Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, Jiangsu, China.
Wuxi Cancer Institute, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China.
PeerJ. 2022 Jul 11;10:e12728. doi: 10.7717/peerj.12728. eCollection 2022.
Pancreatic ductal adenocarcinoma (PDAC) has the worst five-year overall survival rate among all cancer types. Acquired chemoresistance is considered one of the main reasons for this dismal prognosis, and the mechanism of chemoresistance is unknown.
We previously identified a subpopulation of chemoresistant CD44-expressing PDAC cells. Subsequently, we selected the candidate gene, gamma-aminobutyric acid receptor subunit Pi (GABRP), from three Gene Expression Omnibus datasets as the potential CD44 downstream target mediating the gemcitabine resistance. Loss and gain of function such as stable knockdown of CD44 by small hairpin (sh) RNA-mediated silencing technique and overexpression (O/E) of CD44s had been studied for comparing the gemcitabine resistance among CD44-expressing cells, shCD44 cells, CD44-expressing cells and O/E CD44s expressing cells. Functional assays including cell viability, colony formation, invasion, quantitative PCR and western blotting techniques were performed to validate the roles of CD44 and GABRP playing in mediating the gemcitabine resistance in pancreatic cancer cells.
CD44s depletion significantly reduced gemcitabine resistance in shCD44 single clone cells compared to CD44-expressing cells. Knockdown of CD44 cells formed less colonies, became less invasive and remarkably decreased the mRNA level of GABRP. While overexpression of CD44s had the opposite effect on gemcitabine resistance, colony formation and invasive property. Of note, long term gemcitabine resistant pancreatic cancer cells detected increased expression of CD44 and GABRP. Clinically, GABRP expression was significantly upregulated in the tissues of patients with pancreatic cancer compared to the normal samples, and the overall survival rate of patients with low GABRP expression was longer. CD44 and GABRP co-expression was positively correlated in 178 pancreatic cancer patients.
Our findings suggest that GABRP may serve as a CD44s downstream target to diminish gemcitabine resistance in pancreatic cancer, and both CD44s and GABRP molecules have the potential to become prognostic biomarkers for PDAC patients with gemcitabine resistance.
胰腺癌（PDAC）是所有癌症类型中五年总生存率最差的。获得性化疗耐药被认为是这种预后不良的主要原因之一，但其耐药机制尚不清楚。
我们之前发现了一个具有化疗耐药性的 CD44 表达的胰腺癌细胞亚群。随后，我们从三个基因表达综合数据集（Gene Expression Omnibus datasets）中选择候选基因γ-氨基丁酸受体亚基 Pi（GABRP），作为潜在的 CD44 下游靶点，介导吉西他滨耐药。通过小发夹 RNA 介导的沉默技术稳定敲低 CD44（sh）RNA 和过表达 CD44s（O/E）进行了失活和功能获得研究，以比较 CD44 表达细胞、shCD44 细胞、CD44 表达细胞和 O/E CD44s 表达细胞之间的吉西他滨耐药性。进行了细胞活力、集落形成、侵袭、定量 PCR 和 Western blot 技术等功能测定，以验证 CD44 和 GABRP 在介导胰腺癌细胞吉西他滨耐药中的作用。
与 CD44 表达细胞相比，shCD44 单克隆细胞中 CD44s 的耗竭显著降低了吉西他滨耐药性。CD44 敲低细胞形成的集落较少，侵袭性降低，GABRP 的 mRNA 水平显著降低。而 CD44s 的过表达对吉西他滨耐药、集落形成和侵袭特性则产生相反的效果。值得注意的是，长期吉西他滨耐药的胰腺癌细胞检测到 CD44 和 GABRP 的表达增加。临床上，与正常样本相比，胰腺癌组织中 GABRP 的表达显著上调，GABRP 低表达患者的总生存率较长。178 例胰腺癌患者中 CD44 和 GABRP 的表达呈正相关。
我们的研究结果表明，GABRP 可能作为 CD44s 的下游靶点，降低胰腺癌细胞对吉西他滨的耐药性，CD44s 和 GABRP 分子有可能成为具有吉西他滨耐药性的 PDAC 患者的预后生物标志物。
