胰腺癌中MKK7/JNKs/c-Jun通路通过促进施万细胞去分化促进神经浸润

MKK7/JNKs/c-Jun pathway promotes perineural invasion through promoting dedifferentiation of Schwann cells in pancreatic cancer

目的探讨胰腺癌中丝裂原活化蛋白激酶MKK7信号通路对施万细胞去分化以及胰腺癌神经浸润的影响。方法收集我院胰腺癌患者22例癌组织及癌旁组织标本,通过免疫组化检测施万细胞去分化标记物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达情况以及神经浸润情况,检测胰腺癌中MKK7的表达定位及强度。通过RNA干扰(shRNA)下调胰腺癌细胞株PANC-1和BxPC-3中MKK7的表达,构建MKK7低表达细胞株(sh-MKK7)并使用Western blot法检测下游通路JNK/c-Jun的表达情况。将胰腺癌细胞株分为对照组(Con)和MKK7低表达组(sh-MKK7),通过划痕愈合实验观察MKK7对癌细胞迁移能力的影响。将新生大鼠背根神经节(DRG)分别用空白培养基(Con)、正常癌细胞条件培养基(CM)或MKK7低表达癌细胞条件培养基(sh-CM)进行间接共培养实验,观察MMK7对神经节纤维生长的影响。将正常癌细胞(Con)、MKK7低表达癌细胞(sh-MKK7)与神经节进行直接共培养实验,观察MMK7对共培养中神经节纤维生长以及癌细胞向神经迁移能力的影响。结果与癌旁组织相比,癌组织中神经密度、GFAP染色以及MKK7表达均明显升高。成功构建MKK7低表达胰腺癌细胞株,Western blot实验表明MKK7下调可同时下调两株胰腺癌细胞株(PANC-1和BxPC-3)中JNK2以及c-Jun的表达。划痕愈合实验表明,MKK7下调可以抑制两株胰腺癌细胞的迁移能力。神经节与癌细胞条件培养基的间接共培养实验表明,MKK7下调可以抑制神经节纤维的生长(P<0.05)。神经节与癌细胞的直接共培养实验表明,MKK7下调可以抑制癌细胞向神经的迁移能力(P<0.05)。结论胰腺癌细胞通过MKK7/JNK/c-Jun通路,一方面促进胰腺癌细胞自身的迁移能力,另一方面促进施万细胞去分化及神经纤维生长,进而促进胰腺癌中神经浸润的发生。

Objective To investigate the effect of mitogen-activated protein kinase MKK7 signaling pathway on Schwann cell dediffe-rentiation and perineural infiltration of pancreatic cancer.Methods Samples of cancer tissues and paracancerous tissues from 22 pancreatic cancer patients were collected to detect the expression level of Schwann cell dedifferentiation marker glial fibrillary acidic protein(GFAP),nerve infiltration,and MKK7 location and expression in pancreatic cancer by immunohistochemistry.PANC-1 and BxPC-3 cell lines with MKK7 low expression(sh-MKK7)were constructed by RNA interference.The downstream signaling JNKs/c-Jun expression levels were detected by Western blot assay.Pancreatic cancer lines were divided into control group(Con)and low MKK7 expression group(sh-MKK7).Wound healing assay was used to evaluate the influence of MKK7 on the migration ability of pancreatic cancer cells.Newborn rat dorsal root ganglion(DRG)was cultured with control normal medium(Con),normal pancreatic cancer conditioned medium(CM)or MKK7 low expression cancer conditioned medium(sh-CM)for the indirect co-culture experiments to detect the influence of MKK7 on the growth of DRG nerve fibers.Normal cancer cells or MKK7 low expression cells were cultured with DRG for the direct co-culture experiments to detect the influence of MKK7 on the growth of DRG nerve fibers and the migration ability of cancer cells towards nerve.Results Compared with adjacent tissue,the nerve density,GFAP staining and MKK7 expression level were increased in pancreatic cancer tissue.MKK7 knockdown cell lines were successfully constructed.Western blot results showed that MKK7 knockdown downregulated JNK2 and c-Jun expression in two pancreatic cancer cell lines PANC-1 and BxPC-3.Wound healing experiment showed that MKK7 knockdown inhibited the migration ability of pancreatic cancer cells.DRG and cancer conditioned medium indirect co-culture assay showed that MKK7 knockdown inhibited the growth of DRG nerve fibers(P<0.05).DRG and cancer cell direct co-culture experiment showed that MKK7 knockdown inhibited the migration ability of cancer cells towards nerve(P<0.05).Conclusion MKK7/JNKs/c-Jun pathway in pancreatic cancer promotes the perineural invasion through upregulating cancer cell migration ability and promoting Schwann cell dedifferentiation and nerve fiber growth.