The function of miRNA-148a in lymphatic metastases of papillary thyroid cancer and its mechanism were tested. In this investigation, miRNA-148a expression of lymphatic metastases of papillary thyroid cancer patients was inhibited, compared with normal group. We found that miRNA-148a overexpression was effectively reduced cell cell proliferation and metastases, and induced apoptosis of papillary thyroid cancer
Papillary thyroid cancer (PTC) ranks the first among endocrine tumors and head and neck tumors. According to statistics in recent years, the morbidity of PTC shows an increasing and younger trend, which has become one of the common malignancies (
The association of miRNA with tumor has been explained gradually accompanied by increase in research on microRNA (miRNA) (
miRNAs are a class of non-coding single-stranded small RNA molecules with the length of 19–23 nucleotides, which locate in the non-coding region of the genome (
Signal transducer and activator of transcription (STAT) is a kind of signal transduction and transcriptional activator, which is also a type of DNA binding protein (
Research on the correlation of PI3K/Akt/mTOR signaling pathway with malignant tumor has become the focus in recent years (
Written informed consent in the study was obtained from all patients before surgery and volunteer at Department of General Surgery, The Second Affiliated Hospital of Wenzhou Medical University. Peripheral blood (5 ml) of patients with lymphatic metastases of papillary thyroid cancer (n=6) and volunteers (n=6) were selected into this study and serum were collected after centrifuge at 2,000 × g for 10 min and saved at −80°C. This study was approved by the ethics committee of The Second Affiliated Hospital of Wenzhou Medical University. Samples were immediately snap-frozen in liquid nitrogen for store.
Total RNA was extracted from serum by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Genomic DNA was removed using RNase-free DNase RQ1 (10 U; Promega). Total RNA (100 ng) was used to reverse transcripted cDNA and qRT-PCR was performed with the Bio-Rad Real-Time PCR Detection system (CFX Connect™; Bio-Rad) using iQ™ SYBR® Green Supermix (CFX Connect; Bio-Rad). The relative expression of miRNA-148a was calculated using the 2−∆CT method.
Human PTC-derived TPC-1 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco) in a 5% CO2 atmosphere at 37°C. miRNA-148a (50 nM), si-STAT3 (50 nM) and negative control mimics (50 nM) were purchased from and transfected into TPC-1 cell using Lipofectamine 3000 (Invitrogen, Guangzhou, China). After transfected with miRNA-148a for 4 h, 100 nM of LY294002 was added into cells and cultured for 48 h.
After transfection for 24, 48 and 72 h, cells were placed in 96-well polystyrene tissue culture plates and 50 µl 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 1 µg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added and cultured for 4 h at 37°C. Medium was discarded and 150 µl dimethylsulfoxide (DMSO; Invitrogen Co., Australia) were loaded in each well for 20 min at 37°C. Absorbancy was measured at 492 nm with Microplate Reader Model 550 (Bio-Rad Laboratories, Japan).
After transfection for 24 h, TPC-1 cells were seeded into 24-well Transwell plates (Corning, Corning, NY, USA). After incubation for 24 h, cells were fixed and stained with 2% crystal violet solution in ethanol (Beyotime Biotechnology). Cell was observed using an IX71 fluorescence microscope (Olympus, Tokyo, Japan).
After transfection for 48 h, 1×106 cells/ml were counted and washed in PBS, re-suspended in binding buffer. Cells were stained with FITC-V and PI (Pharmingen, Becton-Dickinson Co., San Diego, CA, USA), and incubated for 15 min in the dark at room temperature. Cell apoptosis were analyzed by flow cytometry (FACSCalibur; Becton-Dickinson) using CellQuest software.
After transfection for 48 h, cells were lysed using Radio Immunoprecipitation assay lysis buffer (Beyotime Biotechnology) and protein concentration was determined by using a BCA Protein assay kit (Beyotime Biotechnology). Equal amount of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 6–10%), and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Western blotting was performed using antibodies against Bax, p-STAT3, PI3K, p-Akt and GAPDH (Cell Signaling Technology) at 4°C overnight. Membranes was conducted with a goat anti-rabbit IgG horseradish peroxidase (HRP) conjugated antibody for 1 h at 37°C and visualized using enhanced chemiluminescence (Goodbio Biotechnology). Protein blank was imaged with the Molecular Imager ChemiDoc™ XRSþ (Bio-Rad) and ImageLab software version 4.1 (Bio-Rad). Equal protein was used to measure caspase-3/9 activities using caspase-3/9 activities apoptosis kits (Beyotime Biotechnology). Absorbancy was measured at 405 nm with Microplate Reader Model 550 (Bio-Rad Laboratories, Japan).
Data are presented as the mean ± SD. Statistical comparisons between two groups were made using one-way ANOVA followed by least significant difference (LSD) post hoc test. p<0.05 was considered statistically significant.
To explore the role of miRNAs in gastric cancer, we performed qRT-PCR to analyze miRNA-148a expression. As showed in
We validated the function of miRNA-148a on cell proliferation and metastases of TC cells.
To verify our hypothesis, we analyzed apoptosis rate in TPC-1 cells by miRNA-148a overexpression using flow cytometry. Conversely, the apoptosis rate of TPC-1 cells transfected with miRNA-148a mimics were higher than that of control group (
Then, we analyzed Bax protein expression and caspase-3/9 levels of PTC cells by miRNA-148a overexpression. We found that miRNA-148a overexpression induced Bax protein expression and caspase-3/9 levels of PTC cells (
Western blot analysis was used to measure p-STAT3, PI3K and p-Akt protein expression of PTC cells after miRNA-148a overexpression. As shown in
Then, we downregulated p-STAT3 protein expression in PTC cells following miRNA-148a overexpression. As shown in
Then, we found that apoptosis rate of TPC-1 cells after miRNA-148a overexpression in si-STAT3 were higher than that of miRNA-148a overexpression group (
As expected, Bax protein expression and caspase-3/9 levels of PTC cells after miRNA-148a overexpression were markedly induced by si-STAT3, compared miRNA-148a overexpression group (
We next examined whether the inhibition of PI3K enforced miR-148a expression could suppress tumor growth of PTC. The results showed that PI3K inhibitor inhibits PI3K and p-Akt protein expression of PTC cells after miRNA-148a overexpression, compared with miRNA-148a overexpression group (
Then, PI3K inhibitor increased the anticancer effects of miRNA-148a overexpression on the inhibition of cell proliferation and metastases of PTC cells, compared with miRNA-148a overexpression group (
We determined whether PI3K inhibitor affects apoptosis of PTC cells after miRNA-148a overexpression. As shown in
Lastly, we found that PI3K inhibitor promoted the anticancer effects of miRNA-148a overexpression on the induction of Bax protein expression and caspase-3/9 levels of PTC cells, compared with miRNA-148a overexpression group (
PTC is the most commonly seen malignant endocrine system tumor, which severely impair female health (
So far, >700 miRNAs in human genome have been identified (
Caspase family includes important proteins for executing apoptosis discovered in research on the molecular mechanism of cell apoptosis in recent years. Among them, caspase-3/9 is one of the pro-apoptotic factors locating in the upstream of caspase cascade reaction. Moreover, it is also one of the most critical components in the caspase system that induces cell apoptosis (
STAT3 phosphorylation can result from multiple factors that promote the genesis and development of cancer. The activated STAT3 can inhibit cell apoptosis, promotes cell proliferation and differentiation, and results in carcinogenesis. Therefore, it is defined as an oncogene (
The growth factor receptor, PI3K, Akt and eIF-4E in the PI3K/Akt/mTOR signaling pathway are all proteins encoded by oncogenes (
In conclusion, our results indicate that miRNA-148a inhibits cell growth and metastases of papillary thyroid cancer through STAT3 and PI3K/AKT signaling pathways. To our knowledge, this is the first study to describe a potential role for miRNA-148a as potential targets in future studies on the prevention and treatment of papillary thyroid cancer.
miRNA-148a expression of lymphatic metastases of papillary thyroid cancer patients. Normal, normal group; PTC, papillary thyroid cancer patients group. ##p<0.01 compared with normal group.
miRNA-148a overexpression reduced cell proliferation and metastases of PTC cells. miRNA-148a overexpression (A), metastases (B) and cell proliferation (C) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group. ##p<0.01 compared with control group.
miRNA-148a overexpression reduces apoptosis of PTC cells. miRNA-148a overexpression reduced apoptosis rate (A) and cell apoptosis by flow cytometry (B). Control, control group; miRNA-148a, miRNA-148a overexpression group. ##p<0.01 compared with control group.
miRNA-148a overexpression induces Bax protein expression and caspase-3/9 levels of PTC cells. miRNA-148a overexpression induced caspase-3/9 levels (A and B), Bax protein expression by statistical analysis (C) and Bax protein expression by western blot analysis (D) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group. ##p<0.01 compared with control group.
miRNA-148a overexpression suppresses p-STAT3, PI3K and p-Akt protein expression of PTC cells. miRNA-148a overexpression suppressed p-STAT3, PI3K and p-Akt protein expression by statistical analysis (A-C) and p-STAT3, PI3K and p-Akt protein expression by western blot analysis (D) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group. ##p<0.01 compared with control group.
si-STAT3 inhibits p-STAT3 protein expression, reduced cell proliferation and metastases of PTC cells after miRNA-148a overexpression. p-STAT3 protein expression by statistical analysis (A) and p-STAT3 protein expression by western blot analysis (B), metastases (C) and cell proliferation (D) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group; miRNA-148a+si-STAT3, miRNA-148a overexpression + si-STAT3 group. ##p<0.01 compared with control group; **p<0.01 compared with miRNA-148a overexpression group.
si-STAT3 increased apoptosis of PTC cells after miRNA-148a overexpression. si-STAT3 increased apoptosis rate (A) and cell apoptosis by flow cytometry (B). Control, control group; miRNA-148a, miRNA-148a overexpression group; miRNA-148a+si-STAT3, miRNA-148a overexpression + si-STAT3 group. ##p<0.01 compared with control group; **p<0.01 compared with miRNA-148a overexpression group.
si-STAT3 induces Bax protein expression and caspase-3/9 levels of PTC cells after miRNA-148a overexpression. si-STAT3 induced caspase-3/9 levels (A and B), Bax protein expression by statistical analysis (C) and Bax protein expression by western blot analysis (D) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group; miRNA-148a+si-STAT3, miRNA-148a overexpression + si-STAT3 group. ##p<0.01 compared with control group; **p<0.01 compared with miRNA-148a overexpression group.
PI3K inhibitor, inhibits PI3K and p-Akt protein expression of PTC cells after miRNA-148a overexpression. PI3K and p-Akt protein expression by statistical analysis (A and B) and p-STAT3, PI3K and p-Akt protein expression by western blot analysis (C) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group; miRNA-148a+ PI3K inhibitor, miRNA-148a overexpression + si-STAT3 group. ##p<0.01 compared with control group; **p<0.01 compared with miRNA-148a overexpression group.
PI3K inhibitor reduces cell proliferation and induces metastases of PTC cells after miRNA-148a overexpression. inhibitor reduced metastases (A) and cell proliferation (B) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group; miRNA-148a+ PI3K inhibitor, miRNA-148a overexpression + si-STAT3 group. ##p<0.01 compared with control group; **p<0.01 compared with miRNA-148a overexpression group.
PI3K inhibitor increased apoptosis of PTC cells after miRNA-148a overexpression. PI3K inhibitor increased apoptosis rate (A) and cell apoptosis by flow cytometry (B). Control, control group; miRNA-148a, miRNA-148a overexpression group; miRNA-148a+PI3K inhibitor, miRNA-148a overexpression + si-STAT3 group. ##p<0.01 compared with control group; **p<0.01 compared with miRNA-148a overexpression group.
PI3K inhibitor induced Bax protein expression and caspase-3/9 levels of PTC cells after miRNA-148a overexpression. PI3K inhibitor induced caspase-3/9 levels (A and B), Bax protein expression by statistical analysis (C) and Bax protein expression by western blot analysis (D) of PTC cells. Control, control group; miRNA-148a, miRNA-148a overexpression group; miRNA-148a+ PI3K inhibitor, miRNA-148a overexpression + si-STAT3 group. ##p<0.01 compared with control group; **p<0.01 compared with miRNA-148a overexpression group.