LncRNA MALAT1通过靶向miR-23b-3p调节FGFR2对PDAC转移的影响及其机制研究

Pancreatic duct adenocarcinoma (PDAC) has the characteristics of fast progression and early metastasis, but the mechanism remains unclarifed. Metastasis associated lung adenocarcinoma transcript 1 is a tumor associated long non-coding RNA (lncRNA),which is closely related to invasion and metastasis of malignant tumors. However, there is few report of MALAT1 in PDAC. Previously, we demonstrated that the expression level of lncRNA MALAT1 was upregulated in PDAC tissues. Inhibition of MALAT1 in vitro suppressed the migratory and invasive capacity of PDAC cells. Furthermore, we found that miR-23b-3p was down-regulated in a small number of PDAC tissues as compared with that in the adjacent non-cancerous tissues. The expression of miR-23b-3p was negatively correlated with the expression of MALAT1. Interestingly, microarray analysis revealed that FGFR2 was downregulated when MALAT1 was knocked down. MiRNA target prediction software analysis also revealed that FGFR2 could be a potential target gene of miR-23b-3p. Therefore, we hypothesize that MALAT1 may regulate FGFR2 expression via sponging miR-23b-3p and thus, enhance the invasion and metastasis of PDAC. In the current study, we plan to verify the relationships of MALAT1, miR-23b-3p and FGFR2, and to investigate their molecular mechanisms of regulation on the invasion and metastasis in vitro and in vivo by using CRISPR/Cas9, chicken embryochorioallantoic membrane and nude mouse models.

胰腺导管上皮癌（PDAC）具有进展快、转移早的特点，但机理未阐明。肺腺癌转移相关转录物1（MALAT1）是人类肿瘤相关长链非编码RNA(LncRNA），与恶性肿瘤侵袭转移有关，但鲜见在PDAC中的报道。前期我们发现MALAT1在PDAC中高表达，沉默MALAT1后侵袭及迁移能力减弱；同时miR-23b-3p在PDAC表达低于癌旁组织，与MALAT1呈负相关，而沉默MALAT1后成纤维细胞生长因子受体2（FGFR2）表达下调；预测软件显示miR-23b-3p与FGFR2存在互补序列。据此推测MALAT1可能扮演miR-23b-3p海绵，通过调节FGFR2水平对PDAC的侵袭转移发挥作用。本项目拟在前期基础上通过CRISPR/Cas9技术，CAM 和裸鼠等体内、体外模型、多角度的探讨MALAT1、miR-23b-3p及FGFR2之间的靶向关系，及对PDAC细胞侵袭转移作用的分子机制。

MALAT1在胰腺导管上皮癌（PDAC）中的研究甚少，其作用机制尚不清楚。为阐明其在PDAC发生发展中的作用机制，本研究通过RT-qPCR技术检测发现MALAT1在PDAC组织中表达水平明显高于癌旁。在PDAC多种细胞系中，发现MALAT1在ASPC-1细胞中高表达。应用原位杂交研究发现MALAT1阳性率在PDAC组织明显高于癌旁。且其表达与肿瘤大小、浸润深度、肿瘤临床分期呈正相关，与生存时间呈负相关。.沉默PDAC细胞系MALAT1后，发现细胞增殖、侵袭及迁移能力减弱，凋亡增加。体内实验发现，敲减MALAT1抑制PANC-1细胞的小鼠移植瘤生长。敲减MALAT1后，PDAC细胞中FOS蛋白表达水平下降和CVA1及FGF2的蛋白水平表达增加。由此推测：MALAT1可能通过调节FOS/FGF2或CAV1等基因的表达来激活ERK/MAPK信号通路，从而调控PDAC细胞生物学功能。.同时在PDAC中，miR-23b-3p在PDAC表达明显低于癌旁胰腺组织，与MALAT1表达呈负相关，也与PDAC肿瘤浸润和临床分期密切相关。体外细胞学发现miR-23b-3p抑制细胞增殖、侵袭及迁移能力。同时，鸡胚绒毛尿囊膜(CAM)体内试验表明了miR-23b-3p表达上调可抑制肿瘤形成。使用CRISPR/Cas9、qRT-PCR技术发现miR-23b-3p与MALAT1结合并相互调控，MALAT1可抑制miR-23b-3p在PDAC中的表达。.在此过程中，多个预测平台预测ANXA2是miR-23b-3p的潜在靶基因，经双荧光素酶证实miR-23b-3p可与ANXA2 mRNA的3′UTR进行特异性结合。通过免疫组化的方式证实了ANXA2在PDAC组织中表达显著性升高，且与肿瘤大小、TNM分期、神经侵袭等有关，miR-23b-3p的表达与ANXA2免疫组化染色评分呈负相关。.我们推测MALAT1可能通过下调miR-23b-3p表达，调控其下游靶基因ANXA2促进肿瘤细胞增生及侵袭能力，从而参与PDAC发生、发展的过程。所涉及的通路有望为胰腺癌的发生发展提供研究方向。.在完成既定研究计划的过程中，我们发现胰腺癌自噬相关非编码RNA与胰腺癌的发生发展存在相关性，对其作用机制进行了初步探讨。