BRAF^V600E mutation, BRAF-activated long non-coding RNA and miR-9 expression in papillary thyroid carcinoma, and their association with clinicopathological features

Thyroid cancer is an endocrine malignancy and its incidence is increasing worldwide, especially in developed countries [1]. Papillary thyroid carcinoma (PTC) is the most common pathological subtype of thyroid cancer, accounting for 80–90% of cases [2]. PTC is thought to arise through interactions between genetic and environmental factors. The most common genetic changes in PTC are mutations in B-type RAF kinase (BRAF) of the RAS/BRAF/mitogen-activated protein kinase signaling pathway [3]; the most frequent mutation (95%) is BRAF^V600E [4, 5], which is associated with enhanced extrathyroid extension, lymph node metastasis (LNM), and advanced tumor stage [6‐8]. However, this has not been corroborated by other studies [9], and therefore the relationship between clinicopathological characteristics of PTC and BRAF^V600E mutation remains unclear.

Long non-coding (lnc)RNAs are RNA molecules with a length greater than 200 nt that are transcribed from non-protein-coding sequences in the genome. LncRNAs play an important role in gene regulation at the epigenetic, transcriptional, and translation levels and in post-translational protein modification [10, 11], and have been implicated in processes such as tumorigenesis as well as tumor progression, metastasis, and recurrence [12‐15]. For example, the lncRNA BRAF-activated long non-coding RNA (BANCR) promotes proliferation, inhibits apoptosis and G1 arrest, and stimulates autophagy in IHH-4 thyroid cancer cells [16]. Micro(mi)RNAs are small ncRNAs with length of about 22 nt that negatively regulate the expression of target genes at the post-transcriptional level by inducing the degradation or inhibition of the translation of mRNAs. The interaction between lncRNA and miRNAs has been shown to influence tumor development and progression [17]. The miRNA miR-9 regulates the growth of cancer cells, and BANCR and miR-9 mutually regulate by altering the activity of nuclear factor (NF)-κB in gastric cancer cells [18]. However, it is unclear how this is related to clinicopathological features in PTC patients. Although thyroid tumor diagnosis has been improved by high-frequency ultrasound [19], about 20% of PTC patients show recurrence and have poor prognosis due to distant metastasis [20]. Clarifying the mechanisms underlying PTC progression can lead to the development of more effective treatment strategies.

In this study, we investigated the relationships between the molecular features of PTC including BRAF^V600E mutation and BANCR and miR-9 expression, and clinicopathological characteristics of PTC patients. Our findings provide important insight into the molecular basis for pathophysiological changes leading to PTC progression.

Among malignant thyroid tumors, PTC is a common histopathological subtype [22, 23]. Given that imaging modalities are used for physical examination of thyroid, the diagnosis rate of thyroid tumors is increasing worldwide. However, this has not translated into a decrease in mortality rate, although it is possible that thyroid cancer is overdiagnosed and overtreated [1, 24]. Ito Y et al. [20] reported that up to 20% of patients show recurrence and have poor prognosis due to distant metastasis in PTC metastasis. Therefore, the excessive or inadequacy treatment on PTC is confusing. Thus, the biological characteristics of PTC require clarification for finding suitable PTC patients of surgery through analyzing the relationship between their clinicopathological features, BRAF^V600E mutation status, and BANCR and miR-9 expression.

The BRAF^V600E mutation is the most common genetic change in PTC patients and is not observed in normal thyroid tissue or benign lesions; therefore, we did not evaluate BRAF^V600E mutation status in normal tissue in the present study. We detected the BRAF^V600E mutation in the carcinoma tissue of 76.5% patients; although it was unrelated to sex, age, extrathyroid extension, co-occurrence of Hashimoto’s disease, and TNM stage, patients with the mutation had larger tumors, a higher frequency of bilateral tumor location, multifocality, extracapsular invasion, and lateral LNM compared with those without the mutation. Consistent with our findings, a meta-analysis of 32 studies and 6372 patients found that BRAF^V600E mutation was associated with several of the variables used in prognostic staging systems such as tumor size, multifocality, and LNM [25]. Another study indicated that extracapsular invasion is an indicator of distant metastasis and poor prognosis in patients with PTC [26]. Our results showed that patients with higher expressions of BRAF^V600E mutation and BANCR have higher rates of extracapsular invasion.

In the present study, patients with higher BANCR expression had a higher rate of lateral LNM, and those with higher miR-9 expression had a lower rate of central and lateral LNM. In addition, patients with the BRAF^V600E mutation had a higher frequency of lateral LNM compared with those without the mutation. Previous studies have mainly focused on LNM in the central region [27, 28] based on complete central neck dissection. However, more attention needs to be paid to LNM in the lateral region, not only the confirmed LN should be dissect, but also preventive LN dissection should be performed in the lateral region to prevent the omission and recurrence of tumor. In addition, BANCR and miR-9 expression in adjacent normal tissue was also associated with tumor location, size, and multifocality. Therefore, for patients with high-risk clinicopathological characteristics, surgical treatment can be relaxed from glandular lobectomy to total thyroidectomy as appropriate.

BANCR is a recently identified lncRNA activated by BRAF that plays an important role in the occurrence and progression of PTC. A qRT-PCR analysis revealed that BANCR is highly expressed in PTC tissue compared with normal tissue; it was also found to promote proliferation, inhibit cell apoptosis, alleviate G1 arrest, and stimulate autophagy in IHH-4 thyroid cancer cells [15]. In addition, the expression of miR-9 was downregulated in carcinoma compared with adjacent normal tissue in the present study. Consistent with our finding, Sondermann A et al. [29] observed the significantly downregulated expression of miR-9 in patients with recurrent PTC, which can be used as a prognostic indicator of PTC. Gu Y et al. [30] found that miR-9 may inhibit the activity of PTC cells and tumor growth by directly targeting the expression of BRAF in PTC. In the present study, patients with BRAF^V600E mutation have a higher rate of BANCR overexpression and tended to have a lower rate of miR-9 overexpression (P = 0.057), and there is a negative association were found between BANCR and miR-9 expression levels. One study reported that, in gastric cancer cells, downregulation of BANCR decreased NF-κB activity and inhibited cell proliferation while promoting apoptosis, whereas overexpression of NF-κB—a target of miR-9 that regulates cancer cell growth—and inhibition of miR-9 reversed these effects [18]. Given that BANCR is a lncRNA activated by BRAF, we speculate that BRAF^V600E mutation and BANCR and miR-9 expression together contribute to PTC development and progression. There were some limitations to this study such as the small size of the study population and the fact that BANCR and miR-9 expression was not evaluated in all adjacent tissues. Therefore, additional studies are needed to validate our findings. In addition, BRAF^V600E mutation can be determined by fine-needle aspiration cytology with the current hospital detection technology, while the preoperative sample size is not enough to determine the BANCR and miR-9 expression. The sample size obtained during the operation is enough for determining the BRAF^V600E mutation, BANCR, and miR-9 expression, but there is still a lack of rapid detection and analysis technology at present. Therefore, rapid gene detection before and during operation is not available. By expanding the number of retrospective studies to increase the evidence of clinical inquiry and the development of rapid detection technology, it is expected that the research results will be used in clinical practice in the future.

In conclusion, our results demonstrate that the tumor size, bilateral tumor location, multifocality, extracapsular invasion, and lateral LNM are closely associated with BRAF^V600E mutation, and the BANCR and miR-9 expression, which could be as the high-risk clinicopathological characteristics. Particularly, PTC patients with high-risk clinicopathological characteristics, BRAFV600E mutation, and high BANCR expression and low miR-9 expression needed earlier surgical treatment, and are recommended for total thyroidectomy in primary surgery for reducing the risk of recurrence. These findings provide new insight into the molecular basis for PTC and can inform strategies for the management of PTC.