Silencing of polo-like kinase 2 increases cell proliferation and decreases apoptosis in SGC-7901 gastric cancer cells.

Polo­like kinase 2 (PLK2) is a serine/threonine protein kinase, which has vital roles during mitosis and the centrosome cycle. In acute myeloblastic leukemia and hepatocarcinogenesis, PLK2 acts as a tumor suppressor; however, the function of PLK2 in gastric cancer remains to be elucidated. In the present study, PLK2 was overexpressed in gastric cancer tissues and three types of gastric cancer cells, SGC­7901, MKN­45 and BGC­823. Transfection of SGC­7901 gastric cancer cells with small interfering (si)RNA against PLK2 exerted no effect on the ratio of cells at different stages of the cell cycle compared with that of the untransfected and control siRNA­transfected cells. In addition, silencing of PLK2 significantly enhanced the growth of SGC­7901 cells through inhibiting apoptosis. Furthermore, apoptosis­associated genes Bax and caspase 3 were found to be downregulated at the protein level. In conclusion, these results suggested that PLK2 may act as a tumor suppressor in gastric cancer, therefore indicating its therapeutic potential.

Proteomic Profiling of Invasive Ductal Carcinoma (IDC) using Magnetic Beads-based Serum Fractionation and MALDI-TOF MS.

AIM: To reveal the serum proteomic profiling of intraductal carcinoma (IDC) patients in China, establish a serum proteome fractionation technique for choosing magnetic beads for proteomic analysis in breast cancer research; and identify differentially expressed peptides (m/z; P < 0.0001) as potential biomarkers of early IDCs. METHODS: We used two different kinds of magnetic beads (magnetic bead-based weak cation exchange chromatography (MB-WCX) and immobilized metal ion affinity chromatography (MB-IMAC-Cu)) to analyze 32 patients with early stage (stages I-II) IDC and 32 healthy control serum samples for proteomic profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The mass spectra, analyzed using ClinProTools software, distinguished between IDC patients and healthy individuals based on k-nearest neighbor genetic algorithm. RESULTS: The serum samples purified in the MB-WCX group provided better proteomic patterns than the MB-IMAC-Cu group. The samples purified by MB-WCX had better average peak numbers, higher peak intensities, and better capturing ability of low abundance proteins or peptides in serum samples. In addition, the MB-WCX and MB-IMAC-Cu purification methods, followed MALDI-TOF MS identification and use of ClinProTools software accurately distinguished patients with early stage IDC from healthy individuals. CONCLUSION: Serum proteomic profiling by MALDI-TOF MS is a novel potential tool for the clinical diagnosis of patients with IDC in China.

Mir-126 inhibits growth of SGC-7901 cells by synergistically targeting the oncogenes PI3KR2 and Crk, and the tumor suppressor PLK2.

MicroRNA (miRNA)-126 (miR-126) was reported to be downregulated and to act as a tumor suppressor in cancers of the lung, cervix, bladder and prostate. However, the functions of miR-126 in gastric cancer appear to be diverse and are largely unknown. MiR-126 was reported to act as a tumor suppressor by targeting the Crk gene, or as an oncogene by targeting the SOX2 gene in gastric cancer. We identified that the expression of miR-126 was decreased in gastric cancer cell lines and tissues. PLK2, a tumor suppressor gene, was directly regulated by miR-126 in SGC-7901 cells. Overexpression of miR-126 not only suppressed the growth and clone formation of SGC-7901 cells, but also induced apoptosis in vitro, whereas inhibition of miR-126 slightly promoted SGC-7901 cell proliferation. The cell cycle was not affected by miR-126. Moreover, miR-126 suppressed tumor growth in vivo in a xenograft model. PLK2, PI3KR2 and Crk were regulated by miR-126 in SGC-7901 cells. We infer that the functions of miR-126 in gastric cancer depend on synergistic targeting balance between oncogenes and anti-oncogenes. Our study indicates that miR-126 is a tumor suppressor, which in the future may become a therapeutic target for gastric cancer.

miR-638 suppresses cell proliferation in gastric cancer by targeting Sp2.

BACKGROUND: MicroRNAs play important roles in the development and progression of various cancers. Recent studies have shown that miR-638 was downregulated in several tumors; however, its role in gastric cancer (GC) has not been investigated in detail. AIMS: The purpose of this study was to determine the role of miR-638 and to elucidate its regulatory mechanism in GC. METHODS: The expression levels of miR-638 and specificity protein 2 (Sp2) were detected by real-time PCR and Western blotting in GC. After pcDNA6.2-GW/EmGFP-miR-638 vector, miR-638 inhibitor and Sp2-siRNA transfection, the AGS cell proliferation was investigated by MTT assay and cell cycle, and apoptosis was detected using the Annexin V/PI. In addition, the regulation of Sp2 by miR-638 was evaluated by real-time RT-PCR, Western blot and luciferase reporter assays; cyclin D1 expression was measured by Western blotting. RESULTS: The expression of miR-638 is dramatically down-regulated and Sp2 expression is remarkably up-regulated in GC tissues. Luciferase assays revealed that miR-638 inhibited Sp2 expression by targeting the 3'-UTR of Sp2 mRNA. Overexpression of miR-638 and Sp2-siRNA reduced Sp2 expression at both the mRNA and protein levels in vitro, and inhibition of miR-638 increased Sp2 expression. Moreover, we found that miR-638 overexpression and Sp2-siRNA markedly suppressed cell proliferation with decreasing expression of cyclin D1 and inducing G1-phase cell-cycle arrest in vitro; inhibition of miR-638 significantly promoted cell proliferation by increasing expression of cyclin D1 and leading more cells into the S and G2/M phase. CONCLUSIONS: Our results demonstrated that miR-638 suppressed GC cell proliferation by targeting Sp2 with influence on the expression of cyclin D1. We suggest that miR-638 might be a candidate predictor or an anticancer therapeutic target for GC patients.

Proteomics reveals intersexual differences in the rat brain hippocampus.

It is widely accepted that intersexual differences occur in cognitive domains, e.g., in spatial learning and memory. The hippocampus plays important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation. However, it still remains unknown whether the hippocampal proteomic profiling differs between males and females. In this study, we investigated the intersexual differences in protein expression of hippocampi using the two-dimensional electrophoresis analysis. In all, 33 differentially expressed proteins were characterized by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and validated by Western-blotting analysis. In line with Western-blotting validation, the proteomic identification revealed the overexpression of glial fibrillary acidic protein in female rats' hippocampi, and the overexpression of both creatine kinase B-type and DRP-2 in male rats' hippocampi. The intersexual differences in hippocampal proteomic profiling are probably closely related to those in spatial learning and memory abilities.

Identification of novel low molecular weight serum peptidome biomarkers for non-small cell lung cancer (NSCLC).

AIM: To identify discriminating protein patterns in serum samples among non-small cell lung cancer (NSCLC), chronic obstructive pulmonary disease (COPD), pneumonia, and healthy controls. To discover specific low molecular weight (LMW) serum peptidome biomarkers and establish a diagnostic pattern for NSCLCby using proteomic technology. METHODS: We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify patients with NSCLC, COPD, and pneumonia. A total of 154 serum samples were analyzed in this study, among which there were 60 serum samples from NSCLC patients, 30 from patients with other lung-related diseases (16 pneumonia patients and 14 patients with COPD) as disease controls, and 64 from healthy volunteers as healthy control. The mass spectra, analyzed using ClinProTools software, distinguished between cancer patients and healthy individuals based on GA algorithm model. RESULTS: In this study, we generated numerous discriminating m/z peaks as well as disease-specific discrimination peaks. A set of five potential biomarkers (m/z: 7,763.24, 1,012.61, 4,153.16, 1,450.55, and 2,878.89) could be used as the diagnostic biomarkers to distinguish NSCLCpatients from healthy controls. In the training set, patients with NSCLC could be identified with sensitivity of 97.5% and specificity of 98.8%. Similar results were obtained in the testing set, showing 80.7% sensitivity and 91.2% specificity. CONCLUSION: Our study demonstrated that a combined application of magnetic beads with MALDI-TOF MS technique was suitable for identification of serum biomarkers for NSCLC.

Serum peptidome profiling in patients with gastric cancer.

To identify discriminating protein patterns in serum samples between gastric cancer patients (early and advanced stages) and healthy controls. We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to identify patients with gastric cancer. In total, serum samples from 62 gastric cancer patients (32 in the training set and 30 in the test set; 19 of which had early-stage tumors and 43 of which had advanced-stage tumors) and 64 healthy controls (32 in the training set and 32 in the test set) were analyzed. The mass spectra, analyzed using ClinProTools software, distinguished between cancer patients and healthy individuals based on three different algorithm models. In the training set, patients with gastric cancer could be identified with a mean sensitivity of 94.7% and a mean specificity of 99%. Similar results were obtained with the test set, showing 79.3% sensitivity and 86.5% specificity. Our study demonstrates the high sensitivity and specificity of screening serum protein patterns using MALDI-TOF MS for the identification of patients with gastric cancer.

[STAT1 and STAT2 participate in growth inhibition of human hepatoma HepG2 cells induced by phosphatidylethanolamine].

OBJECTIVE: To investigate the roles of STAT1 and STAT2 in growth inhibition induced by phosphatidylethanolamine (PE) in human hepatoma HepG2 cells. METHODS: The growth of HepG2 cells exposed to 0.125, 0.25, 0.5 and 1.0 mmol/L PE was assessed by MTT assay, and the expressions of STAT1 and STAT2 were analyzed using immunocytochemical assay. RESULTS: PE inhibited the growth of HepG2 cells in a dose-dependent manner and increased the expression of STAT1 and STAT2 in comparison with those in the control group. AG490, an inhibitor of JAKs, partially reversed PE-induced growth inhibition of HepG2 cells. CONCLUSION: STAT1 and STAT2 are involved in the growth inhibition of human hepatoma HepG2 cells induced by PE.

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells.

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine ß-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.

Serum peptidome profiling in patients with lung cancer.

Serum peptide profiling is a promising approach for classification of cancer versus noncancer samples. In this study, we aimed to search for discriminating peptide patterns in serum samples between lung cancer patients and healthy controls. The magnetic beads-based weak cation-exchange chromatography followed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used in this study to identify patients with lung cancer. In total, serum samples from 64 lung cancer patients (32 for training set and 32 for testing set), 64 healthy controls (32 for training set and 32 for testing set), and 10 COPD patients (for disease control) were analyzed in this study. The mass spectra data analyzed with ClinProTools software was used to distinguish between cancer patients and healthy individuals based on three different algorithm models (GA, SNN, and QC). In the training set, patients with lung cancer could be identified with the mean sensitivity of 98.9% and specificity of100%. Similar results could be obtained from testing set, showing 87% sensitivity and 84.8% specificity. Screening for serum peptide patterns using MALDI-TOF MS showed high sensitivity and specificity in identifying patients with lung cancer.

[Progress of polymorphism in microRNA and microRNA target sites].

MicroRNAs (miRNAs), which are evolutionarily well-conserved, 21- to 23-nucleotide-long, small non-coding RNAs, are widely involved in the regulation of multiple biological processes, such as development, organogenesis, cell proliferation, cell differentiation, and apoptosis. Recent studies have shown that polymorphism in miRNAs and their target sites are closely related to various diseases, such as tumor and cardiological diseases. This review introduces recent progresses on polymorphism in microRNAs and their targeting sites and the related diseases.

Small-interfering RNA-mediated silencing of the MAPK p42 gene induces dual effects in HeLa cells.

The genesis and progression of cervical cancer involve the mutation or deviant expression of numerous genes, including the activation of oncogenes (Ha-ras, C-myc, C-erbB2 and Bcl-2) and inactivation of tumor-suppressor genes (p53 and Rb). Previous studies showed that small-interfering RNAs (siRNAs) targeting the MAPK p42 gene partly inhibit proliferation and increase apoptosis in human cervical carcinoma HeLa cells. Results of a microarray analysis showed that MAPK p42 siRNA inhibited cell growth through the regulation of cell cycle control and apoptosis and induced interferon-like response in HeLa cells. In order to confirm the dual effects of MAPK p42 siRNA, we compared the roles of siRNA and U0126, an inhibitor of MAPK p42, in HeLa cells. Short 21-mer double-stranded/siRNAs were synthesized to target MAPK p42 mRNA in HeLa cells. The siRNAs were transfected into HeLa cells using Lipofectamine. The cells were treated with siRNA or U0126 at different concentrations for a period of 48 h. The biological effect of siRNA and U0126 on HeLa cells was measured by MTT and flow cytometry. MAPK1, NUP188, P38, STAT1, STAT2, PML and OAS1 were analyzed by real-time quantitative PCR. HeLa cell growth was inhibited by siRNA or U0126, and the effect of siRNA inhibition was greater than that of U0126. Cell cycle phases were different for siRNA or U0126, but HeLa cell growth was arrested at the S phase by siRNA and at G1 phase by U0126. A down-regulation in MAPK p42 expression by siRNA and up-regulation by U0126 were noted. The results of real-time quantitative PCR showed that P38 was up-regulated and NUP188 was down-regulated by siRNA in comparison with the control groups, and the results were consistent with those of U0126. Expression levels of STAT1, STAT2, PML and OAS1 induced by siRNA differed from those induced by U0126. siRNA-mediated silencing and deactivation induced by U0126 in MAPK p42 led to growth inhibition in the HeLa cells. The effects of siRNA on HeLa cell growth were different from those of U0126. Dual effects of MAPK p42 siRNA-2 on HeLa cell growth were noted: one consisted of a specific effect induced by siRNA-mediated p42 MAPK silencing and the other exhibited a non-specific interferon-like response.

Breviscapine improves functions of spatial learning and memory of focal cerebral ischemia rats.

OBJECTIVE: To investigate the effects of breviscapine on the functions of spatial learning and memory of focal cerebral ischemia rats. METHODS: Rots withl the left middle cerebral artery occluded were made by an intraluminal filament. Then breviscapine (20 mg/kg,40 mg/kg) in experimental group and 10% glucose in control group were administered intraperitoneally once a day for 2 weeks, and the Morris water maze tasks were carried out for 5 days. RESULTS: Compared with sham-operation group,the animals of ischemia-control group exhibited seriously impaired spatial learning and memory in both place navigation test and spatial probe test. In the place navigation test, the mean value of escape latency in breviscapine group was significantly shorter than that in ischemia control group (P < 0.01 for lower-dose and P < 0.05 for higher-dose breviscapine group, respectively). In the spatial probe test,compared with sham-operation (P < 0.01) and breviscapine group (P < 0.01), the rats of ischemia-control group spent more time in the no-former platform quadrant, and showed reduced frequency of crossing former platform site significantly. The numbers of neurons with Nissl staining and choline acetyltransferase (ChAT) immunopositive neurons in ipssilateral cortex in the breviscapine group were significantly more than those in the ischemia-control group (P < 0.01). In hippocampus, the numbers of neurons with Nissl staining and ChAT immunopositive neurons in the stratum pyramidale of CA area were similar among the groups. CONCLUSION: These results indicate that breviscapine can improve the functions of spatial learning and memory of focal cerebral ischemia rats and the protection against the loss of ChAT immunopositive neuron in new cortex may be involved in its mechanisms.

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/Bax pathway.

AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (DeltaPsi m) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a dose- and time- dependent manner. It did not affect the cell cycle, but induced apoptosis. PE significantly decreased DeltaPsi m at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a dose- and time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.

MicroRNA profiling of human gastric cancer.

MicroRNAs are a group of small non-coding RNAs that modulate gene expression. The de-regulation of microRNA expression has been found in several types of cancer. To study the role of microRNAs in gastric cancer (GC), we analyzed the expression profile of 847 microRNAs in GC from Chinese patients. Total RNA was used for hybridization on the miRCURY LNA Array (v. 11.0), which contains probes specific for 847 human microRNAs. The results from the miRNA microarray analysis were validated by real-time RT-PCR. A total of 24 miRNAs with a more than 2-fold change were differentially expressed between normal gastric tissue and GC. Of these, 22 miRNAs (miR-223, miR-106b, miR-147, miR-34a, miR-130b*, miR-106a, miR-18a, miR-17, miR-98, miR-616*, miR-181a-2*, miR-185, miR-1259, miR-601, miR-196a*, miR-221*, miR-302f, miR-340*, miR-337-3p, miR-520c-3p, miR-575 and miR-138) were significantly up-regulated in GC (P<0.05), whereas only miR-638 and miR-378 were significantly down-regulated in GC (P<0.05) compared to normal gastric tissue. The expression of miR-185 and miR-638, as measured by miRNA microarray analysis, was in agreement with the expression level of these microRNAs found by real-time RT-PCR in the same samples. Our results show that microRNAs are de-regulated in GC, suggesting the involvement of these genes in the development and progression of gastric cancer.

Effects of small interfering RNAs targeting MAPK1 on gene expression profile in HeLa cells as revealed by microarray analysis.

The mitogen activated protein kinases (MAPK) signaling cascade plays an important role in cell life. We proved that small interfering RNAs targeting MAPK1 (siRNA-2) could inhibit HeLa cell growth, but the effects of siRNA-2 on gene expression profile were unclear. Using Affymetrix GeneChip HG-U133A 2.0, we identified the long-term changes for 48 h in gene expression profile in HeLa cell treated by siRNA-2. The results showed that expressions of 181 genes were altered by siRNA-2 and were divided into two groups: (i) one group showed downregulation by siRNA-2, including the proliferation associated genes, small G protein, cytoskeleton associated protein and extracellular matrix associated protein; and (ii) the other group showed upregulation by siRNA-2, including interferon response genes, OAS family, TRIM family and apoptosis associated genes. The results of Real-time quantitative PCR for MAPK1, NUP188, P38, STAT1, STAT2, MPL and OAS1 were consistent with that of gene chip. Two networks were found to react substantially to the downregulation of MAPK1 by siRNA-2. One of the networks regulates cell growth through cell-cycle control, apoptosis and cytoskeleton. The other network is related to interferon-like response. Our findings suggest that siRNA-mediated downregulation of MAPK1 could be an attractive strategy for cancer therapy.

[Small interfering RNA-mediated nuclear factor-kappaB P65 suppression induces apoptosis of hepatic carcinoma SMMC-7721 cells].

OBJECTIVE: To study the mechanism of hepatic carcinoma cell apoptosis induced by small interfering RNA (siRNA)-mediated nuclear factor-kappaB (NF-kappaB) P65 silencing. METHODS: Hepatic carcinoma SMMC-7721 cells were exposed to liposome-mediated transfection with NF-kappaB P65 siRNA synthesized by in vitro transcription, and the cells with empty liposome transfection and those without particular treatment served as the control groups. The expression of NF-kappaB P65 in the cells was detected by Western blotting, the cell viability examined by MTT assay, and the cell apoptosis assessed by flow cytometry. Immunohistochemistry was used to examine the expressions of Bcl-2 and Bax. RESULTS: siRNA transfection significantly inhibited the expression of NF-kappaB P65 in SMMC-7721cells, with inhibition rates of 64.74% compared with the untreated cells and of 34.52% compared with the liposome-treated cells. The siRNA-treated SMMC-7721 cells also exhibited significant decrease in cell proliferation by 33.39% and 27.23% in comparison with the untreated and liposome-treated cells, respectively. NF-kappaB P65 siRNA induced obvious cell apoptosis with down-regulated Bcl-2 and up-regulated Bax expressions. CONCLUSION: NF-kappaB p65 siRNA can induce SMMC-7721 cell apoptosis via the Bcl-2/Bax pathway.

[New progress of the highly efficient siRNA design].

RNA interference (RNAi), a conserved evolutionary mechanism widely found in living nature, is considered for defending the alien gene and virus infection. RNAi can induce gene silencing by prohibiting the translation of the target mRNA, which is specially recognized by the corresponding siRNA, and finally degraded by the RISC. So RNAi can be applied as a new technology tool for the functional gene research and gene therapy. But the effects of random designed siRNAs on gene silencing differ greatly from each other. The design of the special, highly efficient siRNA comes to be a crucial problem. This issue gives an overview about the new progress of the siRNA design principle.

[Inhibition of the expression of p42MAPK in HeLa cell line by RNA interference].

OBJECTIVE: To screen for siRNAs that inhibit the expression of p42(MAPK) in HeLa cell line. METHODS: Three p42(MAPK) siRNAs and one random siRNA were synthesized using Silencer siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTT and flow cytometry. RESULTS: Two siRNAs (siRNA-2 and siRNA-3) among three tested were identified to be able to downregulate the p42(MAPK) expression. A concurrent growth retardation of HeLa cell line was observed in comparison with the control. CONCLUSION: Inhibition of p42(MAPK) expression with siRNA technique can inhibit the proliferation of HeLa cells.