研究背景:

胰腺导管腺癌是最常见的胰腺癌，肿瘤进展迅速，致死率高，预期生存期短，对于放疗、化疗以及现有的免疫治疗均不敏感。本课题组前期研究提示ZC3H7A可能参与胰腺癌的转移过程。作为另一个候选蛋白、具有同样结构域的ZC3H15，则有报道表明在不同恶性肿瘤性疾病中，通过偶联TRAF2蛋白，发挥促肿瘤作用。ZC3H7A和ZC3H15在胰腺癌发生发展过程中所发挥的生物学作用及机制尚不明确。

研究目的:

1、研究ZC3H7A及ZC3H15蛋白在人胰腺导管腺癌组织中的表达水平，分析其与胰腺导管腺癌患者临床病理特征及临床预后之间的关系；

2、探索不同ZC3H7A及ZC3H15蛋白表达水平，对胰腺癌细胞增殖、凋亡、侵袭、迁移及耐药等恶性表型的影响；并研究调控胰腺癌细胞恶性表型的机制。

研究方法:

1、应用免疫组织化学及公共数据库数据，探索癌组织、配对癌旁组织中ZC3H7A及ZC3H15的表达水平。分析靶基因各水平表达情况与临床病理特征及胰腺癌患者预后的关系。

2、应用siRNA干扰技术或转染过表达质粒调控细胞内ZC3H15或ZC3H7A的蛋白表达水平；应用CRISPR-Cas9基因编辑技术构建ZC3H7A基因敲除的稳定细胞系。应用CCK8、流式细胞术、平板克隆成型及Transwell法观察细胞的增殖、凋亡、侵袭及迁移能力；在吉西他滨干预下探索ZC3H7A蛋白在不同表达水平时胰腺癌细胞对于药物反应能力。应用敲除ZC3H7A基因的胰腺癌细胞，在小鼠体内验证胰腺癌细胞成瘤及成瘤后吉西他滨干预效果。应用转录组+microRNA测序在ZC3H7A蛋白不同表达水平下，探索下游靶基因。应用免疫共沉淀法，通过质谱分析、Western blot，探索ZC3H15在蛋白水平的作用机制。

研究结果:

1、ZC3H15蛋白在胰腺癌组织中高表达，且高表达患者有更多的淋巴结转移和更差的组织分化情况，是影响胰腺癌患者预后的独立危险因素。ZC3H15与ZC3H7A的表达有显著正相关性。ZC3H7A表达水平与胰腺癌患者预后无直接相关性。

2、肿瘤恶性表型实验结果：

（1）调控ZC3H15表达水平后，结果提示ZC3H15通过干预细胞G0/G1向S期的转化，促进胰腺癌细胞的增殖。敲低胰腺癌细胞中ZC3H15蛋白水平后，在TNF-α的激活下，可显著促进细胞的凋亡。

（2）ZC3H7A基因敲除后显著抑制胰腺癌细胞体外的增殖能力和体内成瘤能力。抑制ZC3H7A蛋白的表达水平或敲除ZC3H7A基因，在体内、体外实验中均能提高吉西他滨的药物作用能力。

（3）调控ZC3H15及ZC3H7A的表达水平对人胰腺癌细胞的侵袭、迁移及单细胞克隆成型能力均无显著影响。

3、MAPK信号通路中关键信号蛋白ERK1/2的激活状态，与ZC3H15的表达水平显著正相关。ZC3H15表达受抑制后，NF-κB信号通路中的关键信号蛋白p65的激活显著受抑制。免疫共沉淀后蛋白质谱分析结果，及公共数据库结果同时显示ZC3H15与DRG1相结合；免疫共沉淀后western blot结果显示，ZC3H15与TRAF2存在相互结合。

4、ZC3H7A公共数据库结果提示ZC3H7A与ZC3H15存在并表达现象。转录组测序，microRNA测序及结合药物干预后转录组测序结果获得多个潜在下游靶点， CD40 和VASH1可能是ZC3H7A关键的下游靶点。

研究结论:

ZC3H15在胰腺癌组织中呈高表达，其高表达是胰腺癌预后的危险因素。ZC3H15通过结合TRAF2，激活MAPK及NF-κB信号通路，促进胰腺癌细胞的增殖抑制细胞凋亡。ZC3H15和ZC3H7A的表达具有正相关性。ZC3H7A基因敲除后显著抑制细胞增殖能力，提高药物作用能力。CD40 和VASH1或是ZC3H7A的重要下游靶点；DRG1则是ZC3H15蛋白影响胰腺癌恶性表型另一潜在功能靶点。

Background

Pancreatic ductal adenocarcinoma, the most common type of pancreatic cancer, is known as rapid tumor progression, high mortality, short expected survival, and resist to radiotherapy, chemotherapy, and existing immunotherapies. Our previous study suggested that ZC3H7A was involved in the metastasis of pancreatic cancer. As another candidate protein, ZC3H15, which has the same structure domain, plays a tumor-promoting role by combining with TRAF2 protein in different tumor diseases. The biological roles and mechanisms of ZC3H7A and ZC3H15 in the development of pancreatic cancer are still unclear

Objective

1. To study the expression levels of ZC3H7A and ZC3H15 in pancreatic ductal adenocarcinoma tissue, and to analyze the relationships among the expression level, the clinicopathological features and prognosis of patients with pancreatic ductal adenocarcinoma.

2. To explore the effects of different ZC3H7A and ZC3H15 protein expression levels on malignant phenotypes such as proliferation, apoptosis, invasion, migration and drug resistance of pancreatic cancer cells. And to explore the mechanism of regulating the malignant phenotype of pancreatic cancer cells.

Methods

1. Expression of ZC3H7A and ZC3H15 in tumor tissues, matched tumor-adjacent pancreas tissues were investigated by immunohistochemistry method, and the association among ZC3H7A /ZC3H15, clinicopathological characteristics and clinical prognosis of patients was analyzed.

2. Protein expression levels of ZC3H15 or ZC3H7A in cells were regulated by siRNA interference or transfection with overexpressing plasmids. The stable cell lines of ZC3H7A gene knockout were constructed by CRISPR-Cas9 gene editing technique. Cell proliferation, apoptosis, invasion and migration were observed by CCK8, flow cytometry, plate cloning and transwell test. The effect of gemcitabine was verified in vitro and in vivo. ZC3H7A downstream target genes were explored by mRNA sequencing and microRNA sequencing. The mechanism of ZC3H15 was investigated by immunoprecipitation, mass spectrometry and Western blot.

Results

1. ZC3H15 protein is over-expressed in pancreatic cancer tissues, and its high expression is significantly correlated with lymph node metastasis and poor differentiation. High ZC3H15 expression level appeared to be an independent risk factor affecting the prognosis of pancreatic cancer patients. The significant positive correlation between ZC3H15 and ZC3H7A could be observed.

2. Experimental results of malignant phenotype:

(1) The results suggested that ZC3H15 could promote the proliferation of pancreatic cancer cells by interfering with the transformation from G0/G1 to S phase. ZC3H15 can protect pancreatic cancer cells form apoptosis.

(2) ZC3H7A gene knockout significantly inhibited proliferation both in vitro and in vivo. The loss of ZC3H7A protein can improve gemcitabine effect both in vivo and in vitro.

(3) ZC3H15 and ZC3H7A had no significant effect on the invasion, migration and the ability of single cell cloning in human pancreatic cancer cells.

3. The activation state of ERK1/2, a key signaling protein in the MAPK signaling pathway, is significantly positively correlated with ZC3H15.Inhibition of ZC3H15 significantly inhibited the activation of p65, a key signaling protein in the NF- B signaling pathway. The results of protein spectrum analysis after immunoprecipitation and the results of public database showed that ZC3H15 was combined with DRG1.Western blot results after immunoprecipitation showed that ZC3H15 was bound to TRAF2.

4. mRNA sequencing and microRNA sequencing results obtained a number of potential downstream targets. CD40 and VASH1 may be the key downstream targets of ZC3H7A.

Conclusions

ZC3H15 is highly expressed in pancreatic cancer tissues, and its high expression is a risk factor for the prognosis of pancreatic cancer. ZC3H15 activates the MAPK and NF- B signaling pathways by binding to TRAF2 to promote proliferation and inhibit apoptosis of pancreatic cancer cells. The expressions of ZC3H15 and ZC3H7A were positively correlated. After the knockout of ZC3H7A gene, the cell proliferation was inhibited and the gemcitabine effect was improved. CD40 and VASH1 may be important downstream targets of ZC3H7A; DRG1 is another potential target of ZC3H15。