Exosomal miR-130b-3p Promotes Progression and Tubular Formation Through Targeting PTEN in Oral Squamous Cell Carcinoma

Abstract

Oral squamous cell carcinoma (OSCC), accounting for two-thirds of head and neck cancer, is characterized by poor prognosis and a high rate of mortality. Exosomes have emerged as potential molecule-shuttle in intercellular communication, thereby regulating the physiological processes of recipient cells. To date, the effect of exosomal microRNAs (miRNAs) on the progression of OSCC has not been fully investigated. In this study, we found that the protein, but not mRNA expression of Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was decreased in OSCC. The results revealed that miR-130b-3p was an important negative regulator for PTEN expression. Additionally, overexpression and knockdown of miR-130b-3p enhanced and inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs), respectively. Also, miR-130b-3p was transferred by exosomes to HUVECs and then promoted angiogenesis and inhibit the expression of PTEN. Furthermore, exosomal miR-130b-3p derived from OSCC cells promoted tumor growth and blood vessel formation in the xenograft mice model. Taken together, we demonstrated that exosome-mediated miR-130b-3p promoted progression and tubular formation in OSCC in vitro and in vivo. These results would provide new insight into exploring biomarkers and effective therapeutic strategies for OSCC.

Keywords: Pten; exosome; miR-130b-3p; oral squamous cell carcinoma; progression; tubular formation.

FIGURE 1

PTEN protein expression is decreased in OSCC. (A,B) Protein expression of PTEN in OSCC tissues and paired adjacent control tissues. (C) mRNA expression of PTEN in OSCC tissues and paired adjacent control tissues. (D) Protein expression of PTEN in OSCC tissues and paired adjacent control tissues, as determined by IHC assay. (E) The expression of seventeen candidate miRNAs in OSCC tissues and the serum of OSCC patients. (F) The expression of miR-130b-3p in OSCC tissues and paired adjacent control tissues. (G) The expression of miR-130b-3p in the serum of OSCC patients and control subjects. (H) The expression of miR-130b-3p in exosomes isolated from the serum of OSCC patients and control subjects. (I) The correlation between expression of PTEN and miR-130b-3p. ***p < 0.001, **p < 0.01.

FIGURE 2

Exosomal miR-130b-3p in the serum reduces PTEN expression. (A,B) The morphology of exosomes and the exosome diameter size distribution, determined by transmission electron microscope. Scale bar = 100 nm. (C) The expressions of exosome markers Tsg101, Alix, and CD63. (D) The expression of miR-130b-3p in exosomes derived from control (Ctr-exo) and OECM1 (OECM1-exo) cells. (E) The expression of miR-130b-3p in OECM1-exo treated with miR-130b-3p inhibitor. (F) The expression of miR-130b-3p in HUVECs treated with OECM1-exo and the combination of OECM1-exo and miR-130b-3p inhibitor. (G,H) The protein expression of PTEN in HUVECs treated with OECM1-exo and the combination of OECM1-exo and miR-130b-3p inhibitor. (I) The mRNA expression of PTEN in HUVECs treated with OECM1-exo and the combination of OECM1-exo and miR-130b-3p inhibitor. **p < 0.01.

FIGURE 3

Exosomal miR-130b-3p directly targets PTEN. (A) Putative binding site of miR-130b-3p in 3’UTR of PTEN. (B) Relative luciferase activity in OECM1 cells transfected with luciferase reporter vector carrying wild-type or mutant binding site of miR-130b-3p in 3’UTR of PTEN and miR-130b-3p mimic or inhibitor. (C) Relative luciferase activity in OECM1 cells transfected with luciferase reporter vector carrying wild-type or mutant binding site of miR-130b-3p in 3’UTR of PTEN and OECM1-exo or the combination of OECM1-exo and miR-130b-3p inhibitor. (D,E) The protein expression of PTEN in HUVECs treated with miR-130b-3p mimic or inhibitor. (F) The mRNA expression of PTEN in HUVECs treated with miR-130b-3p mimic or inhibitor. *p < 0.05.

FIGURE 4

Exosomes derived from OECM1 cells promote angiogenesis. (A) Exosomes derived from OECM1 cells (OECM1-exo) were uptaken by HUVECs. Blue color indicated nuclei stained with DAPI and red color indicated exosomes stained with Edu. (B) Migration ability of HUVECs treated with OECM1-exo or the combination of OECM1-exo and miR-130b-3p inhibitor. (C) Ring formation ability of HUVECs treated with OECM1-exo or the combination of OECM1-exo and miR-130b-3p inhibitor. (D) Proliferation of HUVECs treated with OECM1-exo or the combination of OECM1-exo and miR-130b-3p inhibitor. ^∗∗p < 0.01.

FIGURE 5

MiR-130b-3p is essential in angiogenesis in HUVECs. (A) Migration ability of HUVECs treated with miR-130b-3p mimic or inhibitor. (B) Ring formation ability of HUVECs treated with miR-130b-3p mimic or inhibitor. (C) Proliferation of HUVECs treated with miR-130b-3p mimic or inhibitor. ^∗p < 0.05.

FIGURE 6

Exosomal miR-130b-3p promotes tumor growth in vivo. (A) Representative image of tumor tissues. (B,C) Tumor weight and volume of mice injected with OECM1 cells treated with miR-130b-3p-overexpressing lentivirus (miR-OE), miR-130b-3p-knockdown lentivirus (miR-KD), the combination of miR-KD and OECM1-exo, and TSG101-knockdown lentivirus (TSG101-KD), respectively. (D,E) The morphology of exosomes and the exosome diameter size distribution, determined by transmission electron microscope. Scale bar = 100 nm. **p < 0.01, *p < 0.05.

FIGURE 7

Exosomal miR-130b-3p promotes angiogenesis in vivo. (A,B) The expression of miR-130b-3p in tumor tissues and plasma exosomes in mice injected with OECM1 cells treated with miR-130b-3p-overexpressing lentivirus (miR-OE), miR-130b-3p-knockdown lentivirus (miR-KD), the combination of miR-KD and OECM1-exo, and TSG101-knockdown lentivirus (TSG101-KD), respectively. (C–E) The protein and mRNA expression of PTEN in mice injected with OECM1 cells treated with miR-130b-3p-overexpressing lentivirus (miR-OE), miR-130b-3p-knockdown lentivirus (miR-KD), the combination of miR-KD and OECM1-exo, and TSG101-knockdown lentivirus (TSG101-KD), respectively. (F) Tubular formation in tumor tissues was determined by IHC assay by using anti-CD31 antibody. ***p < 0.001, **p < 0.01, *p < 0.05.