Background
Oral squamous cell carcinoma (OSCC), the sixth mostcommon malignant tumor in humans, accounts for approximately 2% of all malignancies. 90% of oral carcinoma are squamous cells. There are approximately 350,000 new cases of oral carcinoma and up to 170,000 deaths annually worldwide [
1‐
3]. Although treatments for cancer such as surgery and chemoradiotherapy have improved greatly, the five-year survival rate of patients is still less than 70%, and it is difficult to achieve the purpose of a surgery alone or surgery with adjuvant radiotherapy [
4]. Therefore, it is of great significance for the treatment and prognosis to find potential molecular markers and therapeutic targets in the pathogenesis of OSCC.
MicroRNAs (miRNAs) are a class of small non-coding RNAs, which can affect the function of mRNA to regulate gene translation and protein synthesis by complementary pairing with mRNA. Recent studies have reported that multiple miRNAs play important roles in the occurrence and development of tumors by affecting the proliferation, invasion, and migration of tumor cells [
5,
6]. Studies have shown that miR-191 is a type of microRNA that plays a role in the occurrence and development of a variety of tumors through different pathways. In follicular thyroid carcinoma, decreased expression of miR-191 could increase tumor growth and cell migration by targeting CDK6 [
7]. Whereas in breast cancer, the expression of miR-191 increased, and increased expression of miR-191 promotes the proliferation and migration of breast cancer cells by targeting SATB1 [
8]. The expression of miR-191 was also increased in colorectal cancer, and the overexpression of miR-191 promoted the development of human colorectal cancer by targeting C/EBPβ [
9]. Studies have also suggested that overexpression miR-191 was involved in angiogenesis [
10]. These findings suggest that miR-191 in different tissues can produce multiple biological effects through different mechanisms. However, the function and mechanism of miR-191 in OSCC are remains unclear.
This study aimed to detect the expression of miR-191 in OSCC tissues and cell lines, and to explore the correlation between miR-191 and its target gene as well as its downstream signaling, and clarify the role and mechanism of miR-191 in OSCC. This study may provide new evidence for miR-191 as a promising gene therapy target for OSCC.
Materials and methods
Human tissue samples
Samples of OSCC tissues and the adjacent noncancerous tissues (2 cm adjacent to the cancerous tissues) used in this study were obtained from patients with primary OSCC (total of 30 cases) who were hospitalized in the Second Hospital of Shanxi Medical University underwent surgical resection. All patients did not receive chemotherapy or preoperative radiotherapy before surgery. The clinicopathological characteristics of these OSCC tissues are shown in Table
1. This study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (2022YX-219), and informed consent was obtained from all patients.
Table 1
Clinicopathological characteristics of patients with OSCC
Gender |
Male | 18 (60) |
Female | 12 (40) |
Age |
< 60 | 16 (53.3) |
> 60 | 14 (46.7) |
T stage |
T1/T2 | 27 (90) |
T3/T4 | 3 (10) |
TNM stage |
I +II | 22 (73.3) |
III+ V | 8 (26.7) |
Site of primary tumor |
Tongue | 7 (23.3) |
Floor of mouth | 1 (3.3) |
Buccal mucosa | 1 (3.3) |
Gingiva | 10 (33.3) |
Lip | 5 (17.7) |
Jaw | 6 (20.0) |
Histologic differentiation |
Well | 11 (36.7) |
Moderately | 16 (53.3) |
Poorly | 3 (10) |
Tumor size |
≤ 4 cm | 27 (90) |
> 4 cm | 3 (10) |
Lymph node metastasis |
Positive | 5 ( 16.7) |
Negative | 25 (83.3) |
Cell culture
Human OSCC cells (SCC-9 and CAL-27), and Human normal oral keratinocyte line (hHOK) cells used in this study were purchased from GuanDao Biological Engineering Co., Ltd. (Shanghai, China), human OSCC cells (SCC-4, HSC3, and SAS cells) were purchased from BeNa Bio Inc (Heibei, China). All cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% (v/v) penicillin–streptomycin (Gibco, USA). The cells were incubated at 37 ℃ in a constant temperature incubator with 5% CO2 concentration until logarithmic phase.
RNA extraction and real-time quantitative PCR (RT-qPCR)
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA). MiRNA, and mRNA were reverse transcribed into respective cDNAs using Mir-X miRNA First-Strand Synthesis and PrimeScript™ RT Reagent Kit (Takara Bio USA, Inc.), and then quantified using TB Green® Premix Ex Taq™ II (Takara Bio USA, Inc.) following the manufacturer’s instructions on a Quant Studio Real-Time PCR system (Applied Biosystems, CA). The expression levels of miR-191 relative to U6, and target genes of PLCD1 relative to GAPDH were determined as respective comparative cycle thresholds (ΔCt). Specific primers used for PLCD1 and GAPDH were as follows: PLCD1 forward 5’-ATGGTGGGACACGGAGTTTG-3’ and reverse 5’-GAGGTGGACATGGCGGTATC-3’; GAPDH forward 5’-GCACCGTCAAGGCTGAG AAC-3’ and reverse 5’-TGGTGAAGACGCCAGTGGA-3’.
Cell transfection
Following the instructions of transfection reagent Lipofectamine 2000 (Invitrogen, CA, USA), CAL-27 and SCC-9 cells were transfected with miR-191 mimics or miR-191 inhibitor (mimics-NC, and inhibitor-NC was used as a negative control) to increase or inhibit the activity of miR-191. All transfection plasmids were purchased from GenePharma Co., Ltd. (Shanghai, China).
Detection of cell proliferation
After transfection, cells of each group were seeded into 96 well plates at a density of 4 × 103/well. Cells were supplemented with 10 μL Cell Counting Kit 8 (CCK-8, Boster, Wuhan, China) in each well at 24, 48, 72, and 96 h after seeding, followed by incubation at 37 ℃ for 1 h, and the absorbance value was measured at 450 nm with a microplate reader.
Detection of cell invasion and migration
Transwell assay was employed to detect cell invasion and migration according to the manufacturer's protocol (BD Biosciences, Bedford, MA, USA). After 24 h of transfection, cells were resuspended in FBS-free DMEM, 100 μL of DMEM medium containing cells (5×104 for SCC-9,1×105 for CAL-27) were added to the upper chamber, and supplemented with 600 μL DMEM medium containing 10% FBS in the lower chamber. After 24 h of incubation, cells were fixed with methanol for 30 min and were stained with 0.1% crystal violet (Boster, Wuhan, China). Five fields were randomly selected for cell counting under microscope.
Detection of cell cycle
Forty-eight hours after transfection, cells were fixed with 75% ethanol for 24 h. After discarding ethanol, cells were resuspended in PBS and then stained using a cell cycle detection kit (Invitrogen, CA, USA) for 30 min, after which the cell cycle was measured by flow cytometry.
Western blot assay
Total protein was obtained using a protein extraction reagent (ThermoFisher, USA). The proteins were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). PVDF membranes were then blocked using 5% bovine serum albumin and placed at 4 ℃ overnight after the addition of primary antibodies. PVDF membranes were subsequently incubated in Horseradish Peroxidase (HRP) conjugated secondary antibodies (BIO-RAD, USA) for 1 h at room temperature. ECL chromogenic kit (Boster, Wuhan, China) was used for chromogenic development, and the gray values of the bands were analyzed.
Prediction and identification of target genes
Dual Luciferase assay
The target genes of miR-191 were predicted by bioinformatics software. The target gene of miR-191 was predicted by three software (MiRanda, TargetScan, and PicTar) and the human PLCD1 3'-UTR region targeted by miR-191 was amplified by PCR, and cloned into the PLCD1-3'-UTR plasmid obtained from luciferase assay to construct PLCD1-WT. Plasmid PLCD1-MUT with mutant binding site was also constructed as control. OSCC cells were co-transfected with pmiR-PLCD1-WT / pmiR-PLCD1-MUT, and miR-191 mimics or negative controls by using Lipofectamine 2000. Forty-eight hours after transfection, luciferase activity was measured using dual- luciferase reporter assay system according to the instructions of manufacturer.
Regulation of PLCD1 expression by miR-191
To further confirm the regulation of miR-191 on PLCD1 expression, RT-qPCR and western blot assays were used to detect the expression of PLCD1 in OSCC tissues and OSCC cells. MiR-191 mimics, miR-191 inhibitor, and NC were transfected into OSCC cells, and the mRNA expression level of PLCD1 in cells were detected and compared.
Analysis of the effect of miR-191 on proliferation, invasion and migration of OSCC cells by regulating expression of PLCD1
To further verify whether miR-191 inhibitor affect the proliferation, migration and invasion of OSCC cells by upregulating PLCD1, the OSCC cells were co-transfected with miR-191 inhibitor +small interfering RNA (siRNA) -NC, and miR-191 + siRNA-PLCD1. At 24, 48, 72, and 96 h after transfection, MTT assay was used to detect cell proliferation. Transwell assay was used to detect cell invasion and migration at 48 h after transfection.
Determination of the effect of miR-191 on the PLCD1 downstream signaling pathway
In order to further clarify the mechanism of miR-191 regulating OSCC cells growth, the protein expression of β-catenin in PLCD1 downstream signaling pathway was detected in OSCC cells transfected with miR-191 mimics / miR-191 inhibitor. The downstream genes expression of the β-catenin signaling pathway, including C-myc, matrix metalloproteinase-9 (MMP-9), CDK4 and PCNA protein were also detected. Since β-catenin pathway is involved in epithelial-mesenchymal transition (EMT) process, the EMT-related marker N-cadherin was further detected. The siRNA-PLCD1 was used to inhibit mRNA of PLCD1 to verify the effect of miR-191 on β-catenin, MMP-9, C-myc, N-cadherin, CDK4 and PCNA expression through PLCD1.
Xenograft model
Animal experiments in this study were approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (DW2022069) and were performed in accordance with Guide for the Care and Use of Laboratory Animals published by National Institutes of Health. Healthy female BALB/c nude mice (4 weeks old) were purchased from Gempharmatech Co., Ltd. (Jiangsu, China). According to the instruction of Lipofectamine 2000 reagent, OSCC cells were transfected with miR-191 enhancer (miR-191 agomir) and negative control (miR-191 agomir NC), miR-191 inhibitor (miR-191 antagomir), and negative control (miR-191 antagomir NC). At 48 h after cell transfection, 0.2 mL of OSCC cell suspension containing 2 × 106 cells was injected subcutaneously into the right axillary region of nude mice. Treatment was started on 7 day after cell injection. MiR-191 agomir, miR-191 antagomir and negative controls were injected directly into the tumors every 7 days. Tumor size was monitored by measuring tumor length (L) and width (W) with vernier calipers every 7 days prior to subsequent injection, and tumor volume was calculated using the following formula: volume (mm3) = length × width2× 0.5. After 28 days, the nude mice were sacrificed. The tumors in vivo were removed, weighed, and photographed.
Data analysis
All statistical analyses and plots were performed and generated using GraphPad Prism software (version 8.0; GraphPad Software, Inc, CA, USA). Student’s T-test and One-way analysis of variance (ANOVA) were used to compare the difference between groups. Data correlation between the two groups were analyzed by Pearson correlation. The measurement data were presented as the mean ± standard deviation (SD), and a P-value less than 0.05 or 0.01 was considered statistically significant.
Discussion
MiRNAs functions as oncogenes or tumor suppressor genes in many human tumors, affecting various stages of tumor formation and development, which can be used for early diagnosis and prognosis evaluation of tumors [
11]. In recent years, several studies have shown that miR-191 is abnormally expressed in a variety of tumor tissues and plays a role in tumor initiation and progression through different mechanisms [
12‐
18]. However, the function and mechanism of miR-191 in OSCC have not been clarified.
This study showed that expression level of miR-191 in OSCC tissues and cell lines was significantly higher than in adjacent non-neoplastic tissues and hHOK. In vitro results showed that miR-191 overexpression promoted cell proliferation, migration, invasion and cell cycle in OSCC cells. In vivo experiments showed that miR-191 overexpression promoted tumorigenesis of OSCC cells in nude mice. In contrast, downregulation of miR-191 expression inhibited proliferation, migration and invasion of OSCC cells, and inhibited tumorigenesis of OSCC cells in nude mice.
PLCD1 encodes an enzyme involved in energy metabolism, calcium homeostasis, and intracellular movement. It is located at 3p22 in a region that is frequently deleted in multiple cancers [
19]. Several studies have shown that PLCD1 has anti-cancer effects. PLCD1 has been confirmed to play a cancer suppressive effect in colorectal cancer [
20], gastric cancer [
21], esophageal squamous carcinoma [
22], pancreatic cancer [
23], breast cancer [
24], chronic myeloid leukemia [
25]. Studies have confirmed that overexpression of PLCD1 can promote the apoptosis of colorectal tumor cells, arrest the cell cycle at G1 / S phase, and inhibit proliferation, invasion and migration of colorectal tumor cells [
26]. Mu et al. [
27] found that PLCD1 could significantly promote G2 / M cell cycle arrest and apoptosis in human breast cancer cells, thereby significantly inhibiting the proliferation of human breast cancer cells. However, the function and mechanism of PLCD1 in OSCC have not been clarified.
In vitro experiments, this study demonstrated that miR-191 could directly target the 3′-UTR of PLCD1 and the expression of miR-191 was negatively correlated with expression of PLCD1. Inhibiting the expression of miR-191 significantly increased the expression of PLCD1 in OSCC cells, and inhibited the proliferation, migration and invasion of OSCC cells, suggesting that miR-191 promoted the proliferation, migration, invasion and cell cycle of OSCC cells by targeting PLCD1. In vivo animal tests further proved these conclusions that overexpressed miR-191 promoted tumor growth and invasion in nude mice by inhibiting PLCD1, and decreasing miR-191 expression promoted PLCD1 and reduced tumor growth and infiltration in nude mice.
Activation of the Wnt/β-catenin pathway has been found in many human tumors [
28], and activation of its target genes can promote tumor development by affecting cell migration, proliferation, and angiogenesis [
29]. A study from He et al. [
19] proved that PLCD1 could downregulate expression of β-catenin, leading to a decrease in the level of phosphorylated β-catenin. Upregulation of PLCD1 can inhibit the activity of β-catenin pathway in esophageal cancer. Our study demonstrated that overexpressed miR-191 promoted expression of β-catenin, and inhibition of miR-191 expression could decrease activity of β-catenin, suggesting that miR-191 could affect the proliferation, migration and invasion of OSCC cells through β-catenin pathway by targeting PLCD1. C-myc has been recognized as a proto-oncogene that can be malignant by uncontrolled cell proliferation due to its overexpression, and the degradation of extracellular matrix by MMP-9 plays a critical role in tumor invasion and metastasis [
30,
31]. CDK4 overexpression can activate the progression of the cell cycle, and regulates progression from G1 to S phase of the cell cycle. Dysregulated CDK4 contributes to abnormal cell proliferation and tumor development [
32]. Proliferating cell nuclear antigen (PCNA) is closely related to DNA synthesis and cell cycle in cells [
33]. Studies have shown that β-catenin signaling pathway can promote proliferation and metastasis of tumor cells by activating the transcription of various oncogenes, such as MMP9, C-myc, and CDK4 [
32,
34‐
36]. Therefore, we examined the effect of enhancing or inhibiting miR-191 on the expression of MMP9, C-myc, and CDK4 which are the major target genes in the downstream pathway of β-catenin. We further examined the effect of enhancing or inhibiting miR-191 on the expression of PCNA. Western blot assay showed that the expression of MMP9, C-myc, CDK4 and PCNA significantly increased after enhancing miR-191 expression, and significantly decreased after inhibiting miR-191 expression.
EMT was closely related to cell migration and invasion ability [
37,
38]. Since β-catenin pathway is involved in EMT process [
39], we further explored whether miR-191-mediated β-catenin increasing affects EMT-related marker N-cadherin. Western blot results showed that N-cadherin gene expression was significantly increased after overexpression of miR-191, and inhibition of miR-191 significantly decreased the expression of N-cadherin.
In order to verify the effect of downregulation of miR-191 on the expression of β-catenin, MMP-9, C-myc, N-cadherin, CDK4 and PCNA protein in OSCC cells on the basis of downregulation of PLCD1. OSCC cells were co-transfected with siRNA-PLCD1 and miR-191 inhibitor, and the expression of β-catenin, C-myc, MMP9, N-cadherin, CDK4 and PCNA detected by western blot assay. The results showed that downregulation of miR-191 inhibited the expression of β-catenin, MMP-9, C-myc, N-cadherin, CDK4 and PCNA protein in OSCC cells, and downregulation of PLCD1 on the basis of downregulation of miR-191 could partially recover the effect of downregulation of miR-191 on the protein expression of β-catenin, MMP-9, C-myc, N-cadherin, CDK4 and PCNA in OSCC cells, suggesting that miR-191 regulates the expression of β-catenin, MMP-9, C-myc, N-cadherin, CDK4 and PCNA through targeting PLCD1.
These results suggest that miR-191 promotes proliferation, migration and invasion of OSCC cells, which may be associated with the decrease of PLCD1 and then increase of β-catenin, and ultimately lead to the increase of MMP-9, C-myc, N-cadherin, CDK4 and PCNA. Inhibition of miR-191 increase PLCD1, and then decrease β-catenin, MMP-9, C-myc, N-cadherin, CDK4 and PCNA, ultimately leading to the decrease of proliferation, migration and invasion of OSCC cells, thus exerting anti-tumor effects.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.