Hongmei Ding

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer.

Identification of an aptamer targeting hnRNP A1 by tissue slide-based SELEX†

We report a new in situ tissue slide‐based SELEX strategy targeting neoplastic tissues from breast cancer patients. The methodology, using the molecular differences between clinical specimens, can evolve aptamers to all fractions of tissue. The aptamers may be used as new molecular probes for pathological diagnosis and tumour imaging, and also to reveal the molecular differences that are responsible for the diseases. The specific aptamers were enriched by unequal length strand PCR employing a structured (−) strand primer. After 12 rounds of selection, using the paraffin tissue sections from infiltrating ductal carcinomas as targets, and using the adjacent normal tissue from the same case as controls, one of the enriched ssDNA aptamers, BC15, was selected from a nucleic acid library and characterized as recognizing breast cancer cells either within the tissue sections or from the culture medium, but only weakly binding to adjacent normal cells or immortalized breast cell line MCF10A. The calculated equilibrium dissociation constants (Kd) of BC15 bound to MCF7 cells was 111.0 ± 36.9 nM. Through streptavidin magnetic beads mediated affinity purification assay followed by mass spectrometry identification and western blot confirmation, the target of BC15 was characterized to be hnRNP A1, which was further verified to be specifically and highly expressed in cancerous tissues of breast by hnRNP A1 antibody immunostaining as well as western blot. BC15 aptamer was also used to probe cancer cells in tissues from other pathological types of breast cancers including lobular carcinoma, ductal carcinoma complicated with lobular carcinoma, comedo carcinoma, and lymph node metastasis of breast ductal carcinoma origin or breast lobular carcinoma origin. Therefore, tissue slide‐based SELEX holds promise in identifying tumour markers and developing specific molecular probes for cancer diagnosis. Copyright

MicroRNA-200b Is Overexpressed in Endometrial Adenocarcinomas and Enhances MMP2 Activity by Downregulating TIMP2 in Human Endometrial Cancer Cell Line HEC-1A Cells

MicroRNAs (miRNAs) play important roles in tumorigenesis and metastasis. In this study, we investigated miR-200b expression in endometrial adenocarcinomas and normal adjacent tissues and found that miR-200b is more highly expressed in cancer tissues than in normal adjacent tissues. A novel target of miR-200b, tissue inhibitor of metalloproteinase 2 (TIMP2), was predicted using a bioinformatics approach and was confirmed in human endometrial cancer cell line HEC-1A cells by luciferase assay, quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. We found that miR-200b repressed TIMP2 expression at both the messenger RNA and protein levels, although a family member, miR-200a, had no such effect. Using reverse gelatin zymography, we showed that miR-200b enhances matrix metallopeptidase 2 (MMP2) activity by downregulating TIMP2 expression in HEC-1A cells. These data suggest that miR-200b may play an important role in the metastasis of endometrial adenocarcinomas.

MiR-138 downregulates miRNA processing in HeLa cells by targeting RMND5A and decreasing Exportin-5 stability

MicroRNAs (miRNAs) are a class of non-coding small RNAs that consist of ∼22 nt and are involved in several biological processes by regulating target gene expression. MiR-138 has many biological functions and is often downregulated in cancers. Our results showed that overexpression of miR-138 downregulated target RMND5A (required for meiotic nuclear division 5 homolog A) and reduced Exportin-5 stability, which results in decreased levels of pre-miRNA nuclear export in HeLa cells. We also found that miR-138 could significantly inhibit HeLa cell migration by targeting RMND5A. Our study therefore identifies miR-138–RMND5A–Exportin-5 as a previously unknown miRNA processing regulatory pathway in HeLa cells.

Identification and expression of small non-coding RNA, L10-Leader, in different growth phases of Streptococcus mutans.

Streptococcus mutans is one of the major cariogenic bacteria in the oral environment. Small non-coding RNAs (sRNAs) play important roles in the regulation of bacterial growth, stress tolerance, and virulence. In this study, we experimentally verified the existence of sRNA, L10-Leader, in S. mutans for the first time. Our results show that the expression level of L10-Leader was growth-phase dependent in S. mutans and varied among different clinical strains of S. mutans. The level of L10-Leader in S. mutans UA159 was closely related to the pH value, but not to the concentrations of glucose and sucrose in culture medium. We predicted target mRNAs of L10-Leader bioinformatically and found that some of these mRNAs were related to growth and stress response. Five predicted mRNA targets were selected and detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and we found that the expression levels of these mRNAs were closely related to the level of L10-Leader at different growth phases of the bacteria. Our results indicate that L10-Leader may play an important role in the regulation of responses in S. mutans, especially during its growth phase and acid adaption response.

Aptamer BC15 against heterogeneous nuclear ribonucleoprotein A1 has potential value in diagnosis and therapy of hepatocarcinoma.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was reported to be participated in tumor development. The association between hnRNP A1 and liver cancer and the functional role of hnRNP A1 in liver cancer have never been reported. Herein, hnRNP A1-specific single-stranded DNA aptamer, BC15, was used to (a) evaluate hnRNP A1 expression in liver cancer, and (b) treat hepatocarcinoma by acting as an inhibitor of hnRNP A1. Results showed that there is high hnRNP A1 expression in liver cancer including serum α-fetoprotein-negative liver cancer tissues compared with either para-cancer or benign controls. Down regulation of hnRNP A1 expression by RNA interference inhibits the proliferation and migration of cancerous HepG2 cells, while overexpression of hnRNP A1 in normal HL-7702 cells increased the proliferation and migration of the cells. Importantly, BC15 showed a stronger inhibiting effect on the proliferation of cultured hepatoma cells than hnRNP A1 small interfering RNA, strongly suggesting that BC15 could also be a potential drug candidate for an hnRNP A1 inhibitor besides its prospect utility in in situ histological examination.