miR-324-3p promotes gastric cancer development by activating Smad4-mediated Wnt/beta-catenin signaling pathway
miR-324-3p promotes gastric cancer development by activating Smad4-mediated Wnt/beta-catenin signaling pathway
Guang-Li Sun 0 1 2 3
Zheng Li 0 1 2 3
Wei-Zhi Wang 0 1 2 3
Zheng Chen 0 1 2 3
Lei Zhang 0 1 2 3
Qing Li 0 1 2 3
Song Wei 0 1 2 3
Bo-Wen Li 0 1 2 3
Jiang-Hao Xu 0 1 2 3
Liang Chen 0 1 2 3
Zhong-Yuan He 0 1 2 3
Kai Ying 0 1 2 3
Xuan Zhang 0 1 2 3
Hao Xu 0 1 2 3
Dian-Cai Zhang 0 1 2 3
Ze-Kuan Xu 0 1 2 3
0 Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University , No. 300 Guangzhou road, Nanjing, Jiangsu , China
1 & Ze-Kuan Xu
2 Guang-Li Sun , Zheng Li, Wei-Zhi Wang, and Zheng Chen
3 Department of Surgery, Vanderbilt University Medical Center , Nashville, TN , USA
Background Emerging evidence suggested that miRNAs can function as oncogenes or tumor suppressors by regulating downstream target genes. miR-324-3p has been reported to function in several carcinomas, but its role in gastric cancer (GC) is still unknown. This study aims to explore the effects of miR-324-3p on the development of GC. Methods Expression of miR-324-3p was examined in GC cells and tissues by qRT-PCR. Effects of miR-324-3p on GC cells were evaluated by cell vitality assay, colony formation assay, cell migration assay, and flow cytometric assay. The dual luciferase assay was used to verify whether miR-324-3p could interact with the potential target genes. Western blot was used to assess the expression level of Smad4 and beta-catenin. Intracellular ATP level was also examined. The tumor xenografts were established using nude mice. A gastric organoid model was made from fresh stomach tissue. Results miR-324-3p was expressed at higher levels in the tumor tissues compared with adjacent normal tissues. Overexpression of miR-324-3p promoted cell growth, migration, and decreased apoptosis. miR-324-3p repressed the expression of Smad4, and loss of Smad4 activated the Wnt/beta-catenin signaling pathway. Overexpression of Smad4 rescued the effects of miR-324-3p on GC cells. The intracellular ATP level was upregulated with overexpression of miR-324-3p. miR-324-3p facilitated tumor cell colonization and growth in vivo and contributed to the growth of gastric organoids. Conclusions The results suggested that miR-324-3p promoted GC through activating the Smad4-mediated Wnt/beta-catenin signaling pathway. The miR-324-3p/ Smad4/Wnt signaling axis may be a potential therapeutic target to prevent GC progression.
Gastric cancer; miR-324-3p; Smad4; Organoid
Abbreviations
AJCC American Joint Committee on Cancer
cck-8 Cell counting kit-8
DAB 3,30-Diaminobenzidine
FBS Fetal bovine serum
GC Gastric cancer
miRNA micro-RNA
PBS Phosphate buffered solution
PCR Polymerase chain reaction
PVDF Polyvinylidene fluoride
TNM Tumor node metastasis
TUNEL Terminal deoxynucleotidyl
mediated dUTP nick end labeling
30-UTR 30-Untranslational region
Introduction
Gastric cancer (GC) is the fourth most common carcinoma
in men and the fifth in women and is the second leading
cause of cancer-related death [
1
]. Overall, 43% of the GC
patients are in China [
2
]. Most of the patients with gastric
cancer are diagnosed at an advanced stage and they have a
poor prognosis with low 5-year survival rate [
3
]. One of the
reasons is the lack of effective early diagnostic biomarkers.
It is necessary to study the molecular mechanism of gastric
cancer to determine biomarkers for early diagnosis and
novel targets for more effective therapy.
Increasing evidence demonstrates that micro-RNAs
(miRNAs) act either as oncogenes or as tumor suppressors
in the development and progression of tumors [
4
]. miRNAs
are small, non-coding RNAs that bind to the
30-untranslational regions (30-UTRs) of target mRNAs [
5, 6
]. The
target genes usually play a critical role in controlling
cancer-related cellular processes such as proliferation,
apoptosis, migration, differentiation, and cell cycle
progression [
7–9
].
It has been reported that miR-324-3p was significantly
upregulated in plasma of stage I lung squamous cell
carcinoma compared to healthy controls [
10
]. Plasma
miR324-3p level was significantly increased in hepatocellular
carcinoma, so it might act as an early biomarker for
hepatocellular carcinoma [
11
]. Previous studies have shown
that miR-324-3p acted as a tumor suppressor in
nasopharyngeal carcinoma [
12
]. The effect of miR-324-3p on
cancer is still uncertain and the relationship between
miR324-3p and GC remains blank. Whether miR-324-3p could
regulate the biological functions of GC cells and the
mechanism needs to be explored.
It has been reported that Smad4 was inactivated in
different types of carcinomas and acted as a tumor suppressor
in GC [
13, 14
]. Smad4 has been confirmed to suppress
Wnt/beta-catenin signaling activity in colon carcinoma
[15]. The Wnt/beta-catenin signaling pathway is a highly
conserved system during evolution [
16
]. It has been
reported to regulate various processes that are important for
cancer pr (...truncated)