Identification of a novel m6A-related lncRNAs signature and immunotherapeutic drug sensitivity in pancreatic adenocarcinoma
Xia-Qing Li1†, Shi-Qi Yin1†, Lin Chen2†, Aziguli Tulamaiti3 , Shu-Yu Xiao3 , Xue-Li Zhang3 , Lei Shi4 , Xiao-Cao Miao3 , Yan Yang3* and Xin Xing1,2*
Abstract
Background Pancreatic adenocarcinoma (PDAC) ranks as the fourth leading cause for cancer-related deaths worldwide. N6-methyladenosine (m6A) and long non-coding RNAs (lncRNAs) are closely related with poor prognosis and immunotherapeutic effect in PDAC. The aim of this study is to construct and validate a m6A-related lncRNAs signature and assess immunotherapeutic drug sensitivity in PDAC.
Methods RNA-seq data for 178 cases of PDAC patients and 167 cases of normal pancreatic tissue were obtained from TCGA and GTEx databases, respectively. A set of 21 m6A-related genes were downloaded based on the previous report. Co-expression network was conducted to identify m6A-related lncRNAs in PDAC. Cox analyses and least absolute shrinkage and selection operator (Lasso) regression model were used to construct a risk prognosis model.
The relationship between signature genes and immune function was explored by ingle-sample GSEA (ssGSEA). The tumor immune dysfunction and exclusion (TIDE) score and tumor mutation burden (TMB) were utilized to evaluate the response to immunotherapy. Furthermore, the expression levels of 4 m6A-related lncRNAs on PDAC cell lines were measured by the quantitative real-time PCR (qPCR). The drug sensitivity between the high- and low-risk groups
was validated using PDAC cell lines by Cell-Counting Kit 8 (CCK8).
Results The risk prognosis model was successfully constructed based on 4 m6A-related lncRNAs, and PDAC patients were divided into the high- and low-risk groups. The overall survival (OS) of the high-risk groups was more unfavorable compared with the low-risk groups. Receiver operating characteristic (ROC) curves demonstrated that the risk prognosis model reasonably predicted the 2-, 3- and 5-year OS of PDAC patients. qPCR analysis confirmed
the decreased expression levels of 4 m6A-related lncRNAs in PDAC cells compared to the normal pancreatic cells. Furthermore, CCK8 assay revealed that Phenformin exhibited higher sensitivity in the high-risk groups, while Pyrimethamine exhibited higher sensitivity in the low-risk groups.
文章详情请见：https://doi.org/10.1186/s12885-024-11885-8
在该研究中使用了圣尔生物总RNA提取试剂盒、一管式反转录试剂盒、SYBR Green qPCR Premix和CCK8细胞增殖检测试剂盒等产品，用于RNA提取与qPCR实验和IC50实验。
RNA extraction and qPCR
Appropriate Trizol (ShareBio, Shanghai, China) was added into the cells in the dish washed by PBS with 1–2 times (10 cm dish with 2mL, 6 cm dish with 1mL). Then, 1 ml cell suspension was transferred to the 1.5 mL EP tubes after placing at ice for 5 min.
The first-strand cDNA was reverse-transcribed by All-in-One First-Strand Synthesis Master Mix reagent Kit (ShareBio, Shanghai, China).
The 10µL system of qPCR per well was defined as follows: 0.5µL Forward primer, 0.5µL Reverse primer, 5µL SYBR green qPCR Premix (ShareBio, Shanghai, China) and 4µL cDNA.
IC50
After that, Cell viability was measured by 10% CCK8 reagent (Share-Bio, shanghai, China, SB-CCK8L) at the incubator (5% CO2 at 37 °C) with 1h.
