miRNA is a kind of endogenous non-coding short RNA. Mature miRNA was formed through the process of shearing and transporting after genetic transcription. miRNA exhibits many important biological functions through regulating expression and translation of target mRNAs. Different miRNAs may act as oncogenes or antioncogenes, and have tissue specificity. The progress of the tumorigenesis is usually accompanied by expression-profile changes of miRNAs. MiRNA regulates many tumor biological behaviors such as differentiation, proliferation, apoptosis, invasion, metastasis, and drug resistance of tumors. Furthermore, some miRNAs have clinical significance in predicting prognosis of tumor patients.

DNA, as the material basis of all living cells, triggers innate immune responses through TLR9 and other cytosolic recognition receptors. In recent years, the research progress of TLR9 is mainly manifested by the following four aspects: (1) the determinants of TLR9 interacting with its ligands; (2) the mechanisms and the importance of TLR9 translocation from the endoplasmic reticulum to the endosome; (3) the roles of the endosomal acidification and maturation, and subsequent TLR9 cleavage in TLR9 signal transduction pathway; and (4) the possible mechanisms by which the organism distinguish self DNA from microbial DNA. Meanwhile, a series of experiments on TLR9 antagonists and TLR9 deficient mice confirmed the presence of TLR9-independent cytosolic DNA sensors. So far, three TLR9-independent DNA sensors have been found, and they are DAI, AIM2, and RNA polymerase Ⅲ.

Objective: To investigate the effect of nerve growth factor-β(NGF-β) on the proliferation and cell cycle of human pancreatic cancer MIA PaCa-2 cells. Methods: MIA PaCa-2 cells were treated with different concentrations of NGF-β and K252a (inhibitor of NGF-β receptor TrKA) alone or in combination. Clone forming rate, proliferation, and cell cycle of MIA PaCa-2 cells treated with different strategies were examined by clone formation assay, MTT, and flow cytometry, respectively. Results: NGF-β significantly increased the clone formation and proliferation of MIA PaCa-2 cells (P<0.05, P<0.01). K252a significantly inhibited the proliferation of MIA PaCa-2 cells (P<0.05), while NGF-β combined with K252a had no significant effect on the proliferation of MIA PaCa-2 cells. NGF-β arrested MIA PaCa-2 cell cycle in S phase, K252a arrested cell cycle in G_0/ G_1 phase, and NGF-β combined with K252a arrested cell cycle in S phase. Conclusion: NGF-β can enhance the proliferation of pancreatic carcinoma MIA PaCa-2 cells.

Objective: To study the effect of wortmannin (WM), a PI3K/Akt inhibitor, on the proliferation and apoptosis of leukemia cells and the possible mechanism. Methods: Human leukemia cell line K562 was treated with different concentrations of WM. The proliferation of K562 cells was examined by MTT assay. DNA damage in K562 cells was examined by single cell gel electrophoresis assay, and apoptosis of K562 cells was detected by Annexin V-FITC/PI double-staining. The expressions of total Akt, phosphorate-Akt (p-Akt), and NF-κB p65 mRNA and protein were detected by RT-PCR and Western blotting, respectively. Results: WM inhibited the proliferation of K562 cells in a dose- and time-dependent manner, with the IC((50) value of 24 h being 25 nmol/L. WM also induced apoptosis of K562 cells in a dose-dependent manner. DNA damage in K562 cells was demonstrated by appearance of comet tail after treatment with WM, with the rate of DNA tail and the tail length being significantly higher than those in the control group (P<0.01). WM dose-dependently inhibited P-Akt and NF-κB p65, but not the total Akt, mRNA and protein expressions. Conclusion: WM can inhibit proliferation and induce apoptosis of K562 cells in a dose- and time-dependent manner, probably through down-regulation of phosphorate PI3K/Akt signal pathway and NF-κB expression.

Objective: To construct the expression plasmid of a novel gene human NBEAL1 (neurobeachin like 1), and to study its relationship with the pathological grades of glioma. Methods: Total RNA of human glioma cell line U251 was extracted. NBEAL1 expression plasmid pGEX-KG/NBEAL1 was constructed and transferred into E. coli BL21. Recombinant NBEAL1 protein was induced by IPTG and further purified by GST affinity chromatographic column. The purity of recombinant NBEAL1 protein was examined by Western blotting analysis. A NBEAL1 protein specific monoclonal antibody was prepared and was used to study the relationship of NBEAL1 expression with pathological grades of glioma. Results: The NBEAL1 gene fragment was successfully cloned into pGEX-KG expression plasmid and verified by DNA sequencing. The recombinant NBEAL1 protein was expressed in inclusion bodies, with a yield of more than 30% of total bacterial proteins; the purity of purified NBEAL1 protein was above 95%. Western blotting analysis confirmed that the purified protein containing GST tag and NBEAL protein. NBEAL1 protein was lowly expressed in normal brain tissues and highly expressed in low grade glioma tissues; and the expression of NBEAL1 decreased with the increase of glioma malignancy. Conclusion: The NBEAL1 protein has been successfully cloned, expressed and purified. NBEAL1 protein expression in glioma tissues is negatively associated with the pathological grades of glioma.

Objective: To investigate the differentially expressed genes in osteosarcoma cell lines with various metastatic potentialities, and to screen for new candidate genes related to metastasis of osteosarcomas. Methods: The total RNAs of a lowly metastatic and a highly metastatic osteosarcoma cell lines (M6 and M8) were extracted. Differentially expressed genes in the two osteosarcoma cell lines were studied by cDNA microarray. The hybridization signals were scanned with a Generation Ⅲ array scanner and analyzed by Imagequant 5.0 software. Typical differentially expressed genes were further verified by real-time quantitative PCR. Results: There were 330 differentially expressed genes between M6 and M8 cells. In the high-metastasis M8 cells, 178 genes were up-regulated and 152 genes were down-regulated compared to the low-metastasis M6 cells, with 43 extremely up-regulated and 49 extremely down-regulated. The differentially expressed genes were mainly associated with cell proliferation, indicating these genes might be related to the inhibition of M6 cells. Other differentially expressed genes included those associated with the regulation of gene expression and signal transduction, indicating these genes might be correlated with tumor metastasis. Conclusion: cDNA microarray shows an advantage in identifying genes associated with metastasis of osteosarcoma. In M8 subset of MG63 osteosarcoma cells,43 genes are up-regulated and 49 genes are down-regulated, which may be related with metastasis of osteosarcoma.

Objective:To investigate the effect of retinoid-interferon-induced mortality (GRIM-19) gene on the apoptosis of colon cancer. Methods: A GRIM-19 eukaryotic expression vector (pCMV-Flag-GRIM-19) was constructed and transfected into SW480 cells. Expressions of GRIM-19 and apoptosis-related proteins were detected by Western blotting analysis. Apoptosis of SW480 cells was measured by Annexin-V/PI assay and mitochondrial membrane potential JC-1 staining. Results: The GRIM-19 eukaryotic expression vector pCMV-Flag-GRIM-19 was successfully constructed. Expression of GRIM-19 in SW480 cells was up-regulated and that of apoptosis-related protein Bcl-xl was down-regulated after transfection with pCMV-Flag-GRIM-19. Apoptosis rate was (7.7±1.39)% in SW480 cells transfected with pCMV-Flag empty vector and (15.0 ± 2.52)% in pCMV-Flag-GRIM-19 transfected cells (P<0.05). Mitochondrial membrane potential was decreased in (7.5±2.09)% of pCMV-Flag transfected cells and (17.5±3.07)% of pCMV-Flag-GRIM-19 transfected cells (P<0.05). Conclusion: In vitro GRIM-19 transfection can effectively induce apoptosis of colon cancer SW480 cells.

Objective: To explore the apoptosis-inducing effect of salvianolate on hepatoma SMMC-7721 cells and the underlying mechanism. Methods: SMMC-7721 cells were co-cultured in vitro with different concentrations (0.5, 1, 2 mg/ml) of salvianolate for 24 h. The apoptotic SMMC-7721 cells were examined by flow cytometry, and the changes of mitochondrial transmembrane potential were examined by mitochondrial transmembrane potential JC-1 kit. The activities of caspase-8, caspase-9, and caspase-3 were detected by spectrophotometry in the hepatoma SMMC-7721 cells after co-cultured with 1 mg/ml salvianolate. The changes of apoptotic SMMC-7721 cells induced by salvianolate in the presence or absence of caspase-9 inhibitor or caspase-3 inhibitor were measured by flow cytometry. The expressions of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were detected by Western blotting analysis. Results: Salvianolate significantly induced apoptosis of hepatoma SMMC-7721 cells (P<0.05), and the decline of mitochondrial membrane potential increased with the increase of salvianolate concentration (P<0.05). The activities of caspase-9 and caspase-3, but not caspase-8, were increased in hepatoma cells after treatment with 1 mg/ml salvianolate for 24 h (P<0.05). The apoptosis-inducing effect of salvianolate was significantly decreased in the presence of caspase-9 or caspase-3 inhibitors (P<0.05). Western blotting results showed that salvianolate increased pro-apoptotic protein Bax expression and decreased anti-apoptotic protein Bcl-2 expression. Conclusion: Salvianolate can induce the apoptosis of human hepatoma SMMC-7721 cells in a dose-dependent manner, which is probably mediated by mitochondrial apoptosis pathway.

Objective:Toprepare poly-DL-lactide-poly (PELA) microspheres encapsulating recombinant tissue inhibitors of metalloproteinase-1 (TIMP-1) adenovirus, and to investigate their effects on the proliferation of hepatocellular carcinoma HepG2 cells. Methods: The microsphere was constructed by encapsulating recombinant adenovirus containing TIMP-1 in biodegradable PELA. The diameter of the microsphere, quantity of virus encapsulated, loading rate, and releasing kinetics were measured. HepG2 cells were infected with the microspheres; the infection efficiency was examined by fluorescent microscope; and the ultrastructure was observed by TEM. The expression of TIMP-1 mRNA in HepG2 cells was examined by semi-quantitative RT-PCR, and the proliferation of HepG2 cells was detected by MTT assay. Results: The microsphere encapsulating recombinant TIMP-1 adenovirus was successfully constructed, with its diameter, entrapment efficiency, and virus loading rate being 1.965, 60.0%, and 10.5×10~8/mg, respectively. About 60% of the viruses were released within 120 h, and the total releasing time was longer than 240 h. Infection with rAdTIMP-1 PELA microsphere efficiently induced TIMP-1 expression in HepG2 cells, and significantly inhibited the proliferation of HepG2 cells, with the inhibitory rate being 47%. Conclusion: PELA microsphere encapsulating recombinant TIMP-1 adenovirus can markedly inhibit the proliferation of HepG2 cells, which provides an experimental basis for the combining macromolecular chemistry and gene therapy for treatment of hepatocellular carcinoma.

Objective: To investigate the effect of RNA interference (RNAi) targeting AKT1 and PI3K P85 on the proliferation and invasion of breast carcinoma MCF-7 cells. Methods: The recombinant adenovirus expression vector, which contained short hairpin RNA (shRNA) targeting open reading frames of AKT1 and PI3K P85 (rAd5-siAKT1-siPI3K), was transfected into human breast carcinoma MCF-7 cells. AKT1 and PI3K P85 mRNA and protein expressions were detected by real-time PCR and Western blotting analysis. The expressions of PCNA, cyclinD1, and P53 were also detected by Western blotting analysis. The proliferation and apoptosis of MCF-7 cells were measured by MTT, flow cytometry and 2-dementinal and 3-dementional matrigel assay. Results: Recombinant adenovirus vector rAd5-siAKT1-siPI3K dramatically down-regulated AKT1 and PI3K P85 mRNA and protein expressions in MCF-7 cells; the downstream factors PCNA and cyclin D1 were also down-regulated, while P53 was up-regulated. Growth of MCF-7 cells was inhibited by over 50% in rAd5-siAKT1-siPI3K group as measured by MTT assay, and cell cycle was arrested in G_1/G_0 phase compared with untransfected and rAd5-siCtrl transfected groups. Cell growth on matrigel matrix showed normal cell shapes, while the cells in rAd5-siAKT1-siPI3K transfected group were detached from the matrix or grew in scattered clustering patterns, forming only small aggregates. Conclusion: shRNA targeting AKT1 and PI3K P85 can significantly down-regulate the expression of AKT1 and PI3K P85 in breast carcinoma MCF-7 cells, and inhibit the growth of MCF-7 cells in vitro.