NEK6 Promotes the Progression of Osteosarcoma Through Activating STAT3 Signaling Pathway by Down-Regulation of miR-26a-5p.

Background: Osteosarcoma is a malignant tumor originating from the skeletal system. There is no effective treatment other than surgery and chemotherapy, which seriously endangers the health of children and adolescents. NEK6 is a novel discovered Serine/Threonine protein kinase that can regulate cell cycle and activate several oncogenic pathways. Methods: NEK6 expression in pan-cancer including sarcoma was evaluated using analysis tools of TIMER, UALCNA and GEPIA with TCGA database, and its association with overall survival in patients with sarcoma was also analyzed. TargetScan, tarbase, microT-CDS and Starbase online software were used to predict NEK6-targeted miRNAs, including miR-26a-5p. Tumor tissues from patients with osteosarcoma were collected for NEK6 and miRNA detection using RT-qPCR. NEK6 down-regulated by siRNAs or miR-26a-5p in osteosarcoma cells was detected by RT-qPCR, Western blot and Immunofluorescence staining assays. Effects of NEK6 knockdown on proliferation, migration, invasion and apoptosis of osteosarcoma cells were detected by CCK-8, wound healing, transwell and flow cytometry, respectively. The expressions of STAT3, metastasis and apoptosis-related genes were detected by Western blot. Results: High expression of NEK6 and low expression of miR-26a-5p were lowly expressed in osteosarcoma and they were negative correlation. NEK6 has been confirmed as a direct target for miR-26a-5p. In addition, NEK6 down-regulated by siRNAs or miR-26a-5p led to inhibition of cell proliferation, migration and invasion while promoting cell apoptosis. The levels of phosphorylated STAT3 and metastasis genes (MMP-2, MMP-9) were inhibited, while apoptotic gene Bax was promoted and Bcl2 was inhibited by miR-26a-5p upregulation. Conclusion: NEK6 can promote osteosarcoma progression via activating STAT3 signaling pathway, which is inhibited by miR-26a-5p, suggesting that NEK6 is a potential oncogene and miR-26a-5p is a suppressor of osteosarcoma. The strategy of inhibiting of NEK6 by miR-26a-5p may be an effective approach for osteosarcoma therapy.

lncRNA LINC00284 promotes nucleus pulposus cell proliferation and ECM synthesis via regulation of the miR­205­3p/Wnt/ß­catenin axis.

Intervertebral disc degeneration (IDD) is a leading cause of degenerative spinal disease. Long non­coding RNA (lncRNA) LINC00284 is overexpressed in multiple types of cancer and promotes cancer cell proliferation and inhibits apoptosis; however, its role in human IDD and nucleus pulposus (NP) remain unclear. In the present study, intervertebral disc (IVD) tissues were collected from IDD patients for detection of LINC00284 expression using reverse transcription­quantitative PCR, the binding effect between miR­205­3p and LINC00284 was validated by dual­luciferase reporter assay. miR­205­3p and small interfering RNA (siRNA) was used for LINC00240 knockdown to investigate the proliferation, apoptosis of cells in the NP cells measured by Cell Counting Kit (CCK)­8 assay and Annexin V­FITC/Propidium Iodide (PI) staining with flow cytometry receptivity. IDD animal models were constructed for in vivo study of the role LINC00284 in IDD improvement. The results showed that LINC00284 expression was upregulated in IDD tissue and IL­1ß­induced NP cells. LINC00284 knockdown resulted in an increase in IL­1ß­induced NP cell proliferation, a decrease in apoptosis and matrix metalloproteinase­3 expression and an increase in expression of extracellular matrix (ECM) markers aggrecan and collagen II. In vivo experiments and histomorphometric analysis confirmed the protective effect of LINC00284 knockdown in IDD. LINC00284 was also shown to be a target of microRNA (miR)­205­3p, and there was a negative correlation between LINC00284 and miR­205­3p levels in IDD tissue. Additionally, LINC00284 knockdown or miR­205­3p upregulation resulted in inhibition of Wnt/ß­catenin signaling and subsequent degradation of the ECM. The present study demonstrated that LINC00284 activated the Wnt/ß­catenin signaling via sponging miR­205­3p, resulting in inhibition of NP cell proliferation and ECM synthesis. These results suggested that targeting LINC00284 to rescue miR­205­3p expression may be a potential method for IDD management.

Clinical comparison of unilateral biportal endoscopic discectomy with percutaneous endoscopic lumbar discectomy for single l4/5-level lumbar disk herniation.

OBJECTIVE: The purpose of this research was to investigate the outcomes between unilateral biportal endoscopic discectomy (UBE) and percutaneous endoscopic lumbar discectomy (PELD) for the single L4/5-level lumbar disk herniation (sLDH). METHODS: From January 2018 to January 2021, a total of 40 patients with sLDH were retrospectively analyzed in this study. All the patients had received spinal surgeries in Affiliated Hospital of Nantong University and Affiliated Nantong Hospital 3 of Nantong University. Among them, 20 patients were treated with PELD (PELD group), and 20 patients were treated with UBE discectomy (UBE group). Postoperative length of hospital stay, estimated blood loss, operation time, and clinical complications of the patients were compared between the two groups. The visual analog scale (VAS) and Oswestry Disability Index (ODI) were measured before surgeries and 3 days, 1, and 6 months after surgeries. RESULTS: Compared with the UBE group, the PELD group had obviously less intraoperative blood loss, shorter operative time, and shorter hospital stay. The differences in the rate of complications were not statistically significant between the two groups. The VAS score and the ODI score of the two groups had a great reduction after operation. In addition, both the groups had satisfactory clinical outcome; the VAS score and ODI of the PELD group decreased more obviously. CONCLUSION: The UBE for sLDH yielded similar clinical outcomes to PELD as minimally invasive surgeries; however, PELD is superior to UBE in terms of intraoperative blood loss, operative time, postoperative hospitalization, and short-term postoperative pain relief. The advantages and disadvantages of the two surgeries should be circumspectly balanced when evaluating a patient for a minimally invasive surgery for sLDH, selecting the most appropriate surgical method for patients.

Polarization of Helper T Lymphocytes Maybe Involved in the Pathogenesis of Lumbar Disc Herniation.

This study aimed to explore the expression of T helper type 1 (Th1)/T helper type 2 (Th2) in herniated nucleus pulposus (NP) and determine their association with sciatic pain. NP was collected from 12 patients with lumbar disc herniation (LDH) (extrusion group) and 6 patients with a vertebral fracture (control group). The expression of Th1/Th2 and related cytokines in the NP was examined by flow cytometry, Western blot, and immunofluorescent staining. Subsequently, an LDH model was established in male Sprague-Dawley rats, and behavioral testings were carried out. The expression of Th1/Th2 and related cytokines in rat NP and the expression of macrophages in the dorsal root ganglia (DRG) were also examined. The number of Th1 cells in rat NP dramatically increased on day 14 after the surgery, but significantly decreased on day 28. The number of Th2 cells increased on day 28. Chemokine ligand 3(CCL3) and CD86 proteins (M1-specific molecules) were expressed at a relatively low level in naive DRG, markedly increased on day 14 after the surgery, and decreased on day 28. Arg1 and CD206 protein (M2-specific molecules) were expressed at a relatively low level in naive DRG and markedly increased on day 28. The mechanical allodynia and heat hyperalgesia developed after NP application and finally partially alleviated. The results suggested that the polarization of Th cells might be involved in the pathogenesis of LDH, and this might be achieved via the phenotypic shift of macrophages.

Interleukin-21 Is Associated with the Pathogenesis of Lumbar Disc Herniation.

Inflammation is an important reaction underlying lumbar disc herniation (LDH). Th17 cells play a critical role in immune activation. Interleukin (IL)-21 controls the functional activity of effector T-helper cells and the differentiation of Th17 cells, and promotes B-cell differentiation. It plays important roles in chronic inflammation and autoimmune diseases. However, little is known about relationship between IL-21 and LDH. This study was aimed to determine the association between IL-21 levels and pain scores in LDH patients compared to healthy controls.We enrolled 34 LDH patients and 20 healthy controls in this study. The LDH patients underwent surgery. Pain intensity was recorded using visual analogue scale (VAS) scores preoperatively. Serum IL-21 and IL-17 levels in the peripheral blood were determined using enzyme-linked immunosorbent assay. Disc tissue was examined using western blot and quantitative reverse-transcription polymerase chain reaction to determine IL-21, IL-17, and cyclooxygenase (COX)-2 expression, and using immunohistochemistry to assess IL-21 expression.LDH patients exhibited significantly higher levels of serum IL-21 and IL-17 than healthy controls. Moreover, higher expression of IL-21, IL-17, and COX-2 was found in the protein and mRNA levels in disc tissues from LDH patients than in normal disc tissues. Different parameters like VAS pain scores, IL-17, and COX-2 were positively correlated with the IL-21 levels. Enhanced production of IL-21 in disc tissues of LDH patients was also confirmed using immunohistochemical analyses.We concluded that inflammation was responsible for the pain associated with LDH, and that increased IL-21 expression may be associated with the pathogenesis of LDH.