长链非编码RNA KLHL7-DT对人退变髓核细胞增殖和凋亡的影响及其相关机制

目的 探讨长链非编码RNA KLHL7-DT对人退变髓核细胞增殖和凋亡的影响及其相关机制。方法 选取2018年1月—2019年10月南京江北医院骨科脊柱骨折患者手术切除的正常椎间盘标本18例,其中男11例、女7例,年龄为22~46 （38.3±4.3）岁,PfirrmannⅠ级6例、Ⅱ级12例。取椎间盘标本常规分离、培养髓核细胞,使用10 ng/mL IL-1β处理髓核细胞获得退变髓核细胞。将退变髓核细胞分为沉默对照组、KLHL7-DT沉默组、过表达对照组、KLHL7-DT过表达组。4组细胞分别对应转染沉默对照序列、siRNA-KLHL7-DT沉默序列、过表达对照序列、KLHL7-DT过表达序列。取转染后4组退变髓核细胞采用5-乙炔基-2’脱氧尿嘧啶核苷（EdU）法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Western blot检测聚集蛋白聚糖（Aggrecan）、Ⅱ型胶原（Col Ⅱ）蛋白的表达情况。结果 （1）KLHL7-DT过表达组EdU染色阳性细胞/DAPI染色阳性比值（0.147±0.002）低于过表达对照组（0.203±0.007）,而KLHL7-DT沉默组比值（0.428±0.050）高于沉默对照组（0.240±0.032）,差异均有统计学意义（t=14.25、-5.44,P值均<0.05）。（2）KLHL7-DT过表达组细胞凋亡率（19.01%±0.41%）高于过表达对照组（14.38%±0.31%）,KLHL7-DT沉默组细胞凋亡率（16.08%±0.59%）低于沉默对照组（17.42%±0.36%）,差异均有统计学意义（t=15.69、3.36,P值均<0.05）。（3）Western blot结果显示,KLHL7-DT过表达组细胞Aggrecan和Col Ⅱ蛋白的相对表达量（分别为0.34±0.29、0.57±0.11）均低于过表达对照组（1.00±0.22、1.05±0.10）,KLHL7-DT沉默组Aggrecan和Col Ⅱ蛋白的相对表达量（分别为1.77±0.14、1.63±0.12）均高于沉默对照组（1.10±0.18、0.98±0.08）,差异均有统计学意义（t=3.10、5.54、-5.05、-7.66,P值均<0.05）。结论 上调KLHL7-DT的表达可抑制退变髓核细胞的增殖,促进退变髓核细胞的凋亡,其机制可能是通过调节细胞外基质Aggrecan、Col Ⅱ蛋白的合成,进而参与椎间盘退变的发展。

Expression of long non-coding RNA KLHL7-DT in human intervertebral disc and its effect on proliferation and apoptosis of nucleus pulposus cells and its related mechanism

Objective This study aims to evaluate the effect of long non-coding RNA KLHL7-DT on the proliferation and apoptosis of degenerate nucleus pulposus cells and its related mechanism. Methods A total of 18 normal intervertebral disc specimens were selected from orthopedic spine fracture patients in Nanjing Jiangbei Hospital from January 2018 to October 2019, including 11 males and 7 females, with an average age of 22-46 (38.3±4.3) years old. There were 6 and 12 patients with Pfirrmann grades Ⅰ and Ⅱ, respectively. Nucleus pulposus cells were routinely isolated and cultured from normal intervertebral discs removed by the patient. Then, the degraded nucleus pulposus cells were obtained by treating them with 10 ng/mL IL-1β. The degenerated nucleus pulposus cells were divided into silencing control, KLHL7-DT silencing, overexpression control, and KLHL7-DT overexpression groups. The four groups of cells were corresponding to the transfected silenced control, siRNA-KLHL7-DT silenced, overexpressed control, and KLHL7-DT overexpressed sequences, respectively. Cell proliferation was detected by the 5-acetyne-2 'deoxyuracil nucleoside (EdU) method, cell apoptosis was detected by flow cytometry, and the expression of aggrecan and type Ⅱ collagen (Col Ⅱ) protein was detected by Western blot analysis. Results (1) The EdU staining positive cell/DAPI staining positive ratio in the KLHL7-DT overexpression group (0.147±0.002) was lower than that in the control group (0.203±0.007), and the EdU staining positive cell/DAPI staining positive ratio in the KLHL7-DT silencing group (0.428±0.050) was higher than that in the control group (0.240±0.032). The differences were statistically significant (t=14.02,-5.44, P<0.05). (2) The apoptosis rate of the KLHL7-DT overexpression group (19.01%±0.41%) was higher than that of the overexpression control group (14.38%±0.31%), and the apoptosis rate of the KLHL7-DT silenced group (16.08%±0.59%) was lower than that of the silenced control group (17.42%±0.36%). The differences were statistically significant (t=15.69, 3.36, P<0.05). (3) Western blot analysis results showed that the relative expression levels of aggrecan and Col Ⅱ in the KLHL7-DT overexpression group were 0.34±0.29 and 0.57±0.11, which were lower than 1.00±0.22 and 1.05±0.10 in the overexpression control group, respectively. The relative expression levels of aggrecan and Col Ⅱ in the KLHL7-DT silenced group were 1.77±0.14 and 1.63±0.12, respectively, which were higher than those in the KLHL7-DT silenced group (1.10±0.18 and 0.98±0.08). A statistical significance was observed between the two groups (all P values<0.05). Conclusion The upregulation of the KLHL7-DT expression can inhibit the proliferation and promote the apoptosis of degraded nucleus pulposus cells which may be involved in the development of intervertebral disc degeneration by regulating the synthesis of extracellular matrix aggrecan and Col Ⅱ proteins.