AS is a complex chronic disease and an important cause of various secondary diseases 1–2. LP(a) is considered to be a major risk for the occurrence and development of AS by promoting foam cell formation, EPC dysfunction, plaque formation, and inflammatory response24–25. Therefore, LP(a) was transfected in EPCs in this study to simulate EPC damage in vitro. The results indicated that XIST targets the miR-126-3p/PLK2 axis to regulate the LP(a)-mediated EPC damage.
XIST, as a common lncRNA, has been extensively studied in a variety of diseases. XIST was initially discovered to silence X chromosome genes via cis-acting 26. Recent studies have suggested that the abnormal expression of XIST and its "ceRNA" effect as a miRNA sponge are responsible for the dysfunctions of many miRs, and plays a regulatory role in various neoplasms. Chen Zhenzhang et al. reported that XIST is highly expressed in esophageal cancer, and inhibiting XIST significantly attenuates the proliferation, migration and invasion of TE-1 and SKGT-4 cells, and induces their apoptosis. The abnormal expression of XIST may be involved in the occurrence and development of esophageal cancer through regulating the JAK2/STAT3 signaling pathway and miR-494/CDK6 axis27. Meanwhile, the carcinogenic effect of XIST has been demonstrated in thyroid cancer 28, colorectal cancer 29–30, laryngeal squamous cell carcinoma and other tumors 31–34. In recent years, reports on XIST in other non-tumor diseases are also increasing. Zhou Tao et al. showed that XIST promotes myocardial infarction by targeting miR-130A-3p35. Consistent with the results reported by Zhou Tao et al., Yang Xiaoyu et al. found that XIST expression in cartilage specimens of OA patients is distinctly up-regulated, which confirms that XIST regulates the proliferation and apoptosis of OA chondrocytes through the miR-211/CXCR4 axis 36. Studies in spinal cord injury have revealed that XIST gene knockout limited the neuronal apoptosis in rats, and its protective effect is regulated by the phosphorylation of AKT through competitive binding to miR-49437. Thus, it can be seen that XIST could strengthen cell apoptosis in inflammatory diseases, and knocking down XIST plays a protective role in injury. Above all, the role of XIST in EPC is scarcely reported. This study clarified that knocking down XIST significantly reduces LP(a)-induced EPC damage.
As a classical miR, miR-126 has been widely studied. Sabo Alexandru A et al. found that miR-126 is up-regulated in plasma EVS and urine, and is positively correlated with tumor grade 38. Studies in gastric cancer also have confirmed that miR-126 inhibits cell apoptosis and promotes its proliferation by down-regulating the PI3K pathway, such as IGF1R, INSR, PDK1 and AKT1 39. Meanwhile, the carcinogenic effect of miR-126 has been proved in glioblastoma 40, NSCLC 41, porcine granulosa cells 42, and other tumors. There are vast studies on miR-126 in AS, and most scholars believe that miR-126 plays a protective role in ECs. For instance, Ohta Momoka et al. hold that IL-6 reduces the accumulation of miR-126 in EA.hy926 cells, thus increasing the expression of miR-126-targeted genes. Moreover, IL-6 enhances the adhesion of human mononuclear cell line THP-1, accompanied by an increase of intercellular adhesion molecule-1 level 43. Xue Wen-long et al. confirmed that high glucose reduces the endogenous miR-126 level in HUVECs and increases the expression of DNMT1, thus resulting in the dysfunction of HUVEC migration 44. In addition, studies in cerebral ischemia have found that overexpression of miR-126 dampens the expression of pro-inflammatory cytokines and adhesion molecules after ischemic stroke, thus alleviating the destruction of blood-brain barrier 45. Furthermore, miR-126 mimics down-regulate VCAM-1 in the bleeding area and ease the destruction of the blood-brain barrier after cerebral hemorrhage 46. Thus, studies in both hemorrhagic/ischemic stroke and HUVECs show that miR-126 plays a positive role in protecting the function of HUVECs. The results of this study initially demonstrated the protective effect of miR-126 in LP(a)-mediated EPC damage.
Also, this study confirmed that miR-126 is negatively regulated by XIST. Although there have been vast studies on XIST, only Cheng Zhihua et al. reported that XIST regulates the IRS1/PI3K/Akt pathway as the ceRNA of miR-126 in glioma47. Consistent with previous reports, this study also proved that XIST gene knockout inhibits the survival, migration, invasion, anti-apoptosis and glucose metabolism of glioblastoma cells. Therefore, this study initially testified through compensation experiments in non-tumor diseases that XIST sponges miR-126 as a ceRNA, thus regulating the function of EPC, which provides a basis for in-depth exploration of the role and mechanism of XIST in EPCs.
By further exploring the downstream mechanism of the XIST/miR-126 axis, we discovered PLK2 is the target of miR-126. PLK2 also was a cell cycle regulation gene that affects cell cycle progression, cell proliferation and individual bone development 48. Meanwhile, PLK2 plays a carcinogenic role in multiple tumors, such as glioma 49, oral cancer 50 and colorectal carcinomas 51. In contrast, down-regulating PLK2 inhibits synovial damage in KOA rats, promotes synovial cell apoptosis, dampens angiogenesis, cartilage damage, chondrocyte apoptosis and alleviates the inflammatory damage of synovial tissue and cartilage tissue in inflammatory diseases. PLK2 overexpression reverses the effects of miR-27a up-regulation on synovial angiogenesis and chondrocyte injury in KOA rats 52. Ischemia/reperfusion (IR) up-regulates PLK2, but decreases the expression of miR-128. This study proved that PLK2 regulated by miR-128 induces apoptosis/death by activating the nuclear factor κB (NF-κB). MiR-128 and PLK2 are novel targets for the prevention of myocardial I/R or oxidative stress-mediated injury 53. These studies have confirmed that PLK2 is significantly up-regulated in injuries. The findings of this study were consistent with the previous researches. Namely, PLK2 is significantly up-regulated in damaged EPCs, and overexpressing PLK2 further aggravates the damage of EPC, while compensation of miR-126 mimics alleviates the damage of EPC.
In conclusion, our study testified that knocking down XIST ameliorates LP(a)-induced EPC damage. The specific mechanism is that XIST sponges miR-126 as a ceRNA to regulate the expression of PLK2, which provides a new way to explore the treatment of AS.