate 9 (CASC9) has been found to play vital roles in many human tumors. However, the role and the regulatory mechanism of CASC9 have not yet been demonstrated in retinoblastoma (RB). Hence, we performed this study to explore the function and mechanism of CASC9 in RB. CASC9 expression was firstly detected in human RB tissues and cells. The influence of CASC9 on the malignant phenotypes of RB cells, including cell proliferation, invasion, epithelial-mesenchymal transition (EMT) and apoptosis, was analyzed by overexpressing or silencing CASC9. The association between CASC9, miR-145-5p and E2F transcription factor 3 (E2F3) was determined by dual-luciferase reporter assay and RNA immunoprecipitation. We found that CASC9 expression was elevated in RB tissues and cells. Overexpression of CASC9 significantly facilitated the proliferation, invasion and EMT of RB cells. On the contrary, knockdown of CASC9 inhibited the proliferation, invasion and EMT, while enhanced the apoptosis of RB cells. CASC9 acted as a competing endogenous RNA (ceRNA) for miR-145-5p to regulate E2F3. Additionally, miR-145-5p inhibitor and E2F3 overexpression both partly reversed the malignant phenotypes of RB cells affected by CASC9 knockdown. However, miR-145-5p overexpression further strengthened these features induced by CASC9 downregulation. These findings suggested that CASC9 contributed to RB development by regulating E2F3 via sponging miR-145-5p. CASC9 might be a possible therapeutic target for RB.

MicroRNA-124 alleviates the lung injury in mice with septic shock through inhibiting the activation of the MAPK signaling pathway by downregulating MAPK14.

OBJECTIVE: Acute lung injury (ALI) is a severe lung disease with high mortality rate. Research has highlighted that the immune response to ALI is associated with significant changes in the expression of several microRNAs (miRNAs) in the lungs. In our research, we speculated that miR-124 moderated the severity of ALI through comprehensive suppression of the mitogen-activated protein kinase (MAPK) signaling pathway activation by targeting MAPK14. METHODS: A mouse model of ALI was established by array of experiments. The expression of MAPK14 and miR-124 was assessed in the tissues of ALI mice and the expression of inflammatory cytokines in ALI mice was determined. The expression of the related kinases in the MAPK signaling pathway and key cytokines in the pro-inflammatory response were assessed by a series of experiments. Immunohistochemistry and TUNEL staining were adopted to detect lung tissue cell proliferation and apoptosis in mice with ALI. RESULTS: MiR-124 was poorly expressed and MAPK14 was highly expressed in tissues of ALI mice. Overexpression of miR-124 or silence of MAPK14 alleviated the symptoms of ALI by down-regulating inflammatory cytokines expression, which could intrinsically suppress the expression of associated proteins in the MAPK signaling pathway and the downstream pro-inflammatory response factors, promote proliferation and inhibit apoptosis of lung tissue cells. Overexpression of MAPK14 inverted the phenotypic changes induced by overexpressing miR-124. CONCLUSION: These results indicated that miR-124 could alleviate the symptoms of ALI by inhibiting the activation of MAPK signaling pathway via subsequent targeting of MAPK14. Additionally, miR-124 may serve as a useful biomarker to alleviate the severity of septic shock-induced lung injury.

Long non-coding RNA FENDRR attenuates the stemness of non-small cell lung cancer cells via decreasing multidrug resistance gene 1 (MDR1) expression through competitively binding with RNA binding protein HuR.

The roles of long non-coding RNA (lncRNA) FENDRR in non-small cell lung cancer (NSCLC) cells progression have never been revealed. As cancer stem cells (CSCs) act important roles in tumor progression, here, we focused on FENDRR roles in NSCLC cell stemness. We found that lncRNA FENDRR expression was significantly decreased in lung cancer tissues and cells, especially in NSCLC cells. Then we constructed NSCLC cells with FENDRR stable overexpression and revealed that FENDRR overexpression attenuated the stemness of NSCLC cells, evident by decreased stemness markers expression and capacity of cell spheroid formation. Mechanistically, we found that FENDRR could directly and specifically bind to the 3' untranslated region (3'UTR) of multidrug resistance gene 1 (MDR1), hinder the binding of RNA binding protein HuR to MDR1 3'UTR and thus decrease MDR1 expression. Finally, we demonstrated that FENDRR exerted its effects on NSCLC cell stemness through the HuR/MDR1 axis. Our results suggest that FENDRR attenuates NSCLC cell stemness through inhibiting the HuR/MDR1 axis.

Long non-coding RNA-NEF targets glucose transportation to inhibit the proliferation of non-small-cell lung cancer cells.

Long non-coding RNA (lncRNA)-NEF is a newly discovered lncRNA, which exhibits an inhibitory function on the metastasis of hepatocellular carcinoma, while its involvement in other types of malignancy are unknown. In the present study, tumor and adjacent healthy tissues were obtained from patients with non-small-cell lung cancer (NSCLC), and blood was obtained from patients with NSCLC and healthy individuals. Expression levels of lncRNA-NEF in tumor tissue samples, healthy tissue samples and serum were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Receiver operating characteristic curve analysis and survival curve analysis were performed to evaluate the diagnostic and prognostic value of serum lncRNA-NEF for NSCLC. The effects of lncRNA-NEF overexpression in NSCLC cell lines on tumor cell proliferation, glucose uptake, glucose transporter 1 (GLUT1) protein expression and mRNA expression were investigated by Cell Counting kit-8 assay, glucose uptake assay, western blot analysis and RT-qPCR, respectively. It was identified that lncRNA-NEF was downregulated in NSCLC tissues, compared with healthy controls, and the serum level of lncRNA-NEF was negatively associated with primary tumor stage. Therefore, serum lncRNA-NEF may be a sensitive diagnostic and prognostic marker for NSCLC. Overexpression of lncRNA-NEF inhibited NSCLC cell proliferation and glucose uptake, and downregulated GLUT1 expression. Therefore, it can be concluded that lncRNA-NEF can target glucose transportation to inhibit the proliferation of NSCLC cells.

A study on changes and clinical significance of blood glucose, blood lipid and inflammation in patients with ovarian cancer.

PURPOSE: To investigate the changes in blood glucose, blood lipid and inflammation in patients with ovarian cancer and their clinical significance. METHODS: 67 patients diagnosed with ovarian cancer and treated in our hospital from January 2018 to December 2018 constituted the observation group. Fifty healthy women in the corresponding time period were enrolled as the control group. The levels of blood glucose, blood lipid and inflammation were compared between the two groups, and then the changes in those levels in ovarian cancer patients in different clinical stages were analyzed. RESULTS: The levels of fasting blood glucose and 2-h postprandial blood glucose of the observation group were significantly higher than those of the control group, and they were also significantly higher in patients in stage III-IV than those in stage I-II (p<0.05). In the observation group, the levels of total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) were lower than those in the control group. Compared with the patients in stage I-II, the patients in stage III-IV had remarkably lower levels of TC and HDL-C (p<0.05). The levels of C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in the observation group were evidently higher than those in the control group, and they were markedly higher in patients in stage III-IV than those in stage I-II (p<0.05). CONCLUSIONS: Both the metabolism disorders of blood lipid and blood glucose and inflammatory response are more obvious in ovarian cancer patients, indicating that these indicators have a place for early screening and clinical treatment of ovarian cancer.

MicroRNA-155 regulates the proliferation, cell cycle, apoptosis and migration of colon cancer cells and targets CBL.

MicroRNA-155 (miR-155) is a well-studied miR and acts as an oncomiR in numerous cancer types. However, the biological functions of miR-155 in colon cancer as well as its target genes have remained to be fully elucidated. In order to investigate the biological functions of miR-155, MTT, colony formation and wound healing assays, cell cycle analysis and detection of apoptosis were performed. The results demonstrated that miR-155 promoted the proliferation of colon cancer cells and enhanced their colony formation capacity, promoted their cell cycle progression and inhibited apoptosis. miR-155 also promoted the migration of colon cancer cells. In the present study, casitas B-lineage lymphoma was identified as a novel target of miR-155. The present study suggested that miR-155 functions as an oncomiR in colon cancer cells and may become a promising therapeutic target for colon cancer therapy.

Molecular characterization of 20 small supernumerary marker chromosome cases using array comparative genomic hybridization and fluorescence in situ hybridization.

The variability of a small supernumerary marker chromosome (sSMC)-related phenotype is determined by the molecular component, the size, and shape of the marker chromosome. As fluorescence in situ hybridization has limitations regarding the resolution, efficiency, and accuracy. Recently, array comparative genomic hybridization (aCGH) was used for sSMC characterization. In this study, twenty cases with sSMCs were characterized by aCGH and FISH. Chromosomal origin of the marker chromosomes were successfully identified in seventeen of them. For the three cases with negative aCGH results, two of them were more likely due to that the sSMCs only contained centromere heterochromatin, whereas the reason for the remaining case with negative aCGH finding was uncertain. In order to establish a stronger genotype-phenotype correlation for clinical service in the future and avoid miss characterization, more sSMC cases were needed to be detailed characterized. This will help to clarify the variable clinical characteristics of sSMCs and provide additional information to aid clinical service and future research.

RNA-binding protein Dnd1 inhibits epithelial-mesenchymal transition and cancer stem cell-related traits on hepatocellular carcinoma cells.

OBJECTIVES: To investigate the roles of Dead end 1 (Dnd1) in modulating cancer stem cell-related traits of hepatocellular carcinoma (HCC). RESULTS: Dead end (Dnd1) inhibited spheroid formation, suppressed the expression of stemness-related genes, and increased sensitivity to sorafenib in HCC cells. Mechanistically, Dnd1 could bind to 3'-UTR of LATS2, the key kinase of Hippo pathway, thus elevating LATS2 mRNA stability and its expression, subsequently leading to phosphorylation of YAP and its cytoplasmic retention. As a result, epithelial-mesenchymal transition (EMT) was weakened and therefore the generation of HCC stem cell properties was suppressed. CONCLUSIONS: Dnd1 functions as a tumor suppressor by prohibiting CSC-like characteristics via activating Hippo pathway in HCC cells. Dnd1 could thus be a novel therapeutic target for HCC patients.

Left renal failure caused by chronic obstructive uropathy due to a uretero-inguinal hernia combined with contralateral renal pelvic carcinoma: A case report.

RATIONALE: Although rare, cases of renal failure secondary to a uretero-inguinal hernia have been reported. PATIENT CONCERNS: Here, we report a case of left renal failure caused by chronic obstructive uropathy due to a uretero-inguinal hernia combined with contralateral renal pelvic carcinoma. DIAGNOSES: The present case highly indicated that a comprehensive examination is very important when diagnosing an inguinal hernia. In particular, it is necessary to check whether the ureter is involved or not. INTERVENTIONS: A computed tomography scan should be performed to a uretero-inguinal hernia patient. OUTCOMES: Unfortunately, this patient was diagnosed too late to attempt surgical management to restore the left renal functions. LESSONS: In our opinion, a computed tomography scan is highly recommended for an accurate diagnosis.

MicroRNAs-491-5p suppresses cell proliferation and invasion by inhibiting IGF2BP1 in non-small cell lung cancer.

MicroRNAs-491-5p (miR-491-5p) has been found to involve in tumor initiation and development in several tumors. However, the biological function and underlying molecular mechanism of miR-491-5p in non-small lung cancer (NSCLC) remain unclear. This study was therefore to investigate biological role of and underlying molecular mechanisms of in NSCLC. It was found that miR-491-5p expression was significantly downregulated in NSCLC tissues when compared with corresponding adjacent normal tissues (P<0.01), and the value was negatively related to advanced and tumor-node-metastasis (TNM) stage and lymph node metastasis (both P<0.01). We also demonstrate that restoration of miR-491-5p suppressed NSCLC cell proliferation by arresting NSCLC cells in the G1/G0 phase and accelerating apoptosis. miR-491-5p also inhibited cell migration and invasion in NSCLC cells. Mechanically, IGF2BP1 was identified as direct targets of miR-491-5p. And IGF2BP1 expression was significantly upregulated, and correlated negative with miR-491-5p expression in NSCLC tissues. In vivo assay showed thatmiR-491-5p suppressed tumor growth in nude model by repressing IGF2BP1 expression. Collectively, miR-491-5p functioned as a tumor suppressor in NSCLC by targeting IGF2BP1. Restoration of miR-491-5p expression may represent a promising therapeutic approach for targeting malignant NSCLC.

Combined RNAi targeting human Stat3 and ADAM9 as gene therapy for non-small cell lung cancer.

Previous studies have demonstrated that human signal transducer and activator of transcription 3 (Stat3) and disintegrin and metalloproteinase 9 (ADAM9) are promising targets for RNA interference (RNAi)-based gene therapy for human non-small cell lung cancer (NSCLC). Thus, in the present study, the recombinant lentiviral (Lv) small hairpin (sh)RNA expression plasmids Lv/sh-Stat3 and Lv/sh-ADAM9, which targeted Stat3 and ADAM9, respectively, were constructed and subsequently infected into the A549 human NSCLC cell line. Cell proliferation, migration, invasion and apoptosis were determined in vitro in A549 cells following treatment with Lv/sh-Stat3 or Lv/sh-ADAM9 alone or in combination. In addition, the combined effect of Lv/sh-Stat3 and Lv/sh-ADAM9 gene therapy was evaluated in vivo using A549 xenograft models in nude mice. The in vitro experiments demonstrated that A549 cells treated with a combination of Lv/sh-Stat3 and Lv/sh-ADAM9 exhibited a significant additive effect in their cell proliferation, migration, invasion and apoptosis abilities, compared with A549 cells treated with Lv/sh-Stat3 or Lv/sh-ADAM9 alone. The in vivo experiments conducted in A549 xenograft tumor mouse models revealed that the combined treatment with Lv/sh-Stat3 and Lv/sh-ADAM9 exerted an additive effect on tumor growth inhibition, compared with the treatment with Lv/sh-Stat3 or Lv/sh-ADAM9 alone. These results suggested that combined RNAi gene therapy targeting human Stat3 and ADAM9 may be a novel and promising strategy for the treatment of NSCLC.

MicroRNA-92a promotes growth, metastasis, and chemoresistance in non-small cell lung cancer cells by targeting PTEN.

MicroRNA-92a (miR-92a) has been reported to play important roles in tumorigenesis of human various cancers. However, the roles and underlying molecular mechanism of miR-92a in non-small cell lung cancer (NSCLC) have not been totally elucidated. Therefore, the aims of this study were to determine the role of miR-92a and to elucidate its regulatory mechanism in NSCLC. We found that miR-92a was significantly upregulated in NSCLC tissues compared to matched adjacent normal lung tissues, and its expression is significantly associated with clinical characteristics of patients, including tumor, node, and metastasis (TNM) stage; tumor size; and lymph node metastasis (all P < 0.01). Function assays demonstrated that upregulation of miR-92a in NSCLC cells promoted cell proliferation, migration, and invasion, decreased apoptosis and caspase-3 activity, and enhanced chemoresistance of NSCLC cells, whereas downregulation of miR-92a showed the opposite effects. Moreover, phosphatase and tensin homolog (PTEN), a unique tumor suppressor gene, was confirmed as a direct target of miR-92a, and PTEN messenger RNA (mRNA) expression was decreased in NSCLC tissues and was inversely correlated with miR-92a. Downregulation of PTEN could mimic the same effects of miR-92a mimic in NSCLC cells and rescue the effects on NSCLC cells induced by miR-92a inhibitor. Taken together, these findings suggested that miR-92a could promote growth, metastasis, and chemoresistance in NSCLC cells at least partially by targeting PTEN.

RNAi-mediated A disintegrin and metalloproteinase 9 gene silencing inhibits the tumor growth of non-small lung cancer in vitro and in vivo.

A disintegrin and metalloproteinase 9 (ADAM9) is a type I transmembrane protein that has been associated with cancer development and metastasis in various types of cancer. However, little is known about its role in non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate whether downregulation of ADAM9 affects cell proliferation, apoptosis, migration and invasion in NSCLC. Thus, a recombinant lentiviral small hairpin RNA expression vector carrying ADAM9 was constructed and infected into the human NSCLC cell line A549. Cell proliferation, apoptosis, migration and invasion in vitro and tumor growth in vivo were determined following downregulation of ADAM9 by RNA interference-mediated ADAM9 gene silencing. It was found that downregulation of ADAM9 expression using an RNA silencing approach in A549 tumor cells significantly inhibited cell proliferation, migration and invasion, induced cell apoptosis in vitro, as well as suppressed in vivo tumor growth in an experimental mouse model of lung metastasis. These data indicate that ADAM9 is potentially an important new therapeutic target for the prevention of tumor growth in NSCLC.

High-resolution analysis of copy number variants in adults with simple-to-moderate congenital heart disease.

As patients with congenital heart disease (CHD) increasingly survive to childbearing age, it becomes important to understand the genetic origins of CHD. In children, CHD is frequently caused by chromosomal imbalances. We searched for submicroscopic imbalances in adults with CHD focusing on simple-to-moderate phenotypes, without associated dysmorphic features, a group not previously examined. A total of 100 Han Chinese adults with a diverse range of isolated CHD and 65 ethnically matched controls were screened using whole-genome array comparative genomic hybridization. Forty-five large (>100 kb) rare copy number variants (CNVs) were identified in 36/100 patients. These variants were not listed in the Database of Genomic Variants nor found in controls. In three of these genomic imbalances (22q11.2, 18q23, 3q21.3), genes that play an important role in cardiac development were implicated, including CRKL, NFATC1, PLXNA1, the latter has not been associated with human CHD before. This study detected a 0.7 Mb 22q11.2 deletion, which marginally overlapped the common 3 Mb 22q11.2 deletion, in one patient with a perimembranous ventricular septal defect without any extracardiac manifestation. Furthermore, we detected a novel inherited aberration dup (16q23.1). Although a causal relationship with CHD remains to be established, this CNVs profile provides a spectrum of genomic imbalances in this condition, and improves the CNV-phenotype correlations.