miRNA-660-3p inhibits malignancy in glioblastoma via negative regulation of APOC1-TGFβ2 signaling pathway

ABSTRACT Glioblastoma as the most common and aggressive central nervous system tumor in adults. Its prognosis and therapeutic outcome are poor due to the limited understanding of its molecular mechanism. Apolipoprotein C-1 (APOC1) as a member of the apolipoprotein family that acts as a tumor promoter in various cancers. MicroRNA (miRNA) can silence gene expression and suppress tumor progression. However, the role of APOC1 and its upstream miRNA has not been explored in glioblastoma. Two glioblastoma cell lines (U87 and U251) were used to explore the role of APOC1 and its upstream miRNA-660-3p in glioblastoma tumorigenesis in vitro. Cells with APOC1/miRNA-660-3p overexpression or knockdown were assessed for their proliferation, migration, and invasion in vitro, and tumorigenesis in vivo. Gene and protein expression was assessed by qRT-PCR and western blot, respectively. Cell proliferation was assessed by the MTT assay and the EdU and Ki67 staining. Cell migration and invasion were assessed by the transwell assay. Tumorigenesis in vivo was assessed in U87 cells with a xenograft mouse model. APOC1 was overexpressed in glioblastoma compared with normal peritumoral tissue and was inversely related to patient prognosis. APOC1 overexpression promotes cell proliferation, migration, and invasion in vitro. APOC1 inhibition reduced tumor growth in vivo. miRNA-660-3p inhibits tumorigenesis by directly targeting APOC1. Mechanistically, APOC1 drives the malignancy of glioblastoma by activating the TGFβ2 signaling pathway. miRNA-660-3p suppresses tumorigenesis by targeting APOC1. Therefore, miRNA-660-3p/APOC1 axis can serve as potential intervention targets in managing glioblastoma progression.


Introduction
Glioblastoma is one of the most aggressive intracranial tumors in the adult central nervous system (CNS), accounting for approximately 14.3% of all primary CNS tumors. 1 Glioblastoma is characterized by its high invasion and vascularization [2][3][4] and the rapid progression that ultimately leads to poor prognosis. 5The median survival time for most patients under the standard care of surgical resection combined with adjuvant radiotherapy is approximately 12 months. 2,6However, even with the most recent advance of combined therapy involving maximal surgical resection followed by radiotherapy and temozolomide, almost all patients experience tumor progression with a median survival time of fewer than 15 months. 7,8In addition, drug resistance often occurs in glioblastoma patients due to overexpressed antiapoptotic proteins. 5Generally, the expected survival time for glioblastoma patients is about two years after diagnosis. 9The poor prognosis and high mortality are mainly due to a limited understanding of disease progression in glioblastoma.Therefore, it is vital to study the molecular mechanisms underlying tumor progression in glioblastoma, which will help us develop better therapeutic interventions for disease control in these patients.
][12][13] Apolipoproteins are proteins that bind lipids to form lipoproteins, functioning as lipid carriers.Recently, many apolipoproteins have been reported to be involved in tumor pathogenesis. 14or APOC1, early studies have shown that it can promote tumor progression in many malignancies, including gastric cancer, 15 colorectal cancer, 16,17 prostate cancer, 18 breast cancer, 19 cervical cancer, 20 lung cancer, 21 pancreatic cancer, 22 and renal carcinoma. 23However, its role in tumor progression has not been explored in brain tumors, including glioblastoma.
MicroRNA (miRNA) is a cellular endogenous non-coding RNA molecule about 19-24 nucleotides in length 24 regulating gene expression through binding to mRNA.miRNAs silence gene expression by targeting the complementary 3'UTR site of the mRNA. 25Numerous research has indicated that the same miRNA can target and regulate multiple mRNAs in various biological processes such as apoptosis, DNA damage repair, proliferation, cell cycle, senescence, invasion, and angiogenesis. 26For example, in glioblastoma, miRNA-1 inhibited cell proliferation and migration in vitro and tumorigenesis in vivo by directly targeting fibronectin. 27In addition, cytosine-methylation of miRNA-181a-5p was associated with poor prognosis in glioblastoma patients. 28These findings indicate that miRNAs and their downstream genes can serve as potential drug targets for managing tumor progression.However, little research indicated the relationship between miR-660-3p and tumors.Only know is that miR-660-3p can be sponged by circRNA and then regulated drug resistance and malignancy in pancreatic cancer and breast cancer.However, there has not been any study exploring the potential relationship between miRNAs and APOC1 in glioblastoma tumorigenicity.
In this study, two glioblastoma cell lines (U87 and U251) were used to explore the role of APOC1 and miRNAs in glioblastoma tumorigenicity.First, the TCGA database was used to analyze the expression level of APOC1 and its association with prognosis in glioblastoma patients.Second, cells with APOC1 overexpression or knockdown were assessed for their proliferation, migration, and invasion in vitro, and tumorigenesis in vivo using a subcutaneous xenograft mouse model.Third, miRNA targeting APOC1 was identified, and its role in glioblastoma malignancy in vitro was assessed by transfecting cells with its mimic or inhibitor.Lastly, the downstream signaling pathway of APOC1 was uncovered and validated.Our study reveals that miRNA-660-3p inhibits the glioblastoma tumorigenesis by targeting the APOC1-TGFβ2 signaling pathway.

APOC1 upregulation is negatively correlated with glioblastoma patient prognosis
To investigate the pathogenesis of glioblastoma, we screened the GEPIA (http://gepia.cancer-pku.cn/detail.php?gene=&clicktag= boxplot) database for genes encoding the apolipoprotein family in paired glioblastoma and paraneoplastic tissues.The expression of APOC1 was much higher in glioblastoma than in normal paraneoplastic tissue (Figure 1a).Furthermore, the Kaplan- Meier curves from TCGA database revealed a lower survival rate in glioblastoma patients with high APOC1 expression (Figure 1b), suggesting that APOC1 may serve as a potential pro-oncogene in the development of glioblastoma.In order to clarify the expression of APOC1 in glioblastoma, we detected the APOC1 protein expression level in U87, U251 and HEB cells.As predicted, APOC1 protein is upregulated in glioblastoma (Figure 1c).Further, we found APOC1 was consistently overexpressed in tumors when compared with peritumor tissues in samples collected from GBM patients (n = 6, Figure 1d).

APOC1 upregulation exacerbates glioblastoma cell malignancy in vitro
Glioblastoma cells (U87 and U251) transfected with APOC1 plasmid (pAPOC1) showed upregulated APOC1 mRNA and protein (Figure 2a, Figure S1A), increased cell proliferation (Figure 2b-d), and enhanced cell migration and invasion (Figure 2e).Cell proliferation was characterized by cell growth assessed by the MTT assay (Figure 2b) and DNA replication activity assessed by the EdU (Figure 2c) and Ki67 staining (Figure 2d).In addition, cell migration and invasion were detected by the transwell assay (Figure 2e).Conversely, cells transfected with APOC1 shRNA showed the exact opposite (Figure S1B, Figure S2).

miRNA-660-3P -APOC1 axis regulates glioblastoma tumorigenesis in vivo
Glioblastoma U87 and U251 cells with stably APOC1 knocked down (lv-shAPOC1) were constructed by transfecting lentivirus.The efficiency of APOC1 expression was detected by western blot (Figure 5a).Then, the lv-shAPOC1 U87 cell was injected subcutaneously into nude mice to assess tumorigenesis in vivo.Tumors in nude mice inoculated with lv-shAPOC1 U87 cells were smaller and had significantly lower volume and weight than those inoculated with the control plasmid (Figure 5c).In addition, immunohistochemical staining showed decreased expression of APOC1 and Ki67 in tissue sections of tumors originating from mice injected with lv-shAPOC1 U87 cells (Figure 5d).To determine whether miR-340-APOC1 axis affects tumor formation in vivo, we further performed the in vivo tumorigenesis study by subcutaneously injecting Lv-miR-660-3p-U87 and Lv-miR-660-3p-U87 cells into nude mice.The inhibitory effect of miR-660-3p on APOC1 expression was detected by Western blot (Figure 5b).The mice in the Lv-miR-660-3p and control groups were euthanized 28 days after inoculation, and the average tumor volumes and weights were significantly reduced in Lv-miR -660-3p groups (Figure 5(e,)).

miRNA-660-3P-APOC1 axis negatively regulates the TGFβ2 signaling pathway
To explore the potential molecular mechanism by which APOC1 promotes glioblastoma malignancy, we examined some common tumor signaling pathways after the APOC1 was knocked down.Our analysis revealed significant downregulation of TGFβ2 in U87 and U251 cells (Figure 6a).Similarly, cells transfected with miR-660-3p mimic had decreased expression of TGFβ2 (Figure 6b).Meanwhile, the upregulated TGFβ2 caused by APOC1 overexpression was partially dampened after miR-660-3p mimic transfection (Figure 6c).Western blotting showed that both TGFβ2 and its downstream phospho-SMAD3 protein levels were elevated after overexpression of APOC1 and decreased after transfection of miR-660-3p mimic (Figure 6d).These results suggest that the miRNA-660-3P-APOC1 axis negatively regulates the TGFβ2 signaling pathway in glioblastoma cells.The rescue experiments showed the overexpressed APOC1 upregulated the protein expressions of TGFβ2 and p-SMAD3.Meanwhile, miR-660-3p can partially nullified the oncogenic effect of the APOC1 on glioblastoma cells.

Discussion
This study showed that APOC1 is significantly elevated in human glioblastoma tissues, and its inhibition led to decreased cell proliferation, migration, and invasion in vitro and tumor growth in vivo, suggesting that APOC1 may serve as a driver gene in glioblastoma malignancy.In addition, miR-660-3p was found to target APOC1 mRNA and inhibit its expression, which in turn inhibits cell proliferation, migration, and invasion in vitro.Further, we discovered that APOC1 inhibition downregulates TGFβ2 and disrupts its associated downstream SMAD3 and pSMAD3, revealing a new signaling pathway involving the miR-660-3p/APOC1/TGFβ2 axis in the development of glioblastoma.Our findings suggest that miR-660-3p/ APOC1/TGFβ2 axis may serve as a new potential therapeutic target for glioblastoma.
Previous studies had suggested that abnormalities in cholesterol biosynthesis and its downstream signaling can lead to glioblastoma tumorigenesis. 29Cholesterol metabolism in the zbrain is different from that in other organs because cholesterol in plasma cannot cross the blood-brain barrier.Instead, cholesterol in the brain is synthesized in astrocytes and delivered to neurons through the apolipoprotein E (APOE). 30otably, a recent study showed that the demand for cholesterol in glioblastoma cells was mainly exogenous rather than endogenous, 31 which has also been suspected to be the underlying cause of developing drug resistance in glioblastoma. 31his study also highlights the importance of cholesterol sources in glioblastoma progression.Together, these findings call for further exploration of cholesterol utilization in glioblastoma cells to unravel possible molecular mechanisms of tumor progression.
APOC1, as a member of the apolipoprotein family, is the primary apolipoprotein of the very-low-density lipoprotein (VLDL)-cholesterol complex. 32This may explain why APOC1 was commonly overexpressed in glioblastoma, as exogenous cholesterol is required for tumor growth.In addition, APOC1 has been shown to serve as a tumor-promoting factor in other malignancies.For example, in triple-negative breast cancer, APOC1 induced epithelial-mesenchymal transition (EMT) by suppressing E-calreticulin. 19In renal clear cell carcinoma, APOC1 directly enhanced the STAT3 signaling, which in turn increased cell migration and invasion through EMT. 23In pancreatic cancer, APOC1 suppressing through the BET inhibitor(JQ1s) enhanced the therapeutic effect of gemcitabine. 33More recently, APOC1 has been identified as a critical marker related to oncogenesis and prognosis of colorectal, cervical, and renal clear cell carcinoma. 17,20In the present study, we, for the first time, demonstrated that APOC1 promotes cell proliferation, migration, invasion in vitro, and tumor growth in vivo in glioblastoma.We only determined the tumor growth in vivo by the subcutaneous xenograft model in this research, However, evaluate the growth of glioblastoma by orthotopic xenograft model would be a better idea.The non-coding miRNAs, similar to the transcription factors, play essential roles in gene expression regulation. 34any studies have shown that miRNAs expression is closely related to tumorigenesis and malignancy. 35For example, in glioblastoma, miR-301a-3p promotes cell proliferation and invasion by upregulating FOSL1 expression through sponging with long non-coding RNA LncRNA-HOTAIR, 36 and miR-3189 targets GLUT3 to inhibit cell proliferation by regulating glucose metabolism. 37In addition, APOC1 has been reported to serve as a direct target for miR-17 and miR-515 in breast and prostate cancer, respectively, to halt tumor growth. 38More recently, miR-660-3p was reported to be sponged by circFARP1 to upregulate LIF expression and inhibit gemcitabine resistance in pancreatic ductal adenocarcinoma. 39In breast cancer, circWWC3 upregulates multiple oncogenes' expression of the Ras signaling pathway through acting as the sponge of miR-660-3p. 40In the present study, we demonstrated that miR-660-3p directly targeted APOC1 and inhibited APOC1 expression, subsequently leading to reduced cell proliferation, migration, and invasion in glioblastoma cells.The fact that miRNAs can cross the blood-brain barrier via exosome and transport between tumor cells 41 makes miRNAs promising therapeutic targets for glioblastoma.
][47] Earlier studies have revealed that the TGFβ2 signaling pathway can be regulated by PDLIM5 to promote lung cancer migration and invasion. 43Furthermore, the expression TGFβ2 receptor and its signaling in glioblastoma are closely related to the patients' prognosis. 48In addition, TGBβ2 downstream protein SMAD was also associated with glioblastoma malignancy. 49In this study, we revealed that TGFβ2 signaling is a downstream pathway for APOC1 in glioblastoma, and its suppression can reduce tumor progression.
This study revealed the role of APOC1 in promoting glioblastoma malignancy and the potential underlying molecular mechanism.APOC1 expression is negatively correlated to glioblastoma prognosis.APOC1 suppression through shRNA or miRNA-660-3p could lead to reduced cell proliferation, migration, and invasion in glioblastoma cells and reduced tumor growth in tumor xenografts.APOC1 suppression is also associated with the downregulation of the TGFβ2 signaling pathway.In conclusion, the miRNA-660-3p-APOC1 axis could inhibit the glioblastoma malignancy by negatively regulating the TGFβ2 signaling pathway.Targeting APOC1 or miRNA-660-3p may provide a new therapeutic intervention for glioblastoma.

Cell lines and cell culture
Human glioblastoma cell lines (U87 and U251) and HEB cell line were purchased from the ATCC.U87, U251 and HEB cells has been performed authenticated using Short Tandem Repeat (STR) analysis on 2021 in Wuhan Procell Life Science&Technology Co., Ltd.The complete medium for U87, U251, and HEB consists of 90% DMEM medium (Gibco, NY, USA), 10% fetal bovine serum (FBS, Gibco, NY, USA) and 1% penicillin/streptomycin (Gibco, NY, USA).All cells were cultured at 37°C in a 5% CO 2 incubator.

Cell transfection
The control and APOC1 (Hanheng, Wuhan, China) downregulated stable cell lines were established using lentivirus transduction and labeled as sh-control and sh-APOC1, respectively.These cells were confirmed with western blot and RT-PCR after 6 days of culture prior to xenograft injection.
The pcDNA3.1 and pAPOC1 plasmids were obtained from Hanheng (Wuhan, China).All cell transfections were carried out in 6-well plates.Cells were transfected with each plasmid at 5ug per well by Lipofectamine 2000 (Invitrogen, Massachusetts, USA).miR-660-3p mimic and miR-660-3p mimic inhibitor were transfected with 80ng per well in the same way.After 48 h transfection, cells were collected for subsequent analysis.

Cell proliferation, migration, and invasion
Cell proliferation was assessed by the MTT assay as described previously. 50Cell invasion and migration were assessed using the transwell assay according to the manufacturer's protocol (B.D. Bioscience).In brief, cells were pretreated with serumfree DMEM medium for 6 h and then were trypsinized and resuspended in serum-free DMEM medium.Cells of 5 × 10 4 per well were plated onto the upper chamber without (migration) or with matrigel (invasion) and cultured for 15 h.The invaded cells on the submembrane surface were stained with crystal violet staining solution, and the number of cells in five randomly selected fields per well was counted.

Quantitative real-time PCR
RNA extraction and subsequent steps had been described previously. 50The primers used are listed in Table S1.

EdU cell proliferation assay
Cell proliferation was assessed by seeding cells in 24-well plates, incubated with 50 μM EdU for 2 h, and fixed in 4% paraformaldehyde and 0.5% Triton X-100 permeation according to the manufacturer's protocol (EdU Cell Proliferation Kit with Alexa Fluor 488, Meilun, Guangzhou, China).Cells were visualized and imaged using a laser confocal microscope (Olympus, Japan) after counterstaining the nucleus with Hearst dye (Proteintech, Wuhan, China).The semi-quantitative (EdU positive cell percentage) results were detected by ImageJ.

Figure 1 .
Figure 1.APOC1 expression in tumor and paraneoplastic tissues and its relationship with prognosis in glioblastoma patients.a:Expression of APOC1 in glioblastoma and paracancerous tissues; *P < 0.05; b: Kaplan-Meier analysis shows reduced postoperative survival of glioblastoma patients with higher APOC1 expression (data extracted from the TCGA database).c: Expression level of APOC1 was detected in HEB, U87 and U251 cells by western blot.D: Expression of APOC1 mRNA was detected in six GBM patients' tumor and peritumor tissues by RT-qPCR.