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

Effect of miR-511-3p targeting ATP2A2 to regulate endoplasmic reticulum stress on proliferation and apoptosis of lung cancer cells

目的探讨miRNA-511-3p(miR-511-3p)对肺癌细胞增殖和凋亡的影响,及ATP2A2、内质网应激在其中的可能作用。方法回顾性收集2020年1月至2022年3月惠州市中心人民医院69例肺癌患者的肺癌组织和癌旁正常组织(距离肿瘤>2 cm)。采用实时荧光定量聚合酶链反应(qRT-PCR)法检测癌和癌旁组织以及永生化肺上皮细胞株BEAS-2B、人肺细胞株CCD-19Lu、人肺癌细胞株A549、H1975、H1299中miR-511-3p转录水平相对表达量。选择miR-511-3p相对表达水平最低的肺癌细胞株A549进行后续实验。将细胞分为miR对照组(转染miR-511-3p无关序列)、miR-511-3p过表达组(转染miR-511-3p模拟物)、miR-511-3p敲低组(转染miR-511-3p抑制物);另取A549细胞分为ATP2A2过表达对照组(转染空载质粒)、ATP2A2过表达组(转染ATP2A2过表达质粒),以及ATP2A2敲低对照组(转染载有小干扰RNA无关序列的质粒)和ATP2A2敲低组(转染载有ATP2A2小干扰RNA序列的质粒)。采用qRT-PCR法检测各组A549细胞miR-511-3p和ATP2A2转录水平相对表达量;蛋白质印迹法检测各组A549细胞ATP2A2蛋白及内质网应激标志蛋白的相对表达量;双荧光素酶报告基因实验验证miR-511-3p与ATP2A2 mRNA的靶向关系;CCK-8法检测各组A549细胞增殖能力(以吸光度值表示);流式细胞术检测各组A549细胞中凋亡细胞占比;无机磷比色法检测各组A549细胞Ca^(2+)-ATP酶活性;荧光探针法检测各组A549细胞Ca^(2+)浓度。结果69例患者肺癌组织和癌旁组织miR-511-3p转录水平相对表达量分别为0.08±0.03、0.17±0.12,差异有统计学意义(t=6.04,P<0.05)。miR-511-3p过表达组、miR-511-3p敲低组、miR对照组A549细胞中凋亡细胞占比分别为(58.1±6.1)%、(11.0±1.3)%、(22.0±2.1)%,miR-511-3p过表达组较miR对照组高,miR-511-3p敲低组较对照组低,差异均有统计学意义(t值分别为9.70、7.64,均P<0.05)。培养24、48、72 h后,miR-511-3p过表达组A549细胞增殖能力均较miR对照组低,miR-511-3p敲低组均较miR对照组高,差异均有统计学意义(均P<0.05)。ATP2A2敲低组、ATP2A2敲低对照组A549细胞中凋亡细胞占比分别为(58.2±1.5)%、(23.8±1.0)%,差异有统计学意义(t=33.94,P<0.05);ATP2A2过表达组、ATP2A2过表达对照组A549细胞中凋亡细胞占比分别为(13.8±2.0)%、(23.8±1.0)%,差异有统计学意义(t=7.96,均P<0.05)。miR-511-3p过表达组、miR-511-3p敲低组、miR对照组A549细胞ATP2A2转录水平相对表达量分别为0.11±0.01、1.34±0.19、0.51±0.12,miR-511-3p过表达组较miR对照组低,miR-511-3p敲低组较对照组高,差异均有统计学意义(t值分别为5.76、6.40,均P<0.05);miR-511-3p过表达组A549细胞ATP2A2蛋白相对表达量较其对照组低,miR-511-3p敲低组较其对照组高,差异均有统计学意义(均P<0.05)。双荧光素酶报告基因实验验证miR-511-3p与ATP2A2 mRNA存在靶向关系。对照组、miR-511-3p过表达组、ATP2A2过表达组、miR-511-3p过表达+ATP2A2过表达组A549细胞中凋亡细胞占比分别为(21.5±3.0)%、(58.1±5.0)%、(13.3±1.2)%、(20.5±4.0)%,差异有统计学意义(F=73.28,P<0.001);对照组、miR-511-3p敲低组、ATP2A2敲低组、miR-511-3p敲低+ATP2A2敲低组A549细胞中凋亡细胞占比分别为(23.5±3.0)%、(11.3±1.2)%、(60.1±7.0)%、(25.6±5.0)%,差异有统计学意义(F=78.45,P<0.001)。ATP2A2过表达组A549细胞Ca^(2+)-ATP酶活性高于对照组,ATP2A2敲低组低于对照组,差异均有统计学意义(t值分别为4.61、6.07,均P<0.05);ATP2A2过表达组A549细胞内Ca^(2+)浓度低于对照组,ATP2A2敲低组高于对照组,差异均有统计学意义(t值分别为3.30、3.95,均P<0.05)。miR-511-3p过表达组A549细胞Ca^(2+)-ATP酶活性低于miR对照组,miR-511-3p敲低组高于miR对照组,差异均有统计学意义(t值分别为6.54、4.16,均P<0.05);miR-511-3p过表达组A549细胞内Ca^(2+)浓度高于miR对照组,miR-511-3p敲低组低于miR对照组,差异均有统计学意义(t值分别为3.60、6.23,均P<0.05)。敲低ATP2A2或过表达miR-511-3p的A549细胞内质网应激标志GRP78、PERK、p-eIF2a、ATF4、CHOP蛋白相对表达量均较相应对照组高,差异均有统计学意义(均P<0.05);过表达ATP2A2或敲低miR-511-3p的A549细胞各蛋白相对表达量均较相应对照组低,差异均有统计学意义(均P<0.05)。结论miR-511-3p水平变化可能影响肺癌细胞的增殖和凋亡,机制可能是其靶向ATP2A2进而调控内质网应激而影响肺癌细胞凋亡。

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,miR-511-3p knockdown group and miR control group were 0.11±0.01,1.34±0.19 and 0.51±0.12,respectively.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.