miR-378a-3p regulates glioma cell chemosensitivity to cisplatin through IGF1R

Abstract Glioma is a type of common intracranial tumor. In this study, we investigated the molecular mechanism by which miR-378a-3p regulates cisplatin (CDDP) chemosensitivity in glioma cells via insulin-like growth factor 1 receptor (IGF1R). U251/CDDP cells were treated with CDDP and transfected with miR-378a-3p mimics, NC mimics, or pcDNA-IGF1R. qRT-PCR was used to measure the differential level of miR-378a-3p. CCK-8 assay was used to test cell proliferation, and flow cytometry was used to analyze apoptosis. The targeting relationship between miR-378a-3p and IGF1R was tested through a dual-luciferase reporter gene assay. In contrast to normal glial cells, the miR-378a-3p level decreased in human glioma U251 cells and had lower expression in U251/CDDP cells. Compared with the CDDP group, miR-378a-3p significantly caused the inhibition of U251/CDDP cell proliferation and enhanced apoptosis in the miR-378a-3p mimics + CDDP group. Another experiment confirmed that IGF1R was a target gene of miR-378a-3p, and overexpression of miR-378a-3p inhibited IGF1R expression. In addition, co-overexpression of miR-378a-3p and IGF1R induced the upregulation of the U251/CDDP cell proliferation and the inhibition of apoptosis in the miR-378a-3p mimics + pcDNA-IGF1R + CDDP group. This study confirmed that miR-378a-3p promoted the sensitivity of glioma cells to CDDP in glioma patients via targeting IGF1R to increase the therapeutic effect during chemotherapy.


Introduction
Glioma is an intracranial tumor, and patients experience increased intracranial pressure due to its mass effect in space, which can lead to vomiting, vision loss, psychiatric symptoms, or localized epilepsy [1]. Glioma can also result in limb pain and numbness, motor and sensory impairment, language expression, and understanding difficulties in patients due to the effect of glioma on the local brain tissue function [2]. Surgical resection is the main treatment for glioma, while chemotherapy and radiotherapy are important adjuvant therapies for glioma [2]. However, the therapeutic effect of chemotherapy is not ideal since cells from glioma patients are prone to develop drug resistance.
Cisplatin (CDDP) is an effective DNA alkylating agent and is the most common and effective chemotherapeutic drug in clinical use. It has a good therapeutic effect on many solid tumors [3]. However, prolonged use of CDDP can lead to drug resistance in humans. It has been reported that CDDP may cause nephrotoxicity or hepatocardiotoxicity through oxidative stress, DNA damage, and inflammation [3,4]. Therefore, CDDP is of limited use due to its resistance and toxicity to nontargeted tissues. In order to solve the problem of drug resistance to CDDP in glioma patients, it is necessary to further explore the mechanisms underlying the drug resistance of CDDP in glioma and therefore strategically improve the chemosensitivity of glioma cells.
MicroRNAs (miRNAs) are small RNAs of highly conserved endogenous noncoding proteins that can bind to the 3′ untranslated regions (UTR) end of mRNAs for exerting their roles in negatively regulating gene expression [5]. miRNAs play an important regulatory role in drug resistance in various tumors, such as gastric cancer, cervical cancer, and particularly glioma [6,7]. Among them, miR-378a-3p is a suppressor of tumor cells such as colorectal cancer, lung cancer, and breast cancer [8][9][10]. However, the regulatory relationship between miR-378a-3p and the sensitivity of glioma cells to chemotherapy is unknown.
As a growth regulator, insulin-like growth factor 1 (IGF1) has a molecular structure similar to insulin and is an essential active substance for human growth [11]. IGF1 receptor (IGF1R) is able to mediate IGF-1 action, and the IGF1R signaling pathway is closely related to the occurrence, development, and metastasis of tumors, which is an important indicator for clinical cancer diagnosis and prognosis [12]. However, the specific effect of IGF1R on glioma is still unclear.
Therefore, the aim of the present study was to validate the involvement of IGF1R in the regulatory effects of miR-378a-3p on CDDP chemosensitivity in glioma cells.

Cell transfection
Cells at the logarithmic growth stage were taken, counted, and spread in 6-well plates. After cells were plastered, 4 μL (50 pmol/μL) of miR-378a-3p mimics was added to 100 μL of double-free medium (no serum, no antibiotics) and placed at room temperature for 5 min. The diluted miR-378a-3p mimics were mixed with 100 μL of Lipofectamine 2000 diluent and then added to 6-well plates and incubated at 37°C for 1 h. Next, the mixture was aspirated and then added to a complete medium (containing serum and antibiotics) and incubated for another 48 h. The absorbance values at a wavelength of 450 nm were tested using a microplate reader, and the cells were analyzed to determine proliferative ability.

Detection of apoptosis in U251/CDDP cells by flow cytometry
U251/CDDP cells were treated with CDDP or transfection and then were digested and collected using 0.25% trypsin (Sigma) for 48 h. The digested cells were washed twice using 400 μL of pre-chilled 1× PBS, centrifuged, and the supernatant was discarded. The cells were resuspended with the binding buffer, added to 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI), and incubated for 10 min [13]. The apoptotic ratio of cells was surveyed by CyFlow ® Cube 8 flow cytometry (Sysmex-Partec, Germany).

Dual-luciferase reporter gene assay
Cells were co-transfected with IGF1R-3′ UTR wild-type (WT) or IGF1R-3′ UTR mut and miR-378a-3p mimics or NC mimics. Cells were digested with 0.25% trypsin, and the supernatant was then discarded after centrifugation. The luciferase activity was measured based on the Dual Luciferase Assay Kit (Zeye, Shanghai, China). The relative activity of luciferase was equal to the firefly luciferase activity value divided by the sea kidney luciferase activity value.

Statistical processing
The data were analyzed using Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA), and the experimental results were expressed as mean ± standard deviation. The one-way analysis of variance (ANOVA) was used for data analysis among three or more groups, and the least significant difference t-test was performed when significant differences were determined. Differences were considered statistically significant at p < 0.05.

Results
3.1 miR-378a-3p was downregulated in drug-resistant U251/CDDP cells The results in Figure 1a showed that miR-378a-3p expression was reduced in U251 cells (p < 0.01) compared with HA cells, while the lowest expression of miR-378a-3p was seen in U251/CDDP cells (p < 0.01). The resistance of U251 cells and U251/CDDP cells to CDDP was examined under different concentration gradient conditions. The results of the CCK-8 assay in Figure 1b showed that the IC 50 for CDDP was 1.5 μg/mL in U251 glioma cells and 45 μg/mL in U251/CDDP cells, indicating the drug-resistant ability of U251/CDDP cells. Taken together, miR-378a-3p was downregulated in drug-resistant U251/CDDP cells, indicating its potential role in drug resistance.

Effects of miR-378a-3p on the chemosensitivity of U251/CDDP cells
The qRT-PCR results in Figure 2a indicated that the level of miR-378a-3p was significantly increased in the miR-378a-3p mimics group as compared to NC mimics (p < 0.01), which suggested the successful transfection of miR-378a-3p mimics in U251/CDDP cells. The degree of resistance to CDDP after transfection with miR-378a-3p/NC mimics in U251/CDDP cells was examined under different concentration gradient conditions. The results of the CCK-8 assay in Figure 2b showed that the IC 50 for CDDP in the miR-378a-3p mimics group was 10 μg/mL, and the IC 50 for CDDP in the NC mimics group was 45 μg/mL in U251/CDDP cells, indicating that miR-378a-3p mimics decreased the resistance of U251/CDDP cells to CDDP. In Figure 2c, the administration of CDDP suppressed the proliferation of U251/CDDP cells as compared to the control group ( p < 0.05 at 24 h, p < 0.01 at 48 h). Then, the proliferation of U251/CDDP cells was significantly inhibited in the miR-378a-3p mimics + CDDP group compared with the NC mimics + CDDP group ( p < 0.001). The results in Figure 2d revealed that CDDP administration induced U251/CDDP cell apoptosis ( p < 0.01). Then, the apoptosis rate of U251/CDDP cells was clearly increased in the miR-378a-3p mimics + CDDP group compared with the NC mimics + CDDP group ( p < 0.001). To sum up, miR-378a-3p could accelerate the inhibitory effects of CDDP on the proliferation and also aggravated the promotive effects of CDDP on the apoptosis of U251/CDDP cells.

miR-378a-3p targets and negatively regulates IGF1R
As shown in Figure 3a, the bioinformatics online analysis website miRanda and Targetscan predicted that IGF1R might be a target gene of miR-378a-3p. The results in Figure 3b showed that miR-378a-3p mimics remarkably inhibited the luciferase activity of IGF1R-wt (p < 0.01), while miR-378a-3p mimics had no inhibitory effect on the luciferase activity of IGF1R-mut, illustrating that miR-378a-3p could target IGF1R. Figure 3c displayed that IGF1R expression was reduced in the CDDP group compared to the control group (p < 0.01), while IGF1R expression was clearly decreased in the miR-378a-3p mimics + CDDP group as compared to that in the NC mimics + CDDP group (p < 0.001). Therefore, IGF1R was negatively regulated by miR-378a-3p.

miR-378a-3p promotes the chemosensitivity of CDDP/U251 cells by regulating IGF1R
As shown in Figure 4a, IGF1R expression was lower in the miR-378a-3p mimics + CDDP group as compared to the control group (p < 0.001). IGF1R expression was increased in the miR-378a-3p mimics + pcDNA-IGF1R + CDDP group as compared to the miR-378a-3p mimics + pcDNA + CDDP group, indicating the successful transfection of IGF1R overexpression vector in U251/CDDP cells (p < 0.001). The results in Figure 4b and c showed that the proliferation was increased and the apoptosis was inhibited in U251/CDDP cells in the miR-378a-3p mimics + pcDNA-IGF1R + CDDP group as compared to the miR-378a-3p mimics + CDDP group (p < 0.001). Thus, the effects of miR-378a-3p on promoting apoptosis and inhibiting proliferation of U251/CDDP cells were reversed by IGF1R.

Discussion
Glioma accounts for about 80% of primary tumors in the human brain, which is highly malignant with a poor  prognosis [15]. Currently, surgery is the mainstay of treatment supplemented by chemotherapy, radiotherapy, and immunotherapy to further improve the therapeutic effect [16]. CDDP is a widely used and effective broad-spectrum antitumor drug among platinum compounds and is used in the treatment of many tumors, such as glioma, cervical cancer, osteosarcoma, and human oral cancer [17,18]. However, drug resistance is also one of the main reasons for discontinuation during chemotherapy [19]. The reasons for CDDP resistance include immunity of the targeted cells to the drug component and expulsion of CDDP before it has a chance to damage the cancer cell DNA.
Hence, drug resistance limits the clinical application of CDDP and affects the treatment of glioma. Studies have reported that various compounds, such as ascorbic acid, lemon oil, and royal jelly [4,20,21], can improve the toxic effects on the kidneys of mice through antioxidant and oxidative effects. Therefore, the specific mechanism of drug resistance development in glioma needs to be further investigated to explore new therapeutic targets.
miRNAs are currently considered as most used regulators to investigate the mechanism of drug resistance. As an oncogenic factor, miR-378a-3p is able to participate in the development of various tumors. miR-378a-3p was lowly expressed in the development of breast cancer, lung cancer, and colorectal cancer [8][9][10]. Consistently, the present study illustrated that miR-378a-3p was lowly expressed in human glioma U251 cells and U251/CDDP cells. In addition, the degree of CDDP resistance in U251/CDDP cells was examined, and the results suggested that lower levels of miR-378a-3p in U251 cells may be associated with CDDP resistance. miR-378a-3p was found to be downregulated and inhibited the proliferation and cell cycle in colorectal cancer cells [22]. Besides, miR-378a-3p can downregulate MAPK1, thereby inhibiting CDDP sensitivity in ovarian cancer cells [23]. The present study revealed that the cell proliferative ability was inhibited, while the cell apoptosis was significantly increased in the miR-378a-3p mimics + CDDP group as compared to the CDPP group, demonstrating that miR-378a-3p could promote the sensitivity of U251/CDDP cells to chemotherapy.
The present study identified IGF1R as a target gene of miR-378a-3p. IGF1R belongs to the receptor-type tyrosine kinase family and is highly expressed in many tumor cells, such as glioma, breast cancer, gastric cancer, and lung cancer [12,24]. IGF1R has a potential mitogenic role in promoting cell proliferation, regulating malignant transformation of cells, protecting tumor cells from apoptosis and other biological functions [25]. Besides, miRNA-532 was shown to inhibit the development of colorectal cancer by directly targeting IGF1R to inhibit the PI3K/Akt pathway [26]. It was further confirmed that miR-378a-3p was able to target and downregulated IGF1R and thus aggravated the inhibitory effects of CDDP on the proliferative capacity of U251/CDDP cells.
In conclusion, miR-378a-3p can directly inhibit the growth of glioma cells and promote apoptosis by targeting IGF1R expression, thereby enhancing the sensitivity to CDDP. Therefore, miR-378a-3p can be used as a chemoresistant target and provide a new idea for the clinical treatment of glioma. However, we also found that miR-378a-3p could participate in the chemosensitivity of glioma cells to cisplatin by regulating multiple targets in previous studies. Therefore, in future studies, we will conduct more studies on other miR-378a-3p target genes to further understand the mechanism of miR-378a-3p in improving the sensitivity of glioma to CDDP.
Funding information: The authors state no funding involved.
Author contributions: Yunjiang Wang designed the study and supervised the data collection; Jia Du analyzed the data and interpreted the data; Yunjiang Wang and Jia Du prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Conflict of interest:
The authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.