长链非编码RNA PCAT1通过调控BLyS/MAPK信号通路促进多发性骨髓瘤细胞生长的机制研究

Multiple myeloma (MM) is characterized by malignant proliferation of plasmocytes with complex pathogenesis. It is particularly important to find diagnostic and prognostic markers for MM. Recent studies have showed that long non-coding RNA (lncRNA), acting as competing.endogenous RNA (ceRNA), may regulate the expression and function of miRNAs, which is also closely associated with the occurrence and.development of malignancies. Our previous studies has showed that lncRNA-PCAT1 was highly expressed in MM patients’ bone marrow MAPK signaling pathway is active in Multiple myeloma (MM), which is a key factor induce plasma cells malignant transformation, but the specific mechanism of this pathway is not very clear. Our previous study found that B-lymphocyte stimulator (BLyS) enhanced MM cells proliferation and survival by MAPK signaling pathway. Bioinformatics analysis suggests that MAP3K7 and MAPK1 may be as the target gene of miR-129-5p. lncRNA PCAT1 was high in MM patients’ bone marrow and plasma, whereas miR-129-5p was low expression. PCAT1 and miR-129-5p can be combined with each other in RNA-induced silencing complex. We speculate that PCAT1 may act as competing endogenous RNAs (ceRNAs) to regulate the function of miR-129-5p. Therefore, luciferase assay and RNA binding protein immunoprecipitation tests were used at cell and animal level to study the mechanisms of PCAT1 as ceRNAs to regulate the expression of MAP3K7 and MAPK1, and then study the molecular mechanisms underlying a feedback loop of BLyS/MAPK in MM cells. Meanwhile, we also explore clinical value of PCAT1 in plasma as a new biomarker in the early diagnosis of MM. This study will shed some light on the pathogenesis of MM and also provide a new thinking and new molecular targets for clinical diagnosis and treatment.

多发性骨髓瘤（MM）中存在MAPK信号通路的持续活化，是诱发浆细胞恶性转化的关键因素，而该通路具体活化机制仍不十分清楚。前期研究发现，B淋巴细胞刺激因子（BLyS）可激活MAPK通路促进MM细胞增殖与存活。生物信息学预测MAPK通路分子MAP3K7及MAPK1可作为miR-129-5p的靶基因；预实验证实MM中miR-129-5p低表达；而lncRNA-PCAT1高表达，且二者可相互结合于RNA诱导沉默复合物；推测PCAT1可作为竞争性内源RNA（ceRNA）调控miR-129-5p的功能。本项目拟运用荧光素酶试验、RNA拉下试验等技术在细胞及动物水平探讨PCAT1作为ceRNA调控MAP3K7与MAPK1的表达，进而通过BLyS/MAPK正反馈信号促进MM细胞生长的分子机制；同时还探讨血浆PCAT1作为MM潜在诊断标志物的临床价值。本研究可为MM发病机制研究及临床诊疗提供新思路和新靶标。

目的：建立基于实时荧光定量PCR（RT-qPCR）检测血清循环长链非编码RNA PCAT1的方法，检测多发性骨髓瘤（MM）患者及正常人血清PCAT1的表达水平，探讨血清PCAT1在MM辅助诊断中的价值及促进MM细胞增殖和凋亡的机制。方法：通过qRT-PCR检测血清PCAT1的相对表达水平，并评价其线性、重复性以及特异性；分析血清PCAT1表达与MM患者临床病理参数的相关性；ROC曲线评价血清PCAT1、轻链κ浓度、轻链λ浓度与β2M单独及联合检测对MM的诊断价值。构建PCAT1短发夹RNA干扰载体及过表达PCAT1的载体转染入MM细胞；通过CCK-8实验检测细胞增殖能力、软琼脂检测细胞克隆形成、流式细胞术检测细胞周期及凋亡，Western blot检测NF-κB、MAPK信号通路相关蛋白表达情况。结果：（1）LncRNA芯片分析MM骨髓中Top20上调与下调的lncRNAs，发现PCAT1在MM患者中呈现高表达。（2）基于qRT-PCR检测血清PCAT1的线性良好，重复性较好，特异性较高，熔解曲线单峰特异。（3）MM患者血清PCAT1的表达上调，显著高于正常对照组，与轻链κ浓度和轻链λ浓度有显著相关性，与β2M浓度有一定的相关性，而与IgA、IgM、IgG浓度无显著相关性。（4）PCAT1、β2M、轻链κ、轻链λ和LDH的ROC曲线下面积分别为0.892、0.866、0.774、0.774和0.574；三者联合诊断可提高诊断灵敏度。（5）sh-PCAT1转染MM细胞后，可明显抑制细胞增殖和克隆形成能力，同时诱导MM细胞G0/G1期阻滞。（6）生物信息学分析及双荧光素酶报告实验证实PCAT1和miR-129直接结合，Spearman相关性分析证明两者呈负相关。过表达PCAT1可以逆转miR-129对MM细胞增殖、周期、凋亡的影响。（7）生物信息学分析及双荧光素酶报告实验证实miR-129的靶基因为MAP3K7，MAP3K7 mRNA水平和miR-129呈负相关，和PCAT1呈正相关。（8）蛋白芯片及Western blot检测发现过表达PCAT1可上调p-p38 and p-JNK蛋白水平的表达。结论：循环PCAT1浓度对MM辅助诊断有一定价值。PCAT1作为一个竞争性内源性RNA和MAP3K7竞争结合miR-129，通过MAPK信号通路调控MM细胞的恶性表型。