Hanwei Wu

shRNA targeting long non-coding RNA CCAT2 controlled by tetracycline-inducible system inhibits progression of bladder cancer cells

Recent reports show that long non-coding RNAs (lncRNAs) are emerging as significant functional regulators in the development of tumors, including bladder cancer. Here, we found that CCAT2 was upregulated in bladder cancer tissues and cell lines. Through the statistical analyses, we also found that the high expression level of CCAT2 was positively correlated with histological grade and TNM stage of bladder cancer. Further experimental results revealed that knockdown of CCAT2 could decrease cell proliferation and migration as well as induce apoptosis in bladder cancer cells. Besides, using the post-transcriptional device of synthetic biology, we create the tetracycline-inducible double small hairpin RNAs (shRNAs) vector to control the expression level of CCAT2 which was induced by doxycycline in a dosage-dependent manner. In summary, our data indicated that CCAT2 may be an oncogene and a therapeutic target in bladder cancer. The expression of CCAT2 can be quantitatively controlled by the synthetic “tetracycline-on” switch system in bladder cancer in response to different concentrations of doxycycline to inhibit the development of bladder cancer cells.

Synthetic tetracycline-controllable shRNA targeting long non-coding RNA HOXD-AS1 inhibits the progression of bladder cancer

BackgroundLong non-coding RNAs (lncRNAs) have been proved to act as key molecules in cancer development and progression. Dysregulation of lncRNAs is discovered in various tumor tissues and cancer cells where they can serve as oncogenes or tumor suppressors. Long non-coding RNA HOXD-AS (HOXD cluster antisense RNA 1) has recently been identified to be involved in the development of several cancers including neuroblastoma, adenocarcinomas and breast cancer. However, the role of HOXD-AS1 in bladder cancer remains unknown.MethodsThe synthetic tetracycline-controllable shRNA was used to modulate the level of HOXD-AS1 by adding different concentrations of doxycycline (dox). RT-qPCR was used to detect the expression level of HOXD-AS1. Cell proliferation was determined by CCK-8 assay and EdU incorporation experiment when HOXD-AS1 was knocked down. We used wound-healing assay for detecting the effect of HOXD-AS1 on cell migration. Eventually, cell apoptosis was determined by caspase 3 ELISA assay and flow cytometry assay.ResultsIn this study, we found that the expression level of HOXD-AS1 was significantly increased in bladder cancer tissues and cells. Furthermore, high expression of HOXD-AS1 was significantly related to tumor size, histological grade and TNM stage. In vitro assays confirmed that knockdown of HOXD-AS1 suppressed cell proliferation/migration and increased the rate of apoptotic cell in bladder cancer cells. At last, we used the important element of synthetic biology, tetracycline(tet)-controllable switch, to construct tet-controllable shRNA vectors which can modulate the expression of HOXD-AS1 in a dosage-dependent manner.ConclusionsOur research suggested that high expression of HOXD-AS1 may be involved in the bladder cancer carcinogenesis through inhibiting the phenotypes and activating endogenous cancer-related molecular pathways. Therefore, HOXD-AS1 may act as an oncogene and provide a potential attractive therapeutic target for bladder cancer. In addition, the synthetic tetracycline-controllable shRNA may provide a novel method for cancer research in vitro assays.

Inducing cell growth arrest and apoptosis by silencing long non-coding RNA PCAT-1 in human bladder cancer.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that play important roles in cancer development and progression. Prostate cancer-associated transcript 1 (PCAT-1) is a novel lncRNA that promotes cell proliferation in prostate cancer. We hypothesized that PCAT-1 also have roles in bladder cancer. In this study, we found that PCAT-1 was up-regulated in bladder cancer compared to paired normal urothelium. Cell proliferation inhibition and apoptosis induction were also observed in PCAT-1 small hairpin RNA (shRNA)-transfected bladder cancer T24 and 5637 cells. Our data suggest that PCAT-1 plays oncogenic roles and can be used as a therapeutic target for treating human bladder cancer.

Roles of ERβ and GPR30 in Proliferative Response of Human Bladder Cancer Cell to Estrogen.

Bladder cancer belongs to one of the most common cancers and is a leading cause of deaths in our society. Urothelial carcinoma of the bladder (UCB) is the main type of this cancer, and the estrogen receptors in UCB remain to be studied. Our experiment aimed to investigate the possible biological effect of 17β-estradiol on human bladder-derived T24 carcinoma cells and to indicate its related mechanisms. T24 cells were treated with various doses of 17β-estradiol, and cell proliferation was detected using MTT assays. 17β-estradiol promoted T24 cell proliferation independent of ERβ/GPR30-regulated EGFR-MAPK pathway, while it inhibited cell growth via GPR30. Furthermore, the expression levels of downstream genes (c-FOS, BCL-2, and CYCLIN D1) were increased by 17β-estradiol and this effect was independently associated with activity of the EGFR-MAPK pathway. The two estrogen receptors might be potential therapeutic targets for the treatment of bladder cancer.

Targeting cellular mRNAs translation by CRISPR-Cas9.

Recently CRISPR-Cas9 system has been reported to be capable of targeting a viral RNA, and this phenomenon thus raises an interesting question of whether Cas9 can also influence translation of cellular mRNAs. Here, we show that both natural and catalytically dead Cas9 can repress mRNA translation of cellular genes, and that only the first 14 nt in the 5′ end of sgRNA is essential for this process. CRISPR-Cas9 can suppress the protein expression of an unintended target gene without affecting its DNA sequence and causes unexpected phenotypic changes. Using the designed RNA aptamer-ligand complexes which physically obstruct translation machinery, we indicate that roadblock mechanism is responsible for this phenomenon. Our work suggests that studies on Cas9 should avoid the potential off-target effects by detecting the alteration of genes at both the DNA and protein levels.

Synthetic Bax-Anti Bcl 2 combination module actuated by super artificial hTERT promoter selectively inhibits malignant phenotypes of bladder cancer

BackgroundThe synthetic biology technology which enhances the specificity and efficacy of treatment is a novel try in biomedical therapy during recent years. A high frequency of somatic mutations was shown in the human telomerase reverse transcriptase (hTERT) promoter in bladder cancer, indicating that a mutational hTERT promoter might be a tumor-specific element for bladder cancer therapy. In our study, we aimed to construct a synthetic combination module driven by a super artificial hTERT promoter and to investigate its influence on the malignant phenotypes of bladder cancer.MethodsThe dual luciferase assay system was used to verify the driven efficiency and tumor-specificity of the artificial hTERT promoter and to confirm the relationship between ETS-1 and the driven efficiency of the artificial hTERT promoter. CCK-8 assay and MTT assay were used to test the effects of the Bax-Anti Bcl2 combination module driven by the artificial hTERT promoter on cell proliferation. Simultaneously, the cell apoptosis was detected by the caspase 3ELISA assay and the flow cytometry analysis after transfection. The results of CCK-8 assay and MTT assay were analyzed by ANOVA. The independent samples t-test was used to analyze other data.ResultsWe demonstrated that the artificial hTERT promoter had a higher driven efficiency which might be regulated by transcription factor ETS-1 in bladder cancer cells, compared with wild-type hTERT promoter. Meanwhile, the artificial hTERT promoter showed a strong tumor-specific effect. The cell proliferation inhibition and apoptosis induction were observed in artificial hTERT promoter- Bax-Anti Bcl2 combination module -transfected bladder cancer 5637 and T24 cells, but not in the module -transfected normal human fibroblasts.ConclusionThis module offers us a useful synthetic biology platform to inhibit the malignant phenotypes of bladder cancer in a more specific and effective way.

Engineering cell signaling using tunable CRISPR–Cpf1-based transcription factors

The catalytically dead Cpf1 endonuclease from Acidaminococcus sp. BV3L6 (dAsCpf1) has been used to construct effective transcriptional repressors in bacteria and plants. However, it is still unclear if dAsCpf1 can function in human cells as a transcriptional regulator or a signal conductor. Here, we repurpose the dAsCpf1 system in human cells for a variety of functions, including the activation or repression of gene transcription. Moreover, we construct programmable ligand-controlled dAsCpf1 systems either by coupling crRNAs with engineered riboswitches or by fusing dAsCpf1 proteins with G protein-coupled receptors. These generalizable approaches allow us to regulate the transcription of endogenous genes in response to diverse classes of ligands, thus constructing artificial signaling pathways with rewired cellular input–output behaviors. The systems exhibit signal amplification, an important feature in cell signaling, when multiple crRNAs are processed from a single transcript. The results provide a robust and efficient platform for engineering customized cell signaling circuits.Cpf1 has been repurposed as a transcriptional repressor in bacteria and plants. Here, the authors construct activators and repressors in human cells using Cpf1 coupled to riboswitches and GPCRs.

Inhibiting cell migration and cell invasion by silencing the transcription factor ETS-1 in human bladder cancer.

As one of the members of the ETS gene family, the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS-1) plays key role in the regulation of physiological processes in normal cells and tumors. In this study, we aimed to investigate the relationship between the transcription factor ETS-1 and malignant phenotypes of bladder cancer. We demonstrated that ETS-1 was up-regulated in human bladder cancer tissue compared to paired normal bladder tissue. In order to evaluate the functional role of ETS-1 in human bladder cancer, vectors expressing ETS-1 shRNA and ETS-1 protein were constructed in vitro and transfected into the human bladder cancer T24 and 5637 cells. Our results showed that the transcription factor ETS-1 could promote cell migration and cell invasion in human bladder cancer, without affecting cell proliferation and apoptosis. In conclusion, ETS-1 plays oncogenic roles through inducing cell migration and invasion in human bladder cancer, and it can be used as a therapeutic target for treating human bladder cancer.

Importance of the residue 190 on bactericidal activity of the bactericidal/permeability-increasing protein 5

The bactericidal/permeability-increasing protein (BPI) with bactericidal and endotoxin-neutralizing activity is of considerable interest in clinical applications. However, the crucial residues responsible for the bactericidal activity of BPI remain elusive. In previous study, we identified the mutation of mBPI5 associated with the male infertility of mice. Here, the effects of Asp190Ala mutation on the antibacterial activity of mBPI5 have been determined. Substitution of Asp190 by alanine caused significant improvement in cytotoxic effect toward both E.coli J5 and P.aeruginosa. Liposome co-sedimentation assay showed that the ratio of Asp190Ala mutant binding to lipids increased by 8 folds. These results were well consistent with known fact that antibacterial activity of BPI is attributed to its high affinity for lipid moiety of lipopolysaccharides (LPS). The constructed structure of mBPI5 revealed that Asp190 was located close to 6 positively charged residues on the surface of N-terminal domain. When replacing Asp190 with alanine, salt linkages with Arg188 were broken, making the side chain of Arg188 be free to move and form tighter contacts with negatively charged LPS. These findings suggest that residue 190 combined with surrounding positively charged residues largely contribute to bactericidal and endotoxin-neutralizing activities of mBPI5.

A nonsense mutation in Ccdc62 gene is responsible for spermiogenesis defects and male infertility in repro29/repro29 mice

Abstract Phenotype-driven mutagenesis is an unbiased method to identify novel genes involved in spermatogenesis and other reproductive processes. Male repro29/repro29 mice generated by the Reproductive Genomics Program at the Jackson Laboratory were infertile with deformed sperm and poor motility. Using selected exonic capture and massively parallel sequencing technologies, we identified a nonsense mutation in the exon 6 of coiled-coil domain-containing 62 gene (Ccdc62), which results in a formation of a premature stop codon and a truncated protein. Among the tissues examined, CCDC62 was found to be expressed at the highest level in mouse testis by reverse transcriptase-PCR (RT-PCR) andWestern blot analysis.With immunofluorescent staining, we demonstrated that CCDC62 was expressed in the cytoplasm and the developing acrosome in the spematids of mouse testis, and was specifically localized at the acrosome in mature sperm. The complementation analysis by mating repro29/+ mice with Ccdc62 -/- mice (generated by CRISPRCas9 strategy) further provided genetic proof that the infertility of repro29/repro29 micewas caused by Ccdc62 mutation. Finally, it was found that intracellular colocalization and interaction of CCDC62 and Golgi-associated PDZ and coiled-coil motif-containing protein may be important for acrosome formation. Taken together, this study identified a nonsense mutation in Ccdc62, which directly results in male infertility in repro29/repro29 mice. Summary Sentence This study identified a nonsense mutation in coiled-coil domain-containing 62 gene (Ccdc62) in repro29/repro29 mice, which directly resulted in spermiogenesis defects and male infertility in the mice.