MicroRNA-628-3p inhibits glioblastoma cell proliferation and GSC maintenance by directly targeting BTK

doi:10.21203/rs.3.rs-2233362/v1

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MicroRNA-628-3p inhibits glioblastoma cell proliferation and GSC maintenance by directly targeting BTK

https://doi.org/10.21203/rs.3.rs-2233362/v1

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Differentially expressed microRNAs (miRNAs) have been identified as a critical factor of glioblastoma (GBM). Therefore, we analyzed the pathogenic mechanism of a new miRNA (miR-628-3p) in glioblastoma. We identified miR-628-3p expression using qRT-PCR and FISH test, and a lot of in vitro tests looked at how miR-628-3p and BTK work biologically. Using a dual luciferase reporter experiment, the target site and mechanism of miR-628-3p binding to BTK were identified. Subcutaneous carcinogenesis was utilized to demonstrate the functionality of miR-628-3p in vivo. Inhibiting GBM growth and GSC maintenance was miR-628-3p overexpression. In addition, miR-628-3p regulated the expression of BTK, which was found as a promoter of GBM development in the research. Our findings showed that miR-628-3p reduces GBM growth and GSC maintenance, suggesting a potential therapeutic target for glioma treatment.

Glioblastoma

miR-628-3p

proliferation

GSC

BTK

One of the most aggressive tumor kinds is glioblastoma (GBM)[1, 2]. There are many treatment options, but the effect is not ideal, and it is easy to relapse[3, 4]. A few cells known as "glioma stem cells" (GSCs), have stemness characteristics associated with glioma recurrence after surgery [5, 6]. The progression of malignant glioma may be related to GSCs [7, 8].

MicorRNAs (miRNAs), in short non-coding RNAs, usually bind to parts of the 3'-UTR of specific messenger RNAs to down-regulate gene expression [9–11]. According to reports, miRNAs control downstream gene expression in cancer and development by serving as molecular sponges[12]. MiRNAs have been linked to GBM proliferation, invasion, differentiation, apoptosis, and GSC maintenance, according to a wealth of literature. [13–16]. Simultaneously, the dysfunction of miRNAs can cause many cancers, especially GBM[17, 18].

Bruton's tyrosine kinase (BTK), a member of the cytoplasmic tyrosine kinase family, has gradually become a research hotspot in tumors in recent years [19, 20]. Previous research has provided evidence to show a high expression of BTK in a variety of cancers[21–24]. BTK has also been considered a diagnostic biomarker and immunotherapeutic target for glioma patients[25, 26].

In this study, we show that a bio-function of tumor inhibitors, miR-628-3p, plays a critical function in glioma proliferation and GSC maintenance by directly targeting BTK.

Public database and clinical human brain samples

The CGGA database (http://www.cgga.org.cn/portal.phpg) was employed to retrieve the information on miRNA and BTK expression in glioma cells. Twenty high grade glioma tissues, ten low grade glioma tissues, and six normal brain tissues were all supplied by the First Affiliated Hospital of Nanjing Medical University. In addition, tissue samples from six brains were obtained with patient approval from emergency decompressive craniectomy patients. All samples were removed intraoperatively and treated with liquid nitrogen and used for subsequent experiments.

Cell lines and transfection

The Chinese Academy of Sciences Cell Bank (Shanghai, China) offered the glioma cell lines U87, U251, U118, A172, T98, and LN229. After that, the cells were cultured in DMEM (Hyclone, Chicago, IL, USA) with 10% FBS added as a supplement. Lonza (Basel, Switzerland) provided the normal human astrocytes, which were cultured in a mixture that also contained rhEGF, insulin, ascorbic acid, GA-1000, L-glutamine, and 5%FBS. All cells were cultivated in an incubator (ThermoFisher,USA) at 37°C with 5% CO2 and purified air.

RNA isolation and qRT-PCR

Using Trizol reagent (Life Technologies, CA, USA) and according to the manufacturer's recommendations, total RNA was extracted from cells or tissues. The 2^−ΔΔCt technique was used to analyze the data, and U6 DNA was used as a control. Primer sequences(Genepharma, Shanghai, China) for qRT-PCR were as described : miR-628-3p Forward: 5’-tctagtaagagtggcagtcga-3’ Reverse: 5′-gcgagcacagaattaatacgac-3′; U6 Forward: 5′-gctagtgcaggaatatactcaact-3′ Reverse: 5′-cgcttcacgaatttgcgtgtca-3′; BTK Forward: 5'-ggggtttgctcagactgtcc-3' Reverse: 5'-aatcactgcggccatagctt-3' and GAPDH Forward: 5′-tcagtggtggacctgacctg-3′ Reverse: 5′-tgctgtagccaaattcgttg-3′. All experiments were performed and tested in triplicate.

Fluorescence in situ hybridization (FISH)

In order to quantify miR-628-3p expression in the NBTs and GBM samples, FISH (BioSense, Guangzhou, China) was utilized. Sections were fixed with 4% paraformaldehyde for 1h and subsequently washed with PBS. After proteinase K treatment, the cells were dehydrated by soaking in ethanol, respectively. Subsequently, the probe was denatured at 78°C and added to the slides. Experiments were then performed overnight at 42 ° C. On the following day, washes with 50% formamide in 2X SSC at 43°C and with SSC at ambient temperature were used to eliminate repetitive and non-specific RNA hybridizations. Slides counterstained with DAPI(Sigma) for 10min were placed on a confocal microscope (Oberkochen, Germany) for observation.

Western blot analysis

Western blot analysis was carried out as previously shown[16]. The antibodies employed for the western blots were as follow: anti-Cyclin D1 (#55506), anti-CDK2 (#18048), anti-Sox2 (#14962), anti-Nestin (#73349), anti-Nanog (#4903), and anti-Oct-4 (#75463) were all bought from (Cell Signaling Technologies, Danvers, MA, USA); anti-BTK (ab208937) and GADPH (ab8245) were bought from Abcam (Cambridge, UK). Membranes were obtained from SuperSignal® Maximum Sensitivity Substrate.

Plasmid construction and transfection

Ribobio (Guangzhou, China) provided the miR-628-3p mimic and its negative control. GenePharma verified the presence of the plasmid. To create a plasmid for BTK overexpression, the BTK coding area was introduced into the pcDNA3.1 vector. Virions were then prepared and collected following the manufacturer's instructions. In accordance with the manufacturer's recommendations, lentivirus transfection was used to create the U87 and U251 cell lines that had stable BTK knockdown.

Cell proliferation assay

Over the course of the next 24, 48, 72, and 96 h, the steady transfected U87 and U251 cells were monitored using the CCK-8. Finally, by measuring the OD value at 450 nm, the rate of proliferation was assessed.

Plate colony formation

Cell reproduction was evaluated via plate colony formation. U87 and U251 cells that had been transfected were grown for two weeks on 60mm cell culture plates. Following methanol fixation, the cells were treated with 1% crystal violet (Sigma, USA).

5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay

Transfected cells were plated onto 24-well plates at a density of 5000 cells per well for the EdU test. A Cell-Light EDU imaging detection kit (Life Technologies, MA, USA) was used according to the manufacturer’s instructions. Lastly, imaging the samples using a fluorescent microscope (Leica, Wetzlar, Germany).

Flow cytometric analysis of the cell cycle

After lentivirus transfection, cells were cultivated in 6-well plates for 48 h in each group and then trypsinized and collected by centrifugation. Following a PBS wash, the cells were treated with propidium iodide for 20 minutes while also being exposed to RNase A (Sigma-Aldrich). After that, flow cytometry (BD Bioscience, Franklin Lakes, NJ, USA) was used to analyze the cells.

Glioma stem cell culture, spheroid formation assay and clonogenic assays.

Those analysis were performed as described previously[16].

Dual luciferase reporter assay

The pGL3-REPORT luciferase vector (Invitrogen, Shanghai, China) was used to clone the binding sites for wild-type (WT) and mutant miR-628-3p in the 3'-UTR region of BTK. Co-transfected miR-628-3p mimics (RiboBio, Guangzhou, China) were added to the WT or mutant 3'-UTR of BTK before the cells were expanded in 96-well plates. The dual luciferase reporter assay kit (Promega, Madison, USA) measured luciferase activity.

Glioma xenografts

Five-week-old BALB/c-A nude mice were bought from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). To generate xenografts, 6 × 10⁶ U87 cells were subcutaneously injected and measured the tumor volumes by calipers. All mice were assigned to the control and treatment groups until the tumor volumes reached 50 mm³. Each group of mice was treated with miR-NC and miR-628-3p mimic via intratumor injection. For a total of 27 days, this therapy was administered once every three days.

Immunohistochemistry (IHC) assay

To analyze the expression of Ki-67 protein in glioma xenografts, immunohistochemistry (IHC) was used in this study. The process was carried out as previously explained [16]. Fresh xenogliomas were settled in 4% paraformaldehyde, then implanted in paraffin and sliced into 5µm partitions. Using a Ki-67 antibody (1:200, Cell Signaling Technologies, Shanghai, China), sections were blotted immunohistochemically. Finally, the images were scanned and imaged on a light microscope (Leica, Shanghai, China).

Statistical analysis

Utilizing Prism GraphPad version 7.0, statistical analyses were carried out on all experiments in triplicate. The reciprocal association between miR-628-3p and BTK was evaluated using Pearson's correlation coefficient. The Kaplan-Meier analysis evaluated the survival rate of patients and mice. Statistical data evaluation was conducted utilizing the student’s t-test, and a p-value of < 0.05 was regarded as statistically meaningful.

The expression of miR-628-3p is markedly reduced in glioma tissues and glioma cell lines

According to the examination of the CGGA database, glioblastoma patients' expression of miR-628-3p was noticeably downregulated (Fig. 1A) and increased miR-628-3p expression resulted in longer survival (Fig. 1B). The expression trend of miR-628-3p in clinical samples was determined by qRT-PCR and found to be similar to that in the CGGA database. (Fig. 1C). In addition, we examined the expression of miR-628-3p in 6 different GBM cell lines and NHAs. In GBM cell lines, notably U87 and U251 cell lines, miR-628-3p was dramatically negatively regulated in comparison to the control NHAs (Fig. 1D). Furthermore, we examined a representative GBM tissue and a normal brain tissue from clinical patients for FISH analysis, which also demonstrated that the expression of miR-628-3p is downregulated in GBM (Fig. 1E). Overall, our results suggested decreased miR-628-3p expression in glioblastoma.

Overexpression of miR-628-3p prevents the proliferation of glioma cells

We transfected miR-628-3p lentivirus into cell lines and set up a control group and used qRT-PCR to verify (Fig. 2A). The CCK8 assay was also used to measure the growth rate. Cells transfected with miR-628-3p grew at a considerably slower pace than the miR-NC group (Fig. 2B). A similar result from the colony formation assays was obtained in the miR-628-3p lentivirus-transfected U87 and U251 cells (Fig. 2C). which according to EdU pictures, miR-628-3p transfected U87 and U251 had markedly decreased positive rates than the miR-NC group (Fig. 2D). Western blot examination revealed that Cyclin D1 and CDK2 expression were highly negatively correlated with the overexpression of miR-628-3p in U87 and U251 cells (Fig. 2E). A flow cytometric study subsequently confirmed the influences of the overexpressed miR-628-3p. Because miR-628-3p was overexpressed, the fraction of G0/G1 cells rose while the proportion of S and G2/M cells dropped (Fig. 2F and 2G). These findings show that upregulation of miR-628-3p prevents glioma cells from progressing through the cell cycle.

Overexpression of miR-628-3p inhibits GSC maintenance in vitro

Western blot was utilized to further investigate the expression of Sox2, Nestin, and Oct4 in GSC pluripotency factors. (Fig. 3A). Colony sphere formation test findings showed that GSCs-miR-628-3p had a much lower proportion of positive wells than GSCs-miR-NC (Fig. 3B and 3C). MiR-628-3p overexpression inhibited the proliferation and vitality of GSCs (Figs. 3D and 3E), indicating that miR-628-3p may be essential in preventing GSC maintenance.

MiR-628-3p directly targets BTK mRNA

To define whether BTK is a downstream protein of miR-628-3p, using TargetScan, we discovered that miR-628-3p might potentially target BTK (Fig. 4A). It was shown using a luciferase reporter experiment that BTK is a miR-628-3p direct target. Co-transfection of the vectors expressing WT or Mut BTK 3'-UTR and miR-628-3p mimic into U87 and U251 cells was performed (Fig. 4B). Since miR-628-3p mimics greatly reduced the WT reporter gene's luciferase activity, it is likely that miR-628-3p directly targeted the 3-UTR of BTK mRNA (Fig. 4C). Additionally, a Western blot study following miR-NC and miR-628-3p transfection revealed the protein level of BTK (Fig. 4D). The CGGA database revealed the expression of BTK were significantly higher in HGG than LGG (Fig. 4E) However, increased BTK expression resulted in a shorter survival time (Fig. 4F). Western blot indicated that BTK was overexpressed in glioma tissue samples and increased with the increase of grade (Fig. 4G). Meanwhile, qRT-PCR indicated that the level of BTK mRNA were remarkably higher in high grade glioma samples (WHO III and IV), compared with low grade glioma samples (WHO II) (Fig. 4H). The level of miR-628-3p was shown to be inversely correlated with the amount of BTK in GBM tissue samples by Spearman correlation analysis (r = -0.42) (Fig. 4I). These findings demonstrated that miR-628-3p directly controlled BTK expression by attaching to a location in the 3'-UTR (nt 418–425) of BTK mRNA.

BTK induces glioma cell proliferation and GSC maintenance by activating Akt and ERK signaling pathways in vitro

We employed lentiviral transduction of shRNAs targeting BTK in the U87 and U251 cells to investigate the loss-of-function of BTK in order to ascertain the function of BTK in vitro. Western blot exposed the decreased protein levels of BTK in the U87 and U251 after BTK was knocked donwn (Fig. 5A). The effects of BTK knockdown on cell survival and development were assessed using the CCK-8, colony development, and EdU assays (Fig. 5B-5D). In order to determine how cells were distributed following transfection, we also performed a flow-cytometric examination. After silencing BTK, we observed that the subpopulation of cells at the G0-G1 transition increased in comparison to the data obtained from shNC cells, but the numbers of cells in the S phase and at the G2-M transition dropped (Fig. 5E). Similarly, western blot analysis demonstrated that the Cyclin D1 and CDK2 protein levels were lower in the shBTK groups of the U87 and U251 cells than in the shNC groups (Fig. 5F). Clonal cell formation assay indicated that GSCs-shBTK had a lower positive pore rate (Fig. 5G and 5H). Knocking down the expression of BTK also declined GSCs growth and cell viability (Fig. 5I and 5J). The outcomes of western blot assays revealed that the expression protein level of p-Akt and p-ERK were particularly down after BTK knockdown, which means inhibiting the expression of BTK could block the AKT and ERK pathways (Fig. 5K upper). After BTK was knocked down, the levels of the AKT and ERK pathway-dependent factors (Nestin, Sox2, Nanog, and Oct4) were significantly decreased (Fig. 5K bottom). These funds provided evidence that BTK could be essential to maintaining the GSC.

Restoration of BTK attenuates miR-628-3p suppressed glioma cell proliferation and GSC maintenance

Identifying the target of miR-628-3p for BTK, pcDNA3-BTK was transfected into U87 and U251 cells that were stably expressing either miR-628-3p or miR-control. The higher expression level of miR-628-3p induced a reduction in the numeral of cancer cells, as shown in the CCK-8, EdU, and colony formation tests. In addition, the restoration of BTK partially resisted the function of miR-628-3p (Figs. 6A–6E). According to depletion cytometer findings, the effect of miR-628-3p mimics was countered by the addition of BTK again (Fig. 6F). The outcomes of the Western blot revealed that the upregulation of BTK reversed the miR-628-3p-induced reduction in Cyclin D1 protein levels (Fig. 6G). Moreover, the inhibitory influence of upregulation of miR-628-3p on GSC was reversed by the reintroduction of BTK (Fig. 6H-6K). We also found that miR-628-3p overexpression greatly declined the SOX2 protein levels in U87-GSC and U251-GSC cells and inhibited miR-628-3p can rescue the action (Fig. 6L). In conclusion, these experiments indicate that BTK is functional downstream from miR-628-3p.

MiR-628-3p suppresses the growth of tumor in vivo

An U87 model was employed to assess the therapeutic value of miR-628-3p in vivo. As anticipated, the miR-628-3p-treated group's xenograft tumor weight significantly reduced as compared to the miR-NC-treated group (Fig. 7A). The same trend was observed for the tumor weight and volume (Figs. 7B and 7C). Western blot examination revealed that the miR-628-3p-treated group had decreased levels of BTK, Cyclin D1, and SOX2 expression (Fig. 7D). Ki-67 stains also demonstrated fewer proliferative cells in the tumor of the miR-628-3p-treated group than the miR-NC-treated group (Fig. 7E). Moreover, the survival time of mice treated with miR-628-3p was longer than that of mice treated with miR-NC (Fig. 7F). These findings demonstrated that miR-628-3p might successfully inhibit the development of glioma tumors in vivo.

The postoperative recurrence and poor prognosis of GBM patients are strongly associated with the features of fast proliferation and invasive growth, according to an increasing number of clinical studies and biological research[27]. Therefore, it is a milestone to explore its potential mechanism of action and provide help for the treatment of glioma. Many experiments have demonstrated that the dysregulation of miRNA is correlated with cancer cell growth, including glioma cells[28, 29].

We found that miR-628-3p was down-regulated in gliomas relative to normal brain tissue (NBT) and steadily declined with the rise in grade from the CGGA database and clinical samples. The expression of miR-628-3p was elevated in cells, and this modification significantly reduced the proliferation of glioma cells in the Colony formation and EdU experiments. The results of flow cytometry indicated that miR-628-3p overexpression resulted in G0/G1 phase cell arrest. Cell cycle control is greatly aided by protein kinase complexes. Cyclin D1, one of cycle-dependent kinases, may promote glioma cells to shift from G0/G1 phase to S phase [30, 31]. We also found that Cyclin D1 expression levels decreased when miR-628-3p was overexpressed. Considering the link between cell cycle and glioma stemness, we supposed that miR-628-3p regulated glioma proliferation and encouraged glioma stem cell-like phenotypes. A clonogenic and glioma steam cells cell viability assay indicated that overexpression of miR-628-3p inhibited GSC maintenance.

Previous studies have reported that BTK is related to glioma cells [19, 22]. Luciferase activity assays and western blot analysis were utilized to verify the influence of BTK in GBM cells. Thus, we have reason to speculate that BTK is a promoter in glioma cells and related to miR-628-3p. We also used a specific shRNA to knockdown the expression of BTK. From the EdU and colony formation assays, we found that the proliferation of U87 and U251 cells was effectively suppressed with the overexpression of miR-628-3p. Western blot examination exhibited that Cyclin D1 expression levels were downregulated, and the cell cycle was repressed as a result of the low BTK concentration. What’ s more, a clonogenic and glioma steam cells cell viability assay showed that blocking BTK could inhibit the maintenance of GSC. In contrast, the result of miR-628-3p on glioma cells was reversed because of the restoration of BTK. These results confirmed that BTK is a promoter for glioma development and is negatively regulated by miR-628-3p.

We constructed several tumor models to assess the impact of miR-628-3p in vivo. As shown by the Ki-67 stain and western blot studies, which showed that the miR-628-3p group had high levels of Cyclin D1 and SOX2 expression, the volume and weight of the tumors were noticeably smaller in the miR-628-3p group than in the miR-NC group. Moreover, the Kaplan-Meier curves demonstrated that the miR-628-3p-treated mice lived longer than miR-NC-treated mice.

We have verified that BTK plays a crucial function in glioma cells. Previous studies have documented that the BTK inhibitor (ibrutinib) can suppress tumorigenesis, stem cell phenotypes, and induce autophagy in glioblastoma[22, 24]. However, further studies are necessary to determine its mechanism. Collectively, our study decorated the critical function of the miR-628-3p/BTK axis in the GBM development.

In summary, we have demonstrated that miR-628-3p inhibits glioma development and is significantly down-regulated in glioma cells. BTK functions downstream from miR-628-3p and is a promoter in glioblastoma. We propose that the therapeutic target miR-628-3p might be employed to treat glioblastoma.

Funding

This study was sponsored by the Natural Foundation of Jiangsu Province (BK20161119) and the National Natural Science Foundation of China (Grant No. 81602212).

Acknowledgements

We thank the First Affiliated Hospital of Nanjing Medical University for providing relevant technical support.

Ethics approval

The experiment was checked and accepted by Nanjing Medical University's research ethics committee (Nanjing, Jiangsu Province, China), and participants' informed consent was obtained.

Competing interests

The authors claim to have no conflicts of interest.

Authors' contributions

Guanhua Xu (First Author): Conceptualization, Methodology, Software, Investigation, Formal Analysis, Writing - Original Draft;

Yuhang Wang: Data Curation, Writing - Original Draft,Methodology;

Wenping Cao: Visualization, Investigation;

Kun Lv: Resources, Supervision;

Fengqi Zhou: Software, Validation;

Ning Liu: Visualization, Writing - Review & Editing;

Binbin Wang and Rui Zhang (Corresponding Author): Conceptualization, Funding Acquisition, Resources, Supervision, Writing - Review & Editing.

Availability of data and materials

This article has all the data that were created or evaluated during this investigation.

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MicroRNA-628-3p inhibits glioblastoma cell proliferation and GSC maintenance by directly targeting BTK

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Abstract

Figures

Introduction

Materials And Methods

Public database and clinical human brain samples

Cell lines and transfection

RNA isolation and qRT-PCR

Fluorescence in situ hybridization (FISH)

Western blot analysis

Plasmid construction and transfection

Cell proliferation assay

Plate colony formation

5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay

Flow cytometric analysis of the cell cycle

Dual luciferase reporter assay

Glioma xenografts

Immunohistochemistry (IHC) assay

Statistical analysis

Results

The expression of miR-628-3p is markedly reduced in glioma tissues and glioma cell lines

Overexpression of miR-628-3p prevents the proliferation of glioma cells

Overexpression of miR-628-3p inhibits GSC maintenance in vitro

MiR-628-3p directly targets BTK mRNA

BTK induces glioma cell proliferation and GSC maintenance by activating Akt and ERK signaling pathways in vitro

Restoration of BTK attenuates miR-628-3p suppressed glioma cell proliferation and GSC maintenance

MiR-628-3p suppresses the growth of tumor in vivo

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1