Long noncoding RNAs HAND2-AS1 ultrasound microbubbles suppress hepatocellular carcinoma progression by regulating the miR-873-5p/tissue inhibitor of matrix metalloproteinase-2 axis

Abstract

Background: Increasing data indicated that long noncoding RNAs (lncRNAs) were directly or indirectly involved in the occurrence and development of tumors, including hepatocellular carcinoma (HCC). Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues, but its role in HCC progression is unclear. Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.

Aim: To study the role of ultrasound microbubbles (UTMBs) mediated HAND2-AS1 in the progression of HCC, in order to provide a new reference for the treatment of HCC.

Methods: In vitro, we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs, and detected cell proliferation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) by cell counting kit-8 assay, flow cytometry, Transwell invasion assay and Western blotting, respectively. In addition, we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior. Next, the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2 (TIMP2) overexpression vector, and we detected cell proliferation, apoptosis, invasion and EMT. In vivo, we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.

Results: We found that UTMBs carrying HAND2-AS1 restricted cell proliferation, invasion, and EMT, encouraged apoptosis, and HAND2-AS1 silencing eliminated the effect of UTMBs. Additionally, miR-873-5p targets the gene HAND2-AS1, which also targets the 3'UTR of TIMP2. And miR-873-5p mimic counteracted the impact of HAND2-AS1. Further, miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs. We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase (MMP) 2/MMP9. In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.

Conclusion: LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.

Keywords: Hepatocellular carcinoma; Long noncoding RNA HAND2-AS1; Tissue inhibitor of matrix metalloproteinase-2; Ultrasound microbubbles; miR-873-5p.

Figure 1

HAND2-AS1 expression in hepatocellular carcinoma development. A and B: HAND2-AS1 expression levels during hepatocellular carcinoma development were analyzed using bioinformatics techniques online (http://gepia.cancer-pku.cn/index.html; https://tnmplot.com/analysis/); C: HAND2-AS1 expression in tumor and surrounding tissues (35 pairings); D: The phrase represents HAND2-AS1. ^aP < 0.05.

Figure 2

HepG2 cell response to ultrasound microbubbles. Two hundred microliters of ultrasound microbubbles was added to HepG2 cells alone or incubated with si-HAND2-AS1. A: HAND2-AS1 expression; B: The proliferation of cells; C: Apoptosis of cells; D: Invasion of the cell; E: Proteins linked to epithelial-mesenchymal transition are expressed. ^aP < 0.05. UTMBs: Ultrasound microbubbles.

Figure 3

HAND2-AS1 directly targets the miRNA miR-873-5p. A: The target sites of HAND2-AS1 and miR-873-5p were identified (StarBase 3.0; https://starbase.sysu.edu.cn/); B: The HAND2-AS1 sequence; C: The relative luciferase activity value; D: RNA pull-down was employed to examine the strength of the binding; E: Cellular expression of miR-873-5p; F: Tissue expression of miR-873-5p (35 pairings); G: Expression of miR-873-5p. ^aP < 0.05.

Figure 4

The impact of miR-873-5p on HepG2 cells. A: Expression of miR-873-5p; B: HAND2-AS1; C: Cell proliferation; D: Apoptosis of cells Cell invasion; E: Expression of the snail protein, vimentin, and E-cadherin. ^aP < 0.05. UTMBs: Ultrasound microbubbles.

Figure 5

MiR-873-5p targets the 3'UTR of tissue inhibitor of matrix metalloproteinase-2. A: The 3’UTR of tissue inhibitor of matrix metalloproteinase-2 (TIMP2) targeted by miR-873-5p; B: The TIMP2 sequence; C: The relative luciferase activity value; D: Expression of miR-873-5p; E and F: TIMP2 mRNA and protein expression. ^aP < 0.05. TIMP2: Tissue inhibitor of matrix metalloproteinase-2.

Figure 6

Ultrasound microbubbles affect HepG2 cells via the miR-873-5p/tissue inhibitor of matrix metalloproteinase-2 axis. A and B: The expression of the proteins tissue inhibitor of matrix metalloproteinase-2, matrix metalloproteinase (MMP)-2, and MMP9; C: The proliferation of cells; D: Apoptosis of cells; E: Cell invasion; F: The expression of proteins linked to epithelial-mesenchymal transition. ^aP < 0.05. UTMBs: Ultrasound microbubbles; TIMP2: Tissue inhibitor of matrix metalloproteinase-2.

Figure 7

Ultrasound microbubbles inhibit tumor progression in vivo. The ultrasound microbubbles group (mice received injections of HAND2-AS1 microbubbles; 200 g; 1 MHz), and the control group (mice received injections of 0.9% NaCl alone; 1 MHz). A: Illustrations of tumors; B: Size of the tumor; C: Tumor mass; D: Expression of miR-873-5p and HAND2-AS1; E: Illustrations from immunohistochemistry analysis; F: Expression of proteins linked to epithelial-mesenchymal transition. ^aP < 0.05. UTMBs: Ultrasound microbubbles; TIMP2: Tissue inhibitor of matrix metalloproteinase-2; MMP: Matrix metalloproteinase.