Skip to main content
Intended for healthcare professionals
Open access
Research article
First published online May 14, 2019

Clinical significance of tumor miR-21, miR-221, miR-143, and miR-106a as biomarkers in patients with osteosarcoma

Hui Zhao, Peng Yan, Jian Wang, Yuqiang Zhang, Mingchao Zhang, Zaijun Wang, Qiang Fu, and Weiguo Liang https://orcid.org/0000-0003-4152-3775 [email protected]View all authors and affiliations
Volume 34, Issue 2
https://doi.org/10.1177/1724600819843537

Abstract

Objective:

To investigate the expression of miR-21, miR-221, miR-143, and miR-106a in patients’ osteosarcoma samples, and to explore the correlation between these microRNAs (miRNAs) and the clinical stage of osteosarcoma.

Methods:

RNA was extracted from tumor and tumor-adjacent normal bone tissues from 94 patients with osteosarcoma. RNA reverse-transcription was carried out using an miRNA reverse transcription kit. The levels of miR-21, miR-221, miR-143, and miR-106a in osteosarcoma and normal bone tissues were analyzed by real-time polymerase chain reaction using SYBR Premix Ex Taq™II.

Results:

The expression levels of miR-21, mirR-221, and miR-106a were significantly higher in 90.42%, 84.04%, and 92.55 % of the osteosarcoma samples compared to the adjacent normal tissues (P<0.05), respectively. While the expression of miR-143 was significantly lower compared to the adjacent normal tissues (P <0.05). Moreover, the expression levels of miR-21 and miR-221 were positively correlated with the Enneking clinical stage and the presence of lung metastasis (P <0.05), while the expression levels of miR-143 and miR-106a showed a significant inverse and direct correlation respectively, with the tumor grade.

Conclusions:

The upregulation of miR-21, miR-221, and miR-106a, as well as the down-regulation of miR-143 were correlated with the pathological stage, tumor grade, and lung metastasis. Therefore, the levels of these miRNAs can serve as potential biomarkers for the early diagnosis of osteosarcoma, and can be used as potential therapeutic targets.

Introduction

Osteosarcoma, a highly malignant mesenchymal tumor, is the most common primary malignant bone tumor in pediatric patients. Osteosarcoma has a rapid growth, short duration, strong metastasis, and low cure rate. Metastasis is an important cause of death in patients with osteosarcoma. The probability of lung metastases in patients with osteosarcoma at the time of diagnosis is 20%. The probability of metastasis is 40% during the mid-late stage of treatment. Within 6–12 months after surgery, 80% of patients will develop pulmonary metastases.1 The survival rate of osteosarcoma has been increased to 60% with new adjuvant chemotherapy, surgical resection, and other methods.2,3.
microRNAs (miRNAs) are small non-coding regulatory RNA molecules (21-25 nt) that play an important role in tumor formation and progression. miRNAs can control a variety of biological processes, including cell division, proliferation, apoptosis, invasion, metastasis, development, metabolism, and tumorigenesis.4
miR-21 is widely expressed in human cells and tissues and is highly expressed in many human tumor cells, such as gastric cancer, non-small cell lung cancer, colon cancer, and liver cancer.5 miR-21 is involved in the regulation of a variety of tumor suppressor genes and apoptosis-related proteins, including tropomyosin-1 (TPM1), programmed death protein 4 (PDCD4), metalloproteinase inhibitor 3 (TIMP3), Fas ligand (FasL), heterogeneous ribonucleic acid protein K (HNRPK), and phosphatase and tensin homolog (PTEN)5. miR-21 can negatively regulate these genes’ expressions and participate in tumor growth, invasion, and metastasis.6 miR-221 is located in the P11.3 region (~1kb) of the X chromosome and regulates a variety of important physiological, pathological, and cell-mediated pathways.7 A large number of studies have found that miR-221 is highly expressed in a variety of human tumors. It is involved in the tumorigenesis through targeting genes that inhibit apoptosis and promote cell proliferation, such as p27, p53, and pro-apoptotic factors Bim and Bmf. miR-143, also known as hsa-miR-143, is located on chromosome 5q32.8 miR-143 can prevent the proliferation, invasion, metastasis, and drug resistance of malignant tumor cells. It is down-regulated in many human malignant tumors.9 miR-106a is composed of 22 nucleotides, and is located in the q26.2 region of the X chromosome. The miR-106a expression is closely related with tumor cell division, proliferation, apoptosis, and drug resistance.10
Studies show that miR-21, miR-221, miR-143, and miR-106a are upregulated in various tumors;11–13 however, the expression of these miRNAs in osteosarcoma tissue, as well as their correlation with clinical pathology, is still unclear. In this study we compared the expression of these miRNAs between osteosarcoma and normal tissues, and analyzed the relationship between miRNA levels and clinicopathological features. Our goal was to identify whether these miRNAs are involved in the regulation of osteosarcoma, as well as their roles in the diagnosis or prognosis of osteosarcoma.

Material and methods

Clinical data

Inclusion criteria were (a) patients with osteosarcoma confirmed by clinical manifestations, imaging results, and histopathology; and (b) patients who did not receive chemotherapy, cellular immunotherapy, and other treatments before surgery. Patients with systemic infection and other tumors were excluded. A total of 94 patients with osteosarcoma (50 males and 44 females) were enrolled in the Department of Orthopedics and Oncology at the First Affiliated Hospital of Jinzhou Medical University from January 2014 to June 2017. There were 68 primary cases and 26 recurrent cases. A primary case was defined as a tumor caused by the change of the organ itself. A recurrent case was defined as metastatic tumors and secondary tumors. During the tumor growth, tumor cells can spread via the blood and lymph, and form secondary tumors in new locations. The mean age of the 94 patients was 46.8 ± 5.4 years (range 16–72 years). Tumor and adjacent normal bone tissues were collected during surgical resection or biopsy at the First Affiliated Hospital of Jinzhou Medical University. The adjacent normal bone tissues were located more than 5 cm away from the edge of the tumor, and pathologically confirmed as non-tumor tissues. The tumor localization included 35 cases in distal femur, 49 cases in proximal tibiofibular, and 14 cases in other locations. A total of 41 cases had a tumor diameter ⩽5 cm and 58 cases had a tumor diameter> 5cm. There were 58 cases with lung metastases and 36 cases without. Clinical stages of the tumor were divided into stage I, stage IIA and stage III in accordance with the Enneking staging system. The tumor types were classified as osteoblasts, fibroblasts, chondroblasts, and others in accordance with Dahlin’s classification. The specimens collected during surgery were fixed with a 4% formaldehyde solution for 24 hours, and then were paraffin-embedded. The paraffin-embedded specimens were used for pathological examination and detection of the gene expression of miR-21, miR-221, miR-143, and miR-106a. This study was approved by the Ethics Committee of IRB of Medical College, Jinan University (NO. WDKQSPF/SQ-04). All participants signed the informed consent form.

Experimental reagents

The Recover All™ Total Nucleic Acid Isolation Kit was purchased from Ambion (Applied Biosystems, Canada). The microRNA Reverse Transcription Kit was purchased from TAKARA Bio USA, Inc. (Takara Biomedical Technology (Beijing) Co. Ltd., Beijing, China). The DEPC reagent was purchased from Amresco (VWR International, LLC, USA). The SYBR Green PCR Master Mix, mi Script Precursor Assay, primers and internal reference U6 primer were purchased from Bioengineering Co., Ltd (Shanghai, China). The primer sequences were as follows: miR-21 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA-3′; miR-221 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGAA-3′; miR-143 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGTACGACGAGCTACA-3′; miR-106a 5′-GTCGTATCCAGTGCGTGTCGTGGAGTGGCAATTGCACTGGATACGACCTACCTG-3′; U6 5′-CGCTTCACGAATTTGCGTGTCAT-3′. Other reagents were provided by the First Affiliated Hospital of Jinzhou Medical Biological Sample Center.

Total RNA extraction

The paraffin-embedded tissues (20 μm/section and 3–4 sections) from each sample were deparaffinized. RNA was extracted using a RecoverAll™ Total Nucleic Acid Isolation Kit. The 260/280 ratio of each RNA sample was detected by Ultraviolet spectrophotometer. The ratio of 1.8:2.1 was defined as the normal range. RNA samples were stored at −80°C.

RNA reverse transcription

RNA samples were reverse transcripted using the microRNA Reverse Transcription Kit. The reaction system (20μL) included 10μL of 2X miRNA Reaction Buffer Mix, 2μL of 0.1% BSA, 2μL of miRNA Prime Script RT Enzyme Mix, 1μL of total RNA, and 5µL of RNase Free dH2O. The reverse transcription conditions were as follows: miR-21: 37℃, 60 min; 95℃, 5 min. miR-221: 16℃, 30 min; 42℃, 30 min; 75℃, 15 min. miR-143: 16℃, 30 min; 42℃, 30 min; 75℃, 15 min. miR-106a: 16℃, 30 min; 42℃, 40 min; 85℃, 5 min. U6: 25℃, 30 min; 42℃, 30 min; 85℃, 5 min. The final products were stored at −20°C.

Real-time quantitative PCR

The polymerase chain reaction (PCR) amplification reaction was performed using SYBR Premix Ex Taq™ II. The reaction system (20 μL) included 10µL of SYBR Premix Ex Taq™ II, 0.8 μL of PCR forward primer (10 μmoL), 0.8 μL of PCR reverse primer (10 μmoL), 0.4 μL of ROX reference II (50×), 2.0 µL of DNA template, and 6.0 µL of dH2O. The primer sequences were as follows: miR-21 forward F5′-GCGGCGGTAGCTTATCAGACTG-3′, reverse R5′-ATCCAGTGAGGGTCCGAGG-3′; miR-221 forward F5′-CATTGGACCTGGCATACAATGTAGAT -3′, reverse F5′-AACGCTTCACGAATTTGCGT-3′; miR-143 forward F5′-TGTAGTTTCGGAGTTAGTGTCGCGC-3′, reverse R5′-CCTACGATCGAAAACGACGCGAACG-3′; miR-106a forward F5′-ATCCAGTGCGTGTCGTG-3′, reverse F5′-TGCTAAAAGTGCTTACAGTG-3′; U6 forward F5′-GTTTTGTAGTTTTTGGAGTTAGTGTTGTGT-3′, reverse R5′-CTCAACCTACAATCAAAAACAACACAAACA-3′. miR-21 reaction conditions were 95°C, 5 min; 94°C, 15s; 55°C, 30s; 70°C, 30s; 35 cycles. miR-221 reaction conditions were 95°C, 30s; 95°C, 5s; 60°C, 30s; 40 cycles. miR-143 reaction conditions were 95°C, 10 min; 95°C, 15s; 60°C, 1 min; 40 cycles. miR-106a reaction conditions were 95°C, 5 min; 95°C, 10s; 60°C, 20s; 40 cycles. Fluorometric analysis of RNA fragmentation obtained by Applied Biosystems ABI7500 (Thermo Fisher Scientific). U6 was used as the internal reference, which had similar amplification efficiency as that of the target genes. The cycle threshold (Ct value) was detected and N= 2-△△Ct method was used to calculated the expression of the miRNAs in the tumor tissues relative to the normal tissues. △△Ct= (CtmiR-21/221/143/106a - CtU6 ) tumor - (CtmiR-21/221/143/106a - CtU6 ) normal control.

Statistical analysis

SPSS 18.0 statistical analysis software was used to organize and analyze the experimental data. All statistical data were expressed as median and interquartile range. Independent samples t test was used for two-group comparisons. One-way analysis of variance (ANOVA) was used for multi-group comparisons. The expression of miRNAs was divided into a “low-expression” group and a “high expression” group based on the median value. The correlation of miRNAs with tumor clinical stages and pathological features were analyzed. P<0.05 was defined as statistically significant.

Results

The miR-21 expression increased in osteosarcoma tissues

Real-time quantitative PCR was used to analyze the expression of miR-21 in 94 cases of osteosarcoma and the adjacent normal bone tissues. We found that miR-21 was expressed in both primary and recurrent osteosarcoma, as well as the adjacent normal bone tissues. miR-21 expression was significantly higher in the primary and recurrent osteosarcoma compared to the adjacent normal bone tissues in 85 patients with osteosarcoma (90.42%) (P=0.037 and P=0.014, respectively). The expression of miR-21 was significantly higher in the recurrent osteosarcoma compared to the primary osteosarcoma (P=0.026) (Supplemental Figure 1).

The relationship between miR-21 expression and clinicopathological parameters of osteosarcoma

We further analyzed the relationship between miR-21 expression and the clinicopathological parameters of osteosarcoma. We found that the expression of miR-21 in osteosarcoma was positively correlated with the Enneking clinical stages and lung metastasis. The difference was statistically significant (P=0.019 and P=0.027, respectively). There was no significant correlation between miR-21 expression in osteosarcoma and other indicators, such as gender, age, tumor location, tumor size, Dahlin’s classification, and tumor grade (Table 1).
Table 1. The correlation of miR-21 expression and clinicopathological parameters of osteosarcoma.
ParametersnmiR-21χ2P value
Low
expression		High
expression		 
Gender     
Male5013378.0580.478
Female441727
Age (year)     
⩽203814242.8430.194
>20562234
Tumor diameter (cm)     
⩽5419326.8150.067
>5531142
Tumor location     
Distal femur3512232.1500.283
The proximal tibiofibular491831
Other14212
Lung metastasis     
Yes3617199.8560.027*
No58949
Dahlin’s classification     
Osteoblasts3113187.6280.357
Fibroblasts27720
Chondroblasts1899
Other18612
Enneking staging system     
I27151210.7310.019*
ⅡA56749
Ⅲ1129
Tumor grade     
High grade3917224.3610.175
Low grade552134
*
P<0.05, compared with the low-expression group.

The miR-221 expression increased in osteosarcoma tissues

The expression of miR-221 in 94 cases of osteosarcoma was analyzed. We found that miR-221 was expressed in both primary and recurrent osteosarcoma, as well as the adjacent normal bone tissues. The expression of miR-221 in 79 cases of osteosarcoma was significantly higher compared to the adjacent normal bone tissues (84.04%). The expression of miR-221 in the primary osteosarcoma was significantly lower compared to the recurrent osteosarcoma (P = 0.023). The expression of miR-221 in the normal bone tissues was significantly lower compared to the primary and recurrent osteosarcoma (P=0.031 and P=0.012, respectively) (Supplemental Figure 2).

The relationship between miR-221expression and clinicopathological parameters of osteosarcoma

We further analyzed the relationship between the expression of miR-221 and the clinicopathological parameters of osteosarcoma. The expression of miR-221 in osteosarcoma was positively correlated with the Enneking clinical stages and lung metastasis (P = 0.016 and P=0.004, respectively). The expression of miR-221 in osteosarcoma was not correlated with gender, age, tumor location, tumor size, Dahlin’s classification, and tumor grade (P> 0.05) (Table 2).
Table 2. The correlation of miR-221 expression and clinicopathological parameters of osteosarcoma.
ParametersnmiR-221χ2P value
Low expression
(%)		High expression
(%)
Gender     
Male5015 (30)35 (70)4.8610.137
Female4414 (31.8)30 (68.2)
Age (year)     
⩽20389 (23.6)29 (76.4)3.7840.297
>205618 (32.1)38 (67.9)
Tumor diameter (cm)     
⩽54111 (26.8)30 (73.2)5.0630.431
>55315 (28.3)38 (71.7)
Tumor location     
Distal femur3510 (28.6)25 (71.4)3.5690.127
The proximal tibiofibular4919 (38.3)20 (61.2)
Other144 (28.6)10 (71.4)
Lung metastasis     
Yes3626 (72.2)10 (27.8)7.6140.004*
No5815 (25.9)43 (74.1)
Dahlin’s classification     
Osteoblasts3111 (35.5)20 (64.5)6.2390.084
Fibroblasts279 (33.3)18 (66.7)
Chondroblasts189 (50)9 (50)
Other184 (22.2)14 (77.8)
Enneking staging system     
I2711 (40.7)16 (59.3)8.3150.016*
ⅡA569 (16.1)47 (83.9)
Ⅲ114 (36.4)7 (63.6)
Tumor grade     
High grade3912 (30.8)27 (69.2)3.6050.144
Low grade5516 (29.1)39 (70.9)
*
P<0.05, compared with the low-expression group.

The miR-143 expression decreased in osteosarcoma tissues

The expression of miR-143 in 94 cases of osteosarcoma was analyzed. We found that miR-143 was expressed in both primary and recurrent osteosarcoma, as well as normal bone tissues. The expression of miR-143 was significantly lower in 89 cases of osteosarcoma compared to the adjacent normal bone tissues (94.68%) (P<0.05). The expression of miR-143 in the primary and recurrent osteosarcoma was significantly lower compared to the normal bone tissues (P=0.006 and P=0.009, respectively). There was no significant difference in the miR-143 expression between the primary and recurrent osteosarcoma (P=0.389) (Supplemental Figure 3).

The relationship between miR-143 expression and clinicopathological parameters of osteosarcoma

We further analyzed the relationship between the expression of miR-143 and the clinicopathological parameters of osteosarcoma. The expression of miR-143 was significantly correlated with tumor differentiation degree and lung metastasis. The expression of miR-143 was significantly lower in low-grade osteosarcoma compared to high-grade osteosarcoma (P = 0.014). The expression of miR-143 was significantly lower in osteosarcoma with lung metastasis compared to osteosarcoma without lung metastases (P = 0.035). The expression of miR-143 was not correlated with gender, age, tumor location, tumor size, Enneking clinical stage, and Dahlin’s classification (Table 3).
Table 3. The correlation of miR-143 expression and clinicopathological parameters of osteosarcoma.
ParametersnmiR-143χ2P value
Low expression (%)High expression (%)
Gender     
Male5019 (38)31 (62)3.7810.304
Female4417 (38.6)27 (61.4)
Age (year)     
⩽203811 (28.9)27 (71.1)2.4970.154
>205624 (42.9)32 (57.1)
Tumor diameter (cm)     
⩽54113 (31.7)28 (68.3)6.1260.719
>55319 (35.8)34 (64.2)
Tumor location     
Distal femur3513 (37.1)22 (62.9)2.7180.325
The proximal tibiofibular4921 (42.9)28 (57.1)
Other145 (35.7)9 (64.3)
Lung metastasis     
Yes3621 (58.3)15 (41.7)4.1670.035*
No5818 (31)40 (69)
Dahlin’s classification     
Osteoblasts319 (29)22 (71)5.4130.125
Fibroblasts277 (25.9)20 (74.1)
Chondroblasts186 (33.3)12 (66.7)
Other188 (44.4)10 (55.6)
Enneking staging system     
I278 (29.6)19 (70.4)1.3490.016*
ⅡA5625 (44.6)31 (55.4)
Ⅲ114 (36.4)7 (63.6)
Tumor grade     
High grade3929 (74.4)10 (25.6)1.0950.014*
Low grade5513 (23.6)42 (76.4)
*
P<0.05, compared with the low-expression group.

The miR-106a expression increased in osteosarcoma tissues

The expression of miR-106a was analyzed in 94 cases of osteosarcoma. miR-106a was expressed in both primary and recurrent osteosarcoma, as well as normal bone tissues. The expression of miR-106a in 87 cases of osteosarcoma was significantly higher compared to the adjacent normal bone tissues (92.55%) (P<0.05). The expression of miR-143 in primary and recurrent osteosarcoma was significantly higher compared to the normal bone tissues (P=0.033 and P=0.028, respectively). There was no significant difference between primary and recurrent osteosarcoma (P=0.473) (Supplemental Figure 4).

The relationship between miR-106a expression and clinicopathological parameters of osteosarcoma

The expression of miR-106a was significantly correlated with the tumor grade and lung metastasis. The expression of miR-106a was significantly higher in low-grade osteosarcoma compared to high-grade osteosarcoma (P = 0.012). The expression of miR-106a was significantly higher in osteosarcoma with lung metastasis compared to osteosarcoma without lung metastasis (P = 0.006). The expression of miR-106a was not correlated with gender, age, tumor location, and tumor size (Table 4).
Table 4. The correlation of miR-106a expression and clinicopathological parameters of osteosarcoma.
ParametersnmiR-106aχ2P value
Low expression (%)High expression (%)
Gender     
Male5013 (26)37 (74)2.9710.235
Female4415 (34.1)29 (65.9)
Age (year)     
⩽20387 (18.4)31 (81.6)4.3850.143
>205620 (35.7)36 (64.3)
Tumor diameter (cm)     
⩽54114 (34.1)27 (65.9)3.5610.578
>55321 (39.6)32 (60.4)
Tumor location     
Distal femur3513 (37.1)22 (62.9)6.3710.276
The proximal tibiofibular4915 (30.6)24 (36.4)
Other142 (28.6)12 (71.4)
Lung metastasis     
Yes3619 (52.8)17 (47.2)1.6950.006*
No5816 (27.6)42 (72.4)
Dahlin’s classification     
Osteoblasts3112 (38.7)19 (61.3)2.3690.067
Fibroblasts2711 (40.7)16 (59.3)
Chondroblasts186 (33.3)12 (66.7)
Other186 (33.3)12 (66.7)
Enneking staging system     
I2711 (40.7)16 (59.3)8.3150.016*
ⅡA569 (16.1)47 (83.9)
Ⅲ114 (36.4)7 (63.6)
Tumor grade     
High grade3919 (48.7)20 (51.3)3.6050.012*
Low grade5511 (20)44 (80)
*
P<0.05, compared with low-expression group.

The impact of clinicopathological parameters on the prognosis of patients with osteosarcoma

The impact of gender, age, tumor size, metastasis, clinical stage, tumor grade, Dahlin’s classification, miR-21, miR-221, miR-143, and miR-106a on the prognosis of patients with osteosarcoma was analyzed by COX regression with univariate and multivariate analysis. The results are shown in Table 5 and Table 6. Univariate analysis showed that tumor size, metastasis, clinical stage, tumor grade, miR-21, miR-221, miR-143, and miR-106a were prognostic risk factors. Multivariate analysis showed that metastasis, clinical stage, miR-21, miR-221, miR-143, and miR-106a were independent risk factors affecting the prognosis of patients with osteosarcoma.
Table 5. Univariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
ParameterHazard ratios95% CIP value
Gender1.0890.345–2.7560.634
Age0.9030.614–1.9930.564
Tumor size1.8421.038–3.6160.028
Metastasis3.0141.194–5.0210.017
Clinical stage2.6031.203–3.7390.011
Tumor grade1.2410.597–2.6470.023
Dahlin’s classification0.6470.317–1.9470.084
miR-214.3253.029–5.9240.008
miR-2214.1873.015–5.7210.013
miR-1433.9472.168–4.6860.019
miR-106a3.8422.013–4.5990.026
CI: confidence interval.
Table 6. Multivariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
ParameterHazard ratios95% CIP
Gender0.9430.457–1.8750.315
Age0.8120.706–1.7820.327
Tumor size1.4631.697–3.0190.159
Metastasis2.4611.875–4.1580.024
Clinical stage2.0671.943–2.9390.028
Tumor grade0.9830.806–2.0640.076
Dahlin’s classification0.3580.328–1.6850.124
miR-213.9973.221–5.1530.011
miR-2213.6543.309–4.9870.019
miR-1433.1632.375–4.1790.024
miR-106a3.3152.331–3.9950.031
CI: confidence interval.

Discussion

Previous studies have found that miRNA-21, miRNA-221, and miRNA-106a were elevated in the circulation in osteosarcoma patients. However, the correlation between the expression of these miRNAs and the clinicopathological parameters are still unclear. Our study is the first to demonstrate that miRNA-21, miRNA-221, and miRNA-106a are upregulated, while miRNA-143 is down-regulated in osteosarcoma tissues. Moreover, the changes in these miRNA levels are correlated with the tumor stage, degree of differentiation, and lung metastasis of osteosarcoma.
Evidence indicates that miR-21 can inhibit the expression of the tumor suppressor gene PTEN in liver cancer cells.14 The upregulation of miR-21 in the colorectal cancer cell line cob206f can inhibit the expression of PDCD4 and induce tumor cell invasion and metastasis. Studies have shown that miR-21 expression is significantly up-regulated in breast cancer, gastric cancer, lung cancer, colon cancer, and glioma. Moreover, miR-21 expression is positively correlated with lymph node metastasis in colon cancer, and with metastasis in breast cancer.15 One study demonstrates that serum miR-21 increased in patients with osteosarcoma, which is correlated with lung metastasis, but is not correlated with the Enneking clinical stages.16 In the present study, we found that the expression of miR-21 in 85 cases of osteosarcoma was significantly higher compared to the adjacent normal bone tissues (90.42%). Moreover, miR21 expression was significantly correlated (P<0.05) with the Enneking clinical stages and lung metastasis. Our findings are consistent with these previous studies, suggesting that miR-21 may be a good biomarker for the early diagnosis of osteosarcoma. It has been found that miR-21 can regulate the expression of downstream molecules of the RECK signaling pathway or methylation of the RECK gene, affecting the invasion and metastasis of osteosarcoma cells.17 The expression of important effector molecules in the RECK signaling pathway and the mechanisms of osteosarcoma development will be investigated in our future study.
The interaction of miR-221 and mRNA-3′UTR of the tumor suppressor PTEN attenuates PTEN’s ability to control cell proliferation, apoptosis, metastasis, and invasion,7 which, in turn, upregulates the expression of cyclin-dependent kinase inhibitor p27kip1 and promotes the proliferation of cancer cells.18 In this study, the expression of miR-221 in 79 cases of osteosarcoma was significantly higher compared to the adjacent normal bone tissues (84.04%) (P <0.05). Our results are consistent with other studies in which miR-221 is upregulated in pancreatic cancer, papillary thyroid cancer, glioblastoma, breast cancer, and prostate cancer.19 Studies also show that the expression of miR-221 is correlated with the invasion and metastasis of papillary thyroid carcinoma.20 The serum miR-221 level is correlated with tumor recurrence and metastasis after liver transplantation in patients with liver cancer.21 In our study, the expression of miR-221 was not correlated with gender and age, but was closely correlated with the clinical stage of the disease (P <0.05). These results indicate that tumor miR-221 expression may be correlated with the invasion and metastasis of osteosarcoma.
miR-143 activates the expression of caspase-3, caspase-8, and caspase-9, inhibits the protein expression of extracellular regulated protein kinase 5 and Bcl-2, and promotes apoptosis.22 Its target genes include the tumor suppressor genes ERK5, KRAS, and DNA methyltransferase A2.23 We found that miR-143 expression significantly decreased in 89 cases of osteosarcoma (94.68%) (P <0.05). The decreased expression of miR-143 was associated with tumor grade and lung metastasis. Our results are consistent with other studies in which the expression of miR-143 is correlated with the degree of lymph node metastasis and tumor differentiation in gastric cancer24 and cervical squamous cell carcinoma.25 However, the low expression of miR-143 is not correlated with the clinical stages and the degree of differentiation of bladder cancer.26 miR-143 inhibits the expression of the RAS oncogene and its downstream transduction pathway, and prevents the formation of cancer cells.27 In our future research we will investigate the relationship between miR-143 expression and RAS activation to elucidate their synergistic roles in the development of osteosarcoma.
The levels of E2F1 and miR-106a are significantly increased, while the expression of tumor suppressor gene Rb1 is significantly decreased in gastric cancer, which leads to the proliferation of cancer cells.25 miR-106a expression is abnormally higher in lymphoma and colorectal adenocarcinoma.28 We found that the expression of miR-106a in osteosarcoma is 2.23 times higher compared to the normal bone tissues, which is consistent with these previous studies. Studies also show that the expression of miR-106a is correlated with the pathological stage of breast cancer (P <0.05), but there is no significant correlation between miRNA-106a expression and gender, age, tumor diameter, and Fuhrman stage in renal cell carcinoma.29 miR-106a expression is associated with distant metastasis in colorectal tumors, as well as lymph node metastasis and clinical stage in lung cancer.30 Our study confirms that the expression of miR-106a correlates with the tumor grade and lung metastasis in osteosarcoma. It has been speculated that Rb1 is a downstream target of miR-106a. miR-106a can reduce Rb1 expression.31 In our future research we will investigate whether miR-106a upregulation can down-regulate Rbl and promote osteosarcoma proliferation.
Our study demonstrates that the upregulation of miR-21, miR-221, and miR-106a, as well as the down-regulation of miR-143 were correlated with the pathological stage, tumor grade, and lung metastasis. In our future study, we will investigate the downstream targets of miR-21, miR-221, miR-143, and miR-106a in correlation to the proliferation, clonal formation, migration, invasion, cycle, and apoptosis of osteosarcoma. Previous studies only included patients with primary cancers. In this population-based study we also included patients with recurrent cancers. However, this may decrease the homogeneity of the samples, and generate issues regarding the selection of higher risk individuals, which may affect treatment quality. In our future study we will focus on analyzing the clinicopathological factors/biomarkers in patients with primary cancers.
In 1948, Mandel and Metais discovered the circulating nucleic acids (CNAs) in the plasma and serum. miRNAs in cancer cells control the production of circulating nucleic acids. Clinical studies have shown that abnormal expressions of miRNAs are found in patients with lung cancer, prostate cancer, ovarian cancer and liver cancer,32 which suggests that miRNAs can be used as biomarkers for the diagnosis of cancers. In this study we measured the expression of miR-21, miR-221, miR-143, and miR-106a in osteosarcoma tissues, and demonstrated their clinical significance in osteosarcoma. We speculate that these miRNAs can be used as (a) biomarkers for the diagnosis and prognosis of osteosarcoma, and (b) new targets for the treatment of osteosarcoma.

Declaration of conflicting interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

References

1. Zhang H, Yan J, Lang X, et al. Expression of circ_001569 is upregulated in osteosarcoma and promotes cell proliferation and cisplatin resistance by activating the Wnt/beta-catenin signaling pathway. Oncol Lett 2018; 16: 5856–5862.
PubMed
Google Scholar
2. Dong J, Wang M, Ni D, et al. MicroRNA-217 functions as a tumor suppressor in cervical cancer cells through targeting Rho-associated protein kinase 1. Oncol Lett 2018; 16: 5535–5542.
PubMed
Google Scholar
3. Han Y, Guo W, Ren T, et al. Tumor-associated macrophages promote lung metastasis and induce epithelial-mesenchymal transition in osteosarcoma by activating the COX-2/STAT3 axis. Cancer Lett 2019; 440–441: 116–125.
Crossref
PubMed
Google Scholar
4. Cheerla N, Gevaert O. MicroRNA based pan-cancer diagnosis and treatment recommendation. BMC Bioinformatics 2017; 18: 32.
Crossref
PubMed
Google Scholar
5. Zheng W, Zhao J, Tao Y, et al. MicroRNA-21: A promising biomarker for the prognosis and diagnosis of non-small cell lung cancer. Oncol Lett 2018; 16: 2777–2782.
PubMed
Google Scholar
6. Lopezanton M, Lambie M, Lopezcabrera M, et al. miR-21 Promotes Fibrogenesis in Peritoneal Dialysis. Am J Pathol 2017; 187: 1537.
Google Scholar
7. Jia QW, Chen ZH, Ding XQ, et al. Predictive effects of circulating miR-221, miR-130a and miR-155 for coronary heart disease: a multi-ethnic study in China. Cell Physiol Biochem 2017; 42: 808–823.
Crossref
PubMed
Google Scholar
8. Li X, Zhao F, Xin M, et al. Regulation of intraocular pressure by microRNA cluster miR-143/145. Sci Rep 2017; 7: 915.
Crossref
PubMed
Google Scholar
9. He Y, Wang G, Zhang L, et al. Biological effects and clinical characteristics of microRNA-106a in human colorectal cancer. Oncol Lett 2017; 14: 830–836.
Crossref
PubMed
Google Scholar
10. Li X, Zhou Q, Tao L, et al. MicroRNA-106a promotes cell migration and invasion by targeting tissue inhibitor of matrix metalloproteinase 2 in cervical cancer. Oncol Rep 2017; 38: 1774–1782.
Crossref
PubMed
Google Scholar
11. Wang C, Jing J, Cheng L. Emerging roles of non-coding RNAs in the pathogenesis, diagnosis and prognosis of osteosarcoma. Invest New Drugs 2018; 36: 1116–1132.
Crossref
PubMed
Google Scholar
12. Wang SN, Luo S, Liu C, et al. miR-491 inhibits osteosarcoma lung metastasis and chemoresistance by targeting alphaB-crystallin. Mol Ther 2017; 25: 2140–2149.
Crossref
PubMed
Google Scholar
13. Li QC, Xu H, Wang X, et al. miR-34a increases cisplatin sensitivity of osteosarcoma cells in vitro through up-regulation of c-Myc and Bim signal. Cancer Biomark 2017; 21: 135–144.
Crossref
PubMed
Google Scholar
14. Wang X, Gao Y, Tian N, et al. Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice. Drug Des Devel Ther 2018; 12: 2431–2442.
Crossref
PubMed
Google Scholar
15. Han JG, Jiang YD, Zhang CH, et al. A novel panel of serum miR-21/miR-155/miR-365 as a potential diagnostic biomarker for breast cancer. Ann Surg Treat Res. 2017; 92: 55–66.
Crossref
PubMed
ISI
Google Scholar
16. Hua Y, Jin Z, Zhou F, et al. The expression significance of serum MiR-21 in patients with osteosarcoma and its relationship with chemosensitivity. Eur Rev Med Pharmacol Sci 2017; 21: 2989–2994.
PubMed
Google Scholar
17. Loayza-Puch F, Yoshida Y, Matsuzaki T, et al. Hypoxia and RAS-signaling pathways converge on, and cooperatively downregulate, the RECK tumor-suppressor protein through microRNAs. Oncogene 2010; 29: 2638–2648.
Crossref
PubMed
Google Scholar
18. le Sage C, Nagel R, Egan DA, et al. Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation. EMBO J 2007; 26: 3699–708.
Crossref
PubMed
Google Scholar
19. Kawaguchi T, Komatsu S, Ichikawa D, et al. Clinical impact of circulating miR-221 in plasma of patients with pancreatic cancer. Br J Cancer 2013; 108: 361–9.
Crossref
Google Scholar
20. Dai L, Wang Y, Chen L, et al. MiR-221, a potential prognostic biomarker for recurrence in papillary thyroid cancer. World J Surg Oncol 2017; 15: 11.
Crossref
PubMed
Google Scholar
21. Xie D, Yuan P, Wang D, et al. Expression and prognostic significance of miR-375 and miR-221 in liver cancer. Oncol Lett 2017; 14: 2305–2309.
Crossref
PubMed
Google Scholar
22. Anton L, DeVine A, Sierra LJ, et al. miR-143 and miR-145 disrupt the cervical epithelial barrier through dysregulation of cell adhesion, apoptosis and proliferation. Sci Rep 2017; 7: 3020.
Crossref
PubMed
Google Scholar
23. Wang H, Li Q, Niu X, et al. miR-143 inhibits bladder cancer cell proliferation and enhances their sensitivity to gemcitabine by repressing IGF-1R signaling. Oncol Lett 2017; 13: 435–440.
Crossref
PubMed
Google Scholar
24. Jin X, Chen X, Hu Y, et al. LncRNA-TCONS_00026907 is involved in the progression and prognosis of cervical cancer through inhibiting miR-143–5p. Cancer Med 2017; 6: 1409–1423.
Crossref
PubMed
Google Scholar
25. Zhu M, Zhang N, He S, et al. MicroRNA-106a functions as an oncogene in human gastric cancer and contributes to proliferation and metastasis in vitro and in vivo. Clin Exp Metastasis 2016; 33: 509–519.
Crossref
PubMed
Google Scholar
26. Qin Y, Chen X, Liu Z, et al. miR-106a reduces 5-fluorouracil (5-FU) sensitivity of colorectal cancer by targeting dual-specificity phosphatases 2 (DUSP2). Med Sci Monit 2018; 24: 4944–4951.
Crossref
PubMed
Google Scholar
27. Zhang L, Meng L, Fan Z, et al. Expression of plasma miR-106a in colorectal cancer and its clinical significance. Nan Fang Yi Ke Da Xue Xue Bao 2014; 34: 354–357.
PubMed
Google Scholar
28. Tusong H, Maolakuerban N, Guan J, et al. Functional analysis of serum microRNAs miR-21 and miR-106a in renal cell carcinoma. Cancer Biomark 2017; 18: 79–85.
Crossref
PubMed
Google Scholar
29. Hao H, Xia G, Wang C, et al. miR-106a suppresses tumor cells death in colorectal cancer through targeting ATG7. Med Mol Morphol 2017; 50: 76–85.
Crossref
PubMed
Google Scholar
30. Xie X, Liu HT, Mei J, et al. miR-106a promotes growth and metastasis of non-small cell lung cancer by targeting PTEN. Int J Clin Exp Pathol 2015; 8: 3827–3834.
PubMed
Google Scholar
31. Huh JH, Kim TH, Kim K, et al. Dysregulation of miR-106a and miR-591 confers paclitaxel resistance to ovarian cancer. Br J Cancer 2013; 109: 452–461.
Crossref
PubMed
Google Scholar
32. Zhao H, Peng C, Lu X, et al. PDCD5 inhibits osteosarcoma cell metastasis via targeting TGF-β1/Smad signaling pathway and is associated with good prognosis. Am J Transl Res 2019; 11(2): 1116–1128.
PubMed
Google Scholar

Supplementary Material

Supplemental Material

Supplemental material files:

Summary

Supplemental material for this article is available online.

Resources

File (supplementary_1.pdf)

Cite article

Cite article

Cite article

OR

Download to reference manager

If you have citation software installed, you can download article citation data to the citation manager of your choice

Share options

Share

Share this article

Share with email
EMAIL ARTICLE LINK
Share on social media

Share access to this article

Sharing links are not relevant where the article is open access and not available if you do not have a subscription.

For more information view the Sage Journals article sharing page.

Information, rights and permissions

Information

Published In

Volume 34, Issue 2
Pages: 184 - 193
Article first published online: May 14, 2019
Issue published: June 2019

Keywords

  1. Osteosarcoma
  2. tissue
  3. miR-21
  4. miR-221
  5. miR-143
  6. miR-106a
  7. lung metastasis

Rights and permissions

© The Author(s) 2019.
Creative Commons License (CC BY-NC 4.0)
This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Request permissions for this article.
PubMed: 31084400

Authors

Affiliations

Hui Zhao
Orthopedic Department, Guangzhou Red Cross Hospital, Medical College, Jinan University, Guangzhou, China
Orthopedic Department, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
View all articles by this author
Peng Yan
Orthopedic Department, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
View all articles by this author
Jian Wang
Orthopedic Department, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
View all articles by this author
Yuqiang Zhang
Orthopedic Department, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
View all articles by this author
Mingchao Zhang
Orthopedic Department, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
View all articles by this author
Zaijun Wang
Orthopedic Department, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
View all articles by this author
Qiang Fu
Orthopedic Department, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
View all articles by this author
Weiguo Liang
Orthopedic Department, Guangzhou Red Cross Hospital, Medical College, Jinan University, Guangzhou, China
https://orcid.org/0000-0003-4152-3775
[email protected]
View all articles by this author

Notes

Weiguo Liang, Orthopeadic Department, Guangzhou Red Cross Hospital, Medical College, Jinan University, No. 396, Road Tongfu, Distract Haizhu, Guangzhou, 510220, China. Email: [email protected]

Metrics and citations

Metrics

Journals metrics

This article was published in The International Journal of Biological Markers.

VIEW ALL JOURNAL METRICS

Article usage*

Total views and downloads: 2135

*Article usage tracking started in December 2016

Articles citing this one

Receive email alerts when this article is cited

Web of Science: 33 view articles Opens in new tab

Crossref: 28

  1. Effect of miR-21 Inhibitor Coated with Ferric Oxide on Ovarian Cancer ...
    Go to citation Crossref Google Scholar
  2. Overview of miR-106a Regulatory Roles: from Cancer to Aging
    Go to citation Crossref Google Scholar
  3. The promising roles of exosomal microRNAs in osteosarcoma: A new insig...
    Go to citation Crossref Google Scholar
  4. miRNAs as potential game-changers in bone diseases: Future medicinal a...
    Go to citation Crossref Google Scholar
  5. miR‐132‐3p promotes heat stimulation‐induced esophageal squamous cell ...
    Go to citation Crossref Google Scholar
  6. MicroRNA-143 expression inhibits the growth and the invasion of osteos...
    Go to citation Crossref Google Scholar
  7. Non-coding RNAs as potential biomarkers in osteosarcoma
    Go to citation Crossref Google Scholar
  8. Identification of Candidate MicroRNA-mRNA Subnetwork for Predicting th...
    Go to citation Crossref Google Scholar
  9. Association between microRNA 671 polymorphisms and the susceptibility ...
    Go to citation Crossref Google Scholar
  10. MicroRNAs as Potential Biomarkers in the Differential Diagnosis of Lip...
    Go to citation Crossref Google Scholar
  11. Kruppel like factor 16 promotes lung adenocarcinoma progression by upr...
    Go to citation Crossref Google Scholar
  12. Current Advances in RNA Therapeutics for Human Diseases
    Go to citation Crossref Google Scholar
  13. miR-221/222 as biomarkers and targets for therapeutic intervention on ...
    Go to citation Crossref Google Scholar
  14. The Biological Function of MicroRNAs in Bone Tumors
    Go to citation Crossref Google Scholar
  15. Noncoding way of the metastasis
    Go to citation Crossref Google Scholar
  16. DNA methylation and regulation of gene expression: Guardian of our hea...
    Go to citation Crossref Google Scholar
  17. MicroRNA-106a regulates autophagy-related cell death and EMT by target...
    Go to citation Crossref Google Scholar
  18. A Molecular Signature of Circulating MicroRNA Can Predict Osteolytic B...
    Go to citation Crossref Google Scholar
  19. The Impact of Melatonin and NLRP3 Inflammasome on the Expression of mi...
    Go to citation Crossref Google Scholar
  20. MicroRNAs Modulate Signaling Pathways in Osteogenic Differentiation of...
    Go to citation Crossref Google Scholar
  21. Analysis of a Preliminary microRNA Expression Signature in a Human Tel...
    Go to citation Crossref Google Scholar
  22. Small and Long Non-coding RNAs as Functional Regulators of Bone Homeos...
    Go to citation Crossref Google Scholar
  23. Molecular Biology of Osteosarcoma
    Go to citation Crossref Google Scholar
  24. Role of exosomal miR‑21 in the tumor microenvironment and osteosarcoma...
    Go to citation Crossref Google Scholar
  25. Overexpression of miR-425-5p is associated with poor prognosis and tum...
    Go to citation Crossref Google Scholar
  26. The Role of miRNA in the Diagnosis, Prognosis, and Treatment of Osteos...
    Go to citation Crossref Google Scholar
  27. Tumor Suppressor Function of miR-127-3p and miR-376a-3p in Osteosarcom...
    Go to citation Crossref Google Scholar
  28. Prognostic Role of miR-221 and miR-222 Expression in Cancer Patients: ...
    Go to citation Crossref Google Scholar

Figures and tables

Figures & Media

Tables

View Options

View options

PDF/ePub

View PDF/ePub

Get access

Access options

If you have access to journal content via a personal subscription, university, library, employer or society, select from the options below:

Alternatively, view purchase options below:

Purchase 24 hour online access to view and download content.

Subscribe to this journal

Access journal content via a DeepDyve subscription or find out more about this option.

Start 2 week free trial
Need help?

Also from Sage