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Upregulation or downregulation of microRNAs (miRNAs) has been identified in human cervical cancer (CC). However, the character and function of miR-378 in CC remains unknown. In the present study, the authors demonstrated that miR-378 was upregulated in CC used the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay, and promoted cell proliferation by accelerating the progress of cell cycle and repressing cell apoptosis in CC cells. The predicted target genes of miR-378 were determined by enhanced green fluorescent protein (EGFP) reporter assays, RT-qPCR assay and western blot analysis. miR-378 suppressed the expression of suppression of tumorigenicity 7-like (ST7L) by targeting the 3′-untranslated region (3′-UTR) of ST7L mRNA in HeLa and SiHa cells. ST7L was downregulated in CC using the RT-qPCR assay, and the malignant phenotype of HeLa and SiHa cells were inhibited by ST7L overexpression. In addition, miR-378 activated the Wnt/β-catenin pathway by targeting ST7L in CC cells. In short, miR-378 functions as an onco-miRNA by directly downregulating ST7L mRNA and protein level in HeLa and SiHa cells, and serves important roles in the malignancy of CC.
Cervical cancer (CC) is one of the most familiar cancers and a leading cause of cancer-associated mortality in women (
MicroRNAs (miRNAs or miRs) include a number of endogenous small non-coding RNAs (approximately 18–22 nucleotides) that serve important roles in controlling gene-targeted expression at the post-transcriptional level by degradation of mRNA or inhibition of translation (
Suppression of tumorigenicity 7-like (ST7L) was identified based on its similarity to the ST7 tumor suppressor gene (
In the present study, the authors demonstrated that miR-378 might function as an oncogene by promoting cell growth, accelerating the cell cycle and inhibiting cell apoptosis by directly downregulating ST7L in HeLa and SiHa cells. Overexpression of the miR-378-activated Wnt/β-catenin pathway in CC. Collectively, these findings may provide insight into tumorigenesis and a potential biomarker for CC.
A total of 27 pairs of human cervical tissue, consisting of human CC and matched normal cervical tissue from the same patient, were used in the study. Written informed consent was obtained from all enrolled patients, and all relevant investigations were performed according to the principles of the Declaration of Helsinki. The samples were received from the Department of Oncology, Xintai Affiliated Hospital of Taishan Medical University, Taian, China. Total RNA was extracted from the human samples and purified using the miRVana miRNA Isolation kit (Ambion, Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The study was approved by the Ethical Review Committee of Xintai Affiliated Hospital of Taishan Medical University (Ethics approval number: 20150023).
Normal human endocervical epithelial cell lines (Endl/E6E7) was obtained from Shanghai Medical College, Fudan University (Shanghai, China), which were cultured in KER-SFM medium supplemented with 10% calf serum (Biological Industries, Carlsbad, CA, USA) at 37°C with 5% CO2. Other cervical cancer cells used in the present study were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were cultivated in RPMI-1640 (Invitrogen, Thermo Fisher Scientific) supplemented with 8% fetal calf serum (FCS; Biological Industries), 100 U/ml penicillin, and 100
For miR-378, an overexpression vector (pri-miR-378) containing a miR-378 precursor region was amplified from the genomic DNA and inserted into the vector of pcDNA3. Pri-miR-378-S, 5′-CGACGCGTCGGGCTGCG AGGAGTGAGCG-3′ and Pri-miR-718-AS, 5′-CCATCGATGGGAGTTCAAATGGCTTGCTCC-3′. The 2′-O-methyl-modified miR-378 antisense oligonucleotide (ASO-miR-378) was commercially synthesized as an inhibitor of miR-378. ASO-miR-378, 5′-CCUUCUGACUCCAAGUCCAGU-3′ and ASO-NC, 5′-CAGUACUGUAGUGUAGUACTT-3′. The segment of 3′-UTR of ST7L containing the miR-378 targets was acquired by polymerase chain reaction with gene-specific primers, and then cloned into pcDNA3/enhanced green fluorescent protein (EGFP) following the stop codon of luciferase with
Total RNA was extracted with TRIzol reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions. The quality and integrity of acquired total RNA was evaluated by NanoDrop 2000c; Thermo Fisher Scientific, Inc. (Wilmington, DE, USA) and 1% gel electrophoresis, respectively. For RT-qPCR, 2
Cervical cells were seeded into the plates of a 96-well plate at 5,000 cells/well one day prior to transfection. The HeLa and SiHa cells were transfected with pri-miR-378, ASO-miR-378, or the respective control vectors. Cell viability at 24, 48 and 72 h post-transfection was determined by MTT assay. The absorbance values at 490 nm were measured via the Quant Microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).
For the colony formation ability assay, the HeLa and SiHa cells were counted at 24 h post-transfection and seeded into 24-well plates at 500 cells/well. Culture medium was replaced every 72 h. After approximately two weeks, cells were cleaned with 1X phosphate-buffered saline (PBS), stained with common crystal violet dye, and colonies containing >50 cells were counted.
The hypothetical targets of miR-378 were predicted using TargetScan 7.1, RNAhybrid and
The EGFP reporter plasmids with ST7L 3′-UTR or ST7L 3′-UTR-mut were transfected into HeLa and SiHa cells with Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific) and RFP expressing plasmid was integrated as a transfection efficiency control. Cells were lysed 48 h post-transfection, and the intensities of EGFP and RFP fluorescence were determined with a spectrophotometer.
Cell extracts were cleaned with 1X PBS buffer, prepared with RIPA buffer supplemented with cocktail, and protein concentrations were quantified using the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocols. Equal amounts of proteins were separated by 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for ~2 h, then followed by incubation with the primary antibodies against GAPDH (1:2,000; WL01547; Wanlei Biotech Co., Ltd., Beijing, China) and ST7L (1:1,000; 17567-1-AP; Proteintech Co., Ltd., Wuhan, China) overnight at 4°C. After washing with TBST, the blots were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (1:5,000; A0216; Beyotime Institute of Biotechnology) at 37°C for 1 h. Thereafter, the proteins of interest were visualized using enhanced chemiluminescence (ECL; Wanlei Biotech) and densitometric analysis was performed using Gel-Pro Analyzer system (Beijing Liuyi Instrument Factory, Beijing, China). LabWorks™ Image Acquisition and Analysis software UVP EC3 (UVP, LLC, Upland, CA, USA) were used to quantify band intensities.
At 48 h after transfection, transfected CC cells were harvested by trypsinization and resuspended in cold PBS for analysis. For the analysis of cell cycle, cells stained with propidium iodide (PI) according to the manufacturer's manual. The rate of cell apoptosis was detected using an Annexin V-FITC/PI apoptosis detection kit (Nanjing Kaiji Biotechnology Development Co., Ltd., Nanjing, China). These analyses were conducted according to the protocol provided by Nanjing Kaiji Biotechnology Development.
To assay the transcriptional activity of Wnt pathway, pri-miR-378, pST7L, or pri-miR-378 and pST7L treated cells were co-transfected with either the Wnt signaling reporter TopFlash or the negative control FopFlash according to the protocol (EMD Millipore). HeLa cells were transiently transfected with either 2
Data are presented as the means ± standard deviation (SD) from at least three independent experiments. Statistical analyses were performed using Student's t-tests. P<0.05 was considered to indicate a statistically significant difference.
To investigate the role and clinical significance of miR-378 in cervical cancer, the authors first detected the level of miR-378 by RT-qPCR assay in 27 pairs of human cervical tumors and matched normal cervical tissues. Compared with the corresponding non-tumorous counterparts, the miR-378 expression level was significantly upregulated in cervical tumor tissues (
The roles of miR-378 in cervical cancer cells were evaluated by transfection of miR-378 overexpression or knockdown plasmids in HeLa and SiHa cells (
miR-378 significantly promoted the growth of HeLa and SiHa cells, so it was speculated that miR-378 could accelerate the cell cycle process in cervical cancer cells. The authors reported that overexpression of miR-378 obviously decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S and G2 phase in both HeLa and SiHa cells compared with cells transfected with the controls; however, knockdown of miR-378 increased the percentage of cells in the G1 phase and decreased the percentage of cells in the S and G2 phase in HeLa and SiHa cells by flow cytometry assay. In addition, cells treated with pri-miR-378 markedly increased the proliferation index of HeLa and SiHa cells, cells treated with ASO-miR-378 decreased the proliferation index of HeLa and SiHa cells, respectively (
miRNAs serve important roles in cell growth, cell differentiation, cell apoptosis, cell cycle and other physiological and pathological processes by binding to the 3′-UTR of target genes, thus, regulating its expression of mRNA and protein level. According to TargetScan 7.1, miRDB and
First, the authors detected the mRNA level of ST7L by RT-qPCR in 27 pairs of cervical tumors and matched normal cervical tissues and in cervical cancer cells and human normal cervical epithelium cell. The mRNA level of ST7L was significantly decreased in human cervical cancer tissues (
Inactivation of Wnt/β-catenin signaling caused by ST7L by repressing β-catenin expression could explain this phenomenon to a certain extent in epithelial ovarian cancer (
Previously, miRNAs have appeared as a highlighted class of gene regulators and dysregulation of miRNAs plays important roles in the genesis and development of various cancers including cervical cancer (
It is well known that miRNAs can play roles by relying on the level of complementarities with the 3′-UTR of their target mRNAs (
ST7L, also as known as ST7R, STLR and FAM4B, was confirmed with its similarity to the ST7 tumor suppressor gene found in the chromosome 7q31 region and clustered with the WNT2B gene in a tail-to-tail manner in a chromosomal region known to be deleted and rearranged in many cancers, including germ cell tumors, breast cancer and non-small cell lung cancer, amongst others (
In conclusion, these reports confirmed that upregulation of miR-378 is a common phenomenonin CC tissues and CC cells, and the authors have identified that miR-378 takes effect in regulating cell proliferation, cell cycle, cell apoptosis and the Wnt/β-catenin pathway of CC cells. miR-378 inhibits ST7L expression at both the mRNA and protein level by directly targeting the 3′-UTR of the ST7L transcript. With its regulation of cervical cancer cell malignant phenotype, knockdown of miR-378 functions as a novel therapeutic strategy in CC.
miR-378 was upregulated in CC tissues and CC cells. (A) The levels of miR-378 in CC and normal tissues were examined by RT-qPCR assay. (B) The levels of miR-378 in C33A, HeLa, SiHa, CaSKi cells and End1/E6E7 cells were examined by RT-qPCR assay. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. the adjacent normal tissues; #P<0.05, ##P<0.01, ###P<0.001 vs. End1/E6E7 cell line. CC, cervical cancer; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
miR-378 served as an onco-miRNA in cervical cancer cells. (A) The efficiency of miR-378 overexpression and knockdown plasmids was confirmed by reverse transcription-quantitative polymerase chain reaction assay. HeLa and SiHa cells were transfected with the pri-miR-378 or ASO-miR-378 and the control groups, respectively. (B) The cell viability of miR-378 on HeLa and SiHa cells were determined by MTT assay. Overexpression of miR-378 promoted cell viability and knockdown of miR-378 inhibited cell viability. (C) Relative colony formation rate of HeLa and SiHa cells with indicated treatment was determined by colony formation assay. Overexpression of miR-378 promoted colony formation ability and knockdown of miR-378 inhibited colony formation ability. Original magnification, ×1. (D) Flow cytometry cell cycle assays demonstrated that miR-378 increased the number of HeLa and SiHa cells in the S and G2 phases and decreased the number of HeLa and SiHa cells in G1 phase. Overexpression of miR-378 enhanced the proliferation index. (E) Flow cytometry cell apoptosis assays revealed that miR-378 overexpression suppressed the apoptosis of HeLa and SiHa cells and miR-378 knockdown promoted cell apoptosis. (F) The expression levels of cleaved PARP and caspase-3 were examined by western blot assay in HeLa cells under the described condition. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. the pcDNA3 group; #P<0.05, ##P<0.01, ###P<0.001 vs. the ASO-NC group. miR, microRNA; ASO, antisense oligonucleotide; NC, negative control; ns, not significant; pri, primary.
miR-378 directly targets ST7L in cervical cancer cells. (A) The predicted miR-378 binding sites using TargetScan 7.1 in ST7L mRNA 3′-UTR and the sites of ST7L mRNA 3′-UTR-mut is shown. (B and C) HeLa and SiHa cells were co-transfected with pcDNA3/EGFP-ST7L 3′-UTR or 3′-UTR-mut and pri-miR-378 or ASO-miR-378. EGFP intensity was determined by spectrophotometer, and the value of the control group (pcDNA3 or ASO-NC) was set to one. (D) HeLa and SiHa cells were transfected with pri-miR-378 or ASO-miR-378. ST7L mRNA level in HeLa and SiHa cells were measured by reverse transcription-quantitative polymerase chain reaction assay. (E) ST7L protein level in HeLa and SiHa cells transfected with pri-miR-378 or ASO-miR-378 and the respective controls was determined by western blot assay. Data are presented as mean ± SD (n=3). **P<0.01, ***P<0.001 vs. the pcDNA3 group; #P<0.05, ##P<0.01 vs. the ASO-NC group. ns, not significant; miR, microRNA; 3′-UTR, 3′-untranslated region; mut, mutant; pri, primary ASO, antisense oligonucleotide; EGFP, enhanced green fluorescent protein; NC, negative control.
The mRNA level of ST7L was downregulated in CC tissues and CC cells. (A) The mRNA levels of ST7L in CC and normal tissues were examined by RT-qPCR assay. (B) The mRNA levels of ST7L in C33A, HeLa, SiHa, CaSKi cells and End1/E6E7 cells were examined by RT-qPCR assay. (C) The protein levels of ST7L in C33A, HeLa, SiHa and CaSKi cells and End1/E6E7 cells were examined by western blot assay. (D) The correlation of the expression of miR-378 and ST7L in tumor tissues was presented using SPSS 19.0. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. adjacent normal tissues; #P<0.05, ##P<0.01, ###P<0.001 vs. the End1/E6E7 cells. CC, cervical cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR, microRNA.
ST7L functions as a tumor suppressor gene in cervical cancer cells. (A) Reverse transcription-quantitative polymerase chain reaction assay indicated that overexpression and knockdown of ST7L in HeLa and SiHa were efficient. (B) MTT assay showed that overexpression of ST7L inhibited cell viability and knockdown of ST7L increased cell viability in HeLa and SiHa cells. (C) Colony formation rates were lower following transfection with pST7L and higher following transfection with shR-ST7L compared with the control groups. Original magnification was ×1. (D) The cell cycle profiles after treatment with pST7L or shR-ST7L were detected by flow cytometry assay. (E) Cell apoptosis assay showed that overexpression of ST7L promoted the apoptosis and that knockdown of ST7L inhibited the apoptosis of HeLa and SiHa cells. (F) Western blot analysis showed that overexpression of ST7L increased the levels of cleaved caspase-3 and PARP and the silencing of ST7L had the opposite effect. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. the pcDNA3 group; #P<0.05, ##P<0.01, ###P<0.001 vs. the pSilencer group.
miR-378 positively regulates the Wnt/β-catenin pathway. (A–C) HeLa cells were transfected with the indicated combinations of pri-miR-378, pST7L, pri-miR-378 and pST7L or the control groups. (A) Western blot assay was used to detect the expression level of β-catenin, C-myc and cyclin D. (B) Immunofluorescence assay was used to examine the nuclear distribution of β-catenin. Original magnification was ×1000. (C) Top/Fop luciferase reporter assays were performed to detect the β-catenin activity. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. the pcDNA3+pcDNA3 group; ##P<0.01, ###P<0.001 vs. the pcDNA3+pST7L group. miR, microRNA; pri, primary.