Ultrasound-Targeted Microbubble Destruction Enhances the Inhibitive Efficacy of miR-21 Silencing in HeLa Cells

Abstract

BACKGROUND Previous studies have shown that miR-21 upregulation is related to the aggressive development of cervical cancer. Ultrasound-targeted microbubble destruction (UTMD) is a method that increases the absorption of targeted genes or drugs by cells. We focus on the role of UTMD-mediated miR-21 transfection in HeLa cells, a cervical cancer cell line. MATERIAL AND METHODS The effects of different ultrasound intensities on the transfection efficiency of miR-21-enhanced green fluorescent protein (EGFP) and miR-21 inhibitor-EGFP plasmids were determined by flow cytometry. The effects of UTMD-mediated miR-21 transfection on HeLa cell proliferation, apoptosis, migration, and invasion were measured by CCK-8, flow cytometry, wound healing experiments, and transwell migration assay, respectively. Western blot and real-time quantitative PCR were used to detect the expression of tumor-related genes. RESULTS When the ultrasound intensity was 1.5 W/cm², the miR-21 plasmid had the highest transfection efficiency. Exogenous miR-21 promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis in HeLa cells. Treatment of cells with UTMD further enhanced the effects of miR-21-EGFP and miR-21 inhibitor-EGFP. In addition, miR-21 overexpression significantly increased the expression of p-Akt, Akt, Bcl-2, Wnt, ß-catenin, matrix metalloprotein-9 (MMP-9), and epidermal growth factor (EGFR) levels, and decreased Bax expression. The regulatory role of miR-21 inhibitor-EGFP was opposite to that of miR-21-EGFP. After UTMD, miR-21-EGFP and miR-21 inhibitor-EGFP had more significant regulatory effects on these genes. CONCLUSIONS Our research revealed that an ultrasound intensity of 1.5 W/cm² is the best parameter for miR-21 transfection. UTMD can enhance the biological function of miR-21 in HeLa cells, and alter the effect of miR-21 on apoptosis, metastasis, and phosphorylation genes.

Figure 1

UTMD-mediated plasmid transfection of HeLa cells. (A) The influence of ultrasound intensity of 0.5 W/cm², 1.5 W/cm², and 2.5 W/cm² on the transfection rate of the miR-21-EGFP plasmid was determined by flow cytometry. (B) The effect of ultrasound intensity of 0.5 W/cm², 1.5 W/cm², and 2.5 W/cm² on the transfection efficiency of miR-21 inhibitor-EGFP plasmid was determined by flow cytometry. The microbubble concentration was 300 μL/mL, the plasmid concentration was 15 μg/mL, the ultrasound duration was 45 s, the ultrasound frequency was 1 MHz, and the load ratio was 20%. UTMD – ultrasound-targeted microbubble destruction; EGFP – enhanced green fluorescent protein.

Figure 2

Effects of UTMD-mediated miR-21 transfection on HeLa cell viability and apoptosis. (A) RT-qPCR was used to determine the expression of miR-21 in the untreated, mimic-EGFP, mimic-EGFP+UTMD, inhibitor-EGFP, and inhibitor-EGFP+UTMD groups. U6 was used as an internal reference. The ultrasonic intensity was 1.5 W/cm². (B) CCK-8 was used to detect the effect of miR-21 plasmid on the survival rate of HeLa cells (ultrasonic intensity was 1.5 W/cm²). (C) The effect of miR-21 plasmid on the apoptosis rate of HeLa cells was selected by flow cytometry (ultrasonic intensity was 1.5 W/cm²). * P<0.05 and *** P<0.001 vs untreated; ^@ P<0.05 and ^@@@ P<0.001 vs mimic+EGFP; ^## P<0.01 and ^### P<0.001 vs inhibitor+EGFP. UTMD – ultrasound-targeted microbubble destruction; RT-qPCR – reverse transcription quantitative polymerase chain reaction; EGFP – enhanced green fluorescent protein.

Figure 3

Effects of UTMD-mediated miR-21 transfection on HeLa cell migration and invasion. (A) Wound healing experiments were performed to detect the effects of miR-21 plasmid on HeLa cell migration in the untreated, mimic-EGFP, mimic-EGFP+UTMD, inhibitor-EGFP, and inhibitor-EGFP+UTMD groups (ultrasonic intensity was 1.5 W/cm²). (B) The transwell migration assay was used to detect the effect of the miR-21 plasmid on HeLa cell invasion (ultrasonic intensity was 1.5 W/cm²). UTMD: Ultrasound-targeted microbubble destruction; EGFP: enhanced green fluorescent protein. * P<0.05 and ** P<0.01 vs untreated; ^@ P<0.05 and ^@@ P<0.01 vs mimic+EGFP; ^# P<0.05 and ^### P<0.001 vs inhibitor+EGFP. UTMD – ultrasound-targeted microbubble destruction; EGFP – enhanced green fluorescent protein.

Figure 4

Effects of UTMD-mediated miR-21 transfection on apoptosis, metastasis, and Wnt/Akt pathway-related genes. (A) Western blot was used to detect the effects of the miR-21 plasmid on phosphorylated (p)-Akt, Akt, Bax, Bcl-2, Wnt, β-catenin, MMP-9, and EGFR expression in HeLa cells; β-actin was an internal reference (ultrasonic intensity of 1.5 W/cm²). (B) RT-qPCR was used to detect the effects of miR-21 plasmid on Akt, Bax, Bcl-2, Wnt, β-catenin, MMP-9, and EGFR expression in HeLa cells; β-actin was an internal reference (ultrasonic intensity of 1.5 W/cm²). * P<0.05, ** P<0.01, and *** P<0.001 vs untreated; ^@ P<0.05, ^@@ P<0.01 and ^@@@ P<0.001 vs mimic+EGFP; ^## P<0.01 and ^### P<0.001 vs inhibitor+EGFP. UTMD – ultrasound-targeted microbubble destruction; RT-qPCR – reverse transcription quantitative polymerase chain reaction; EGFR – epidermal growth factor receptor; EGFP – enhanced green fluorescent protein.