miR‑660‑5p is associated with cell migration, invasion, proliferation and apoptosis in renal cell carcinoma
Retraction in: /10.3892/mmr.2024.13210
- Authors:
- Published online on: November 14, 2017 https://doi.org/10.3892/mmr.2017.8052
- Pages: 2051-2060
Abstract
Introduction
Renal cancer is the seventh most common cancer in men and the tenth most common cancer in women, accounting for 5 and 3% of all malignant tumors in men and women, respectively (1). Renal cell carcinoma (RCC) is a major subtype of renal cancer, and accounts for ~90% of all renal cancers (2,3), with a male-to-female ratio of ~2:1 (4). Clear cell RCC is an aggressive form with a prevalence of 85% and is the most common histological type of RCC (5,6). Although the etiology of RCC is not well understood, some risk factors of RCC are well known, including hypertension, obesity and smoking (7). There were 5,900 new cases of RCC diagnosed in Canada in 2012 and >65,150 new cases were reported in the United States in 2013 (4,8). Following surgical treatment, recurrence and metastasis still occur in 50% of patients with RCC (9). It is inefficient to use chemotherapy or radiotherapy to treat RCC (10); therefore, it is important to identify a useful tumor marker to assist the diagnosis of RCC.
MicroRNAs (miRNAs) are non-coding RNAs that are 20-23 nucleotides in length, which are cleaved from hairpin-shaped pre-miRNA (11). Through binding to the 3′-untranslated regions of messenger RNA, miRNAs serve a crucial role in a number of biological processes, including cell growth, proliferation, apoptosis, differentiation, migration and metabolism (11–14). Previous studies have demonstrated that dysregulated miRNA expression occurred in a variety of cancers and miRNAs may act as oncogenes when upregulated or tumor suppressors when downregulated (15–17). Owing to the imperfect complementarity between miRNAs and mRNAs, one mRNA may be regulated by several miRNAs and one miRNA is able to regulate several mRNAs (16). Therefore, miRNAs have a potential value in clinical practice, such as tumor markers for diagnosis, prognosis and possibly novel treatments.
Previous studies have revealed that miRNA (miR)-660-5p expression is dysregulated in many human malignancies, such as lung cancer (18), breast cancer (19), multiple myeloma (20) and chronic lymphocytic leukemia (21). However, the clinical significance and function of miR-660-5p in RCC remained to be explored. Four previous microarray chip studies have demonstrated that miR-660-5p was downregulatd in RCC (18,19,22,23). In the present study, the expression of miR-660-5p in RCC tissues and cell lines was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), followed by functional analyses of miR-660-5p in RCC cell migration, proliferation, invasion and apoptosis.
Materials and methods
Human patient sample collection
A total of 25 paired RCC tissues and normal adjacent tissues (NATs; which were located 2 cm outside of the visible RCC lesions) were collected in the Peking University Shenzhen Hospital (Shenzhen, China) between 2012 and 2014. Clinicopathological and histological diagnostics for patients with RCC were determined according to the 2009 American Joint Committee on Cancer staging system (Table I) (24). Patients with RCC enrolled in the present study received neither chemotherapy nor radiotherapy prior to tissue sampling. Once removed, all tissue samples were immediately immersed in RNAlater (Qiagen GmbH, Hilden, Germany) and frozen in liquid nitrogen (−80°C) for further study. The present study was approved by the Ethics Committee of Peking University Shenzhen Hospital, and written informed consent was obtained from all patients.
Cell culture
The human RCC cell lines 786-O and ACHN line were obtained from American Type Culture Collection (Manassas, VA, USA), and were seeded and grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% glutamate (Gibco; Thermo Fisher Scientific, Inc.) and 1% antibiotics (100 U/ml penicillin; 100 mg/ml streptomycin). All cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C.
Cell transfection
A total of 3×105 cells were seeded into 6-well plates and cultured for 24 h at 37°C prior to transfection. To upregulate or downregulate miR-660-5p expression, 200 pmol synthesized miR-660-5p mimic or inhibitor (Shanghai GenePharma Co., Ltd., Shanghai, China; Table II), respectively, as well as 200 pmol mimic and inhibitor negative controls (NCs), were transfected into cells (once the cells had reached 60–80% confluence) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h, which were mixed in the Opti-MEM I Reduced Serum Medium (Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After transfection for 6 h, transfection efficiency was verified by RT-qPCR subsequently (25). The different concentrations of RNA used for transfection in subsequent assays have been used according to the manufacturer's protocol for use with different size plates.
Total RNA extraction, cDNA synthesis and RT-qPCR
Total RNA was extracted from cells (2.0×106 cells) and tissues (25 paired RCC samples and normal tissue) with TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and purified with the RNeasy Maxi kit (Qiagen GmbH), according to the manufacturer's protocol. RNA concentration was measured on a NanoDrop 2000c (Thermo Fisher Scientific, Inc.); RNA samples with a 260/280 ratio between 1.8 and 2.1 were used for further investigation. Total RNA (1 µg) from each sample was reverse transcribed into cDNA using the miScript Reverse Transcription kit (Qiagen GmbH), according to the manufacturer's protocol. miR-660-5p expression levels were detected with miScript SYBR-Green PCR kit (Qiagen GmbH) and qPCR using the Roche LightCycler 480 Real-Time PCR system (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's protocol. Primer sequences used in the present study are shown in Table II; U6 small nuclear RNA was used as the internal control. PCR thermocycling conditions were as follows: Initial denaturation at 95°C for 15 min, followed by 40 cycles of 94°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec. The expression levels of miR-660-5p were analyzed using the 2−ΔΔCq method (26). qPCR was performed in triplicate for each set. The log2 ratio (RCC/NAT) expression of miR-660-5p was calculated by log2 fold-change (FC)=the above FC in log2 scale=log2 of ratio of treatment and control data=log2 (treatment/control) (2−ΔΔCT: ΔCT=CTmiR-660-5p-CTU6, ΔΔCT=CTRCC-CTNAT) (24).
Wound-healing assay
The wound-healing assay was performed to examine the migratory ability of 786-O and ACHN cells in vitro. Cells (3×105 cells/well) were seeded into each well of 12-well plate. Cells were grown to 80–85% confluence at 37°C and subsequently transfected with 40 pmol of chemically synthesized miR-660-5p inhibitors, miR-660-5p mimic, inhibitor NC or mimic NC using Lipofectamine 2000. Following 6 h of transfection at 37°C, a sterile 200 µl pipette tip was used to scratch a clear line through the cell monolayer. Cells were rinsed with PBS and cultured in serum-free DMEM in a humidified chamber containing 5% CO2 at 37°C. Images of the scratches were acquired with a digital camera system at 0 and 24 h post-scratch. The experiments were performed in triplicate and repeated at least three times. Two parallel lines were made on the edge of the scratch, and then the distance between two parallel lines was measured on the Adobe Photoshop CS6 software package (Adobe Systems, Inc., San Jose, CA, USA). Relative migratory distance of cells was calculated as follows: (D1-D2)/(D3-D4). The percentage change was calculated as follows: -%=[(D1-D2)-(D3-D4)]/(D3-D4). D1, the average distance of mimic/inhibitor at 0 h; D2, the average distance of mimic/inhibitor at 24 h; D3, the average distance of mimic NC/inhibitor NC at 0 h; D4, the average distance of mimic NC/inhibitor NC at 24 h.
MTT assay and Cell Counting kit-8 (CCK-8) assay
The MTT assay was performed to examined cell proliferative ability of 786-O and ACHN cells in vitro. Cells (5×103 cells/well) were seeded in each well of 96-well plate and transfected with 5 pmol miR-660-5p inhibitor, miR-660-5p mimic, inhibitor NC or mimic NC for 0, 24, 48 or 72 h at 37°C according to the manufacturer's protocol. Cell growth was examined by adding 20 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) into each well post-transfection, followed by incubation at 37°C for 4 h. Formazan crystals were dissolved with the addition of 150 µl dimethylsulfoxide (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature with agitation. The optical density of each well was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 490/630 nm.
Cell proliferation was also assessed with the CCK-8 assay (Beyotime Institute of Biotechnology, Haimen, China), following the manufacturer's protocol. Cells (5×103) were seeded into each well of 96-well plate and incubated for 24 h at 37°C. The cells were transfected with 5 pmol miR-660-5p inhibitor, miR-660-5p mimic, inhibitor NC or mimic NC for 6 h at 37°C. At 0, 24, 48 and 72 h post-transfection CCK-8 solution (15 µl) was added into the wells and cells were incubate an additional 2 h at 37°C. The optical density of each well was measured using microplate reader (Bio-Rad Laboratories, Inc.) at a wavelength of 490/630 nm. The percentage changes for both the CCK-8 and MTT assays were performed as follows: -%=(OD1-OD2)/OD2, where OD1=the average optical density value of mimic/inhibitor and OD2=the average optical density value of mimic NC/inhibitor NC.
Transwell assay
Transwell assays with or without Matrigel were performed to assess the cell invasion and migration, respectively, of 786-O and ACHN cells in vitro. Transwell chamber inserts (BD Biosciences, Franklin Lakes, NJ, USA) with (for invasion) or without (for migration) Matrigel (BD Biosciences) were used in the assay according to the manufacturer's protocol. Cells (3×105 cells/well) were seeded into 6-well plates for 24 h at 37°C until 70–80% confluent, and were transfected with 200 pmol miR-660-5p inhibitors, miR-660-5p mimic, inhibitor NC or mimic NC for 6 h at 37°C. Subsequently, transfected cells (1×104 cells) were seeded in the upper chamber of the insert in 200 µl serum-free DMEM in 24-well plates. The bottom of the inserts was incubated in the medium containing 10% FBS. Cells were allowed to migrate for 40 h and to invade for 60 h at 37°C. An extended incubation period was used as the condition of the cells it was deemed to be poor. The cells that had migrated or invaded to the bottom of the inserts were stained with crystal violet and counted using a Leica DMIRB inverted microscope (DP70; Olympus Corporation, Tokyo, Japan). The relative cell number/field was calculated as follows: The cell number/field=the mean ± standard deviation from three independent experiments; mimic/inhibitor group: Relative cell number per field=N1/N2; mimic NC/inhibitor NC group: Relative cell number per field=N2/N2 (N1=the cell number/field of mimic/inhibitor; N2=the cell number/field of mimic NC/inhibitor NC). The experiments were performed in triplicate and repeated at least three times.
Flow cytometry
Flow cytometry was performed to evaluate the early apoptotic rate of 786-O and ACHN cells cultured with the various treatments, according to the manufacturer's protocol. Cells (3×105 cells/well) were seeded into 6-well plates for 24 h at 37°C until ~70% confluent, and were transfected with 200 pmol miR-660-5p inhibitors, miR-660-5p mimic, inhibitor NC or mimic NC for 6 h at 37°C. At 48 h post-transfection, all cells, including floating and adherent cells, were harvested by centrifugation at 503.1 × g for 5 min at 37°C and washed with cold PBS for twice. Cells were resuspended in 100 µl 1X binding buffer, 5 µl Annexin V-fluorescein isothiocyanate (FITC) and 3 µl propidium iodide (PI; Invitrogen; Thermo Fisher Scientific, Inc.) were added into each cell suspension. Following 15 min incubation at room temperature, 400 µl binding buffer was added to each tube, and cells were examined on an EPICS XL Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA) to analyze the apoptotic rates. Experiments were performed in triplicate and repeated at least three times.
Statistical analysis
Paired t-test was used to compare the expression levels of miR-660-5p in matched RCC and NAT samples. The relative expression of miR-660-5p in tissues is presented as mean ± standard error of the mean as this improved the clarity of the graphical representation. Other data are presented as the mean ± standard deviation from three independent experiments. All the statistical analyses were carried out with SPSS 19.0 statistical software package (IBM Corp., Armonk, NY, USA). Statistical significance was determined with Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
miR-660-5p expression is downregulated in RCC tissues compared with NATs
miR-660-5p expression in the 25 RCC tissues and NATs was examined by RT-qPCR. The log2 ratio (RCC/NAT) expression of miR-660-5p is provided in Fig. 1A, and the overall relative expression levels of miR-660-5p in RCC tissues compared with NATs is demonstrated in Fig. 1B. These results demonstrated that the expression of miR-660-5p in RCC tissues (mean relative expression, 3.44) was lower than in the NATs (P<0.01).
Validation of cell transfection efficiency
The relative transfection efficiency of miR-660-5p inhibitors or mimic compared with inhibitor NC or mimic NC was performed by RT-qPCR. miR-660-5p expression levels in the ACHN and 786-O cells transfected with miR-660-5p inhibitors were 0.01 and 0.13, respectively, whereas expression in cells transfected with miR-660-5p mimic was 590.19 and 1762.10, respectively (Fig. 1C).
Effects of miR-660-5p mimic and inhibitor treatments RCC cell proliferation
MTT and CCK-8 assays were performed to examine cell proliferative ability of 786-O and ACHN cells in vitro. MTT assay results indicated that proliferation in 786-O cells treated with the miR-660-5p inhibitor was promoted by 12.14% (P<0.01), 20.14% (P<0.01) and 27.04% (P<0.01), at 0, 24, 48 and 72 h post-transfection, respectively, compared with the proliferation rates of cells transfected with inhibitor NC (Fig. 2A). Proliferation in the miR-660-5p mimic-treated group was decreased by 10.84% (24 h; P<0.01), 24.83% (48 h; P<0.01) and 34.71% (72 h; P<0.01) compared with mimic NC-treated cells (Fig. 2A). Similarly, the MTT assay demonstrated that cell proliferation of ACHN cells in the miR-660-5p inhibitor group was promoted by 7.04% (P<0.01), 23.59% (P<0.01) and 42.16% (P<0.01) at 0, 24, 48 and 72 h, respectively, compared with cells in the inhibitor NC-treated group (Fig. 2B); proliferation was decreased by 6.80% (P<0.01), 20.61% (P<0.01) and 25.35% (P<0.01) in cells treated with miR-660-5p mimic compared with mimic NC-treated cells (Fig. 2B).
In the CCK-8 assay, 786-O cell proliferation was promoted by 14.59% (P<0.05), 15.80% (P<0.05) and 12.77% (P<0.01) in the miR-660-5p inhibitor group at 24, 48 and 72 h post-transfection, respectively, compared with inhibitor NC-treated cells (Fig. 2C), whereas proliferation in the miR-660-5p mimic-treated group decreased by 14.55% (24 h; P<0.05), 16.03% (48 h; P<0.01) and 30.74% (72 h; P<0.01), compared with those transfected with mimic NC. Similarly, the CCK-8 assay demonstrated that proliferation of ACHN cells in the miR-660-5p inhibitor group was promoted by 12.57% (24 h; P<0.01), 24.40% (48 h; P<0.01) and 39.39% (72 h; P<0.001), compared with cells in the inhibitor NC group (Fig. 2D); proliferation in the miR-660-5p mimic-treated group was decreased by 9.26% (P<0.01), 15.30% (P<0.01) and 22.95% (P<0.01) at 24, 48 and 72 h post-transfection, respectively, compared with the mimic NC-treated group.
Effects of miR-660-5p mimic and inhibitor treatments on RCC cell migration
Wound-healing and Transwell assays were performed to examine the migratory ability of 786-O and ACHN cells in vitro. In the wound-healing assay, cells transfected with miR-660-5p inhibitors for 24 h exhibited significantly increased migration compared with those transfected with inhibitor NC (Fig. 3): The migratory distance was increased by 27.67% for 786-O cells (P<0.01; Fig. 3A) and 56.26% for ACHN cells (P<0.01; Fig. 3B). By contrast, compared with cells transfected with mimic NC (Fig. 3), cell migration was significantly reduced in cells transfected with miR-660-5p mimic, for which the migratory distance was reduced by 21.87% for 786-O cells (P<0.01; Fig. 3A) and 36.24% for ACHN cells (P<0.01; Fig. 3B).
In Transwell assays, the migratory ability of 786-O cells transfected with miR-660-5p inhibitors was increased by 29.18% (P<0.01; Fig. 4A) and reduced by 36.71% (P<0.01; Fig. 4B) in cells transfected with miR-660-5p mimic compared with the respective inhibitor NC- or mimic NC-treated cells. In ACHN cells, the migratory ability of cells transfected with miR-660-5p inhibitors was promoted by 45.99% (P<0.01; Fig. 5A) and reduced by 56.55% (P<0.01; Fig. 5B) following transfection with miR-660-mimic compared with cells transfected with inhibitor NC or mimic NC.
Results from the Transwell and wound-healing assays revealed that miR-660-5p may inhibit the migratory ability of RCC cell.
Effects of miR-660-5p mimic and inhibitor treatment on RCC cell invasion
Transwell assay with Matrigel was performed to assess the invasive ability of 786-O and ACHN cells in vitro. For 786-O cells, the results indicated that the invasive ability was promoted by 60.99% (P<0.01) when transfected with miR-660-5p inhibitors for 24 h, compared with cells transfected with inhibitor NC (Fig. 6A). By contrast, the invasive ability of 786-O cells transfected with miR-660-5p mimic was significantly reduced by 26.39% (P<0.01) compared with cells transfected with mimic NC (Fig. 6B). In ACHN cells, the results indicated that the invasive ability of cells transfected with miR-660-5p inhibitors was increased by 31.76% (P<0.01) and decreased by 40.09% (P<0.01) in cells transfected with miR-660-5p mimic compared with the respective inhibitor NC- or mimic NC-treated cells (Fig. 7A and B, respectively). The results of the Matrigel assays demonstrated that miR-660-5p expression may inhibit the invasive ability of RCC cells.
Effects of miR-660-5p on RCC cell apoptosis
Flow cytometry was used to evaluate the early apoptotic rates of 786-O and ACHN cells under the various transfection treatments. The average apoptotic rates of 786-Ocells transfected with miR-660-5p inhibitor or inhibitor NC were 11.60 and 18.50%, respectively (P<0.01; Fig. 8A). The apoptotic rates of cells transfected with miR-660-5p mimic or mimic NC were 21.08 and 13.47%, respectively (P<0.01; Fig. 8B). In ACHN cells transfected with miR-660-5p inhibitor, the average apoptotic rate was 6.95% and cells transfected with inhibitor NC exhibited an apoptotic rate of 11.40% (P<0.01; Fig. 9A). The apoptotic rates of cells transfected with miR-660-5p mimic or mimic NC were 14.49 and 9.24%, respectively (P<0.01; Fig. 9B). These results suggested that miR-660-5p expression may stimulate RCC cell apoptosis.
Discussion
RCC accounts for ~30% of all malignancies in adults and has a high mortality rate (27); there are no specific characteristic clinical features in the early stage of RCC, and ~30% of patients with RCC exhibit metastatic symptoms at presentation (28). As advanced-stage RCC is not sensitive to the traditional treatments, such as chemotherapy and radiation, it is essential to explore the underlying molecular mechanisms of RCC metastasis, and to identify a useful tumor biomarker that may aid in the early diagnosis of RCC.
Oncogenes or tumor suppressor genes serve an important role in the initiation and development of cancer. miRNAs have been demonstrated to serve important roles in different types of cancers by regulating gene expression (14). Upregulated miRNA expressions may be considered as oncogenes, whereas downregulated miRNA expressions may be regarded as tumor suppressors. Recent studies have demonstrated that several miRNAs functions as either oncogenes or tumor suppressors in the progression of RCC. For example, miR-130b, miR-886-3p and miR-16 have been reported to be oncogenes that are associated with cellular migration, proliferation and apoptosis in RCC (29–31). By contrast, miR-30a-5p, miR-149-5p and miR-125a-5p have been identified as tumor suppressors in RCC progression (32–34). Results from the present study indicate that miR-660-5p is a tumor suppressor in RCC.
Previous studies have revealed that miR-660-5p is dysregulated in a number of human malignancies. For example, miR-660 was reported to be downregulated in patients with lung cancer, and the overexpression of miR-660-5p in lung cancer cells transfected with miRNA mimic inhibited the migration, invasion and proliferation properties and induced apoptosis in p53 wild-type lung cancer cells by targeting MDM2 (18). Another study reported that miR-660-5p is upregulated in breast cancer and that it may be a potential novel prognostic marker for breast cancer (19); miR-660 expression was also revealed to be downregulated in chronic lymphocytic leukemia (21). In addition, dysregulated miR-660-5p expression was also reported in Hodgkin lymphoma (35) and multiple myeloma (20). These results suggested that miR-660-5p may be a novel biomarker that is closely related with tumorigenesis, which serves a role as an oncogene or tumor suppressor. However, the expression and function of miR-660-5p in RCC remains unclear.
Based on previous microarray chip results (22), the present study performed RT-qPCR to quantify the relative expression levels of miR-660-5p in 25 paired RCC tissues and NATs, as well as in human RCC cell lines. The functions of miR-660-5p on cellular migration, invasion, proliferation and apoptosis were analyzed by performing wound-healing assay, Transwell and Matrigel assays, MTT and CCK-8 assays and flow cytometry. The results demonstrated that miR-660-5p expression was significantly downregulated in RCC tissues compared with NATs. In addition, downregulation of miR-660-5p expression by treating cells with a chemically synthesized miR-660-5p inhibitor significantly promoted cell migration, invasion and proliferation, and reduced apoptosis in both 786-O and ACHN cells. By contrast, overexpression of miR-660-5p by miR-660-5p mimic transfections inhibited 786-O and ACHN cell migration, invasion and proliferation, and induced apoptosis. These results suggested that miR-660-5p may act as a tumor suppressor in RCC. However, the miR-660-5p-mediated molecular pathways that affect cell migration, proliferation and apoptosis remains to be further explored.
miR-660-5p has also been reported to serve roles in other diseases besides tumors. For example, miR-660-5p was revealed to be downregulated in thyroid tissues of patients with Graves' disease, which suggested a potential involvement of miR-660-5p in the pathogenesis of this disease (36). Aberrant expression of miR-660-5p, as detected by RT-qPCR, demonstrated a relationship with the prediction or diagnosis of myocardial infarction or chronic heart failure (37,38).
In conclusion, to the best of our knowledge, the present study was the first to demonstrate that miR-660-5p acted as a tumor suppressor in RCC and may regulate cell migration, proliferation and apoptosis. Further analyses are needed to determine the target genes of miR-660-5p and to elucidate the molecular mechanisms in RCC, and may be used as a biomarker to aid in the early diagnosis of RCC.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant no. 81101922), The Science and Technology Development Fund Project of Shenzhen (grant nos. JCYJ20150403091443329 and JCYJ20170307111334308), the fund of “San-ming”project of medicine in Shenzhen and the fund of Guangdong Key medical subject.
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