microRNA-378a-3p regulates the progression of hepatocellular carcinoma by regulating PD-L1 and STAT3 - PubMed (original) (raw)

microRNA-378a-3p regulates the progression of hepatocellular carcinoma by regulating PD-L1 and STAT3

Yaqin Li et al. Bioengineered. 2022 Mar.

Abstract

Programmed death ligand 1 (PD-L1) plays an essential role in the development or progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression during normal and pathophysiological events. Here, we explored the functions and detailed mechanisms of miR-378a-3p and PD-L1 in HCC progression. First, miR-378a-3p was selected by analyzing miRNA levels in two HCC Gene Expression Omnibus datasets. We found that miR-378a-3p levels exhibited a downward trend in HCC and were negatively correlated with PD-L1 levels. Additionally, a dual luciferase assay predicted that miR-378a-3p directly targets PD-L1. Moreover, the transfection of miR-378a-3p mimics into Li-7 and HuH-7 cells effectively decreased the PD-L1 mRNA and protein expression levels, and inhibited Treg differentiation in co-culture models by modulating the expression levels of certain cytokines. Furthermore, the overexpression of miR-378a-3p hindered cell proliferation and migration but facilitated apoptosis by repressing STAT3 signaling in HCC cells. In conclusion, miR-378a-3p appears to inhibit HCC tumorigenesis by regulating PD-L1 and STAT3 levels. Thus, miR-378a-3p may be a potential target for HCC therapy.

Keywords: Hepatocellular carcinoma; PD-L1; STAT3; immune escape; miR-378a-3p.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

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Graphical abstract

Figure 1.

Figure 1.

miR-378a-3p is lowly expressed in HCC tissues. (A) HCC GEO datasets (GSE12717 and GSE57555) were analyzed and one lowly expressed gene was identified from the intersection. (B) qRT-PCR analysis of miR-378a-3p expression in 48 pairs of HCC and normal tissues obtained from HCC patients. (C) Negative correlation between miR-378a-3p and PD-L1 expression in HCC samples. ***P < 0.001.

Figure 2.

Figure 2.

PD-L1 solved as the direct target of miR-378a-3p. (A) Schematic miR-378a-3p putative target sites in 3’UTRs of PD-L1. (B) Dual luciferase reporter assays. (C) The expression level of miR-378a-3p was evaluated using qRT-PCR in HCC cells after transfection. (D) qRT-PCR, (E) Western blot, and (F) flow cytometry were used to evaluate the expression level of PD-L1 after transfection. Data were shown as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.

Figure 3.

miR-378a-3p overexpression reduced HCC cell viability and promoted cell apoptosis. (A) EdU assay showed that miR-378a-3p reduced HCC cell viability. (B) Colony formation assay showed that miR-378a-3p inhibited HCC cell to form colonies. (C) miR-378a-3p transfection effectively induced apoptosis in HCC cell. Data were shown as mean ± SD from three independent experiments. **P < 0.01, and ***P < 0.001.

Figure 4.

Figure 4.

The potential molecular mechanisms of the regulation of miR-378a-3p to the cell viability and apoptosis of HCC cells. (A) C-MYC, as a major gene involving in cell growth, was detected with qRT-PCR. (B) C-MYC was detected with Western blot. (C, D) The levels of major genes related to apoptosis were evaluated using qRT-PCR and Western blot. Data were shown as mean ± SD from three independent experiments. **P < 0.01.

Figure 5.

Figure 5.

miR-378a-3p overexpression reduced HCC cell invasion and migration. (A) A wound healing assay was performed to investigate cell migration. (B) Transwell assay was employed to investigate cell invasion status. (C) The mRNA level of MMP-9 was detected in HCC cells using qRT-PCR. (D) The protein level of MMP-9 was detected in HCC cells. Data were shown as mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01.

Figure 6.

Figure 6.

miR-378a-3p overexpression led to down-regulation of STAT3. (A) qRT-PCR and (B) Western blotting were used to evaluate the expression level of STAT3 in HCC cells after transfection. Data were shown as mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01.

Figure 7.

Figure 7.

miR-378a-3p modulated the cell differentiation of CD25+ Foxp3+ Treg. (A) The percentage of CD4+CD25+ Foxp3+ Tregs in the CD4+ population was detected using FACS. (B, C) Up-regulation of miR-378a-3p lead to a decrease in Treg induction compared to the negative controls. (D) Detecting cytokine levels with qRT-PCR in co-culture medium. Data were shown as mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01.

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This work was supported by the Sanming Project of Medicine in Shenzhen under Grant number (SZSM201612071); Shenzhen Key Medical Discipline Construction Fund under Grant number (SZXK078); and the Cell Technology Center and Transformation Base, Innovation Center of Guangdong-Hong Kong-Macao Greater Bay Area, Ministry of Science and Technology of China (Grant No. YCZYPT[2018]03-1）, the Scientific Research Foundation of PEKING UNIVERSITY SHENZHEN HOSPITAL KYQD202100X.

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