Effect of miR-511-3p targeting ATP2A2 to regulate endoplasmic reticulum stress on proliferation and apoptosis of lung cancer cells
10.3760/cma.j.cn115355-20230731-00030
- VernacularTitle:miR-511-3p靶向ATP2A2调控内质网应激对肺癌细胞增殖和凋亡的影响
- Author:
Jixing ZHAO
1
;
Wencong HUANG
;
Wu YAN
;
Yongsheng LI
Author Information
1. 惠州市中心人民医院胸外科，惠州 516001
- Keywords:
Lung neoplasms;
MicroRNAs;
Endoplasmic reticulum stress;
Sarcoplasmic reticulum calcium-transporting ATPases;
Apoptosis;
Cell proliferation
- From:
Cancer Research and Clinic
2024;36(6):421-428
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effect of miRNA-511-3p (miR-511-3p) on the proliferation and apoptosis of lung cancer cells, and the possible role of ATP2A2 and endoplasmic reticulum stress therein.Methods:Lung cancer tissues and paracancerous normal tissues (>2 cm from the tumor) were retrospectively collected from 69 lung cancer patients who were admitted to Huizhou Central People's Hospital from January 2020 to March 2022. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the transcript-level relative expression of miR-511-3p in cancer and paracancerous tissues, as well as immortalized lung epithelial cell line BEAS-2B, human lung cell line CCD-19Lu, and human lung carcinoma cell lines A549, H1975 and H1299. The lung cancer cell line A549 with the lowest relative expression level of miR-511-3p was selected for subsequent experiments. The cells were divided into miR control group (transfected with miR-511-3p irrelevant sequence), miR-511-3p overexpression group (transfected with miR-511-3p mimic), and miR-511-3p knockdown group (transfected with miR-511-3p repressor); and the additional A549 cells were taken and divided into ATP2A2 overexpression control group (transfected with its empty plasmid), ATP2A2 overexpression group (transfected with ATP2A2 overexpression plasmid), and ATP2A2 knockdown control group (transfected with irrelevant small interfering RNA plasmid) and ATP2A2 knockdown group (transfected with ATP2A2 small interfering RNA plasmid). The transcript-level relative expression of miR-511-3p and ATP2A2 in A549 cells in each group was detected by qRT-PCR; the relative expressions of ATP2A2 protein and endoplasmic reticulum stress marker proteins in each group were detected by Western blotting; the targeting relationship between miR-511-3p and ATP2A2 mRNA was verified by dual-luciferase reporter gene assay; CCK-8 assay was used to detect the proliferation ability of A549 cells in each group (expressed as absorbance value); flow cytometry was used to detect the percentage of apoptotic cells in A549 cells in each group; inorganic phosphorus colorimetry was used to detect the activity of Ca 2+-ATPase in A549 cells in each group; fluorescent probe assay was used to detect the Ca 2+ concentration in A549 cells in each group. Results:The transcript-level relative expression of miR-511-3p in lung cancer tissues and paracancerous tissues of 69 patients were 0.08±0.03 and 0.17±0.12, respectively, and the difference was statistically significant ( t= 6.04, P<0.05). The percentage of apoptotic cells in A549 cells in the miR-511-3p overexpression group, miR-511-3p knockdown group and miR control group was (58.1±6.1)%, (11.0±1.3)% and (22.0±2.1)%, respectively. The percentage of apoptotic cells in the miR-511-3p overexpression group was higher than that in the miR control group, and the percentage of apoptotic cells in the miR-511-3p knockdown group was lower than that in the control group, and the differences were statistically significant ( t values were 9.70 and 7.64, respectively, both P<0.05). After 24, 48 and 72 h of culture, the proliferation ability of A549 cells in the miR-511-3p overexpression group was lower than that in the miR control group, the proliferation ability of A549 cells in the miR-511-3p knockdown group was higher than that in the miR control group, and the differences were statistically significant (all P < 0.05). The percentage of apoptotic cells in A549 cells of ATP2A2 knockdown group and ATP2A2 knockdown control group were (58.2±1.5)% and (23.8±1.0)%, respectively, and the difference was statistically significant ( t= 33.94, P < 0.05); the percentage of apoptotic cells in A549 cells of ATP2A2 overexpression group and ATP2A2 overexpression control group were (13.8±2.0)% and (23.8±1.0)%, respectively, and the difference was statistically significant ( t= 7.96, P < 0.05). The transcript-level relative expression of ATP2A2 in A549 cells of miR-511-3p overexpression group was lower than that of miR control group, the transcript-level relative expression of ATP2A2 in A549 cells of miR-511-3p knockdown group was higher than that of control group, and the differences were statistically significant ( t values were 5.76 and 6.40, respectively, both P < 0.05); the relative expression of ATP2A2 protein in A549 cells of miR-511-3p overexpression group was lower than that of its control group, and the relative expression of ATP2A2 protein in A549 cells of miR-511-3p knockdown group was higher than that of its control group, and the differences were statistically significant (both P < 0.05). Dual-luciferase reporter gene assay verified the targeting relationship between miR-511-3p and ATP2A2 mRNA. The percentage of apoptotic cells in A549 cells in the control group, miR-511-3p overexpression group, ATP2A2 overexpression group, and miR - 511 - 3p overexpression+ ATP2A2 overexpression group were (21.5±3.0)%, (58.1±5.0)%, (13.3±1.2)%, and (20.5±4.0)%, respectively, and the difference was statistically significant (F= 73.28, P < 0.001); the percentage of apoptotic cells in A549 cells in the control group, miR-511-3p knockdown group, ATP2A2 knockdown group, and miR-511-3p knockdown+ ATP2A2 knockdown group were (23.5±3.0)%, (11.3±1.2)%, (60.1±7.0)%, and (25.6±5.0)%, respectively, and the difference was statistically significant ( F= 78.45, P < 0.001). The Ca 2+-ATPase activity in A549 cells of ATP2A2 overexpression group was higher than that of its control group, the Ca 2+-ATPase activity in A549 cells of ATP2A2 knockdown group was lower than that of its control group, and the differences were statistically significant ( t values were 4.61 and 6.07, respectively, both P < 0.05); the intracellular Ca 2+ concentration in A549 cells of ATP2A2 overexpression group was lower than that of its control group, the intracellular Ca 2+ concentration in A549 cells of ATP2A2 knockdown group was higher than that of its control group, and the differences were statistically significant ( t values were 3.30 and 3.95, respectively, both P < 0.05). The Ca 2+-ATPase activity in the miR-511-3p overexpression group was lower than that in the miR control group, the Ca 2+-ATPase activity in the miR- 511-3p knockdown group was higher than that in the miR control group, and the differences were statistically significant ( t values were 6.54 and 4.16, respectively, both P < 0.05); the intracellular Ca 2+ concentration in A549 cells of miR-511-3p overexpression group was higher than that of miR control group, the intracellular Ca 2+ concentration in A549 cells of miR - 511 - 3p knockdown group was lower than that of miR control group, and the differences were statistically significant ( t values were 3.60 and 6.23, respectively, both P < 0.05). The relative expressions of endoplasmic reticulum stress markers GRP78, PERK, p - eIF2a, ATF4, and CHOP proteins in A549 cells with knockdown of ATP2A2 or overexpression of miR-511-3p were higher than those in the corresponding control groups, and the differences were statistically significant (all P < 0.05); the relative expressions of all proteins in A549 cells with overexpression of ATP2A2 or knockdown of miR-511-3p were lower than those in the corresponding control groups (all P < 0.05). Conclusions:Changes in miR-511-3p level may affect the proliferation and apoptosis of lung cancer cells, and the mechanism may be that it affects the apoptosis of lung cancer cells by targeting ATP2A2 to regulate the endoplasmic reticulum stress.
