LncRNA UNC5B-AS1 promotes proliferation and epithelial-mesenchymal transition of cervical cancer cells by regulating Toll-like receptor signaling pathway / 中国药理学通报

Aim To investigate the effects of long non-coding RNA(lncRNA)UNC5B-AS1 on the proliferation and epithelial mesenchymal transformation(EMT)of cervical cancer. Methods GEO and TCGA databases were used to download data sets and differential expression analysis was performed. qRT-PCR was used to verify the differential expression of lncRNA UNC5B-AS1 in normal and cancerous cervical tissues.The interference and overexpression of lncRNA UNC5B-AS1 were transfected into cervical cancer cell lines, and plate cloning, CCK-8 and EdU experiments were used to detect the effect of lncRNA UNC5B-AS1 on the pro-liferation of cervical cancer cells.Transwell assay was used to detect its effect on migration and invasion of cervical cancer cells.The expression levels of EMT-related genes E-Cadherin, N-Cadherin and Vimentin were detected by Western blot. Transcriptome sequencing was used to obtain the signal pathway regulated by lncRNA UNC5B-AS1, and to verify the expression level of related genes. Results RNA microarray and bioinformatics analysis showed that the expression level of lncRNA UNC5B-AS1 in cervical cancer was significantly higher than that in normal cervical tissue, and correlated with the overall survival time of patients.Compared with the negative control group, knockdown lncRNA UNC5B-AS1 could reduce the proliferation, migration and invasion of cervical cancer cells, while overexpression could promote the proliferation, migration and invasion of cervical cancer cells. Western blot showed that lncRNA UNC5B-AS1 could regulate EMT of cervical cancer cells. Transcriptome sequencing showed that lncRNA UNC5B-AS1 could regulate Toll like receptor(TLR)signaling pathway. qRT-PCR and Western blot results showed that the expression levels of TLR-related genes IL-6 and TICAM2 in the knockdown and overexpression lncRNA UNC5B-AS1 group were significantly changed(P<0.05). Conclusions LncRNA UNC5B-AS1 is highly expressed in cervical cancer. Overexpression of lncRNA UNC5B-AS1 may enhance TLR signaling pathway activity, thereby promoting proliferation and EMT of cervical cancer cells.

Selective elimination of host cells harboring replication-competent human immunodeficiency virus reservoirs: a promising therapeutic strategy for HIV cure / 中华医学杂志(英文版)

Many seminal advances have been made in human immunodeficiency virus (HIV)/AIDS research over the past four decades. Treatment strategies, such as gene therapy and immunotherapy, are yielding promising results to effectively control HIV infection. Despite this, a cure for HIV/AIDS is not envisioned in the near future. A recently published academic study has raised awareness regarding a promising alternative therapeutic option for HIV/AIDS, referred to as "selective elimination of host cells capable of producing HIV" (SECH). Similar to the "shock and kill strategy," the SECH approach requires the simultaneous administration of drugs targeting key mechanisms in specific cells to efficiently eliminate HIV replication-competent cellular reservoirs. Herein, we comprehensively review the specific mechanisms targeted by the SECH strategy. Briefly, the suggested cocktail of drugs should contain (i) latency reversal agents to promote the latency reversal process in replication-competent reservoir cells, (ii) pro-apoptotic and anti-autophagy drugs to induce death of infected cells through various pathways, and finally (iii) drugs that eliminate new cycles of infection by prevention of HIV attachment to host cells, and by HIV integrase inhibitor drugs. Finally, we discuss three major challenges that are likely to restrict the application of the SECH strategy in HIV/AIDS patients.

HPV caused pathological changes in genital system of mice / 病毒学报

The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7, Ad-K14 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5% Avertint and the vagina, mammary gland, ovaries and uterus were dissected and fixed in 3.75% paraformaldehyde overnight at 4 degrees C. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.

Melanoma differentiation-associated gene-7/interleukin 24 inhibits invasion and migration of human cervical cancer cells in vitro

In this study, we used an adenoviral vector -melanoma differentiation-associated gene-7 [Ad-mda7] to examine the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human cervical cancer cells. The study took place in the Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing, China, between April 2006 and November 2006. The change of metastasis of cervical cancer cells [CaSki] cells were detected by Cell Migration Assay and Cell Invasion Assay after treated with Ad-mda7. The production of proteins associated with cell migration and invasion were detected by western blot. Cervical cancer cells treated in vitro with Ad-mda7 migrated and invaded less than cells treated with phosphate-buffered saline [PBS] or Ad-Luc [vector control]. Melanoma differentiation-associated gene-7 /IL-24 inhibited migration and invasion by down-regulating the production of matrix metalloproteinase-2 [MMP-2] and by up-regulating the production of p38 mitogen-activated protein kinase. relative to PBS and Ad-Luc. These results show that MDA-7/IL-24 inhibits invasion and migration by cervical cancer cells by down- or up-regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis

An analysis on transcriptional regulation activity of human XBP1 gene 5' upstream DNA sequences / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.</p><p><b>METHODS</b>Six kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.</p><p><b>RESULTS</b>The reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.</p><p><b>CONCLUSION</b>The XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.</p>

A study of the down-regulation effect of hepatitis B virus pre-protein S2 on inducible nitric oxide synthase gene promoter / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the regulating effect of HBV pre-S2 protein on iNOS gene promoter and the molecular biological mechanisms of pre-S2 protein in HBV pathogenicity.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) technique was employed to amplify the sequence of iNOS promoter and 3 deletion mutants using HepG2 genomic DNA as the template, and the products were cloned into the pGEM-T vector. The iNOS gene and 3 deletion mutants were cut from T- iNOS by Kpn I and Xho I, and then cloned into pCAT3-Basic. The resulting vectors were named p1-iNOSp, p2-iNOSp, p3-iNOSp, and p4-iNOSp. Each of the reporter vectors was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-pre-S2 by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-Basic were used as a negative control. The activity of CAT in HepG2 cells transfected was detected by an ELISA kit 48 hours after the transfection, which reflected the regulating effect of HBV pre-S2 protein on iNOS gene promoter activity.</p><p><b>RESULTS</b>The expressive vector pcDNA3.1(-)-pre-S2 and report vector pCAT3-iNOSp were constructed and confirmed by restriction enzyme digestion and sequencing. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells could down-regulate the activity of p1-iNOSp, p3-iNOSp, and the inhibition rate was 54.7% and 79.5%, respectively. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells had no regulatory effects on p2-iNOSp and p4-iNOSp.</p><p><b>CONCLUSION</b>It is suggested that HBV pre-S2 protein can down-regulate iNOS gene promoter.</p>

The study of bidirectional regulation effect on inducible nitric oxide synthase gene promoter by hepatitis B virus preS1 promoter DNA-binding protein 1 / 中华传染病杂志

Objective To investigate the regulating effect of hepatitis B virus(HBV)SPⅠbinding protein 1(SBP1)on inducible nitric-oxide synthase(iNOS)gene promoter activity.Methods Polymerase chain reaction(PCR)technique was employed to amplify the coding sequence of iNOS promoter DNA by using HepG2 genomic DNA as template,and 3 deletion mutants were amplified. The products were cloned into pGEM-T vector,respectively.The iNOS gene and 3 deletion mutants were cut from T-iNOS by KpnⅠand XhoⅠ,and then were cloned into pCAT3-Basic.The construc- ted vectors were named as p1-iNOSp,p2-iNOSp,p3-iNOSp and p4-iNOSp,respectively.Each of the vectors containing different iNOSp DNA fragments was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-SBP1 by FuGENE 6 transfection reagents.The HepG2 cells transfected with pCAT3-Basic were used as negative control.The activity of chloram- phenicol acetyltransferase(CAT)in transfected HepG2 cells was detected by an enzyme-linked immu- nosorbent assay(ELISA)kit after 48 hours,which would reflect the regulating effect of SBP1 on iNOS gene promoter activity.Results The expression vector pcDNA3.1(-)-SBP1 and report vector pCAT3-iNOS were constructed and confirmed by restriction enzyme digestion and sequencing.The expression of pcDNA3.1(-)-SBP1 in HepG2 cells up-regulated the activity of p1-iNOSp and down- regulated the activity of p3-iNOSp.The inhibiting rate was 31.3%.Conclusions It is suggested that SBP1 can regulate iNOS gene promoter bidirectionally by influencing the binding sites of nuclear factor (NF)-IL6,A activator domain binding site and NF-?B in iNOS gene promoter.