MiR-21/RASA1 axis affects malignancy of colon cancer cells via RAS pathways

Abstract

Aim: To determine how the oncogene miR-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.

Methods: RAS p21 GTPase activating protein 1 (RASA1) protein expression in six colon cancer cell lines was assessed by Western blot. Colon cancer RKO cells were chosen for transfection because they are KRAS wild type colon cancer cells whose RASA1 expression is significantly decreased. RKO cells were transfected with vectors overexpressing or down-regulating either miR-21 or RASA1. Furthermore, a luciferase reporter assay was used to determine whether RASA1 is a gene target of miR-21. Then, changes in mRNA and protein levels of RASA1, RAS-GTP, and other components of the RAS signaling pathways were assessed in transfected RKO cells by real-time quantitative reverse transcription-polymerase chain reaction, Western blot and immunoprecipitation. Finally, cell proliferation, apoptosis, invasion, and tumor formation ability were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay, flow cytometry, transwell assay, and animal experiment, respectively.

Results: RASA1 protein levels were significantly decreased in RKO cells compared with the other 5 colon cancer cell lines, and RASA1 was confirmed as a target gene of miR-21. Interestingly, RASA1 mRNA and protein levels in pre-miR-21-LV (up-regulation of miR-21) cells were lower than those in anti-miR-21-LV (down-regulation of miR-21) cells (P < 0.05). In addition, pre-miR-21-LV or siRASA1 (down-regulation of RASA1) cells showed higher cell proliferation, reduced apoptosis, increased expression of RAS-GTP, p-AKT, Raf-1, KRAS, and p-ERK1/2, and higher invasion and tumor formation ability, compared with control, anti-miR-21-LV or pcDNA3.1-RASA1 (up-regulation of RASA1) cells (P < 0.05).

Conclusion: RASA1 is a target gene of miR-21, which promotes malignant behaviors of RKO cells through regulation of RASA1 expression.

Keywords: Colon cancer; RAS; RAS signaling pathways; RASA1; miR-21.

Figure 1

RAS p21 GTPase activating protein 1 expression in different colon cancer cell lines was detected by Western blot. RAS p21 GTPase activating protein 1 (RASA1) expression in RKO cells was the lowest among the cell lines studied.

Figure 2

Validation of transfection efficiency. A: The expression of miR-21 in cells with up/down-regulated miR-21 was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (n = 4, ^aP < 0.05 vs control; ^cP < 0.05 vs control); B: qRT-PCR was used to quantify the expression of RASA1 mRNA (n = 3,^aP < 0.05 vs control; ^cP < 0.05 vs control); C: Western blot detection of RASA1 protein levels; 1: Control; 2: pcDNA3.1-RASA1 NC; 3: pcDNA3.1-RASA1; 4: siRASA1 NC; 5: siRASA1. RASA1: RAS p21 GTPase activating protein 1; NC: Non-coding; LV: Lentivirus.

Figure 3

MiR-21 negatively regulates RAS p21 GTPase activating protein 1. A: Luciferase reporter gene assay showed lower RLU values in pre-miR-21-LV cells co-transfected with pGL3-promoter-LucifeRASe-RASA1 in comparison with cells co-transfected with pGL3-promoter-LucifeRASe (n = 3, ^bP < 0.01); B: qRT-PCR detection of RASA1 mRNA levels in cells with up/down-regulated miR-21 (n = 3, ^aP < 0.05 vs control; ^cP < 0.05 vs control); C: Western blot was used to assess RASA1 protein levels in cells with up/down-regulated miR-21. 1: anti-miR-21-LV; 2: anti-NC-LV; 3: control; 4: pre-miR-21-LV; 5: pre-NC-LV. RASA1: RAS p21 GTPase activating protein 1; NC: Non-coding; LV: Lentivirus.

Figure 4

MiR-21 and RAS p21 GTPase activating protein 1 regulate RAS-GTP activity. Immunoprecipitation was used to assess the change in RAS-GTP activity in cells with up/down-regulated miR-21 or RASA1. A: 1: Control; 2: anti-NC-LV; 3: anti-miR-21-LV; 4: pre-NC-LV; 5: pre-miR-21-LV; B: 1: Control; 2: RASA1NC; 3: pcDNA3.1-RASA1; 4: siRASA1 NC; 5: siRASA1.

Figure 5

Role of miR-21 and RAS p21 GTPase activating protein 1 in the regulation of RAS signaling pathways. Western blot was used to assess the regulatory effect of miR-21 and RASA1. 1: pcDNA3.1-RASA1; 2: RASA1 NC; 3: siRASA1; 4: siRASA1NC; 5: Control; 6: pre-miR-21-LV; 7: pre-NC-LV; 8: anti-miR-21-LV; 9: anti-NC-LV. RASA1: RAS p21 GTPase activating protein 1; NC: Non-coding; LV: Lentivirus.

Figure 6

MiR-21 and RAS p21 GTPase activating protein 1 regulate RKO cell proliferation. A: Cell proliferation was assessed by MTT after miR-21 up/down-regulation; B: Cell proliferation was assessed by MTT after RASA1 up/down-regulation. Down-regulation of miR-21 or up-regulation of RASA1 inhibited cell proliferation (n = 6). RASA1: RAS p21 GTPase activating protein 1; NC: Non-coding; LV: Lentivirus.

Figure 7

MiR-21 modulates apoptosis via RAS p21 GTPase activating protein 1 regulation. An annexin V-PE/7-AAD apoptosis detection kit was used to assess cell apoptosis in RKO cells in each group by flow cytometry. n = 3, ^aP < 0.05 vs pre-miR-21-LV; ^cP < 0.05 vs siRASA1. RASA1: RAS p21 GTPase activating protein 1; NC: Non-coding; LV: Lentivirus.

Figure 8

Role of miR-21 and RAS p21 GTPase activating protein 1 in cell invasion was detected by Transwell assays. Down-regulation of miR-21 or up-regulation of RASA1 inhibited cell invasion. n = 3, ^aP < 0.01 vs control. RASA1: RAS p21 GTPase activating protein 1; NC: Non-coding; LV: Lentivirus.

Figure 9

Oncogenicity of miR-21 and RAS p21 GTPase activating protein 1 in a nude mouse tumor transplantation model. A: The tumors were excised 30 d post-implantation; B: Tumor weights at 30 d post-implantation. ^aP < 0.05 vs control; n = 3, ^cP < 0.05 vs control; ^eP < 0.05 vs control; C: Tumor volumes at 30 d post-implantation. n = 3, ^gP, ⁱP, ^kP < 0.05 vs control. RASA1: RAS p21 GTPase activating protein 1; NC: Non-coding; LV: Lentivirus.