Increased local miR-21 expressions are linked with clinical severity in lumbar disc herniation patients with sciatic pain.

BACKGROUND: The miR-21 has been implicated in the process of neuroinflammation as well as neuropathic pain. OBJECTIVES: To explore the relationship between the plasma and local expression of miR-21 with disease severity of lumbar disc herniation (LDH) patients with sciatic pain. MATERIAL AND METHODS: Ninety-two LDH patients with sciatic pain and 25 scoliosis patients as painless controls were enrolled in the current study. Samples from nucleus pulposus (NP), annulus fibrosus (AF) and soft tissues around nerve root (STANR) were obtained. The plasma and local expressions of miR-21 were detected with quantitative reverse transcription polymerase chain reaction (qRT-PCR). The visual analogue scale (VAS) for lumbar pain and leg pain, and Japanese Orthopedic Association (JOA) score were selected to evaluate the clinical severity. The degree of disc compression on nerve was evaluated using the Pfirrmann grade based on the magnetic resonance imaging (MRI) findings. For the convenience of analysis, LDH patients with sciatic pain were classified into a severe pain (SP) group (VAS ≥ 6) and a mild-moderate pain (MP) group (VAS < 6). Receiver operating characteristic (ROC) curve analysis was performed to detect the potential diagnostic power of miR-21 with regard to the Pfirrmann grade. RESULTS: There were no significant differences in serum miR-21 expressions among SP LDH patients, MP LDH patients and scoliosis painless controls. Local expressions of miR-21 in STANR, AF and NP were all drastically upregulated in the SP group in comparison with the MP group and scoliosis painless group. Local NP and STANR miR-21 expressions were positively associated with the Pfirrmann grade. Local miR-21 expressions in STANR and AF were positively associated with VAS score and negatively related to JOA score. The ROC curve analysis indicated that both STANR and AF miR-21 expressions may serve as significant diagnostic factors for the Pfirrmann grade. CONCLUSIONS: Increased local miR-21 expressions are linked with clinical severity of LDH in patients with sciatic pain.

SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing.

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5'- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications.