Clinical significance of tumor miR-21, miR-221, miR-143, and miR-106a as biomarkers in patients with osteosarcoma
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Hui Zhao, Peng Yan, Jian Wang, ...
First Published May 14, 2019 Research Article
https://doi.org/10.1177/1724600819843537
Article information
Open Access Creative Commons Attribution, Non Commercial 4.0 License
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.
Keywords Osteosarcoma, tissue, miR-21, miR-221, miR-143, miR-106a, lung metastasis
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
Table 1. The correlation of miR-21 expression and clinicopathological parameters of osteosarcoma.
Table 1. The correlation of miR-21 expression and clinicopathological parameters of osteosarcoma.
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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
Table 2. The correlation of miR-221 expression and clinicopathological parameters of osteosarcoma.
Table 2. The correlation of miR-221 expression and clinicopathological parameters of osteosarcoma.
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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
Table 3. The correlation of miR-143 expression and clinicopathological parameters of osteosarcoma.
Table 3. The correlation of miR-143 expression and clinicopathological parameters of osteosarcoma.
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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
Table 4. The correlation of miR-106a expression and clinicopathological parameters of osteosarcoma.
Table 4. The correlation of miR-106a expression and clinicopathological parameters of osteosarcoma.
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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
Table 5. Univariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
Table 5. Univariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
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Table
Table 6. Multivariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
Table 6. Multivariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
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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.
Supplemental material
Supplemental material for this article is available online.
ORCID iD
Weiguo Liang https://orcid.org/0000-0003-4152-3775
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View Abstract
Show all authors
Hui Zhao, Peng Yan, Jian Wang, ...
First Published May 14, 2019 Research Article
https://doi.org/10.1177/1724600819843537
Article information
Open Access Creative Commons Attribution, Non Commercial 4.0 License
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.
Keywords Osteosarcoma, tissue, miR-21, miR-221, miR-143, miR-106a, lung metastasis
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
Table 1. The correlation of miR-21 expression and clinicopathological parameters of osteosarcoma.
Table 1. The correlation of miR-21 expression and clinicopathological parameters of osteosarcoma.
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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
Table 2. The correlation of miR-221 expression and clinicopathological parameters of osteosarcoma.
Table 2. The correlation of miR-221 expression and clinicopathological parameters of osteosarcoma.
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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
Table 3. The correlation of miR-143 expression and clinicopathological parameters of osteosarcoma.
Table 3. The correlation of miR-143 expression and clinicopathological parameters of osteosarcoma.
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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
Table 4. The correlation of miR-106a expression and clinicopathological parameters of osteosarcoma.
Table 4. The correlation of miR-106a expression and clinicopathological parameters of osteosarcoma.
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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
Table 5. Univariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
Table 5. Univariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
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Table
Table 6. Multivariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
Table 6. Multivariate COX regression analysis of the impacts of the clinicopathological parameters on the prognosis of osteosarcoma.
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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.
Supplemental material
Supplemental material for this article is available online.
ORCID iD
Weiguo Liang https://orcid.org/0000-0003-4152-3775
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