Basic Study
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. Apr 15, 2024; 16(4): 1547-1563
Published online Apr 15, 2024. doi: 10.4251/wjgo.v16.i4.1547
Long noncoding RNAs HAND2-AS1 ultrasound microbubbles suppress hepatocellular carcinoma progression by regulating the miR-873-5p/tissue inhibitor of matrix metalloproteinase-2 axis
Qiang Zou, Hao-Wen Wang, Xi-Liang Di, Yuan Li, Hui Gao
Qiang Zou, Department of Interventional Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China
Qiang Zou, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
Hao-Wen Wang, College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China
Xi-Liang Di, Yuan Li, Department of Hematology and Oncology, Linyi People’s Hospital, Linyi 251500, Shandong Province, China
Hui Gao, Department of Comprehensive Oncology, Baotou Cancer Hospital, Baotou 014030, Inner Mongolia Autonomous Region, China
Co-first authors: Qiang Zou and Hao-Wen Wang.
Author contributions: Di XL, Li Y were responsible for conducting the experiments; Di XL, Li Y, Gao H were responsible for data analysis; Gao H were responsible for writing and revising the manuscript. All authors read and approved the final manuscript. The reasons for designating Zou Q and Wang HW as the co-first authors are that they made crucial and indispensable contributions towards the completion of the project, played important and indispensable roles in the experimental design, data interpretation and ensuring effective communication post submission. Zou Q proposed, designed, and conducted analysis, performed data analysis, and prepared the first draft of the manuscript. Wang HW was responsible for patient screening, enrollment, collection of clinical data and revision of the manuscript. Further, the overall research team all plays important contributions to complete the study and the resultant paper. Zou Q and Wang HW as co-first authors of is fitting for our manuscript as it accurately reflects our team's collaborative spirit, contributions, and diversity.
Institutional review board statement: The study was reviewed and approved by the Tianjin Medical University Cancer Institute and Hospital ethics committee approved this investigation.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Tianjin Medical University Cancer Institute and Hospital.
Conflict-of-interest statement: All other authors have nothing to disclose.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Hui Gao, Department of Comprehensive Oncology, Baotou Cancer Hospital, No. 18 Tuanjie Street, Baotou 014030, Inner Mongolia Autonomous Region, China. gaohui145@163.com
Received: December 8, 2023
Peer-review started: December 8, 2023
First decision: December 22, 2023
Revised: January 8, 2024
Accepted: February 7, 2024
Article in press: February 7, 2024
Published online: April 15, 2024
ARTICLE HIGHLIGHTS
Research background

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence and poor prognosis. Studies have confirmed that long noncoding RNAs (lncRNAs) are directly or indirectly involved in the occurrence and development of tumors and the regulation of various biological functions, including HCC, in which the expression of lncRNA HAND2-AS1 is downregulated in HCC tissues, but the specific mechanism of its involvement in HCC progression still needs to be further explored. In addition, ultrasound targeted microbubble destruction mediated gene transfection is a promising new method in recent years. Therefore, studying the role of ultrasound microbubbles (UTMBs) mediated HAND2-AS1 in HCC progression can provide a new reference for the treatment of HCC.

Research motivation

lncRNA HAND2-AS1 expression was downregulated in HCC tissues and cells, which may be involved in tumor progression. We tried to transfect lncRNA HAND2-AS1 into HCC cell line HepG2 by ultrasound targeted microbubble destruction mediated gene transfection technology to detect the effect of HAND2-AS1 on the proliferation, invasion, epithelial mesenchymal transition and apoptosis of HepG2 cells, and further explore the specific regulatory mechanism. In addition, we established a subcutaneous tumor xenograft mouse model to observe the effect of lncRNA HAND2-AS1 on the tumor forming ability of mice. We aimed to clarify the role of lncRNA HAND2-AS1 in HCC progression through in vivo and in vitro studies, in order to provide new ideas for the treatment of HCC.

Research objectives

We transfected lncRNA HAND2-AS1 into HepG2 cells through ultrasound targeted microbubble destruction mediated gene transfection technology, and detected the effect of lncRNA HAND2-AS1 on the biological behavior of HepG2 cells through a series of experiments in vitro, and found the downstream target genes of lncRNA HAND2-AS1 through online bioinformatics data retrieval, and further clarified the specific mechanism of lncRNA HAND2-AS1 participating in HCC cell growth. In addition, we successfully established a subcutaneous tumor xenograft mouse model and verified the inhibitory effect of lncRNA HAND2-AS1 on tumor formation in vivo in mice. Our results clarify the feasibility of ultrasound targeted microbubble destruction mediated gene transfection technology, and provide a new idea for finding gene therapy for HCC.

Research methods

We detected the expression levels of lncRNA HAND2-AS1 and miR-873-5p in tumor cells and tumor tissues by real-time quantitative polymerase chain reaction. The proliferation, apoptosis and invasion of HepG2 cells were detected by cell counting kit-8 assay, flow cytometry and Transwell cell invasion assay, respectively. Western botting was used to detect the protein expression levels of tissue inhibitor of matrix metalloproteinase-2 (TIMP2), matrix metalloproteinase (MMP)-2, MMP9 and epithelial mesenchymal transition related proteins in tumor cells and tumor tissues. In addition, immunohistochemistry was used to detect the expression of TIMP2, MMP2, MMP9 and Ki67 in tumor tissues. Luciferase reporter gene analysis was used to verify the targeting relationship of lncRNA HAND2-AS1 and miR-873-5p, as well as miR-873-5p and TIMP2. The SPSS program (version 21.0; SPSS, Chicago, IL) was used for all statistical analyses.

Research results

UTMBs loaded with lncRNA HAND2-AS1 inhibited the proliferation, invasion, and epithelial mesenchymal transition of HepG2 cells, and promoted apoptosis. We found that miR-873-5p was a target gene of lncRNA HAND2-AS1, and overexpression of miR-873-5p abolished the inhibitory effect of lncRNA HAND2-AS1 on tumor cell growth. In addition, miR-873-5p targeted the 3'UTR of TIMP2, and TIMP2 again reversed the promoting effect of miR-873-5p on tumor cell growth, and the mechanism study showed that this was mediated by blocking the MMP2/MMP9 signaling pathway. In the subcutaneous tumor xenograft mouse model, we observed that UTMBs carrying lncRNA HAND2-AS1 inhibited tumor formation in mice. Our results provide a new idea for gene therapy of HCC. Considering that this study only uses HepG2 cells, we will verify the results of this study in a variety of HCC cell lines later.

Research conclusions

We delivered lncRNA HAND2-AS1 into HeGp2 cells by UTMBs, and found that UTMBs carrying lncRNA HAND2-AS1 suppressed the cell invasion, proliferation and epithelial-mesenchymal transition, and the mechanistic findings indicated that lncRNA HAND2-AS1 suppressed the MMP2/MMP9 signaling pathway and then suppressed tumor progression by upregulating TIMP2 via targeting miR-873-5p. Furthermore, in vivo results demonstrated that tumor formation was inhibited in xenograft mice injected with lncRNA HAND2-AS1-bearing UTMBs.

Research perspectives

We identified the regulatory role of lncRNA HAND2-AS1/miR-873-5p/TIMP2 axis in HCC progression, which is a classic ceRNA pattern. Subsequently, we will take lncRNA HAND2-AS1 as a starting point to explore whether it is involved in tumor immune evasion microenvironment, or its relationship with tyrosine kinase inhibitors and immune checkpoint inhibitors, which were mentioned in the discussion section of the manuscript.