Characterization of MicroRNA Expression Profiles in Patients with Giant Cell Tumor

Abstract

Objective

Giant cell tumors of bone (GCTs) are bone destructive neoplasms, the bone resorption being mediated by osteoclasts. Given that microRNAs are crucially involved in tumorigenesis and the modulation of cell fate and behavior, they are promising candidates for regulation of osteoclastogenesis. However, no reliable miRNAs profile for GCT is available. Our study aimed to evaluate osteoclastogenesis‐related miRNA expression in GCTs of Chinese patients.

Methods

From January 2013 to December 2014, 11 patients with GCTs were treated in our department and grouped into a GCT group. A control group comprising four patients with benign tumors of the iliac bone was established. The diagnoses were initially established by imaging examinations and intraoperative frozen sections and later confirmed by standard histologic examination. The GCT group (five male and six female patients) were aged from 17 to 61 years (mean, 32.9 years; SD, 12.8 years). Six patients with GCT underwent intralesional curettage surgery and the other five wide resection. According to Campanacci grading, four patients had Grade I tumors, three Grade II, and three Grade III. The average age of the control group was 28.75 years (SD, 14.24 years); all of them were diagnosed as having benign tumors and underwent iliac grafting. The morphology of the excised tissue was evaluated by examining standardized hematoxylin and eosin (HE) stained paraffin‐embedded samples. In all, three osteoclastogenesis‐related RNAs and 20 microRNAs (miRNAs) were extracted from the patients. The strength of expression was assessed by quantitative reverse transcription polymerase chain reaction (PCR ) and the results assessed by a Student's t test.

Results

Examination of HE stained sections revealed that the higher the Campanacci grade, the more numerous and bigger the osteoclasts (P < 0.05). PCR results indicated large amounts of osteoclast‐related mRNA (cathepsin K, tartrate‐resistant acid phosphatase and matrix metalloproteinase9) in GCTs (P < 0.05). Expression of six miRNAs was significantly weaker in the GCT than the control group (P < 0.05). The expression of has‐mir‐16‐5p and has‐let‐7a‐5p was correlated with Campanacci grade in the GCT patients (P = 0.009 and 0.034, respectively). The expression of these two miRNAs may indicate severity of bone destruction.

Conclusion

Overall, the clinical utility of six novel miRNA markers for GCTs was demonstrated. Of these, strength of expression of hsa‐mir‐16‐5p and hsa‐let‐7a‐5p may indicate the grade of bone resorption.

Introduction

Giant cell tumors (GCTs) are bone destructive neoplasms that generally run a benign course. They represent about 5% of all primary bone tumors and are frequently located in the meta‐epiphyseal regions of long bones, including the distal femur, proximal tibia and radius1, 2, 3. Less common but well‐documented sites of involvement include the sacrum (5%), pelvis and spine. GCTs are capable of causing significant bone destruction. Bone is a highly stable, fiber‐reinforced, calcified tissue. Its resorption depends on a combination of the action of specific proteases that can remove the organic matrix (predominantly fibrillar type I collagen) and creation of the acidic microenvironment necessary for solubilization of the inorganic mineral component (hydroxyapatite). Curettage and adjunctive nonspecific cytotoxic treatment of the surgical margins followed by bone grafting has reduced local recurrence rate to 10%–35%; however, the final prognosis is highly dependent on localization of the process4, 5. Malignant transformation and development of lung metastases has rarely been observed6, 7. Although a number of studies have focused on the causes of GCT, such as genetic and environmental factors, the underlying pathology is not yet fully understood. Thus, further understanding of the mechanisms leading to significant localized osteolysis may be helpful in developing new treatment options for this tumor.

The three main cellular components of GCT resemble constituents of the normal bone microenvironment: a mesenchymal fibroblast‐like stromal cell; a monocytic, mononuclear cell of myeloid lineage; and the characteristic osteoclast‐like, multinucleated giant cell8, 9, 10. Spindle‐like stromal cells of GCTs, which originate from mesenchymal stem cells in the bone marrow, are the neoplastic component of GCTs and play a crucial role in the occurrence and progression of GCTs by secreting various chemokines11. Human mesenchymal stem cells have been identified as multipotent mesoderm‐derived stromal cells that have the ability to self‐renew and differentiate and have been used as clinical treatments for bone and other tissue defects. In addition, there are multinucleated osteoclast‐like cells that contribute to bone destruction12, 13, 14.

There is growing evidence that microRNAs (miRNAs) play a pivotal role in the development of tumors. MiRNAs are small (−20 nt) non‐coding, single‐stranded RNA molecules that negatively regulate their target genes by inducting mRNA degradation or inhibiting translation15. MiRNAs function by partially or completely binding to the 3′‐untranslated region of their target mRNAs and thereby triggering either inhibition of translation or degradation of mRNA16, 17, 18. They have been shown to be involved in regulation of a wide range of biological functions, including cell growth, differentiation and apoptosis. Genes coding for miRNAs are frequently located in fragile chromosomal regions associated with the development of various tumors. Accordingly, dysregulation of miRNA expression has been observed in several tumors, including breast cancer, adenocarcinoma19, ovarian cancer20 and glioma tumor21. Together with the fact that miRNAs themselves can function as tumor‐suppressors and oncogenes, these data demonstrate that aberrant miRNA expression is strongly implicated in tumorigenesis22, 23, 24. Dysregulation of these miRNAs is likely to be involved in the initiation and formation of cancer. Thus, miRNAs are a possible link to GCT development.

Huang et al. reported that miR‐30a is downregulated in GCT whereas RunX2 is strongly expressed. Further research has shown that miR‐30a can regulate the expression of RunX2 by binding to its 30‐untranslated region, which influences osteoclast differentiation and osteolysis formation25. Wu et al. identified that miR‐126‐5p is significantly downregulated in spindle‐like stromal cells of GCT and affects osteoclast differentiation and bone resorption by repressing matrix metalloproteinase (MMP)‐13 expression at the post‐transcriptional level26. Apparently, there are more miRNAs that may modulate the production and function of osteoclasts. However, because of the diversity of samples, there is as yet no unified understanding of the expression profile of miRNAs in osteoclast‐related diseases, which could have great diagnostic and treatment potential.

Osteoclastogenesis is an intricate multi‐step process, beginning with the proliferation and commitment of mononucleated precursors and culminating in the formation of large bone‐resorbing polykaryons. Some osteolytic related proteins that are produced during this procedure may be the targets of regulation by miRNAs.

A number of MMPs, namely gelatinases (MMP‐2 and MMP‐9) and stromelysin (MMP‐3) have been implicated in the locally aggressive behavior of GCT27. Han et al. have reported that the strength of expression of MMP‐9 in GCTs of bone is correlated with the development and relapse of these cancers28.

Cathepsin K, a unique and potent collagenase, is primarily expressed in osteoclasts and is responsible for degradation of the collagen matrix in bone. The critical involvement of cathepsin K in bone remodeling is supported by the finding that cathepsin K deficiency causes the bone‐sclerosing disorder pycnodysostosis29, and by the ability of specific cathepsin K inhibitors to alleviate bone resorption in a primate model of hypogonadism30.

Our study had the following three aims: (i) to analyze the miRNA expression profiles of GCTs and healthy bones; (ii) to identify differentially expressed candidate miRNAs that might be promising candidates; and (iii) to investigate the possible relationship between strength of expression of GCT=related miRNAs and degree of bone destruction.

Materials and Methods

This study was approved by the Human Ethics Committees Review Board at Shanghai Jiaotong University. Written informed consent was obtained from each study patient.

Sample Collection

Eleven GCTB specimens (six from intralesional curettage and five from wide resection of the tumor) were freshly obtained in accordance with the study protocol. The control group samples were collected from the iliums of four patients with benign tumors who needed iliac grafting and did not have history of GCT. Relevant clinical characteristics are shown in Table 1.

Table 1.

Clinical characteristics of patients in GCT group

Sample	Sex	Age (years)	Location	Surgical procedure	Campanacci grade
1	Male	45	R. P. Tibia	Intralesional curettage	I
2	Female	40	R2. Metatarsal	Intralesional curettage	III
3	Male	21	L. D. Femur	Wide resection	III
4	Female	22	R. D. Femur	Intralesional curettage	III
5	Female	36	L. D. Femur	Wide resection	III
6	Male	61	R. D. Femur	Wide resection	II
7	Male	29	L. Humerus	Intralesional curettage	I
8	Male	17	R. P. Tibia	Wide resection	II
9	Female	24	R. D. Femur	Intralesional curettage	I
10	Female	37	L. P. Femur	Wide resection	II
11	Female	30	R. P. Tibia	Intralesional curettage	I

R/L, Right/Left; D/P, Distal/Proximal.

All samples were collected in our department between January 2014 and July 2015. The diagnoses were initially established by X‐ray films, CT and MRI scans and intraoperative frozen sections and were later confirmed by standard histologic examination. Campanacci grades were assigned based on radiologic features and confirmed by at least two board‐certified radiologists31. MRIs of the ilium showed no evidence of GCTs in the control group. A board‐certified pathologist reviewed each sample to confirm viability (>80% by nucleus counts on hematoxylin and eosin [HE] stained sections) and tumor content (>90%) for each sample. Planned analyses were performed on each specimen as sample size allowed.

HE Staining

Standardized HE stained paraffin‐embedded samples were examined to evaluate the morphology of GCT tissue. Fixed tissues were dehydrated in graded ethanol solutions and embedded in paraffin. For each specimen, three serial sections (4 μm thick) were cut on a microtome and stained with HE. The specimens were examined and photographed using a high‐quality microscope. Osteoclasts were counted in four high power (HP, ×200) fields for each GCT patient and the areas were measured using the Osteomeasure Analysis System (Osteometrics, Atlanta, GA, USA).

RNA Extraction and Quality Assessment

Total RNA from each sample was individually isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and an miRNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. This procedure efficiently recovered all RNA species, including miRNAs. RNA quality and quantity were measured using a NanoDrop spectrophotometer (ND‐1000; NanoDrop Technologies, Wilmington, DE, USA).

Quantitative Reverse Transcription Polymerase Chain Reaction (PCR)

Twenty miRNAs from the array data analysis (data not shown) were selected for validation using an SYBR‐based quantitative PCR method. Expression of three osteoclast‐specific genes (cathepsin K, MMP‐9 and tartrate‐resistant acid phosphatase [TRAP]) was also analyzed. Total RNA (100 ng) was reverse transcribed to cDNA using miRNA‐specific stem‐loop RT primers in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed using SYBR‐Green (Invitrogen) according to the manufacturer's instructions in a Rotor‐Gene 3000 Real‐time PCR instrument (Corbett Research, Brisbane, QLD, Australia). miRNA expression was normalized to U6 as an internal control. RNA results were normalized to the expression of glyceraldehyde phosphate dehydrogenase. The relative abundance of each RNA was calculated using a comparative Ct (2‐ΔΔCt) method and the results were assessed by Student's t‐test.

Statistical Analysis

Statistical analysis was performed using SPSS 16.0 (SPSS, Chicago, IL, USA). All data are presented as mean ± SD. Comparisons of two‐group variables were performed using Student's t test. Spearman's test was used to analyze correlations between expression of miRNAs and GCT. P values of less than 0.05 were considered statistically significant.

Results

Patient Characteristics

The GCT group consisted of five male and six female subjects, ranging from 17 to 61 years in age (mean age 32.9 years; SD, 12.8 years). Six of these patients underwent intralesional curettage surgery and the other five wide resection. Four patients had Campanacci Grade I tumors, three Grade II and three Grade III (Table 1).

The average age of the control group was 28.75 years (SD, 14.24 years). All of them were diagnosed as having benign tumors and underwent iliac grafting (Table 2).

Table 2.

Clinical characteristics of patients in control group

Sample	Sex	Age (years)	Location	Diagnosis
1	Male	27	R. P. Femur	Enchondrosis
2	Male	16	R. Humerus	Aneurysm like bone cyst
3	Female	49	R. Humerus	Aneurysm like bone cyst
4	Male	23	L. P. Tibia	Enchondrosis

R/L, Right/Left; D/P, Distal/Proximal.

Radiological and Morphological Changes in GCTs

X‐ray films and HE staining demonstrated the following features in patients with GCTs (Fig. 1). X‐ray films of the GCT group (Fig. 1a,b) showed varying degrees of osteolytic bone destruction with or without a thin border of sclerotic bone, the cortex being expanded or breached depending on the stage of GCT. Campanacci Grade I (Fig. 1a) GCT tumors had well defined borders of a thin rim of mature bone and the cortex was intact or slightly thinned but not deformed. Grade II tumors (Fig. 1b) had relatively well defined margins but no radiopaque rim; the combined cortex and rim of reactive bone, though rather thin and moderately expanded, was still present.

Figure 1.

Radiological and morphological changes in GCT. Plain films of subjects in GCT group showing different degrees of bone destruction (arrow). (A) Campanacci grade I showing a well‐defined border of thin rim of mature bone and an intact or slightly thinned cortex that is not deformed. (B) A grade II tumor showing relatively well defined margins but no radiopaque rim; the combined cortex and rim of reactive bone is rather thin and moderately expanded but still present. (C, D) HE stained photomicrographs showing obvious increased numbers of enlarged osteoclasts (arrows).

HE staining (Fig. 1c,d) showed three main components: spindle‐like stromal cells, monocytic round cells and obviously increased osteoclast‐like multinucleate giant cells in the GCTs. The numbers and average areas of osteoclasts’ are shown in Table 3. Campanacci grades were assigned by at least two board‐certified radiologists assessments of X‐ray films according to the established criteria (Table 1). HE staining demonstrated that GCTs of higher Campanacci grade had more numerous and larger osteoclasts (P < 0.05).

Table 3.

Morphological changes in patients in the GCT group

Sample	Campanacci grade	OC/HP	OC area
1	I	19	0.8
2	III	43	1.66
3	III	46	1.78
4	III	31	2.19
5	III	39	1.72
6	II	27	1.29
7	I	25	1.12
8	II	29	1.32
9	I	21	0.91
10	II	25	1.59
11	I	24	1.17

OC/HP, number of osteoclasts per high power field; OC area, average osteoclast area relative to average Campanacci Grade I.

Expression of Osteoclast‐specific Genes is Increased in GCTs

Reverse transcription PCR was performed to evaluate the osteoclast‐related genes: cathepsin K, MMP‐9 and TRAP. All these three genes showed significantly stronger expression than the controls (Fig. 2).

Figure 2.

Osteoclast‐specific genes expression is stronger in GCTs than in controls. **, P < 0.01.

MiRNAs are Differentially Expressed in GCTs and Control

Microarrays were initially used to screen osteoclast specific miRNAs by comparing the miRNA expression profiles of pre‐osteoclasts and osteoclasts (data not shown). Based on the microarray data, 20 miRNAs were selected for further study. Among the 20 osteoclastogenesis‐related miRNAs, six miRNAs were differentially expressed to a statistically significant degree (Fig. 3A–F). As demonstrated by quantitative real‐time PCR, all six of them show significantly decreased expression in most GCTs (P < 0.05). For specific miRNAs the probabilities of the lower values being significant versus the controls were as follows: for has‐let‐7a‐5p P = 0.0001; for has‐mir‐10b‐5p P < 0.0001; for has‐mir‐16‐5p P = 0.0002; for has‐mir‐106b‐5p P = 0.0055; for has‐mir‐224‐5p P = 0.0001; and for has‐mir‐876‐5p P < 0.01.

Figure 3.

MiRNAs expression in GCTs are all significantly weaker than in controls (P < 0.01, vs. control). The relative expression of (A) has‐let‐7a‐5p, (B) has‐mir‐10b‐5p, (C) has‐mir‐16‐5p, (D) has‐mir‐106b‐5p, (E) has‐mir‐224‐5p and (F) has‐mir‐876‐5p in iliac bone samples of patients in the control and GCT groups are shown. ** P < 0.01.

Association of the Severity of Bone Resorption and Strength of Expression of Osteoclast‐related miRNA

To investigate which miRNAs indicate the severity of bone destruction in patients with GCTs, the relationship between miRNA expression and severity of bone destruction according to the Campanacci grading system was evaluated. Spearman's test was then performed using SPSS. Correlations were identified between the strength of expression of has‐mir‐16‐5p and Campanacci grade (P = 0.009). The same was true for has‐let‐7a‐5p and Campanacci grade (P = 0.034). Reduced expressions of these two miRNAs may indicate more severe bone destruction (Table 4).

Table 4.

Spearman correlations between Campanacci grade and miRNA (n = 11)

Index		7a‐5p	224‐5p	10b‐5p	876‐5p	106b‐5p	16‐5p	Campanacci
7a‐5p	r	—	0.709	0.745	0.655	0.645	0.755	−0.640
	P		0.015	0.008	0.029	0.032	0.007	0.034
224‐5p	r	0.709	—	0.436	0.536	0.736	0.482	−0.405
	P	0.015	—	0.180	0.089	0.010	0.133	0.217
10b‐5p	r	0.745	0.436	—	0.727	0.764	0.755	−0.405
	P	0.008	0.180	—	0.011	0.006	0.007	0.217
876‐5p	r	0.655	0.536	0.727	—	0.491	0.427	−0.135
	P	0.029	0.089	0.011	—	0.125	0.190	0.693
106b‐5p	r	0.645	0.736	0.764	0.491	—	0.727	−0.371
	P	0.032	0.010	0.006	0.125	—	0.011	0.262
16‐5p	r	0.755	0.482	0.755	0.427	0.727	—	−0.742
	P	0.007	0.133	0.007	0.190	0.011	—	0.009

According to the Spearman test, expression of has‐let‐7a‐5p and has‐16‐5p correlates with Campanacci grade (P = 0.034 and 0.009, respectively).

Discussion

Six miRNAs Were Downregulated in GCTs

In the present study, we collected 11 samples from patients with GCTs. Because it is not ethical to obtain bone tissue from completely normal persons to serve as normal controls, we obtained control samples from four patients with benign tumors requiring iliac grafting. We quantified their miRNA expression patterns and evaluated differences in their miRNA expression profiles by RT‐PCR analysis. We demonstrated that six miRNAs were downregulated in the GCT group compared with the control group: has‐mir‐876‐5p, has‐mir‐10b‐5p, has‐mir‐106b‐5p, has‐let‐7a‐5p, has‐mir‐16‐5p and has‐mir‐224‐5p.

Prediction of Osteoclastogenesis‐related miRNA Functions

Analysis of the three most popular database, TargetScan32, miRanda33 and miRDB34 revealed that these miRNAs have been reported to function in various pathological processes. Has‐mir‐16‐5p reportedly targets several mRNAs, including SMAD3. Li et al., reported that expression of miR‐16‐5p was significantly greater in osteoarthritis cartilage than in healthy cartilage, indicating that miR‐16‐5p is an important regulator of SMAD3 expression in human chondrocytes and may contribute to the development of osteoarthritis35. Given that the abovementioned biological processes and genes are involved in the pathological changes observed in osteoclastogenesis, we predict that has‐mir‐16‐5p may play an important role in the occurrence and development of GCTs. In GCT, osteoclasts are excessively differentiated and thus cause bone destruction36.

Warnecke‐Eberz et al. showed that miR‐224‐5p is significantly downregulated in adenocarcinoma compared with normal tissue and barely or not detectable in exosomes37. Zhao et al. reported that the PRKCD gene is one of the targets of miR‐224‐5p in mediating the primary chemoresistance of ovarian cancer patients and quantitative PCR showed reciprocal expression of miR‐224‐5p and PRKCD38. In a rat study, Zhang et al. reported that has‐let‐7a‐5p may target 1259 MTGs, which functions in lipid biosynthesis and influences lactation39.

Mangolini et al. have report that high serum concentrations of miR‐10b‐5p are associated with clinicobiological markers of poor prognosis in subjects with breast cancer18.

Main Gene of Osteoclastogenesis and Osteoclast Function

MMPs comprise more than 20 enzymes, which degrade the basement membrane and extra‐cellular matrix components in numerous physiological and pathological situations40, 41, 42. MMP‐9 acts as an important oncogene, thereby increasing the invasiveness of cancer cells43, 44. MMP‐9 also plays a role in the resorption of the organic matrix because large amounts of this protein are present in resorption lacunae45.

Cathepsin K, a unique and potent collagenase, is primarily expressed in osteoclasts and is responsible for degradation of the collagen matrix in bone. The critical involvement of cathepsin K in bone remodeling is supported by the finding that cathepsin K deficiency causes the bone‐sclerosing disorder pycnodysostosis46 and by the ability of specific cathepsin K inhibitors to alleviate bone resorption in a primate model of hypogonadism47. During osteoclast‐mediated bone resorption, an acidic microenvironment is created through proton extrusion mediated by a proton pump belonging to the class of vacuolar H‐adenosine triphosphate (ATP)ases (V‐H‐ATPases). Co‐expression of V‐H‐ATPases and cathepsin K has also been found in multinucleated giant cells; hence these cells are able to create the acidic microenvironment required for cathepsin K activity and stability14.

Mature osteoclasts secrete TRAP, which aids in degradation of the bone matrix48. Disruption of the cathepsin K and TRAP genes have been shown to lead to osteopetrosis (a congenital disorder characterized by overly dense bones)49, 50.

In addition, MMP‐9 and cathepsin K are released by mature osteoclasts to degrade bone by removing bone‐lining collagen51, 52, 53, 54.

Thus, cathepsin K, MMP‐9 and TRAP are the main markers of osteoclastogenesis and the function of osteoclasts55. Hence their strong expression in GCTs indicates excessive production and enhanced function of osteoclasts. Our results suggest that miRNAs may be the upstream regulators of these vital osteoclast‐specific genes in GCTs.

In conclusion, our results demonstrate that, compared with normal bone tissue, six miRNAs are downregulated in the tumor tissue of GCT patients. Bioinformatics analysis predicted the target genes and signaling pathways of these miRNAs, which may enhance our understanding of the involvement of miRNAs in the occurrence and development of GCT. Further studies on miRNA functions and target gene verification would provide an experimental basis for the diagnosis and treatment of GCT; this remains an important area for future investigation.