miR-100-5p downregulates mTOR to suppress the proliferation, migration and invasion of prostate cancer cells

Background Previous studies have shown that miR-100-5p expression is abnormal in prostate cancer. However, the role and regulatory mechanism of miR-100-5p requires further investigation. Thus, the aim of this study was to observe the effects of miR-100-5p on the proliferation, migration and invasion of prostate cancer (PCa) cells and to explore the potential related regulatory mechanism. Methods Differential miRNA expression analysis was performed using next-generation sequencing (NGS) in the PCa cell line LNCaP and the normal prostatic epithelial cell line RWPE-1. The expression levels of miR-100-5pwere detected using real-time fluorescence quantitative PCR (qRT-PCR). LNCaP cells were transfected with NC-mimics or miR-100-5p mimics by using liposome transfection. Moreover, the CCK-8 proliferation assay, cell scratch assay and Transwell assay were used to detect the effects of miR-100-5p on cell proliferation, migration and invasion. In addition, the potential target gene of miR-100-5p was predicted, and the influence of miR-100-5p on the expression of mTOR mRNA by qRT-PCR and the expression of mTOR protein was detected by western blot and immunohistochemical staining. Results Differential expression analysis of high-throughput sequencing data showed low expression of miR-100-5p in the PCa cell line LNCaP. It was further confirmed by qRT-PCR that the expression of miR-100-5p in LNCaP cells was significantly lower than that in RWPE-1 cells (P<0.01). miR-100-5p expression in LNCaP cells was markedly upregulated after transfection with miR-100-5p mimics (P<0.01), while cell proliferation, migration and invasion capacities were clearly reduced (P<0.01), and mTOR mRNA and protein expression was also substantially lowered (P<0.01). Finally, we further confirmed by immunohistochemical staining that miR-100-5p regulated the expression of mTOR. Conclusion miR-100-5p is expressed at low levels in LNCaP cells, and it can suppress LNCaP cell proliferation, migration and invasion, the mechanism of which is related to downregulating the expression of mTOR.


Introduction
PCa is the most common malignant tumor of the male reproductive system.
According to the latest statistics, a projected 191,930 new cases of PCa will be diagnosed, accounting for more than 1 in 5 new diagnoses of male tumors. An estimated 33,330 men may die of the disease in the USA in 2020, and mortality due to PCa accounts for 10% of all cancer deaths. 1 With an increasingly aging population and changes in diet, the incidence of prostate cancer in Asia is increasing year by year. 2 MicroRNAs (miRNAs) are highly conserved noncoding single-stranded RNAs consisting of 21-24 nucleotides that regulate the expression of target genes through complete or incomplete complementarity binding with target genes and play an important role in the gene regulatory network. 3,4 Recent studies have found that miRNAs, as oncogenes or tumor suppressor genes, play an important role in the development and progression of malignant tumors. 5 A large number of studies have confirmed that miRNA expression is dysregulated in the occurrence and development of prostate cancer, and there is differential expression between prostate cancer patients and the normal population. 6,7 MiR-100-5p is an important member of the miRNA family, located on chromosome 11q24.1 and highly conserved. MiR-100-5p has abnormal expression in many malignant tumors, including prostate cancer and is involved in biological behaviors such as proliferation, migration and invasion of tumor cells, but the mechanism of action is still unclear. 8 In this study, we observed changes in the expression of miR-100-5p in the PCa cell line LNCaP and its effect on cell proliferation, migration and invasion ability and explored its potential molecular mechanism.
All cells were routinely cultured in a humidity incubator at 37°C and 5% CO2, and logarithmic-phase cells with good growth conditions were selected for subsequent experiments.

Construction of the cDNA library
MiRNAs were isolated and purified by gel electrophoresis, and cDNA was synthesized by reverse transcription. The miRNA sequencing library was obtained by PCR amplification. cDNA libraries were tested using an Illumina Hi Seq 2500 sequencer (GUANGZHOU RIBOBIO CO., LTD).

Differential expression analysis of miRNA
Edger analysis was used to analyze the significance of differences in miRNA expression in each group and calculate the P value, for which the -log10p value was calculated and the differentially expressed miRNAs were screened.

RNA preparation and RT-PCR
We isolated total RNA and total miRNA from cells by using TRIzol (Life Technologies) reagent and the mirVana miRNA Isolation Kit (Ambion). We treated total RNA with TURBO DNase (Ambion) after purification and used a high-capacity cDNA reverse transcription kit (Takara) to reverse transcribe RNA into first-strand cDNA. We purchased primers against mRNAs from Shanghai Sangon Biotech Co., Ltd. Each experiment was carried out in triplicate, and the mean value of the three-cycle threshold was used for further analysis. Cel-miR-39 was used as an internal control. The expression values of miRNAs were normalized to the U6 values, and the relative quantification analysis was performed using the 2 -ΔΔCt method. The primers for miR-100-5p were F: 5'-GAACCCGTAGATCCGAACT-3' and R:

Cell proliferation assay
The cells were proportionally diluted with culture medium and the OD value was measured after adding CCK-8 reagent for 8 h to generate a standard curve. The cell suspension (5000 cells/100 µl/well) was inoculated in a 96-well plate, and 10 µl CCK-8 solution was added to each well. The sample was incubated for 4 h, and the OD value was measured at 450 nm.

Cell scratch assay
LNCaP cells (5 × 10 3 cells/well) were cultured in a 24-well plate in RPMI-1640 culture medium with 10% FBS. The culture plate was incubated overnight at 37°C in a humidified CO2 incubator. Next, the culture medium was removed, and the adherent cell layer was scratched with a sterile 200 μL pipette tip. We washed away the cell fragments with phosphate-buffered saline (PBS). Images of the scratch area at 0 h and 24 h were taken using a built-in camera in the microscope (40x magnification).
Data were evaluated using TScratch imaging software (CSE Lab., ETH, Zurich) to calculate the percent wound area. 9 Transwell assay

Western blotting
The cell harvesting and extraction of proteins followed the method described previously. 10

Differential expression analysis of miRNAs
Compared with those in the normal prostate epithelial cells RWPE-1, 29 miRNAs in prostate cancer cells LNCaP were significantly differentially expressed ( figure 1).

Expression of miR-100-5p in LNCaP cells after transfection
The expression of miR-100-5p in the mimics group was significantly higher than that in the NC-mimics group (P<0.01) (figure 4), indicating that the transfection efficiency of miR-100-5p mimics was high.

Proliferation activity of LNCaP cells after transfection
The

Migration capacity of LNCaP cells after transfection
Compared with that of the NC-mimics group, the scratch healing rate of miR-100-5p mimics cells after transfection was significantly reduced (P<0.01), suggesting that the migration capacity of LNCaP cells after transfection was significantly reduced (figures 6) (figures 7).

Invasion ability of LNCaP cells after transfection
The results of the Transwell assay showed that the number of membrane-penetrating cells in the miR-100-5p mimics group was significantly lower than that in the NC-mimics group (P<0.01), suggesting that the invasion capacity of transfected cells was significantly reduced (figure 8).

Target gene prediction for miR-100-5p
A bioinformatics method was used to predict the potential target genes of miR-100-5p.
By analysis in miRanda (http://www.microrna.org), mTOR was predicted to be a target gene of miR-100-5p, and there were complementary binding sites of seed sequences between miR-100-5p and the 3UTR of mTOR ( figure 9).

Expression of mTOR mRNA and protein
RT-PCR results showed that mTOR mRNA expression in LNCaP cells transfected with miR-100-5p mimics was significantly lower than that in the NC-mimics group (P<0.01). Western blot analysis showed that mTOR protein expression in LNCaP cells transfected with miR-100-5p mimics was significantly lower than that in the NC-mimics group (P<0.01), as shown in figure 10.

Staining of bone tissue
Bone histopathological staining showed that miR-100-5p could significantly inhibit the occurrence and development of osteogenic bone metastases in prostate cancer

Discussion
MiRNA was first discovered by Lee in 1993 and is a kind of ncRNA of approximately 19-24 nt. 11,12 MiRNAs can bind to multiple target mRNA 3'UTRs and regulate gene expression, resulting in abnormal expression of target genes [13,14].
Abnormal miRNA expression has been associated with the occurrence and development of tumors. Studies have confirmed that differential expression of miRNAs in tumor tissues can be used as a biomarker for early detection, typing and prognosis of tumors. 15,16 Studies have demonstrated altered levels of miRNAs in the development of PCa, with differences between PCa patients and healthy individuals. 17,18 Studies have found that the expression of miR-100-5p is downregulated in prostate cancer, which is believed to be an important role of tumor suppressor genes in the development and progression of PCa. 19,20 It has been reported that the absence of miR-100-5p leads to the upregulation of AGO2 expression levels, thus promoting the migration, invasion and EMT of cancer cells and promoting the metastasis of prostate cancer. 21 In this study, the results of the NGS differential expression analysis showed that compared with those in RWPE-1 cells, miR-375, miR-200c-3p and miR-141-3p were upregulated, while miR-100-5p, miR-584-5p, and miR-125b-1-3p were found that the 3'UTR of miR-100-5p and mTOR has complementary seed sequence binding sites by using a bioinformatics method. Therefore, we predicted that mTOR might be a functional target gene of miR-100-5p. MTOR is a serine/threonine protein kinase that participates in physiological processes and pathological reactions by regulating protein synthesis and is related to the pathogenesis of cancer. 24 The MTOR signaling pathway mainly regulates cell proliferation and metabolism involved in tumor development and is an important signaling pathway related to human cancer. 25 In this study, we found that the expression levels of mTOR mRNA and protein decreased obviously in LNCaP cells after upregulation of miR-100-5p expression.
Next, nude mice in the two groups were administered by intraosseous injection

Disclosure
The authors report no conflicts of interest in this work.