The lnc‐CITED2‐2:1 inhibits metastasis via inhibiting CITED2 and epithelial‐mesenchymal transition in gallbladder cancer

摘要

Dear Editor Up to now, the mechanism of gallbladder cancer (GBC) is unclear. In this study, the prognostic and functional role of lnc‐CITED2‐2:1 in GBC has been investigated, which suggested that lnc‐CITED2‐2:1 might regulate GBC cell invasion via suppressing the expression of CITED2 and the EMT process. GBC, the most common malignancy of the biliary tract, is an uncommon but highly lethal malignant tumor. ~(1) Despite the surgery, effective treatment for the advanced GBC patients is still lacking, ~(2) , ~(3) which results in a dismal prognosis. Besides, the underlying mechanism of the initiation of GBC remains unknown. Herein, figuring out the mechanism of disease progression of GBC to develop a potential therapy target is essential to improve the treatment for GBC patients. Currently, accumulating evidence supported that long non‐coding RNAs (lncRNAs) were essential for the development and progression of GBC. ~(4) , ~(5) , ~(6) For instance, it has been reported that lncRNA‐CCAT1 could competitively inhibit miRNA‐218‐5p, and promote the expression of Bmi1 to promote the progression of GBC. ~(6) In this study, the function and the underlying mechanism of lnc‐CITED2‐2:1 in GBC were investigated. Details of the method are listed in the Supporting Information Materials and Methods. Microarray analysis was initially conducted to identify the differently expressed lncRNAs in GBC (Figure? 1A ). Then, the top 10 downregulated lncRNAs with a fold change of?&2.0 in descending order were selected out and validated by using quantitative PCR, which suggested that lnc‐CITED2‐2:1 was the only significantly downregulated lncRNA in the GBC tissues ( P ?&?.01, Figure? 1B ). Besides, this lncRNA was found to be downregulated in another two lncRNA microarray datasets of GBC (GSE62335, P ?&?.01; GSE76633, P ?&?.01; Figure? 1C,D ). Therefore, lnc‐CITED2‐2:1 was chosen for further investigation. Details of this lncRNA are presented in Figure S1. FIGURE 1 The identification and the expression of the lnc‐CITED2‐2:1 in gallbladder cancer (GBC). A, Heatmap representing the expression level of the expression profiles of lncRNAs specific to GBC in GBC tissue and normal tissue. B, The top 10 downregulated and upregulated lncRNAs in gallbladder cancer, sorting by P‐value and fold change (FC) and the qPCR results of the top 10 downregulated long non‐coding RNA in GBC tissues and matched adjacent normal tissues. ~(*) P ?&?.05; ~(**) P ?&?.01; ~(***) P ?&?.001. C and D, Lnc‐CITED2‐2:1 transcript in GBC and corresponding non‐tumor tissues were analyzed in two GEO datasets (GSE62335 and GSE76633), which indicated that this long non‐coding RNA was downregulated in tumor tissues. β‐Actin was used as an internal control. ~(*) P ?&?.05; ~(**) P ?&?.01; ~(***) P ?&?.001. E, qPCR result for lnc‐CITED2‐2:1 in GBC and corresponding non‐tumor tissues, which indicated that this lncRNA was downregulated in GBC tissues. 18s was used as an internal control. ~(*) P ?&?.05; ~(**) P ?&?.01; ~(***) P ?&?.001. F, qPCR result suggested that lnc‐CITED2‐2:1 was downregulated in GBC patients with lymph node metastasis. 18s was used as an internal control. ~(*) P ?&?.05; ~(**) P ?&?.01; ~(***) P ?&?.001. G, The median of the lncRNA expression value was identified as the cut‐off value. Accordingly, patients were stratified into high and low groups. Patients with low expression of lnc‐CITED2‐2:1 were associated with poor prognosis ( P ?=?.044). H, The relative expression level of lnc‐CITED2‐2:1 in four GBC cell lines, including SGC‐996, GBC‐SD, OCUG, and NOZ Then, the mRNA expression level of lnc‐CITED2‐2:1 was examined in 69‐paired GBC tissues and non‐tumor tissues from different patients, and the clinical significance of this lncRNA was investigated. Decreasing expression level of lnc‐CITED2‐2:1 was observed in tumor tissue ( P ?&?.001, Figure? 1E ) and patients without lymph node metastasis ( P ?&?.001, Figure? 1F ). Besides, a low expression level of lnc‐CITED2‐2:1 indicated shorter overall survival ( P ?=?.044, Figure? 1G ). Furthermore, it was found that a high expression level of lnc‐CITED2‐2:1 positively correlated with a lower incidence of lymph node metastases ( P ?&?.001), smaller tumor size ( P ?&?.001), and early TNM stage ( P ?=?.020; Table? 1 ). TABLE 1 Correlations between tumor lnc‐CITED2‐2:1 expression and clinicopathological characteristics in GBC Characteristics Lnc‐CITED2‐2:1 Low (n?=?35) High (n?=?34) P ‐value Age, years &60 20 16 .402 &60 15 18 Gender Female 21 19 .729 Male 14 15 Diabetes mellitus No 18 20 .537 Yes 17 14 CA 19‐9 (U/mL) ≤37 19 15 .398 &37 16 19 Tumor size (cm) ≤2.5 15 25 &.001 &2.5 20 9 Lymph node metastasis No 10 28 &.001 Yes 25 6 Grade Well‐differentiated 5 10 .3082 Moderately differentiated 18 15 Poorly differentiated 12 9 TNM stage IIA+IIB 15