Hsa_circ_0005729 enhances accuracy in diagnosing parathyroid carcinoma

Human tissue specimens

All tissue samples were collected immediately from the patients after resection, snap-frozen in liquid nitrogen, and transferred to a −80°C refrigerator until RNA extraction, from the Beijing Chaoyang Hospital between October 2017 and September 2020. This study was approved by the Ethics Review Board of Beijing Chaoyang Hospital, Capital Medical University, and all the patients written informed consent was taken.

A total of 14 patients with PC and 40 patients with PA were enrolled, and the clinicopathological characteristics of the cohort are described in Table 1. Two patients were diagnosed PC for the first time in our institution, one was pathologically confirmed with PC based on tumor capsular invasion and the other one based on tumor infiltration of striated muscle and adipose tissues. We collected the two specimens of the primary parathyroid tumors. Other 12 patients with PC were admitted to our institution because of local relapse after 1–6 times operations in other hospitals because of parathyroid tumors. All the 12 patients with PC underwent an extended en bloc resection in our institution (28), and we collected 12 specimens of the cervical tumor recurrence. The detailed information of the 14 patients with PC is shown in Table 2.

A total of 6 patients with PC had distant metastases, including the 4 cases with lung metastases, 1 with bone metastases, and 1 with liver and lung metastases simultaneously. We collected 59 tissue samples from the 14 PA and 40 PC patients, including 5 matched distant metastases, with 4 lung metastases and 1 liver metastases. All tissue samples were confirmed with PC pathologically according to the criteria of the WHO Classification of Tumors of Endocrine Organs, 4th Edition, Volume 10 (29).

None of the recruited patients had any other type of cancer or had previously undergone chemotherapy, radiotherapy, or targeted therapy. Patients with multiple endocrine neoplasms, secondary hyperparathyroidism, tertiary hyperparathyroidism, or hyperparathyroidism-jaw tumor syndrome were also excluded according to familial medical histories, preoperative physical examination, laboratory studies, and imaging examinations, such as serum calcitonin, prolactin, insulin, cortisol, aldosterone, brain MRI, abdominal ultrasound, or abdominal contrast-enhanced CT.

RNA extraction and RNA quality estimation

First, three tissue samples from three patients with PC and three tissue specimens from three patients with PA were used for high-throughput circular RNA sequencing. All three patients with PC underwent their first operation in other institutions and were pathologically diagnosed with PC before visiting our institution. They were diagnosed with PC pathologically in our institution due to cervical relapses and distant metastases. One patient had lung metastases, one had bone metastases, and another had lung and liver metastases simultaneously. All tumor tissue samples used for high-throughput circular RNA sequencing were from the cervical relapse cases.

All the fresh-frozen tissues were ground using a tissue homogenizer. rRNA was removed by using an Epicenter Ribo-zero™ rRNA Removal Kit (Epicenter, Madison, WI, USA) and further treated with RNase R (Epicenter). The total RNA from each tissue specimen was isolated by RNA extraction using TRIzol (Invitrogen) according to the manufacturer’s recommendations. The concentration and purity of RNA were evaluated by a Nanodrop 2000 (Wilmington, DE, USA), and RNA integrity was assessed by native agarose gel electrophoresis by inspecting the 28S and 18S rRNA bands.

High-throughput circular RNA sequencing

After removal of rRNA and treatment with RNase R, six RNAs from three PC and three PA tissue samples were prepared for a high-throughput circular RNA sequencing with Illumina sequencing technology from Novegene Inc. Briefly, mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase; second-strand cDNA synthesis was performed using DNA polymerase I and RNase H. PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and index (X) primer. The PCR products were purified, and the library quality was assessed on an Agilent Bioanalyzer 2100 system. The index-coded samples were clustered, and the library preparations were sequenced on an Illumina HiSeq platform, generating 125 bp/150 bp paired-end reads. The detailed methods are described in Supplementary Method 1 (see section on supplementary materials given at the end of this article).

Data analysis of the high-throughput circular RNA sequencing

Clean data (clean reads) were obtained by removing reads containing adapters and poly-N and those of poor quality from the raw data. At the same time, the Q₂₀, Q₃₀, and GC contents of the clean data were calculated. All downstream analyses were based on high-quality clean data. The expected number of fragments per kb of the transcript sequence per million base pairs sequenced (FPKM) is currently the most used method for estimating gene expression levels. The number of FPKM of each gene was calculated based on the length of the gene and the read count mapped to this gene. Genes with an adjusted P-value less <0.05 were considered as differentially expressed. A corrected P-value of 0.005 and log 2 (fold change, FC) of one were set as the thresholds for a significantly differential expression. The detailed data analysis of the high-throughput circular RNA sequencing is described in the Supplementary Method 2.

Quantitative real-time PCR (qRT-PCR) validation

The primers for the circular RNAs were designed according to the Oligo 7 primer analysis software, and the human β-actin (ACTB) served as the endogenous control. The details of the primer sequences are shown in Supplementary Table 1. Total RNAs were isolated from fresh-frozen tissues, and the mRNAs were used for the synthesis of cDNAs after removing rRNAs and treatment with RNase R. The PCR was implemented to determine the primer specificity of circular RNA novel_circ_0024633, hsa_circ_0027093, hsa_circ_0005729, novel_circ_0026113, novel_circ_0026101, which are the top five upregulated circular RNAs according to the results of high-throughput circular RNA sequencing.

The expression levels of hsa_circ_0027093, hsa_circ_0005729, and ACTB were further measured in 14 PC tissues and 40 PA tissues using qRT-PCR. The qRT-PCR assays were performed with PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa Code.DRR047) and SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Code.DRR820) in TaKaRa TP600 PCR Thermal Cycler Dice and ABI StepOne Real-Time PCR System.

First, cDNAs were synthesized using the PrimeScript RT reagent Kit with a gDNA Eraser. Each reaction consisted of 1.5 µg of total RNA, 2 µL of 5× gDNA eraser buffer, 1 µL gDNA eraser, and sufficient RNase-free dH₂O to reach a total volume of 10 µL and was performed at 42°C for 2 min. Secondly, RT products were synthesized by using SYBR Premix Ex Taq II (Tli RNaseH Plus). Each reaction consisted of 10 µL of cDNA, 4 µL of 5×PrimerScript Buffer 2 (for real-time), 1 µL of PrimeScript RT Enzyme Mix I, 1 µL of RT Primer mix, and 4 µL of RNase-free dH₂O and was performed at a temperature of 37°C for 15 s and then 85°C for 5 s. The PCR products were further synthesized with 2 µL of the RT products, 10 µL of SYBR Premix Ex Taq II (2×), 1 µL of forward primer (10 μM), 1 µL of reverse primer (10 Mm), and 6 µL of RNase-free dH₂O at 95°C for 30 s, followed by the 40 cycles of 95°C for 5 s and then 60°C for 30 s. All the reactions were performed in triplicate.

The expression levels of the circular RNAs were calculated using the ΔCT method as follows: expression_{hsa_circ_0005729} = −ΔCT = −(CT_{hsa_circ_0005729} – CTACTB), expression _{hsa_circ_0027093} = −ΔCT = −(CT_{hsa_circ_0027093} – CTACTB), and expression_{RNF138 mRNA} = −ΔCT = −(CT_{RNF138 mRNA} – CT_ACTB).

Clinical, laboratory data collection, and analysis

Clinicopathological features and laboratory data of 14 PC and 40 patients with PA were collected and analyzed. Information was collected including age, sex, serum calcium, calcium on the first postoperative day, the decline in calcium levels (calcium on the first postoperative day relative to preoperative), serum intact PTH (iPTH), iPTH on the first postoperative day, the decline in the iPTH (iPTH on the first postoperative day relative to preoperative), phosphorus, alkaline phosphatase (ALP), creatinine, 25-hydroxy vitamin D, 24-h urinary calcium, relapse times, the date of the initial operation, date of the operation in our institution, and sites of the distant metastases. We analyzed the differences of the above clinical parameters between 14 PC and 40 patients with PA. We also examined the above indexes between 8 patients with PC without the distant metastases and 6 PC cases with the metastases. The factors with the significant differences between patients with PC and patients with PA were further analyzed together with hsa_circ_0005729 and hsa_circ_0027093.

ROC curves and AUC analysis of laboratory parameters were used to assess the diagnostic efficiency of PC

Laboratory parameters that were significantly different between the patients with PC and patients with PA were further used to assess the diagnostic power of PC by these parameters alone and combined with hsa_circ_0027093 and hsa_circ_0005729 (30) according to the logistic regression, ROC curve analysis, and the AUC calculation. The cutoff values were identified according to the Euclidean index (31), along with the sensitivity, specificity, and 95% CIs.

Analysis of the correlation between hsa_circ_0005729 and RNF138 mRNA in PC and patients with PA

RNF138 is the host gene of hsa_circ_0005729, and we measured the expression of RNF138 mRNA by qRT-PCR in 14 patients with PC and 40 patients with PA. The relationship between the hsa_circ_0005729 and RNF138 mRNA in 54 patients was analyzed by a Pearson’s correlation analysis. In addition, the relationship between the hsa_circ_0005729 and RNF138 mRNA in 14 PC and 40 patients with PA were analyzed, respectively.

Statistical analysis

Statistical analyses were performed with SPSS version 23.0, GraphPad Prism 8.0, and SigmaPlot 14.0 (Systat Software Inc, Beijing, China). All the measurements were repeated three times in triplicate. Differences in hsa_circ_0027093, hsa_circ_0005729, RNF138 mRNA, and laboratory parameters between patients with PC and patients with PA were determined using the Mann–Whitney U-test. The clinicopathological features of different groups were evaluated using chi-square tests. Logistic regression, ROC curve, and AUC calculation were performed to assess the diagnostic value of serum calcium, ALP, creatinine, hsa_circ_0027093, and hsa_circ_0005729 alone, or a combination of them in PC. P < 0.05 was considered as statistically significant.

Clinical characteristic features of PC and patients with PA

This study included 14 patients with PC and 40 patients with PA. The PC group had an equal number of males and females in our institution (7 vs 7), and the mean age was 42.9 ± 15.1 years (median: 42 years, range: 19–69 years) at the time of initial operation. The PA group had a female-to-male ratio of 2.3:1 (28 vs 12), and the mean age was 49.4 ± 11.2 years (median: 52, range: 28–66). There was no significant difference in either age or the sex distribution between the patients with PC and patients with PA, but the age at the initial operation among the patients with PC was approximately 7 years younger than that among the patients with PA. Shaha et al. (15) and Schulte et al. (32) established different TNM staging systems that differ in the T classification for identifying PC. The T classification is associated with the tumor size in the Shaha system, while the Schulte system utilizes the histopathological features. The Schulte system was used because most patients with PC in this study had experienced recurrence, and the histopathological features were considered as the better indicators (Table 1).

Overview of the results of the high-throughput circular RNA sequencing

A total of three PC and three PA tissues were used for screening the differentially expressed circular RNAs by a high-throughput circular RNA sequencing on the Illumina HiSeq 2500 Platform. In total 550,623,586 raw reads were generated, including the 279,097,882 raw reads for PC tissues and 271,525,704 raw reads for the PA tissues. A total of 524,800,460 clean reads (266,092,644 for PC and 258,707,816 for PA) were filtered after removing the poor quality, poly-N-containing, and adapter-containing reads from the raw data. Finally, 16,038 circular RNAs were identified as differentially expressed between the PC and PA according to the find_circ software and then used for further analysis.

Analysis of differentially expressed circular RNAs PC vs PA

A total of 274 significant differentially expressed circular RNAs were identified according to the threshold log 2(FC) greater than 1 or lesser than −1 and corrected P < 0.005, including 118 upregulated and 156 downregulated circular RNAs in PC relative to PA. Clustering heatmaps and volcano plots were constructed to show the differentially expressed circular RNAs (Fig. 1A andB). The top ten upregulated circular RNAs in PC are shown in the Supplementary Table 2.

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Heat map and volcano plot were constructed to show the differentially expressed circular RNAs between three parathyroid carcinoma (PC) and three parathyroid adenoma (PA) tissues. (A) Heat map: differentially expressed circular RNAs in three PC (PC1+PC2+PC3) and three PA (PA1+PA2+PA3) tissues were screened using high-throughput circular RNA sequencing. Each column represents one specimen, and each row shows one circular RNA. (B) Volcano plot: a total of 274 significant differentially expressed as circular RNAs were identified, including 118 upregulated and 156 downregulated circular RNAs in PC relative to PA, according to the threshold log 2 (FC) > 1 or <−1 and P < 0.005.

Citation: Endocrine Connections 11, 2; 10.1530/EC-21-0605

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Hsa_circ_0005729 and hsa_circ_0027093 were significantly higher in 14 PC than patients with PA

PCR was performed to assess the expression levels of the top five upregulated circular RNAs in the PC tissues, and only hsa_circ_0027093 and hsa_circ_0005729 were chosen to study further because of the good shape of the PCR amplification curve and the specific melt peak. Furthermore, PCR products were detected by gel electrophoresis, and the sequences of hsa_circ_0027093 and hsa_circ_0005729 were also identified by sequencing using gel extraction. qRT-PCR was performed to measure the expression of hsa_circ_0027093 and hsa_circ_0005729 in 14 patients with PC and 40 patients with PA. The results showed that both hsa_circ_0005729 and hsa_circ_0027093 were significantly upregulated in 14 patients with PC specimens compared to the 40 PA tissue specimens, with P  = 0.0073 and P  = 0.0359, respectively (Fig. 2A and B).

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Validation of hsa_circ_0005729 and hsa_circ_0027093 expression in 14 PC and 40 PA tissues by qRT-PCR. (A and B) Boxplots: Hsa_circ_0005729 and hsa_circ_0027093 were verified significantly higher in PC than in the patients with PA by qRT-PCR using the −ΔCT on the y-axis. (C and D) Boxplots: There was no significant difference in hsa_circ_0005729 expression between patients with PC with distant metastases (PC-M1) (n  = 6) and without distant metastases (PC-M0) (n  = 8), as well as hsa_circ_0027093. (E) Scatter plot: Hsa_circ_0005729 expression was higher in the local relapses than in the matched distant metastases of the same patient. (F) Scatter plot: there was no significant difference in hsa_circ_0027093 expression between the local relapses and the matched distant metastases.

Citation: Endocrine Connections 11, 2; 10.1530/EC-21-0605

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Hsa_circ_0005729 expression was higher in local relapses than in matched distant metastases of the same PC patients

There was no significant difference of hsa_circ_0005729 expression between patients with PC with (n  = 6) and without distant metastases (n  = 8) (P  = 0.2824), as well as hsa_circ_0027093 (P  = 0.8518) (Fig. 2C and D). In addition, we compared the hsa_circ_0005729 expression between local relapse tissues (n  = 4) and matched distant metastases specimens (n  = 5), including the four lung metastases and one liver metastases. The results showed that hsa_circ_0005729 expression was higher in the local relapses than in the matched distant metastases of the same patient (P  = 0.0226) (Fig. 2E), but there was no significant difference of hsa_circ_0027093 expression between the two groups (P  = 0.2764) (Fig. 2F).

There were 2 specimens from the first-operated patients with PC and 12 specimens from the multifold-operated patients with PC. Hsa_circ_0005729 and hsa_circ_0027093 between the first-operated and multifold-operated patients with PC were analyzed, and the results showed no significant differences in the hsa_circ_0005729 and hsa_circ_0027093 between the first-operated patients with PC and the multifold-operated PC patients, with P  = 0.440 and P  = 0.659, respectively.

Several laboratory parameters were expressed higher in the 14 patients with PC than that in 40 patients with PA

We analyzed several laboratory parameters between PC and patients with PA, and the results showed that the levels of serum calcium (P  = 0.0447), ALP (P  = 0.0481), and creatinine (P  = 0.0358) were significantly higher in the 14 patients with PC than in 40 patients with PA (Fig. 3A, B and C). However, there was no significant difference in calcium on the first postoperative day (P  = 0.1047), the decline in calcium levels (P  = 0.4058), iPTH (P  = 0.0562), iPTH on the first postoperative day (P  = 0.0698), the decline in iPTH (P  = 0.5913), phosphorus (P  = 0.0745), 25-hydroxy vitamin D (P  = 0.2510), 24-h urinary calcium (P  = 0.8052) between the PC and patients with PA (Supplementary Fig. 1).

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Hsa_circ_0005729 enhanced the accuracy in diagnosing PC by combination with serum calcium, alkaline phosphatase (ALP), and creatinine. (A) Boxplot: serum calcium was significantly higher in 14 patients with PC than in the 40 patients with PA using the −ΔCT on the y-axis. (B) ROC curve: AUC was 0.81 when hsa_circ_0005729 was combined with serum calcium (AUC 0.68). (C) Boxplot: ALP was significantly higher in the 14 patients with PC than in 40 patients with PA. (D) ROC curve: AUC was 0.81 when hsa_circ_0005729 was combined with ALP (AUC 0.68). (E) Boxplot: creatinine was significantly higher in 14 patients with PC than in 40 patients with PA. (F) ROC curve: AUC was 0.83 when hsa_circ_0005729 was combined with creatinine (AUC 0.69). (G) ROC curve: the maximum AUC was 0.86 when hsa_circ_0005729 was combined with ALP, serum calcium, and creatinine.

Citation: Endocrine Connections 11, 2; 10.1530/EC-21-0605

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ROC curve analysis of laboratory parameters in PC patients

Serum calcium, creatinine, and ALP had the AUC of 0.68, 0.69 and 0.68, respectively, while the AUC of hsa_circ_0005729 and hsa_circ_0027093 was 0.74 and 0.69, separately. The AUC was 0.86 when hsa_circ_0005729 was combined with serum calcium, creatinine, and ALP (Fig. 3D, E, F and G), while AUC was 0.79 when hsa_circ_0027093 was combined with the above three indexes. The median levels and the cutoff values according to the Euclidean index of all parameters are shown in Table 3.

The correlation between hsa_circ_0005729 and RNF138 mRNA in PA and PC patients

Hsa_circ_0005729 is translated from the RNF138 gene, and we measured RNF138 mRNA in 14 patients with PC and 40 patients with PA. The results showed no significant difference in RNF138 mRNA expression between patients with PC and patients with PA (Fig. 4A). Additionally, we analyzed the relationship between hsa_circ_0005729 and RNF138 mRNA, and the results revealed that hsa_circ_0005729 expression was positively correlated with RNF138 mRNA (Pearson’s r = 0.375, P  = 0.005) slightly in all 54 patients (Fig. 4B). Subgroup analysis showed a positive correlation between hsa_circ_0005729 and RNF138 mRNA in 40 patients with PA (Pearson’s r = 0.468, P  = 0.002) (Fig. 4C). However, there was no correlation between hsa_circ_0005729 and RNF138mRNA in 14 patients with PC (Pearson’s r = 0.318, P  = 0.268) (Fig. 4D).

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Hsa_circ_0005729 expression was positively correlated with RNF138 mRNA in patients with PA but not in PC patients. (A) Boxplot: RNF138 mRNA, the corresponding linear transcript of hsa_circ_0005729, was measured by qRT-PCR, and there was no significant difference in RNF138 mRNA expression between 14 patients with PC and 40 patients with PA using the −ΔCT on the y-axis. (B) Scatter plot with trend lines: the relationship between hsa_circ_0005729 and RNF138 mRNA was analyzed by Pearson’s correlation analysis, and hsa_circ_0005729 expression was positively correlated with RNF138 mRNA (Pearson’s r = 0.375, P= 0.005) slightly in all 54 patients. (C) Scatter plot with trend lines: Hsa_circ_0005729 expression was positively correlated with RNF138 mRNA in 40 patients with PA (Pearson’s r = 0.468, P = 0.002). (D) Scatter plot with trend lines: there was no significant correlation between hsa_circ_0005729 and RNF138 mRNA in 14 patients with PC (Pearson’s r = 0.318, P = 0.268).

Citation: Endocrine Connections 11, 2; 10.1530/EC-21-0605

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The differences in the laboratory parameters between patients with PC with metastases and without metastases were analyzed

The laboratory parameters between patients with PC with and without metastases were analyzed. The results showed no significant difference in preoperative serum calcium (P  = 0.6870), calcium on the first postoperative day (P  = 0.3277), the decline in serum calcium (P  > 0.9999), preoperative iPTH (P  = 0.4136), the decline in iPTH (P  = 0.0813), ALP (P  = 0.9497), creatinine (P  = 0.5728), phosphorus (P  = 0.6870), and 25-hydroxyvitamin D (P  = 0.2949) between the patients with PC with metastases and patients with PC without metastases. However, there was a significant difference in iPTH on the first postoperative day between the patients with PC with and without metastases (P  = 0.020), which showed the level of iPTH on the first postoperative day was higher in the patients with PC with metastases than that in patients with PC without the metastases (Fig. 5A, B, C, D, E, F, G, H, Iand J). In addition, we analyzed the relationship between the hsa_circ_0005729 and iPTH on the first postoperative day, and the results showed no correlation between hsa_circ_0005729 and iPTH on the first postoperative day in 14 patients with PC (Pearson’s r = 0.149, P  = 0.612).

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The differences in laboratory parameters between patients with PC with metastases and without metastases were analyzed. (A) Scatter plots: the results showed a significant difference in the iPTH on the first postoperative day between 6 patients with PC with the metastases and 8 patients with PC without metastases (P = 0.0200), which showed the level of iPTH on the first postoperative day was higher in patients with PC with the metastases than in patients with PC without metastases. (B, C, D, E, F, G, H, I and J) Scatter plots: there was no significant difference in preoperative serum calcium (P = 0.6870), calcium on the first postoperative day (P = 0.3277), the decline in serum calcium (P> 0.9999), preoperative iPTH (P = 0.4136), the decline in iPTH (P = 0.0813), phosphorus (P = 0.6870), ALP (P = 0.9497), creatinine (P = 0.5728), 25-hydroxyvitamin D (P = 0.2949) between 6 patients with PC with metastases and 8 patients with PC without the metastases.

Citation: Endocrine Connections 11, 2; 10.1530/EC-21-0605

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