超声微泡介导miR-1290通过调节DKK3表达对卵巢癌细胞增殖、凋亡、侵袭的影响

Effects of miR-1290 mediated by ultrasound microbubbles on proliferation,apoptosis and invasion of ovarian cancer cells by regulating the expression of DKK3

目的探讨超声微泡介导miR-1290通过调节DKK3表达对卵巢癌细胞增殖、凋亡、侵袭的作用机制。方法将对数期SKOV3细胞分为对照组(Control)、miR-1290 NC组、微泡处理(MB)组、miR-1290 inhibitor组、miR-1290 inhibitor-MB组。双荧光素酶实验验证miR-1290与DKK3的靶向关系;RT-PCR检测SKOV3细胞中miR-1290与DKK3 mRNA的表达;MTT法检测SKOV3细胞活性;流式细胞仪检测SKOV3细胞凋亡情况;细胞划痕实验和Transwell实验检测SKOV3细胞迁移侵袭能力;Western blot检测蛋白表达。结果双荧光素酶实验证明,miR-1290与DKK3有靶向关系,miR-1290可负靶向调控DKK3;与miR-1290 NC组[24、48、72 h:(0.53±0.05)、(0.82±0.06)、(1.24±0.06)]比较,MB组[24、48、72 h:(0.43±0.06)、(0.71±0.03)、(1.03±0.03)]、miR-1290 inhibitor组[24、48、72 h:(0.41±0.03)、(0.66±0.04)、(0.78±0.05)]、miR-1290 inhibitor-MB组[24、48、72 h:(0.33±0.04)、(0.54±0.05)、(0.67±0.06)]SKOV3细胞增殖活性明显下降(P<0.05)。与miR-1290 NC组SKOV3细胞迁移率、侵袭数目[(45.98±4.11)%、(235.14±5.78)个]比较,MB组[(36.77±4.24)%、(189.57±4.58)个]、miR-1290 inhibitor组[(32.14±3.78)%、(165.35±5.01)个]、miR-1290 inhibitor-MB组SKOV3细胞迁移率、侵袭数目[(20.40±3.01)%、(86.21±4.23)个]明显下降,SKOV3细胞中DKK3 mRNA及蛋白表达、细胞凋亡率、促凋亡蛋白C-Caspase-3、Bax表达显著上升(P<0.05);miR-1209 NC组与Control组SKOV3细胞上述指标无显著差异(P>0.05)。结论miR-1290可负向调DKK3表达,而超声微泡可下调miR-1290表达,上调DKK3表达,从而抑制卵巢癌SKOV3细胞增殖和迁移侵袭,促进癌细胞凋亡。

Objective To investigate the mechanism of miR-1290 mediated by ultrasound microbubbles on the proliferation,apoptosis and invasion of ovarian cancer cells by regulating the expression of DKK3.Methods Logarithmic SKOV3 cells were divided into Control group,miR-1290 NC group,microbubble treatment(MB)group,miR-1290 inhibitor group and miR-1290 inhibitor-MB group.The targeting relationship between miR-1290 and DKK3 was verified by double luciferase assay;RT-PCR was used to detect the expression of miR-1290 and DKK3 mRNA in SKOV3 cells;the activity of SKOV3 cells was detected by MTT assay;the apoptosis of SKOV3 cells was detected by flow cytometry;cell scratch test and Transwell test were used to detect the migration and invasion abilities of SKOV3 cells;Western blot was used to detect the expression of protein.Results The double luciferase experiment showed that miR-1290 had a targeting relationship with DKK3,and miR-1290 could negatively target DKK3;Compared with miR-1290 NC group[24,48,72 h:(0.53±0.05),(0.82±0.06),(1.24±0.06)],MB group[24,48,72 h:(0.43±0.06),(0.71±0.03),(1.03±0.03)],miR-1290 inhibitor group[24,48,72 h:(0.41±0.03),(0.66±0.04),(0.78±0.05)],miR-1290 inhibitor MB group[24,48,72 h:(0.33±0.04),(0.54±0.05),(0.67±0.06)]SKOV3 cell proliferation activity decreased significantly(P<0.05),compared with the migration rate and invasion number of SKOV3 cells in miR-1290 NC group[(45.98±4.11)%,(235.14±5.78)],the migration rate and invasion number of SKOV3 cells in MB group[(36.77±4.24)%,(189.57±4.58)],miR-1290 inhibitor group[(32.14±3.78)%,(165.35±5.01)],and miR-1290 inhibitor MB group[(20.40±3.01)%,(86.21±4.23)]decreased significantly,the expression of DKK3 mRNA and protein,apoptosis rate,apoptosis promoting protein C-Caspase-3 and Bax in SKOV3 cells increased greatly(P<0.05);the above indexes of SKOV3 cells in miR-1209 NC group and Control group had no great difference(P>0.05).Conclusion MiR-1290 can negatively regulate the expression of DKK3,while ultrasound microbubble can down regulate the expression of miR-1290 and up regulate the expression of DKK3,thereby inhibiting the proliferation,migration and invasion of ovarian cancer SKOV3 cells and promoting the apoptosis of cancer cells.