Objectives: To explore the effect of miR-92a-3p on the proliferation and metastasis of pancreatic cancer cells via targeting phosphatase and tension homolog deleted on chromosome ten (PTEN).
Methods: MiR-92a-3p expression and PTEN protein levels were quantified in a normal pancreatic cells (HPDE6-C7) and 5 pancreatic cancer cell lines (Panc-1, BxPC-3, AsPC-1,MIA Paca-2, and Capan-2) by real-time PCR and Western blotting, respectively. BxPC-3 and Panc-1 cells were selected for further experiment. After transfection of normal control (NC) mimics (NC mimics group), miR-92a-3p mimics (miR-92a-3p mimics group), NC inhibitor or miR-92a-3p inhibitor (NC inhibitor group or miR-92a-3p inhibitor group), the proliferation of BxPC-3 and Panc-1 cells was measured by cell counting kit-8 (CCK-8), and the migration of them was measured by Transwell assay, and the levels of PTEN protein were measured by Western blotting. In addition, wild-type PTEN 3'-UTR (wt-PTEN 3'UTR) and mutant-type PTEN 3'-UTR (mut-PTEN 3'UTR) luciferase reporter vectors were constructed and co-transfected with NC mimics, miR-92a-3p mimic, NC inhibitor or miR-92a-3p inhibitor into 293T tool cells, and then the dual luciferase reporter assay was performed to examine the regulative correlation between miR-92a-3p and PTEN. The BxPC-3 cells were divided into 4 groups: a NC inhibitor+si-NC group, a miR-92a-3p inhibitor+si-NC group, a NC inhibitor+si-PTEN group, and a miR-92a-3p inhibitor+si-PTEN group. The Panc-1 cells were also assigned into 4 groups: a NC mimics+NC group, a miR-92a-3p mimics+si-NC group, a NC mimics+ PTEN group, and a miR-92a-3p mimics+PTEN group. The proliferation of Panc-1 cells was measured by CCK-8; the cell migration was measured by Transwell assay, and the levels of PTEN protein were measured by Western blotting.
Results: The miR-92a-3p was highly expressed in pancreatic cancer cell lines (allP<0.01), while the PTEN protein levels were lower in pancreatic cancer cell lines (allP<0.05)compared with that in the HPDE6-C7 cells. Compared with the NC mimics group, the cell viability of BxPC-3 and Panc-1 cells were both increased in the miR-92a-3p mimics group (bothP<0.01); compared with the inhibitor group, the cell viability of BxPC-3 and Panc-1 cells were both decreased in the miR-92a-3p inhibitor group (bothP<0.01). Compared with the NC mimics group, the cell number of BxPC-3 and Panc-1 cells through micropores were increased in the miR-92a-3p mimics group (bothP<0.01); compared with the inhibitor group, the cell number of BxPC-3 and Panc-1 cells through micropores were decreased in the miR-92a-3p inhibitor group (bothP<0.01). Compared with NC mimics group, the activity of dual luciferaseof wt-PTEN3'-UTR was inhibited in the miR-92a-3p mimics group (P<0.01); compared with the NC inhibitor group, the activity of dual luciferase of wt-PTEN3'-UTR was promoted in the miR-92a-3p inhibitor group (P<0.01). Compared with the miR-92a-3p inhibitor+si-NC group, the suppressive effects of miR-92a-3p on the proliferation and metastasis of BxPC-3 cells was restored in the miR-92a-3p inhibitor+si-PTEN group; while compared with the miR-92a-3p mimics+NC group, the positive effects of miR-92a-3p overexpression on the proliferation and metastasis of Panc-1 cells was restored in the miR-92a-3p mimics+PTEN group.
Conclusions: The highly expressed miR-92a-3p in pancreatic cancer cells can decrease the protein levels of PTEN, thereby enhancing the proliferation and metastasis activity of pancreatic cancer cells.
目的: 探讨微小RNA-92a-3p(microRNA-92a-3p，miR-92a-3p)靶向第10号染色体缺失的磷酸酶及张力蛋白同源基因(phosphatase and tension homolog deleted on chromosome ten，PTEN)调控胰腺癌细胞增殖和转移的作用机制。方法: 采用Real-time PCR检测人正常胰腺导管上皮细胞株(HPDE6-C7)与胰腺癌细胞株(Panc-1，BxPC-3，AsPC-1，MIA Paca-2，Capan-2)中的miR-92a-3p表达；同时，采用蛋白质印迹法检测上述细胞中PTEN的表达。选取胰腺癌细胞株BxPC-3和Panc-1的细胞进行实验，分为转染对照模拟物组(NC mimics组)、miR-92a-3p模拟物组(miR-92a-3p mimics组)、对照拮抗剂组(NC inhibitor组)、miR-92a-3p拮抗剂(miR-92a-3p inhibitor组)，采用细胞计数试剂盒-8(cell counting kit-8，CCK-8)检测细胞增殖，Transwell小室检测细胞转移能力，蛋白质印迹法检测PTEN蛋白表达水平。将野生型PTEN的3’-非编码区(untranslated regions，UTR)(wt-PTEN 3’UTR)或突变体PTEN的3’-UTR(mut-PTEN 3’UTR)分别与NC mimics，miR-92a-3p mimics，NC inhibitor，miR-92a-3p inhibitor共转染293T细胞，采用双萤光素酶报告实验检测miR-92a-3p与PTEN的靶向结合关系。在BxPC-3细胞的回复实验中，实验又分NC inhibitor+si-NC组、miR-92a-3p inhibitor+si-NC组、NC inhibitor+si-PTEN组和miR-92a-3p inhibitor+si-PTEN组；在Panc-1细胞的回复实验中，实验又分为NC mimics+NC组、miR-92a-3p mimics+NC组、NC mimics+PTEN组和miR-92a-3p mimics+PTEN组，再分别采用CCK-8检测细胞增殖，Transwell小室检测细胞转移能力，蛋白质印迹法检测PTEN蛋白的表达。结果: 与HPDE6-C7细胞相比，miR-92a-3p在胰腺癌细胞株中均呈高表达(均P<0.01)，PTEN在胰腺癌细胞株中均呈低表达(均P<0.05)。与NC mimics组相比，miR-92a-3p mimics组BxPC-3和Panc-1细胞活力均升高(均P<0.01)；与NC inhibitor组相比，miR-92a-3p inhibitor组BxPC-3和Panc-1细胞活力均降低(均P<0.01)。与NC mimics组相比，miR-92a-3p mimics组穿过微孔的BxPC-3和Panc-1细胞数均增多(均P<0.01)；与NC inhibitor组相比，miR-92a-3p inhibitor组穿过微孔的BxPC-3和Panc-1细胞数均减少(均P<0.01)。与NC mimics组相比，miR-92a-3p mimics组的wt-PTEN 3'UTR荧光素酶活性被抑制(P<0.01)；与NC inhibitor组相比，miR-92a-3p inhibitor组的wt-PTEN 3'UTR荧光素酶活性被增强(P<0.01)。与miR-92a-3p inhibitor+si-NC组相比，miR-92a-3p inhibitor+si-PTEN组恢复了miR-92a-3p inhibitor对BxPC-3细胞增殖和转移的影响；与miR-92a-3p mimics+NC组相比，miR-92a-3p mimics+PTEN组恢复了miR-92a-3p mimics对Panc-1细胞增殖和转移的影响。结论: MiR-92a-3p可靶向抑制PTEN的表达，促进胰腺癌细胞的增殖和转移。.
Keywords: metastasis; miR-92a-3p; pancreatic cancer; phosphatase and tension homolog deleted on chromosome ten; proliferation.