The Role of Non-Coding RNAs in Breast Cancer Drug Resistance.

Breast cancer (BC) is one of the commonly occurring malignancies in females worldwide. Despite significant advances in therapeutics, the mortality and morbidity of BC still lead to low survival and poor prognosis due to the drug resistance. There are certain chemotherapeutic, endocrine, and target medicines often used for BC patients, including anthracyclines, taxanes, docetaxel, cisplatin, and fluorouracil. The drug resistance mechanisms of these medicines are complicated and have not been fully elucidated. It was reported that non-coding RNAs (ncRNAs), such as micro RNAs (miRNA), long-chain non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) performed key roles in regulating tumor development and mediating therapy resistance. However, the mechanism of these ncRNAs in BC chemotherapeutic, endocrine, and targeted drug resistance was different. This review aims to reveal the mechanism and potential functions of ncRNAs in BC drug resistance and to highlight the ncRNAs as a novel target for achieving improved treatment outcomes for BC patients.

miR-30c-5p inhibits glioma proliferation and invasion via targeting Bcl2.

BACKGROUND: Glioma is a highly malignant brain tumor, characterized by the poor prognosis and high recurrence rates. Previous studies have confirmed that miRNA-30c-5p is closely associated with tumor cell biological properties. The present study explored the biological role of miR-30c-5p in human glioma malignant behavior and underlying mechanisms. METHODS: Levels of miR-30c-5p were detected in glioma tissues and adjacent normal tissues. Two glioma cell lines including U87 and U251 were transfected with miR-30c-5p mimic or inhibitors. Cell proliferation was evaluated by MTT assay and colony formation assay. Cell apoptosis and invasive potential of glioma cells were assessed by flow cytometry and transwell assays, respectively. Luciferase reporter assay was performed to validate the target gene of miR-30c-5p. RESULTS: Levels of miR-30c-5p were dramatically decreased in glioma tissues as compared to the adjacent normal tissues. Upregulation of miR-30c-5p significantly suppressed cell growth and colony formation, and induced apoptosis in glioma cells. In contrast, inhibition of miR-30c-5p promoted the proliferation and inhibited apoptosis in tumor cells. Furthermore, miR-30c-5p strongly suppresses the invasion of glioma cells. Western blot showed that Bcl-2 was significantly decreased following treatment with miR-30c-5p mimics and increased after miR-30c-5p inhibitor treatment. Moreover, luciferase reporter assays indicated that transfection of miR-30c-5p led to a marked reduction of luciferase activity, but had no effect on Bcl-2 3'-UTR mutated fragment. Mechanically, miR-30c-5p promoted the activation of caspase 3 and caspase 9 in glioma cells. Furthermore, miR-30c-5p promoted apoptosis and inhibited colony formation and migration, and knockdown of Bcl2 further increased the number of apoptotic cells and suppressed colony formation and migration of glioma cells. By contrast, miR-30c-5p inhibitors decreased apoptosis and increased colony formation and migration, and restored Bcl2 expression further suppressed glioma cell apoptosis and enhanced colony formation and migration. CONCLUSIONS: These results demonstrated that miR-30c-5p regulated growth, apoptosis and migration in glioma cells by targeting Bcl2, suggesting that miR-30c-5p might serve as a novel target for glioma therapy.

Associations of CXCL16, miR­146a and miR­146b in atherosclerotic apolipoprotein E­knockout mice.

Atherosclerosis is the primary cause of cardiovascular and cerebrovascular diseases. Recent studies have revealed that C­X­C motif chemokine ligand 16 (CXCL16), microRNA (miR)­146a and miR­146b may have important roles in atherosclerotic diseases. However, the associations of CXCL16, miR­146a and miR­146b in atherosclerotic diseases in vivo remain unclear. Previous studies have demonstrated that miR­146a and miR­146b may negatively regulate the toll like receptor (TLR4)/nuclear factor (NF)­κB signaling pathway to repress the inflammatory response. The present study investigated the associations of CXCL16, miR­146a and miR­146b in atherosclerotic apolipoprotein E (ApoE)­/­ mice in vivo. The expression levels of CXCL16, TLR4/NF­κB signaling pathway, miR­146a and miR­146b in the control and atherosclerotic ApoE­/­ mice were investigated via reverse transcription­quantitative polymerase chain reaction and western blot analysis. The present study demonstrated that the expression of CXCL16 was significantly upregulated in atherosclerotic ApoE­/­ mice compared with control ApoE­/­ mice. The expression levels of TRL4, interleukin­1 receptor­associated kinase 1, tumor necrosis factor receptor associated factor 6, NF­κB, tumor necrosis factor­α and interleukin­1ß were also significantly upregulated in atherosclerotic ApoE­/­ mice compared with control mice. However, the present study revealed that the expression levels of miR­146a and miR­146b were significantly downregulated in atherosclerotic ApoE­/­ mice compared with control ApoE­/­ mice. Overall, the results of the present study suggested that CXCL16 may regulate the TRL4/NF­κB/CXCL16 signaling pathway, and that miR­146a and miR­146b may negatively regulate CXCL16 via this pathway in atherosclerosis in vivo.

miR-142 suppresses proliferation and induces apoptosis of osteosarcoma cells by upregulating Rb.

It has been reported that microRNA-142 (miR-142) is a tumor suppressor gene. The present study primarily investigated whether the overexpression of miR-142 was able to inhibit the proliferation, apoptosis and expression of apoptosis-associated proteins in osteosarcoma (OS) cells. Different concentrations of miR-142 were transfected into the OS MG-63 cell line using Lipofectamine 2000. The cell lines were divided into three groups: Normal group (non-transfected group), miR-142 transfected group, and negative group, which were transfected with random miR-142 fragment. The proliferation of cells was detected by MTT assay. The expression of miR-142 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). DAPI staining was performed to investigate the influence of miR-142 on the morphology of MG-63c ells. The apoptotic cell percentages were determined by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Expression of tumor suppressors, phosphatase and tensin homolog (PTEN) and Retinoblastoma-associated protein (Rb), and apoptosis-associated proteins were evaluated by western blotting. RT-qPCR indicated a higher expression of miR-142 in the transfected group (miR-142 was transfected into the MG-63 cell line) compared with that in the normal (non-transfected group) and negative control groups. The proliferation of miR-142 transfected cells was significantly lower compared with that in the normal and negative groups. Furthermore, an increased apoptosis rate accompanied by a statistically significant upregulation of PTEN, Rb phosphorylation, cleaved caspase-3 and cytochrome c protein levels were detected in the transfected group, indicating an internal apoptosis pathway was involved in this process. Furthermore, no significant changes were identified between the normal and negative groups (P>0.05). The present study demonstrated that miR-142 overexpression by liposomal transfection resulted in an inhibitory effect on MG-63 cell proliferation. The underlying mechanisms may relate to the upregulation of tumor suppressor and activation of caspase signaling pathway, which may provide a novel horizon in short nucleotide drugs on the management of OS.

MicroRNA-552 promotes tumor cell proliferation and migration by directly targeting DACH1 via the Wnt/ß-catenin signaling pathway in colorectal cancer.

The purpose of the present study was to analyze the crucial role of microRNAs (miRNAs/miRs) involved in the proliferation and migration of colorectal cancer (CRC) and to investigate their underlying mechanisms. The present study discusses the expression and function of miR-552 in CRC. The expression level of miR-552 in CRC cells and tissues was observed, and it was suggested that the high expression of miR-552 accelerated the proliferation and migration of CRC cells in vitro. Notably, a result of the present study was that the cell fate determination factor Dachshund family transcription factor 1 (DACH1) was identified as a direct target of miR-552. Suppressing miR-552 expression in CRC cells increased endogenous DACH1 mRNA and protein levels, which was negatively correlated with miR-552. DACH1 performs an important role in the development of a number of neoplasms, and has the ability to regulate the Wnt/ß-catenin signaling pathway as a novel predictive and diagnostic biomarker. Accordingly, it was concluded that miR-552 exerted a tumor-promoting role in CRC development by targeting DACH1, which may contribute to the increase in the rates of CRC proliferation and migration. miR-552 may serve as a potential diagnostic and prognostic biomarker for CRC.

Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells.

AIM: To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin (IL)-24 in hepatocellular carcinoma (HCC) cells. METHODS: γ-secretase inhibitors (GSIs) were used to down-regulate Notch1. HepG2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h. Cell viability was measured by MTT assay. The cellular and nuclear morphology was observed under a fluorescence microscope. To further verify the apoptotic phenotype, cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining. The expression of Notch1, SNAIL1, SNAIL2, E-cadherin, IL-24, XIAP and VEGF was detected by Western blot. The invasion and migration capacities of HCC cells were detected by wound healing assays. Notch1 and Snail were down-regulated by RNA interference, and the target proteins were analyzed by Western blot. To investigate the mechanism of apoptosis, we analyzed HepG2 cells treated with siNotch1 or siCON plus IL-24 or not for 48 h by caspase-3/7 activity luminescent assay. RESULTS: GSI-I at a dose of 2.5 µmol/L for 24 h caused a reduction in cell viability of about 38% in HepG2 cells. The addition of 50 ng/mL IL-24 in combination with 1 or 2.5 µmol/L GSI-I reduced cell viability of about 30% and 15%, respectively. Treatment with IL-24 alone did not induce any cytotoxic effect. In SMMC7721 cells with the addition of IL-24 to GSI-I (2.5 µmol/L), the reduction of cell viability was only about 25%. Following GSI-I/IL-24 combined treatment for 6 h, the apoptotic rate of HepG2 cells was 47.2%, while no significant effect was observed in cells treated with the compounds employed separately. Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in HepG2 cells. Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I. Furthermore, the increased GSI-I and IL-24 in HepG2 cell was associated with downregulation of MMP-2, XIAP and VEGF. In the absence of treatment, HepG2 cells could migrate into the scratched space in 24 h. With IL-24 or GSI-I treatment, the wound was still open after 24 h. And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I. Treatment of Notch-1 silenced HepG2 cells with 50 ng/mL IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination. Caspase-3/7 activity was increased in the presence of siNotch1 plus IL-24 treatment. CONCLUSION: Down-regulation of Notch1 by GSI-I or siRNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of HepG2 cells.

Expression of IER3 in primary hepatocarcinoma: correlation with clinicopathological parameters.

BACKGROUND: Studies indicate the immediate early response gene 3 (IER3) is involved in many biological processes. Recently, it was discovered that IER3 plays an important role in tumorigenesis and tumor progression. Thus it may be a valuable biomarker in tumor. This study was designed to investigate the expression status of IER3 in primary hepatocarcinoma (PHC) and correlation with clinicopathological parameters. MATERIALS AND METHODS: Real-time PCR was performed to evaluate the expression levels of IER3 in 62 pathologically diagnosed human PHC specimens. RESULTS: A statistically significant association was disclosed between the expression of IER3 and P53 mutant protein (short for P53), Ki-67, EGFR and the biggest diameter, differentiation grade of tumor. CONCLUSIONS: This work is the first to shed light on the potential clinical usefulness of IER3, as an efficient tumor biomarker in PHC.

Prediction of lung cancer based on serum biomarkers by gene expression programming methods.

In diagnosis of lung cancer, rapid distinction between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) tumors is very important. Serum markers, including lactate dehydrogenase (LDH), C-reactive protein (CRP), carcino-embryonic antigen (CEA), neurone specific enolase (NSE) and Cyfra21-1, are reported to reflect lung cancer characteristics. In this study classification of lung tumors was made based on biomarkers (measured in 120 NSCLC and 60 SCLC patients) by setting up optimal biomarker joint models with a powerful computerized tool - gene expression programming (GEP). GEP is a learning algorithm that combines the advantages of genetic programming (GP) and genetic algorithms (GA). It specifically focuses on relationships between variables in sets of data and then builds models to explain these relationships, and has been successfully used in formula finding and function mining. As a basis for defining a GEP environment for SCLC and NSCLC prediction, three explicit predictive models were constructed. CEA and NSE are frequently- used lung cancer markers in clinical trials, CRP, LDH and Cyfra21-1 have significant meaning in lung cancer, basis on CEA and NSE we set up three GEP models-GEP 1(CEA, NSE, Cyfra21-1), GEP2 (CEA, NSE, LDH), GEP3 (CEA, NSE, CRP). The best classification result of GEP gained when CEA, NSE and Cyfra21-1 were combined: 128 of 135 subjects in the training set and 40 of 45 subjects in the test set were classified correctly, the accuracy rate is 94.8% in training set; on collection of samples for testing, the accuracy rate is 88.9%. With GEP2, the accuracy was significantly decreased by 1.5% and 6.6% in training set and test set, in GEP3 was 0.82% and 4.45% respectively. Serum Cyfra21-1 is a useful and sensitive serum biomarker in discriminating between NSCLC and SCLC. GEP modeling is a promising and excellent tool in diagnosis of lung cancer.

Downregulation of both EGFR and ErbB3 improves the cellular response to pemetrexed in an established pemetrexed­resistant lung adenocarcinoma A549 cell line.

Epidermal growth factor receptor (EGFR) and ErbB3 (HER3) play important roles in the regulation of cell proliferation, differentiation, anti-apoptosis and chemoresistance; however, their dysregulation in pemetrexed (PEM) resistance remains unclear. The aim of the present study was to clarify the relationship between PEM resistance and gene expression of EGFR and ErbB3, by establishing the PEM-resistant lung adenocarcinoma A549 cell line, A549/PEM. Compared with A549 cells, the A549/PEM cells were significantly more resistant to PEM (P=0.0024). The downregulation of S phase and arrest at G1 stage were detected in the A549/PEM cell line when compared to the A549 cells (P<0.05). The apoptosis rate of A549/PEM cells was much lower than that of the A549 cells after a 24 h continuous exposure to PEM (P<0.001). Real-time PCR and western blotting demonstrated the overexpression of EGFR and ErbB3 in A549/PEM cells. However, downregulation of EGFR or ErbB3 by lentiviral delivered shRNAs in A549/PEM cells showed no significant correlation with PEM sensitivity while silencing both EGFR and ErbB3 increased the cellular response to PEM in the A549/PEM cells and significantly decreased phosphorylation of STAT3, AKT and ERK. Together, these data suggest that either high expression of EGFR or ErbB3 plays a critical role in the cellular response to PEM in human lung adenocarcinoma cells though EGFR/ErbB3-dependent pathways.

Construction and expression of an eukaryotic expression vector containing the IER3 gene.

BACKGROUND: More and more research indicate that the immediately early response gene 3 (IER3) is involved in many biological processes, such as apoptosis and immunoreaction, as well as viral infection, tumorigenesis and tumour progression. METHODS: Here we describe the construction of an eukaryotic expression vector containing IER3 gene and its expression in A549 cells as assessed through fluorescence microscopyand Western- blotting. RESULTS: Fluorescence detection displayed that GFP in cytoplasm was high during 48 and 72 hours post-transfection. In addition, Western blotting showed significant increase in IER3 gene expression in the transfected cells compared with controls. CONCLUSION: The recombinate plasmid expression vector was constructed successfully, which may provide a basis for further exploration of function of IER3 in lung cancer.

Construction of lentivirus vector containing human homeobox gene HOXB4 and its expression in human umbilical cord mesenchymal stem cells.

This study was purposed to construct lentivirus vector containing human homeobox gene HOXB4 and explore changes of human umbilical cord mesenchymal stem cells (HUCMSC) after infected with HOXB4 mediated by lentivirus. PCR amplification was performed to obtain HOXB4, which was cloned in lenti-shuttle vector. Four-plasmid lentivirus packaging system was used to transfect HEK293T cells. After 48 h, lentivirus Lenti-HOXB4 was harvested and lentivirus titer was determined. Lenti-HOXB4 was used to infect HUCMSC. The infected cells were observed under inverted fluorescence microscope to determine the optimal multiplicity of infection (MOI). Meanwhile, RT-PCR, immune fluorescence staining, CCK-8 and flow cytometry (FCM) were used to determine the expression of HOXB4 and its effect on cell growth. The results indicated that lenti-HOXB4 was successfully obtained by co-transfecting the 293T cells with four plasmids. The determined virus titer was 3×10(8) TU/ml; when MOI was 20. Lenti-HOXB4 had a high transfection rate in HUCMSC, over 80%. In HUCMSC infected with lenti-HOXB4, the expression of target gene could be detected both at mRNA and protein levels. It could promote the proliferation of HUCMSC. FCM results indicated HOXB4 gene did not significantly influence the surface marker of HUCMSC. It is concluded that HOXB4 gene can promote the high proliferation of HUCMSC and does not significantly influence the expression of the surface marker of HUCMSC.

[High temperature reduces the proliferation of and occludin expression in rat Sertoli cells in vitro].

OBJECTIVE: To explore the action mechanisms of temperature in male infertility or subfertility by observing the effects of different temperatures on the proliferation of and occludin (OCLN) expression in rat Sertoli cells in vitro. METHODS: We isolated Sertoli cells from the testis of male Wistar rats, and performed oil red O staining and immunohistochemistry to identify their FasL. We cultured the Sertoli cells at 34 degrees C (control group) and at 35, 36, 37, 38 and 39 degrees C (experimental groups) for 4 days. Then we measured their proliferation by CCK-8 assay, observed their morphology and structure by hematoxylin-eosin staining, and determined their OCLN expression level by Western blotting and immunofluorescence. RESULTS: The purity of the isolated Sertoli cells was (96.20 +/- 1.95)%. CCK-8 assay indicated that the proliferation of the Sertoli cells was increased between 34 and 36 degrees C, and decreased at 36-39 degrees C. The pyknotic nuclei and fragmentation of the Sertoli cells were more obvious at > 36 degrees C. Western blot and immunofluorescence showed the highest level of OCLN expression at 36 degrees C, which, however, decreased while the temperature rose above 36 degrees C (P < 0. 01). CONCLUSION: High temperature (> 36 degrees C) inhibited the proliferation of rat Sertoli cells in vitro, and decreased the expression of OCLN, which suggests that a higher temperature above 36 degrees C may reduce male fertility by affecting the proliferation of Sertoli cells and integrity of the tight junction among Sertoli cells or Sertoli cells and other cells.

[Construction and identification of a eukaryotic expression vector containing human homeobox gene].

In order to investigate the special role of HOXB4 in expansion and self renewal of hematopoietic stem cells, the cDNA of HOXB4 was extracted and cloned from umbilical cord blood mononuclear cells by using RT-PCR. Then the eukaryotic expression bicistronic plasmid vector pIRES2-EGFP/HOXB4 was designed and constructed after cutting HOXB4 and pIRES2-EGFP respectively by restriction enzyme EcoRI and BamHI. The recombinant plasmid was delivered into competent cells of Escherichia coli. The successful construction of plasmid was confirmed by the identification of endonuclease cutting and sequencing. The results showed that the HOXB4 cDNA was cloned successfully from umbilical cord blood mononuclear cells and the recombinant eukaryotic expression bicistronic plasmid vector was constructed, and then introduced it into 293T cells successfully. It is concluded that a pIRES2-EGFP/HoxB1 eukaryotic expression bicistronic plasmid vector has been constructed successfully, which results provide a useful material basis for exploration of HoxB4 function in the proliferation and differentiation of hematopoietic cells.

[Study on construction of chimeric adenovirus vector Ad5/11 carrying human eGFP and endostatin-K5 and its experimental investigation in vitro].

AIM: To construct chimeric adenoviral vector Ad5/11 carrying reporter gene eGFP and human endostatin-K5. METHODS: Chimeric adenoviral backbone vector expressing eGFP was generated by overlap PCR and homologous recombination in E.coli BJ5183. Then chimeric adenoviral vector Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP carrying eGFP and human endostatin-K5 was constructed by co-transfecting Pac I linearized chimeric adenoviral backbone and adenoviral E1 shuttle vector expressing human endostatin-K5 into HEK 293 cells. The expression of eGFP was observed under fluorescent microscope. The expression of human endostain-K5 in U87MG cells infected by chimeric adenoviral vector was detected by RT-PCR. The infection efficiency between chimeric adenovirus and unmodified control adenovirus for human glioblastoma cell line A172 and breast cancer cell line MDA-MB-231 in vitro was evaluated by the comparison of the expression of eGFP. RESULTS: Chimeric adenovirus Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP could successfully express eGFP and endostatin-K5. Chimeric adenoviral vector significantly enhances the infection efficiency for human glioblastoma cell line A172 and breast cancer cell line MDA-MB-231 compared with unmodified adenoviral vector Ad5 E1-CMV-eGFP. CONCLUSION: Chimeric adenoviral vector Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP can significantly improve the infection efficiency for human glioblastoma cell line A172 and breast cancer cell line MDA-MB-231.

[Microsurgical treatment of posterior cranial fossa tumors via keyhole approaches].

OBJECTIVE: To explore the surgical outcome and skills of keyhole approaches to posterior cranial fossa tumors. METHODS: A retrospective analysis of the clinical data of 43 consecutive patients with posterior cranial fossa tumors, including acoustic neurinoma, petroclival meningioma, pons tumor, fourth ventricular tumor, etc. was conducted. Subtemporal, retromastoid, or middle suboccipital keyhole approach was chosen respectively according to the anatomic positions of those different tumors. The length of the incision was about 4 cm, and the diameter of bone window was 2.0 - 2.5 cm. Normally dura was sutured tightly and no catheter was placed in the surgical fields. RESULTS: The tumors were totally removed in 37 of the 43 patients (86.0%), subtotally removed in 5 (11.6%) and mostly removed in 1 (2.3%). There were no complications obviously related to the limited exposure resulting from keyhole approaches. All of the 18 acoustic neurinomas (100%) were totally removed and the facial nerves of 15 patients (83.3%) were preserved anatomically, however, one patient died of brain stem edema on the 2nd postoperative day. Out of the 8 petroclival meningiomas, 5 (62.5%) were resected totally, 2 (25%) subtotally, and 1 (12.5%) grossly. Postoperatively, 2 patients still remained slight hemiparesis, and 1 presented with mild facial paralysis. Among the 6 pons tumors 3 were removed totally and 3 subtotally. There were no postoperative neurological deficits. All the other tumors were resected completely without neurological dysfunction observed, however, one patient with a cholesteatoma failed to demonstrate apparent improvement in his diplopia. CONCLUSION: Microsurgical treatment of posterior cranial fossa tumors via keyhole approaches, with safe, succinct and minimally invasive property, is one of the promising directions in modern neurosurgery.