Laryngeal cancer (LC) is found in the upper respiratory tract of the human body and is the most common type of head and neck cancer globe wide. LC mainly develops in males, but with exposure to such as smoking, alcohol, and air deterioration, LC is becoming more frequent and de-sexualized ( 1 , 2 ). The current treatment protocols for LC include surgical resection, radiotherapy, and chemotherapy, etc. However, due to the high recurrence after surgery and damage caused by radiotherapy and chemotherapy to the weak body, the clinical treatment outcome is not satisfactory ( 3 , 4 ). To eliminate the pain of patients, there is an urgent requirement to find specific biological targets in the development of LC, to provide a theoretical basis for the development of specific drugs and the optimization of clinical treatment strategies.
MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 bases that are involved in regulating various life activities such as apoptosis, cell division, and cell proliferation ( 5 , 6 ). By binding to the 3ʹUTR of target messenger RNA (mRNA), miRNAs repress/promote specific mRNA translation and participate in post-translation regulation ( 7 ). MiRNAs are involved in cancer initiation, drug escaping, and cancer metastasis as well ( 8 , 9 ). For example, miR-107 inhibits the development of hepatocellular carcinoma by increasing drug sensitivity ( 10 ); high expression of miR-125b in pancreatic cancer accelerates cancer progression and metastasis ( 11 ), and miR-139 promotes prostate cancer progression by acting on RIG-1 ( 12 ). Currently, the disordered expression of multiple miRNAs like miR-376a ( 13 ), miR-892a ( 14 ), miR-29a-3p ( 15 ), miR-145-5p ( 16 ) and others are involved in the development of LC. Also, miR-106a-5p is involved in the development of a variety of cancers and has anti- or promote roles in different cancers. In hepatocellular carcinoma (HCC), the FER1L4-miR-106a-5p regulatory axis was found to promote drug sensitivity in HCC cells ( 17 ); mesenchymal stem cell-derived miR-106a-5p promoted cancer growth via exosomes delivery to breast cancer cells ( 18 ), and miR-106a-5p expression promoted proliferation and invasion of ovarian cancer cells ( 19 ). However, the specific role of miR-106a-5p in LC remains unclear.
We found that the level of miR-106a-5p was considerably elevated in LC tissues compared to adjacent tissues. However, the exact mechanism remains unclear. We conducted a preliminary work using commercial cell lines in vitro. This study is an initial validation of miR-106a-5p, and provides a theory to reveal the mechanism of LC carcinogenesis and progression.
To demonstrate the significance and specific mechanisms of miR-106a-5p in the development and metastasis of LC through clinical sample testing and cell biology approaches.
3. Materials and Methods
3.1. Clinical Specimens
The LC tissues and paired normal adjacent tissues (>0.5 cm from the safe border of the tumor) were collected from 30 patients who underwent surgery from Jan 2019 to Oct 2020. All patients had not received any treatments before sampling (details presenting in supplement Table 1). All specimens were placed into liquid nitrogen quickly. The study was approved by the ethics committee of First Affiliated Hospital of Jinzhou Medical University (No. 202288), informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.
3.2. Cell Lines and Culture
Human LC cell line AMC-HN8, TU212 (AC338377, HTX2130) was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The cells were cultured in DMEM medium (Gibco, USA) + 10% fetal bovine serum (FBS, Gibco, USA) and incubated at 37 °C and 5% CO2.
3.3. Cell Transfection
Logarithmically grown cancer cells were digested with 0.5% trypsin and inoculated into 6-well plates (2-3×105 cell), then miR-106a-5p sponge virus (Hanheng, Shanghai; 5’-CUACCUGCACUGUAAGCACUUUU-3’)or miR-106a-5p mimic (Hanheng, Shanghai, 5’-UCUA CUCUUUCUAGGAGGUUGUGA-3’) and liposome transfection reagent (Polyplus, France) were used in serum-free medium. The stable infections were screened by using puromycin and then detected by RT-PCR.
3.4. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Tissues or cells were washed with PBS and then lysized (1 mL Buffer RLM). RNA was then extracted using an RNA extraction kit (TaKaRa, Japan) and miR-106a-5p was synthesized using the Takara One Step PrimeScript miRNA cDNA Synthesis Kit (TaKaRa, Japan). Real-time PCR (ABI) was applied to amplify miR-106a-5p, U6 was used as the internal reference, and the 2-∆∆CT values were used to represent the fold difference of miR-106a-5p in the experimental group relative to each group.
3.5. Dual Luciferase Reporting Assay
AMC-HN8 (TU212) cells were incubated in 24-well plates for 24 hours. The pGL3-AKTIP-3’-UTR wt and pGL3-AKTIP-3’-UTR mutant reporter plasmids were constructed in advance. AMC-HN8 and TU212 cells were mixed with miR-106a-5p mimics or miR-106a-5p inhibitors with 0.1 μg reporter plasmids and transiently transfected using Lipofectamine 2000 according to the manufacturer’s instructions. 48 h later, the dual luciferase reporter gene assay system (Promega, USA) was used to detect luciferase activity and recorded using a 96-microplate reader (Promega, USA).
3.6. Western Blot
The treated cells were fully lysed to extract proteins and prepare suspensions, 10% SDS-PAGE were prepared, electrophoresed at 40V for 4-5h, then transferred to nitrocellulose membranes and blocked, antibody incubation was performed, and finally visualized and image analysis was carried out. PI3K/p-PI3K (Abcam, ab154598, UK), AKT/p-AKT (Abcam, ab38449, UK), mTOR/p-mTOR (Abcam, ab134903, UK), Bcl-2 (Invitrogen, PA5-11379, USA), Bax (Invitrogen, 14-6997-82, USA), GAPDH (Invitrogen, 39-8600, USA).
3.7. Colony Formation Assay
The treated AMC-HN8 and TU212 LC cells were prepared into single-cell suspensions. The cell suspensions of each group were inoculated in 6-well culture plates (400 cells/well) and incubated at 5% CO2 and 37 °C for 48h. The cells were washed twice with PBS, fixed with 4% paraformaldehyde liquid for 20min then discarded, and an appropriate amount of Jimson’s staining solution was added to them and stained for 30min, washed, and dried at room temperature. Microscopically observe the number of cell clones.
3.8. Cytometry Analysis
Cell cycle: Cells were harvested and washed with PBS. 5mL or more of pre-cooled 70-80% ethanol was added drop by drop, mixed the cells overnight at 4 °C (>18 hours). The cells were Centrifuged at 1000-1500 rpm for 10 minutes and discard the supernatant. Cell I/RNase staining: Cells are resuspended in 0.5 mL PI/RNase (Invitrogen, USA) staining solution and incubated for 15 minutes at room temperature and avoiding light. The samples were stored at 4°C and protected from light before analysis and tested by flow cytometry within 1 hour.
Apoptotic: The cells were collected after digestion by EDTA-free trypsin (Life, USA, trypsin digestion time should not be too long) and then washed twice with PBS (2000 rpm, 5 min), 1-5×105 cells were prepared; 500 μL of Binding Buffer was added to resuspend the cells; 5 μL of Annexin V-FITC (Life, USA,) was added and mixed, then 5 μL of PI (Life, USA,) was added and mixed; the reaction was carried out for 5-15 min at 25 °C and protected from light; the cells were observed and detected by flow cytometry within 1 hour.
3.9. Wound Healing Assay
The LC cells were planted into 6-well plates at concentration of 2×106, cells were cultured with DMEM medium containing 10% FBS. When the cells spread across the bottom of wells, a 200ul pipettor was taken and scratched across the diameter of each well of the 6-well plate, washed 3 times with PBS, and recorded under the microscope. Serum-free medium added and keep culture. 8h or 24h after, the scratch distance was observed and measured. Calculate the migration rate respectively. Image results were taken with the microscope at 10x.
3.10. Transwell Assay
LC cells were digested with trypsin, and then the single-cell suspension was prepared. 2 × 104 LC cells were added to the upper compartment, and the lower compartment added culture medium containing 10% FBS and HGF (20 ug.mL-1) (GlpBio, USA). Under the incubation condition of 37 °C and 5% CO2 ,the compart-ment was put into the incubator for 24 h. The LC cells of each group were fixed with 1 mL 4% formaldehyde for 10 min then washed, then stained with 1 mL 0.1% crystal violet solution for 30 min, and then washed with PBS. Count under a microscope after drying (20x).
3.11. Statistical Analysis
All statistical analyses were carried out using SPSS (13.0). Statistical analyses were performed using the One-way ANOVA. Data were showed with mean + SEM. P-values less than 0.05 were considered to be statistically different.
4.1. MiR-106a-5p is Over-Expressed in LC Tissues and Cell Lines
To investigate the role of miR-106a-5p in the development of LC, we first examined the expression of miR-106a-5p using clinical tissues (in paired cancer and adjacent tissues). RNA was extracted from the tissues and cDNA was obtained by reverse transcribed. Using qRT-PCR experiments we found that miR-106a-5p expression was significantly higher in LC tissues (5.71±1.65 foldchange) than in adjacent cancer tissues (2.66±0.87 foldchange), p<0.001 (Fig. 1A). We also examined LC cell lines, AMC-HN8 and TU212, for the expression of miR-106a-5p. The results showed that the expression of miR-106a-5p was notably higher in LC cell lines compared to normal cells (Fig. 1B), consistent with the clinical tissue test findings. To investigate whether the high expression of miR-106a-5p promotes the development of LC, miR-106a-5p was knockdown by miR-106a-5p inhibitor transfection in AMC-HN8 and TU212 cells. PCR results showed that intracellular miR-106a-5p was accurately targeted and reduced the miR-106a-5p expression (Fig. 1C, D).
The above results tentatively showed that miR-106a-5p expression was significantly increased in LC tissues and cells, but the exact role of miR-106a-5p needs further investigation.
4.2. MiR-106a-5p Promote LC Cells Proliferation In Vitro
We first studied the effect of miR-106a-5p on the proliferation of LC cells. By clonogenesis assay we found that after miR-106a-5p was inhibited, the number of clonogenesis of LC cells was significantly decreased compared to control and NC groups (Fig. 2A, D), indicating that the growth of LC cells was inhibited. Clonogenesis rates of AMC-HN8 cells were: 49.02±0.63 % (control), 48.83±1.53 % (NC), 37.17±0.76 % (miR-inhibitor), respectively. The clonogenic rates of TU212 cells were: 50.96±2.56 % (control), 52.33±2.36 % (NC), and 35.50±2.65 % (miR-inhibitor), respectively. In addition, we further analyzed the cell cycle distribution and apoptosis of cancer cells after miR-106a-5p inhibition using cell flow cytometry assays.
The results show that AMC-HN8 and TU212 cells transfected with miR-106a-5p inhibitor presenting a significant decrease in S-phase cells and a significant increase in G1-phase cells (Fig. 2B, E), indicating that the transition from G1 to S-phase of tumor cells was blocked after miR-106a-5p inhibition. The apoptosis assay revealed that the apoptosis rate of cancer cells after miR-106a-5p inhibition was significantly higher than that of the control and NC groups (Fig. 2C, F). The apoptosis rates of AMC-HN8 cells were: 9.19±0.21 % (control), 9.08±0.14 % (NC), 10.76±0.6 % (miR-inhibitor); the apoptosis rates of TU212 cells were: 4.4±0.29 % (control), 4.5±0.12 % (NC) and 25.35±0.17 % (miR-inhibitor), respectively. These results indicated that miR-106a-5p inhibition could prevent cancer cells from progressing from the G1 phase to the S phase, which in turn led to blocked cell proliferation and increased apoptosis, suggesting that miR-106a-5p could promote the growth and development of LC.
4.3. MiR-106a-5p Facilitate LC Cells Migration and Invasion
Next, we investigated the role of miR-106a-5p in the migration and invasion of LC cells by wound healing and Transwell assays. In AMC-HN8 cells, we found that the migration rate of cancer cells was significantly reduced by transfection with miR-106a-5p inhibitor (Fig. 3A), and the cell migration rates in control, NC, and miR-inhibitor groups were 64.10±5.94 %, 71.43±8.45 %, and 38.96±5.87 %, respectively. The results of the Transwell assay showed that the invasive ability of the cells was significantly decreased after miR-106a-5p inhibition comparing to control and NC (Fig. 3B). We have observed the same results in TU212 cells, where cells transfected with miR-inhibitor had significantly lower migratory and invasive abilities than both control and NC groups (Fig. 4A, B). These results suggest that miR-106a-5p plays a role in promoting cell metastasis and invasion in LC cells.
4.4 MiR-106a-5p Activate PI3K/AKT/mTOR Pathway Through AKTIP
Further, the specific mechanism of miR-106-5p in LC was studied. By TargetScan, we found that the AKT interacting protein (AKTIP) was one of the specific targets of miR-106-5p. In vitro, the expression of AKTIP in LC cells was dramatically decreased after miRNA inhibitor addition in culture medium (Fig. 5A). To verify the specificity of this interaction, sequence mutations were modified in the miRNA binding region (Fig. 5B, GCACUUU) and a dual luciferase reporting assay was performed (Fig. 5C). The figures show that the luciferase activity was decreased in the wt group rather than the mut group demonstrating that the specific interaction between miR-106-5p and AKTIP mRNA. AKTIP (encode by FT1/FTS) plays a role in cell apoptotic and activates the protein kinase/AKT pathway by enhancing the phosphorylation state. As shown in Figure. 5D, the phosphorylated PI3K/AKT/mTOR pathway was decreased when miR-106-5p was knockdown, and meanwhile, the Bcl-2 was declined and Bax was up-regulated, indicating that the cell was apoptotic.
It has been widely documented that miRNA play an important role in cancer progression ( 10 ). MiR-106a-5p, a member of the miR-17 family, is abnormally expressed in several tumors and plays a pro- or anti-cancer role ( 20 ). However, the contribution of miR-106a-5p in LC has not been described. In the present study, we found that miR-106a-5p was generally overexpressed in LC clinical tissues and commercial cell lines, as well as that there was a significant reduction in both cell proliferation and migration capacity and an increase in apoptosis after inhibition of miR-106a-5p expression in LC cells. Mechanismly, AKTIP was down-regulated and PI3K/AKT/mTOR pathway was inactivated by miR-106a-5p inhibition, demonstrating that the miR-106-5p-AKTIP- PI3K/AKT/mTOR axis mediates LC development.
Many miRNAs are involved in LC modulation. Recently, it was reported that miR-892a was found to be elevated in LC tissues and that inhibition of its expression significantly reduced cell proliferation ( 14 ). CeRNA (competing endogenous RNAs) are thought to be a new mechanism by which miRNAs function. LncNEAT1 (Long noncoding RNA NEAT1), a novel oncogene in LC, competitively binding to miR-29a-3p, thereby inhibits the regulation of downstream targets by miRNAs ( 15 ). Previous study shows that miR-106a-5p increases the sensitivity of head and neck cancers to radiotherapy by directly acting on RUNX3, thus exhibiting an anti-cancer efficacy ( 21 ). Furthermore, in a recent study in nasopharyngeal carcinoma, the anti-cancer potency of miR-106a-5p was also found. The LncRNA SMAD5-AS1 was act as a sponge on miR-106a-5p to promote the epithelial-mesenchymal transition (EMT) of cancer cells and accelerate cancer metastasis, but abrogated by over-expression of miR-106a-5p ( 22 ). In another study it was similarly demonstrated that miR-106a-5p was at a low level in kidney cancer tissues and cells, however, their experiments revealed that miR-106a-5p exhibited anti-oncogenic effects through the regulation of VEGFA ( 23 ). However, what we found in LC was quite the contrary. We found that miR-106a-5p was significantly more abundant in LC tissues than adjacent tissues in clinical specimens. We then constructed LC cell lines that inhibited miR-106a-5p expression in AMC-HN8 and TU212 cells, and found that the clonogenic ability of LC cells was significantly decreased after miR-106a-5p inhibition. Flow cytometric analysis showed that most of the cells stopped at the G1 phase, and apoptosis increased when cell proliferation decreased. The migration and invasion ability of LC cells were then examined by wound healing assay and Transwell assay. The results showed that the migration rate and invasion ability of AMC-HN8 and TU212 cells were reduced with different degrees after miR-106a-5p inhibition. Therefore, our results demonstrated that miR-106a-5p promote the development of LC. Most recently, and consistent with our results, Li (J Cell Mol Med. 2021 Oct;25(19):9183-9198) found that serum levels of exosomal miR-106a-5p were higher in chemotherapy-resistant and final-cycle nasopharyngeal carcinoma patients than in non-resistant and first-cycle patients, and that exosomal miR-106a-5p enhanced the proliferation of nasopharyngeal carcinoma cells.
As we know, the mechanism of miRNAs action is to target specific genes and regulate downstream genes expression and to signal pathway responses. Based on Targetscan databases, AKTIP was found and selected for mechanism study. AKTIP associated with telomerase complex and mediate cell growth and programmed death ( 24 ). Here, we have proved that AKTIP was down-regulated by miR-106a-5p inhibition, and their interaction have been verified by dual luciferase reporting assay. In previous reports, AKTIP has been proved promotes cell’s proliferation and cell connection through PI3K/AKT/mTOR signaling pathway ( 25 ). To determine whether the promotion of LC by miR-106a-5p was mediated by the PI3K/AKT/mTOR pathway, we examined the activation status of this pathway using WB assays after miR-106a-5p inhibition and found that the levels of p-PI3K, p-AKT, and p-mTOR were significantly reduced, and the expression of Bcl-2, a protein associated with cell proliferation, was also reduced; In contrast, the expression of the apoptotic protein Bax was increased. These results confirm our suspicions about the mechanism of miR-106a-5p in LC development.
The pro-cancer role of PI3K/AKT/mTOR signal pathway was fully studied as one of the major cellular signaling pathways. The PI3K/AKT/mTOR pathway regulates cell growth, metabolism and motility. Genetic members of this pathway have been extensively studied and found to be frequently activated in human cancers ( 26 ). Recently, Wang et al., recently, have found that miR-214 regulates the activation of PI3K/AKT/mTOR pathway in cervical cancer ( 27 ). In another hand, m ( 6 ). A modification also mediated the enhanced phosphorylation in PI3K/AKT/mTOR pathway ( 28 ). Inhibition of the PI3K/AKT/mTOR pathway has been shown to benefit human tumors regression and has been validated in numerous clinical trials ( 29 ). Meanwhile, a number of molecular inhibitors specifically targeting this pathway, such as Idelalisib, Arqule, Everolimus, etc. were approved by the Food and Drug Administration after demonstrating excellent performance and safety in clinical trials ( 30 ). In this study, we identified an important role of miR-106a-5p-AKTIP-PI3K/AKT/mTOR pathway axis in the development of LC for the first time, providing a theoretical basis for personalized clinical treatment and the development of new molecular inhibitors in LC.
In this study, we found that the level of miR-106a-5p was elevated in LC tissue than normal, further experiments were conducted using cell lines, and we demonstrated that the expression of miR-106a-5p was significantly raised in LC cell lines. Moreover, we found that miR-106-5p mediated the activation of PI3K/AKT/mTOR pathway by interacting with AKTIP, thus promoting the growth and invasion of LC. This study is a first step in the study of miR-106a-5p in LC carcinogenesis and development. The specific mechanism of miR-106a-5p in LC needs to be further explored.
We appreciate the support from the Instructional Science & Technology project of Jinzhou and the Project of Jinzhou Medical University. Meanwhile, a great thanks to the First Affiliated Hospital of Jinzhou Medical University for the research platform offered in this study.
This work was supported by the Instructional Science & Technology project of Jinzhou City (NO: JZ2022B051); Crosswise Research Project of Jinzhou Medical University (NO: 2022007)
Conflict of Interest
All authors have completed the ICMJE uniform dis- closure form. The authors have no conflicts of interest to declare.
The study was approved by the ethics committee of First Affiliated Hospital of Jinzhou Medical University (No. 202288); informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.
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