Background
Renal cell carcinoma (RCC) is one of the common urological cancers usually with poor prognosis [
1]. RCC accounts for approximately 3% of adult malignancies and for approximately 90–95% of neoplasms arising from the kidney [
2,
3]. Surgical treatment can cure 60–70% of localized RCC but only prolongs survival in most metastatic RCC patients [
4]. RCC is relatively resistant to radiation and chemotherapy [
5]. Considerable progress has been made in the therapy of patients with localized RCC; however, the treatment options for patients with metastatic RCC are very limited [
6]. Therefore, identification of new biomarker and anti-tumor agents, particularly with better efficiency against metastatic RCC, remains a high priority.
MicroRNAs (miRNAs) are a large gene family of short (21–23 nucleotides) non-coding RNAs. The single-stranded miRNA binds through imperfect base pairing with the 3′ untranslated region (3′-UTR) of target mRNAs and causes either repression of translation or degradation of mRNAs [
7]. Each of the hundreds of known miRNAs can epigenetically downregulate many target genes that participate in various biological processes including cell proliferation, apoptosis, migration, differentiation and development [
8].
It is increasingly apparent that the interplay between cancer cells and their stroma is of great importance to tumorigenesis and progression [
9]. Tumor-associated stroma is composed of multiple stromal cell types, such as cancer-associated fibroblasts, immune inflammatory cells, and endothelial cells, as well as a variety of extracellular matrix (ECM) proteins, such as fibronectin and collagen. The tumor-associated stroma constitutes an important compartment of tumor microenvironment, which can enable primary, invasive, and then metastatic growth of tumor through crosstalk with cancer cells [
10]. Recent studies have shown that the regulatory role of miRNAs during cancer progression is not limited to cancer cells, and that miRNAs are also involved in the activation and transition of tumor stromal cells [
11]. Thus, miRNAs have emerged as a potent regulator in the crosstalk between cancer and stromal cells in the tumor microenvironment.
The let-7 family, originally identified in
Caenorhabditis elegans, consists of 13 family members that are highly conserved across the animal phylogeny from
C. elegans to human. Nine members of the let-7 family have been identified in humans [
12]. Let-7 functions as a heterochronic gene in many species. It is undetectable in human and mouse embryonic stem cells, but increases during embryogenesis and differentiation [
13]. High let-7 expression levels are subsequently maintained in a variety of adult tissues [
14]. Conversely, let-7 is frequently downregulated in many human malignancies, such as lung cancer, breast cancer, and hepatocellular carcinoma [
15‐
17], possibly reflecting the reverse embryogenesis process that occurs during oncogenesis [
18].
Recently, we have identified a subset of miRNAs that are low expressed in RCC relative to adjacent normal tissues by using microarray (unpublished data). The current study was designed to explore the function of one of these miRNAs, let-7d, in RCC progression.
Discussion
Let-7d belongs to the let-7 family that functions as tumor suppressor in many types of cancer [
12]. Reported direct targets of let-7 include oncogenes such as RAS, MYC, and HMGA2 [
25]. In this study, we demonstrated the tumor suppressive role of let-7d in RCC and validated that the targets of let-7d were COL3A1, an important stroma component, and CCL7, a chemokine attracting monocytes to tumor tissue. Let-7d expression was negatively correlated with COL3A1, CCL7 and CD68
+ cells in RCC tissues.
Collagen is the most abundant ECM protein in stroma, and contributes to the tumor progression in tumor stroma [
10]. Elevated deposition of collagen has been particularly associated with an altered stroma during breast tumorigenesis and correlated with increased breast cancer risk [
26]. Lysyl oxidase is an ECM crosslinking enzyme. Collagen crosslinking-mediated matrix stiffening increases integrin clustering, which leads to phosphorylation of focal adhesion kinase and activation of extracellular signal-regulated kinase [
27]. These changes are all involved in cell migration, invasion and proliferation, leading to tumor progression [
28]. COL3A1, also known as collagen, type III, alpha 1, is a fibrillar collagen found in extensible connective tissues. The increase of COL3A1 and COL1A1 are found in epithelial ovarian cancers and are prognostic markers of poor prognosis [
29]. Interestingly, COL3A1 is the target of miR-29 family, and downregulation of this miRNA family is responsible for the increased invasiveness of lung cancer [
30,
31]. Here, we validated through
in vitro and
in vivo research that COL3A1 is a functional direct target gene of let-7d. Importantly, COL3A1 expression was inversely correlated with let-7d levels in RCC clinical specimens. Our findings suggest the involvement of the let-7d-COL3A1 regulatory pathway in RCC growth and metastasis.
Macrophages, which are abundant in the tumor microenvironment, were proven to promote cancer initiation and malignant progression by persuasive clinical and experimental evidence [
32]. Tumor-associated macrophages are also involved in tumor progression of RCC and can be used as a potential therapeutic target for metastatic RCC [
33]. CCL7 (monocyte chemotactic protein-3, MCP-3) is a member of the C-C chemokine subfamily. CCL7 has been found to be overexpressed in gastric cancer tissues and is associated with tumor lymph node metastasis and poor prognosis [
34]. Furthermore, CCL7 is more abundant in metastatic tumor site than in the primary site, and is associated with macrophage infiltration in tumor [
35]. For example, CCL7 is higher in brain metastatic RCC than in primary RCC [
36]. CCL2 is another potent macrophage chemoattractant chemokine belonging to the same chemokine family with CCL7. CCL2 recruits macrophages to facilitate metastasis of breast cancer [
37]. In RCC [
38], however, CCL2 was shown to be of minor importance in the recruitment of macrophages that preserve diverse tumor-promoting functions. CCL7 promotes the invasion and migration of oral squamous cell carcinoma cells through directly binding to its receptor [
39]. Interestingly, we found that the suppressed proliferation and migration in let-7d overexpressing RCC cells could not be restored by the addition of exogenous CCL7 alone, probably due to the multiple pathways downstream of let-7d or the lack of functional CCL7 receptor in RCC cells. However, a strong inverse correlation between let-7d expression and number of infiltrated macrophage was found in our clinical RCC samples. Therefore, there is an indirect role of CCL7 in RCC malignancy via the let-7d-CCL7-macrophage chain. The negative correlation between let-7d expression and CCL7 in T3 stage RCC tissues additionally highlights the role of CCL7 in tumor invasion and metastasis. CCL7 may be functionally involved in RCC malignant progression and may be used in chemokine target therapy for RCC.
In summary, our results indicate that the tumor suppression role of let-7d in RCC may be partially ascribed to its ability to decrease collagen expression and macrophage recruitment through targeting COL3A1 and CCL7 mRNAs. If sufficient let-7d is present in RCC, tumor stroma will be remodeled and cancer cells will be suppressed. It should be noted that let-7d may possess various functions owing to its pleiotropic regulation of genes. It is our expectation that more functional let-7d target genes will be identified in the near future. Further studies are required to fully illustrate their functional roles and interactions with tumor-associated stroma and to determine whether let-7d can be used for the therapy of metastatic RCC, as described in other cancers [
23].
Methods
Clinical samples and cell lines
A total of 80 paired RCC tissues and adjacent normal tissues were obtained with informed consent from patients who underwent surgical resection at Peking University First Hospital between 2012 and 2013. The study was approved by Review Board of Peking University First Hospital. All histological subtypes were classified by the Heidelberg classification [
40]. Pathological T stage was classified according to the 2010 TNM classification system [
41]. Tumor grade was assessed according to Fuhrman nuclear grade [
42]. The presence of vascular invasion includes either microvascular invasion, renal vein invasion or inferior vena cava invasion. Immortalized renal proximal tubule epithelial cell line of HK-2 and human RCC cell lines of A498, 769P, 786O, Caki-1 and Caki-2 were purchased from ATCC (MA, USA). Human RCC cell line OS-RC-2 was obtained from the cell bank at Chinese academy of sciences. Transformed human embryonic kidney cells 293FT were purchased from Invitrogen. A498, 769P, 786O, and OS-RC-2 cells were maintained in RPMI 1640 medium, HK-2, Caki-1 and Caki-2 cells in DMEM/F12 (50:50, v/v) medium, and 293FT cells in DMEM. These media were supplemented with 10% fetal bovine serum (HyClone), and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Invitrogen).
Peripheral blood mononuclear cell (PBMC) isolation
Heparinized venous blood obtained from RCC patients with informed consent was diluted 1:5 with phosphate buffer saline (PBS) and the 40 mL diluted blood was then underlaid on 10 mL of Ficoll (Seromed, Berlin, Germany) in 50 mL plastic tube. After centrifugation at 400 ×
g for 20 minutes, PBMC were aspirated from the interface, washed with PBS and resuspended to 4 × 10
6 cells/mL in complete RPMI 1640 medium [
43].
In vivo assay
All mice were maintained in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals with the approval of Review Board of Peking University First Hospital, Beijing. Patient derived xenograft (PDX) model was established as previously described [
23]. RCC surgical samples were gently grinded, labeled with Cell Tracker™ CM-Dil dye (MoBiTec, Göttingen, Germany) and subcutaneously injected into NOD/SCID mice (Vitalriver, Beijing, China). The xenograft was then harvested, minced into pieces and transplanted into successive mice. For establishment of RCC cell derived xenograft (CDX) model, 5-week old female BALB/C nude mice (Chinese Academy of Sciences, Shanghai, China) were subcutaneously inoculated with 786-O cells labeled with Dil dye and stably transfected with 3 × 10
6 pri-let-7d or vehicle control lentivirus. Growth of established xenografts was monitored every two days by a caliper for length (L) and width (W) measurement. Tumor volumes were calculated using the formula (L × W
2) / 2.
In vivo treatment of miRNA mimics in PDX model was performed as previously described [
23]. 20 nM chemically-modified mi-Ribo™ hsa-let-7d mimics or mi-Ribo™ hsa-let-7d control (Ribobio Co., Guangzhou, China) in 50 μL PBS mixed with 50 μL
in vivo transfection reagent Entranster™-
in vivo (Engreen, Beijing, China) were locally injected into the tumor mass once every 3 days for 3 weeks. Quantification of the RCC cell lung metastatic colonies were obtained by examining the mice lung using the TCS 4D laser scanning confocal microscope (Leica, Heidelberg, Germany).
RNA extraction and real-time RT-PCR
Total RNA was extracted using miRNeasy Mini Kit (Qiagen, Hilden, Germany). For miRNA quantification, 100 ng total RNA was either reverse transcribed directly using stem-loop primers [
44], or was polyadenylated with polyA polymerase (NEB, Beverly, MA, USA) then reverse-transcribed with an oligo-dT adapter primer into cDNAs for quantitative real-time PCR [
45]. Although both reverse-transcription methods yield reliable and comparative results, the polyA polymerase tailing method was used in this experiments unless specified, given that it allows measuring multiple target miRNAs with one RT reaction. For mRNA analyses, cDNAs were synthesized from 2 μg total RNA, using oligo(dT)
15 primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) in a final volume of 10 μL in ABI 7500 Fast PCR machine. The expression of miRNA and mRNAs were normalized to U6 and GAPDH, respectively. Data are presented as relative quantification (RQ) based on the calculation of 2
-ΔCt. ΔCt was derived from subtracting the Ct value of reference cDNA from the Ct value of the cDNA of interest. (For a list of all the primers, see Supporting Data, Additional file
2: Table S1)
Lentiviral transduction
The human pri-let-7d (primary transcript of let-7d) cDNA sequence was synthesized and inserted into the lentiviral shuttle vector plenti6 (Invitrogen) to generate human plenti6-pri-let-7d plasmid. Plenti6 control and human pri-let-7d lentivirus were generated by transfecting 3 μg of plenti6 or plenti6- pri-let-7d and 9 μg of ViraPower Packaging Mix (Invitrogen) into 293FT packaging cells using Lipofectamine 2000. After overnight exposure to the transfection mixture, the medium was changed, and the virus-containing supernatant was harvested 48 h later. The infected cells were selected with 5 μg/mL blasticidin. The antibiotic-resistant clones were pooled and used for subsequent assays.
In vitro cell proliferation, migration, wound healing, and chemotaxis assays
The cell proliferation assay was performed by using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Cell migration was evaluated by Boyden chamber assay and wound healing assay [
46]. Monocyte chemotaxis was assayed in 24-well transwell plates (Costar #3421) with 5 μm pore polycarbonate filter membrane. Briefly, 1 × 10
7 RCC cells were cultured in 5 mL complete medium for 24 h, the cultured media were collected after centrifugation and used as conditioned media. PBMC were resuspended in 0.1% BSA-RPMI medium, 4 × 10
5 cells in 100 μL medium were added to the upper chamber of the 24-transwell apparatus, and 800 μL conditioned medium were added in the lower chamber. After incubation for 8 h, cells that migrated though the membrane were fixed with100% methanol, stained by Giemsa dye, and counted under a microscope. Five high-power fields (×200) were randomly selected and manually counted for each well. The experiment was performed in triplicate (3 wells) with three independent tests. Human recombinant COL3A1 (Fitzgerald, Sudbury, MA) (0.2 μg/mL), human recombinant CCL7 (PeproTech, Rocky Hill, NJ) (10 ng/mL), CCL7 neutralizing antibody and normal goat IgG control antibody (R&D Systems, Minneapolis, MN) (1 μg/mL) were used in these assays.
Elisa
Cultured media of RCC cells were used for detection of CCL7 by CCL7 ELISA kits (Ray Biotech, Inc). The optical density (OD) at 450 nm was quantified with a Multiskan microplate spectrophotometer (Thermo LabSystems, Milford, MA).
Quantitative detection of human tumor cell metastasis
The detection of RCC metastasis in mice lung was performed as described previously [
47]
. Genomic DNA was extracted from mouse lung tissues using the EasyPure Genomic DNA Kit (Transgen Biotech, Beijing, China). Quantitative real-time PCR was used to measure human Alu-sequences specific for the most conserved region of humans. The primers for Alu-sequences and PCR conditions were used as previously described [
47]. The level of human Alu-sequence was normalized to the amount of mouse/human GAPDH genomic DNA sequence amplified by using mouse/human GAPDH primers [
48].
Immunohistochemistry (IHC) and toluidine blue staining
Paraffin embedded tissues were analyzed using immunohistochemical staining [
49] with the following primary antibodies: anti-CD68 antibody (DAKO, Carpinteria, CA), anti-α-SMA antibody (DAKO, Carpinteria, CA), anti-CCL7 antibody (Gen Way Biotech, San Diego, CA), anti-COL3A1 antibody (Bioss, Beijing, China), anti-FOXP3 antibody (Biolegend, San Diego, CA) and rabbit anti-Mouse CD68 antibody (Bioss, Beijing, China). For quantification of tumor stromal cells within the tumor area, CD68 was used as a pan-macrophage marker, α-SMA was used to detect cancer activated fibroblasts adjacent to RCC cells [
50], FOXP3 was used as a specific marker for regulatory T cells (Tregs) [
22], and mast cells were assessed using the routine toluidine blue staining method [
21]. Each tumor section was evaluated by using 20× objective lens, and five independent areas with the most abundant positive cells were selected, digitally photographed, and manually counted under a microscope. The average positive cell counts for each patient were used for statistical analysis. For quantification of CD68
+ cells in CDX xenografts, four sections from each xenograft were randomly selected and quantified as described above, the average positive cells for each mouse were used for statistical analysis. Results were confirmed by two pathologists in a double-blind analysis.
Western blot analysis
The lysates were obtained by lysing cells in lysis buffer containing 50 mM Tris, pH7.4, 150 mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM PMSF, and complete protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of total protein were subjected to 10% SDS-PAGE and blotted onto PVDF membranes (Pall, Pensacola, FL). Western blotting was performed using rabbit anti-human COL3A1 antibody (Bioss, Beijing, China). The blotting membranes were scanned using GeneSnap acquisition software (Syngene, Cambridge, UK) and band densities were quantified with the GeneTool program (Syngene, Synoptics). GAPDH were used as internal control.
Dual luciferase activity assay
The 3′-UTR of human COL3A1 and CCL7 containing the putative binding sites and the mutant binding sites of the mature hsa-let-7d were chemically synthesized and inserted immediately downstream of the luciferase cDNA in the pGL3-control vector (Promega, Madison, WI) by GenePharma (Shanghai, China) to form pGL3-COL3A1, pGL3-CCL7 and pGL3-COL3A1-Mut, pGL3-CCL7-Mut constructs. Twenty-four hours before transfection, 786O and 293FT cells were plated at 1.5 × 105 cells/well in 24-well plates. 0.5 μg of pGL3 constructs plus 0.08 μg of pRL-TK (Promega) were transfected in combination with 60 pmol of either a stability-enhanced nontargeting RNA control oligonucleotide or stability-enhanced hsa-let-7d oligonucleotides (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well. The results were obtained from three independent experiments and each one was performed in triplicate.
Inhibition of let-7d with a miRNA inhibitor
The chemically-modified mi-Ribo™ hsa-let-7d inhibitor or mi-Ribo™ hsa-let-7d negative control oligonucleotides were synthesized by Ribobio Co. (Guangzhou, China). RCC cells were transfected using Lipofectamine 2000. Cells were collected and assayed at 72 h post-transfection.
Statistical analysis
Data are presented as mean ± SD and were analyzed using the statistical package SPSS17.0 or GraphPad Prism software 5.0. The significance of differences between two groups was determined using a two-sided Student’s t-test. In case of multiple tests, one-way ANOVA followed by Bonferroni-Holm procedure was applied. Correlation was performed using two-tailed Spearman’s test. P ≤ 0.05 was considered statistically significant.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
B.Su, B.Shi, Z.Z, Y.X., F.X., Z.G., X.L., W.Z., Z.Q.Z. and L.Z. conceived the experiments and analysed the data; Q. S. and J. W. examined the tumor sections. B.Su, W.Z., B.Shi, Z.Z, Y.X., F.X., X.Z., J.L. and Z.G. carried out experiments; B.Su and Y.X. drafted the manuscript. Z.Q.Z. and L.Z. supervised research project, acted as corresponding authors and did the revisions. All authors were involved in writing the paper and approved the submitted manuscript.