Long non‐coding RNA TNRC6C‐AS1 promotes methylation of STK4 to inhibit thyroid carcinoma cell apoptosis and autophagy via Hippo signalling pathway

Abstract The role of long non‐coding RNAs (lncRNAs) in thyroid carcinoma (TC), the most frequent endocrine malignancy, has been extensively examined. This study investigated effect of interaction among lncRNA TNRC6C‐AS1, serine/threonine‐protein kinase 4 (STK4) and Hippo signalling pathway on TC. Initially, lncRNA TNRC6C‐AS1 expression in TC tissues was detected. To explore roles of lncRNA TNRC6C‐AS1, STK4 and Hippo signalling pathway in TC progression, their expressions were altered. Interaction between lncRNA TNRC6C‐AS1 and STK4, STK4 promoter methylation, or Hippo signalling pathway was verified. After that, a series of experiments were employed to evaluate in vitro ability of apoptosis, proliferation and autophagy of TC cells and in vivo tumorigenicity, and tumour growth of TC cells. lncRNA TNRC6C‐AS1 was highly expressed while STK4 was poorly expressed in TC tissues. LncRNA TNRC6C‐AS1 promoted the STK4 methylation and down‐regulated STK4 expression, which further activated the Hippo signalling pathway. STK4 silencing was observed to promote the proliferation ability of TC cells, inhibit the apoptosis and autophagy abilities, as well as enhance the tumorigenicity and tumour growth. Moreover, the in vitro proliferation ability as well as the in vivo tumorigenicity and tumour growth of TC cells were inhibited after the blockade of Hippo signalling pathway, while the apoptosis and autophagy abilities were promoted. The results demonstrate that the lncRNA TNRC6C‐AS1 increases STK4 promoter methylation to down‐regulate STK4 expression, thereby promoting the development of TC through activation of Hippo signalling pathway. It highlights that lncRNA TNRC6C‐AS1 may be a novel therapeutic target for the treatment of TC.


| INTRODUC TI ON
Thyroid carcinoma (TC) is considered as one of the most common malignancy of endocrine system. 1 In the last decade, the incidence of TC has dropped by about 2% annually in men, while the mortality rate dropped by about 1.5% annually in both men and women. 2

TC
can be subdivided into several types, including differentiated (papillary, follicular, Hurthle cell), which accounts for about 90% of TC cases, medullary TC and anaplastic TC. 3 Radiation to the thyroid gland during our childhood, age and family disease history are the several main risk factors to high-level differentiated TC. 4 Despite the progress for the treatment of TC, the 5-year cancer-specific survival rate remains low. 5 Therefore, it is important to identify the specific biomarkers that can be used in diagnosis of prognosis in TC patients.
Long non-coding RNA (lncRNA) dysfunction is related to a wide range of diseases, such as cancer, neurodegeneration, cardiovascular disease and neurasthenia. 6 For example, overexpressed lncRNA NR_036575.1 would lead to the proliferation and migration of papillary TC. 7 In order to discover the genes that might be correlated with TC, the Gene Expression Omnibus (GEO) database was employed to retrieve the lncRNA trinucleotide repeat containing 6C (TNRC6C)-AS1 was overexpressed in TC cells. Therefore, lncRNA TNRC6C-AS1 was the main focus of our study. LncRNA TNRC6C-AS1 has been reported to play a tumorigenic role in the tumour occurrence and invasive ability of papillary thyroid carcinoma (PTC). 8 A new study has found that lncRNA TNRC6C-AS1 affects the development of TC by regulating UNC5B expression as a competitive endogenous RNA of microRNA-129-5p (miR-129-5p). 9 The complementary pairing sites in the promoter regions of lncRNA TNRC6C-AS1 and serine/ threonine-protein kinase 4 (STK4) genes were analysed by bioinformatics. STK4, a multifunctional protein, is an essential kinase of the Hippo signalling pathway for tumour inhibition in the treatment of cancers with poor prognosis. 10 STK4 is considered as a tumour suppressor in hepatocellular carcinoma, breast cancer and lymphoma that regulates cell apoptosis. [11][12][13] Hippo signalling pathway was initially discovered by genetic studies in Drosophila as a regulator of organ size, limiting cell number via the regulation of cell proliferation and apoptosis. 14 Hippo signalling pathway is responsible for controlling organ size by regulating cell proliferation, apoptosis, stem-cell self-renewal and tumorigenesis of TC cells. 15 From all that mentioned above, we hypothesize that lncRNA TNRC6C-AS1 in TC can affect the proliferation, apoptosis and autophagy in TC through STK4 regulation and the Hippo signalling pathway. Thus, this study was aimed to determine the roles of lncRNA TNRC6C-AS1, STK4 and Hippo signalling pathway in apoptosis and autophagy of TC cells.

| Ethical statement
All patients in this study signed an informed consent, which was in accordance with the Helsinki Declaration and approved by the ethics committee of the Sun Yat-Sen Memorial Hospital. This experiment animal-related programme has been approved by the experimental animal ethics Committee and conforms to the relevant regulations of the national experimental animal welfare ethics. Significant efforts were made in order to minimize the number of animals used as well as their respective suffering. The study was conducted with the approval of the Animal Ethics Committee of the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University.

| Microarray analysis
The GEO database was used to retrieve the TC-related microarray data and annotation probe file, obtained by examination of Affymetrix Human Genome U133 Plus 2.0 Array. Each microarray data set was processed using the Affy package of R software. 16 Then, the linear model-Empirical Bayes statistical method in the Limma package was combined with the traditional t test to perform non-specific filtering of the microarray data and to screen the differentially expressed miRs and lncRNAs. 17   The cells were cultured in Roswell Park Memorial Institute-1640 medium (Gibco) containing 10% foetal bovine serum (FBS) (Gibco), placed and subcultured in a 5% CO 2 incubator (Thermo) at 37°C.

| Tissue samples and cell lines
When the cell density reached 90%, cells were detached with 0.25% trypsin and subcultured at a ratio of 1:3.

| Fluorescence in situ hybridization (FISH)
The subcellular localization of lncRNA TNRC6C-AS1 in SW579 cells was identified by FISH. The following steps were performed using Ribo™ lncRNA FISH Probe Mix (Red) (Ruibo Biotech Co., Ltd.) according to the instructions from manufacturer's protocol. The SW579 cells were inoculated into a 6-well culture plate covered with a coverslip for 1 d to reach about 80% confluency. Next, the cells were fixed in 1 mL 4% paraformaldehyde at room temperature.
The cells were first treated with protease K (2 μg/mL), glycine and ethanolamine, then added with 250 μL pre-hybridization solution and then incubated at 4°C for 1 hour. Subsequently, the cells were added with 250 μL hybridization solution containing probe (300 ng/ mL) and hybridized at 42°C overnight. After the cells were washed with Phosphate-Buffered Saline/Tween (PBST) 3 times, cells were stained using 4′,6-Diamidino-2-Phenylindole (DAPI) (1:800) diluted with PBST, and then added to a 24-well culture plate for 5 minutes.
The cells were sealed with anti-fluorescence quencher, and five different fields of view were selected to observe and photograph under the fluorescence microscope (Olympus).

| Chromatin immunoprecipitation (ChIP)
ChIP kit (Millipore) was used to identify the enrichment of DNA meth-yltransferase1 (DNMT1), DNMT3a and DNMT3b in the promoter region of STK4. When the thyroid cells reached 70%-80% fusion rate, 1% formaldehyde were added to fix the cells at room temperature for 10 minutes to immobilize and cross-link the DNA and protein in the cells. After cross-linking, the cells were randomly broken by ultrasonic treatment, 10 seconds each time with an interval of 10 seconds for 15 cycles to break cells into fragments of appropriate size.
After that, the cells were centrifuged at 30 237 g at 4°C, the supernatant was then collected and aliquoted into three tubes. The positive control antibody RNA polymerase II, Immunoglobulin G (IgG) of normal mice with NC antibody and specific antibody DNMT1 of target protein (Abcam Inc, ab13537, Rabbit anti), DNMT3a (Abcam Inc, ab2850, Rabbit anti), DNMT3b (Abcam Inc, ab2851, Rabbit anti) were separately added to the cells and incubated overnight at 4°C. The protein agarose/sepharose was utilized to precipitate the endogenous DNA-protein complexes. After centrifugation, the supernatant was aspirated and the non-specific complex was washed, de-cross-linked at 65°C overnight, and then, DNA fragments were purified by phenol/chloroform extraction. The binding of DNMT1, DNMT3a and DNMT3b with STK4 promoter region was examined by STK4 promoter region specific primers (Table 1).

| RNA-binding protein co-immunoprecipitation (RIP)
According to the instructions of Magna RIP RNA-Binding Protein Immuno Preparation Kit (Millipore), the specific steps were as follows. Cells from the blank group, the oelncRNA TNRC6C-AS1 group and the shlncRNA TNRC6C-AS1 group were collected by cell scraper, washed twice with pre-cooled PBS and added with 100 μL (1:100, ab2850) and rabbit anti-human DNMT3B (1:100, ab2851) and were mixed at room temperature for 30 minutes. Rabbit antihuman IgG (1:100, ab109489) was used as NC. The antibodies were purchased from Abcam Inc.

| Methylation-specific PCR (MSP)
The DNA of cells was subjected to hydrosulfite treatment, whereas Wizard purified resin (Promega Corporation) was used to purify DNA samples. DNA was precipitated with ethanol and resuspended in water after NaOH treatment. PCR primers were designed according to Herman method. The sequences are shown in Table 1. STK4 methylation-specific primers (M) and STK4 non-methylation specific primers (U) were purchased from Invitrogen (Invitrogen). The MSP amplification system procedure was as follows: pre-denaturation at

| Immunofluorescence staining
After conventional treatment of transfected cells, the cells were

| RNA isolation and quantitation
Total RNA was extracted from tissues and cells by using TRIzol (Invitrogen). The nanodrop2000 micro ultraviolet spectrophotometer (1011U, nanodrop Technologies Inc) was used to detect the A260/A230 value, which was then used to determine the total RNA concentration and purity. Then, RNA was reversely transcribed into cDNA using the TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, Inc). Primers for lncRNA TNRC6C-AS1, STK4, large tumour suppressor kinase1 (LATS1) and Beclin1 were designed and synthesized using TaKaRa Biotechnology Ltd., as shown in Table 2. ABI7500 quantitative PCR instrument (7500, ABI Company) was employed to perform RT-qPCR. The relative level of lncRNA TNRC6C-AS1 used U6 as the internal reference.
The relative expression of STK4, LATS1 and Beclin1 used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. LATS1 was the protein of involved in Hippo signalling pathway.

| Monodansylcadaverine (MDC) staining
After cell grouping and treatment, the cells were inoculated into a 6well plate containing coverslips before staining. After the treatment, the culture medium was discarded.

| 5-Ethynyl-2′-deoxyuridine (EdU) assay
Cells in logarithmic growth phase were inoculated into a 96-well plate at 4 × 10 3 cells per well and cultured to normal growth stage.
The EdU solution was diluted using cell culture medium at the ratio

| Tumour xenografts in nude mice
Female BALB/C nude mice (4-6 weeks old, no specific pathogen) were purchased from Sichuan University Medical Laboratory Animal Center). The cells were inoculated into a low-adhesion culture plate for 7 d and centrifuged. After the supernatant was discarded, the cells were added with 1 mL 0.5% trypsin and cultured in a 37°C incubator. After that, the cell spheres were dissociated into single

| Statistical analysis
Data were analysed using SPSS 21.0 (IBM Corp) statistical software.
The measurement data were expressed by mean ± standard deviation. The t test was used for data analysis between two groups. One- A P value < .05 indicated that the difference was statistically significant.

| LncRNA TNRC6C-AS1 may affect STK4 expression and regulate the Hippo signalling pathway to regulate apoptosis and autophagy of TC
Gene expression data of TC in TCGA database showed that the expression of lncRNA TNRC6C-AS1 in TC was significantly higher than that in the normal control ( Figure 1). The target gene and function of lncRNA TNRC6C-AS1 were predicted through MEM and KEGG websites. Results showed that lncRNA TNRC6C-AS1 might regulate the expression of STK4 and the Hippo signalling pathway to regulate apoptosis and autophagy of TC cells in vivo (Table 3).

| The silencing of lncRNA TNRC6C-AS1 inhibits proliferation as well as promotes apoptosis and autophagy of TC cells
RT-qPCR of 54 TC tissues and adjacent tissues (Figure 2A The results showed that delivery of oelncRNA TNRC6C-AS1 induced significantly lower levels of Beclin1, LC3-II/I and Casepase-3 ( Figure 2F) along with larger volume and weight ( Figure 2G). Opposite changing tendency was observed following delivery of shlncRNA TNRC6C-AS1. Therefore, lncRNA TNRC6C-AS1 could promote proliferation and inhibit apoptosis and autophagy of TC cells.
Based on FISH experiment results, lncRNA TNRC6C-AS1 was further verified that its localization in the nucleus ( Figure 3B). Blast comparison results showed that the promoter regions of lncRNA TNRC6C-AS1 and STK4 had base complementary pairing binding sites ( Figure 3C). In order to verify the binding of STK4 promoter region with methyltransferase, ChIP analysis of STK4 promoter region in SW579 cells was performed. The expression of lncRNA TNRC6C-AS1 in the enriched product was identified and found that the expression of lncRNA TNRC6C-AS1 was significantly higher than that of IgG NC ( Figure 3D). The binding of lncRNA TNRC6C-AS1 to methyltransferase was also analysed by RIP experiment. The results showed that when compared with the blank group, the combination with DNMT1, DNMT3a and DNMT3b was significantly increased in the oelncRNA TNRC6C-AS1 group (P < .05), but decreased significantly in the sh-lncRNA TNRC6C-AS1 group (P < .05) ( Figure 3E). It was also found that the promoter region of STK4 existed variable methylation in the cytosine-phosphate-guanine (CpG) island by bioinformatics analysis ( Figure 3F). MSP ( Figure 3G) verified that the STK4 was highly methylated in SW579 cells. The above results showed that the expression of STK4 was regulated by lncRNA TNRC6C-AS1, and lncRNA TNRC6C-AS1 silenced STK4, hence allowing STK4 promoter methylation.
F I G U R E 1 LncRNA TNRC6C-AS1 is highly expressed in TC tissues. The expression of lncRNA TNRC6C-AS1 in TC tissues (n = 510) and adjacent tissues (n = 58) analysed by TCGA database. The comparison between the two groups was analysed by t test, and data were expressed by mean ± standard deviation Expression level of TNRC6C-AS1

| LncRNA TNRC6C-AS1 silencing blocks the activation of Hippo signalling pathway via STK4
The , the comparison between the two groups was analysed by t test (mean ± standard deviation). B, The expression of lncRNA TNRC6C-AS1 in 5 TC cell lines was determined by RT-qPCR, with normal human thyroid cell Nthy-ori 3-1 as a reference. *P < .05, **P < .01, ***P < .001 vs Nthy-ori 3-1. C, EdU assay was used to detect the proliferation ability of SW579 cells transfected with oelncRNA TNRC6C-AS1 and shlncRNA TNRC6C-AS1, respectively. D, TUNEL method was used to detect the apoptosis rate of SW579 cells transfected with oelncRNA TNRC6C-AS1 and shlncRNA TNRC6C-AS1, respectively. E, MDC method was used to detect the number of autophagic vesicles in SW579 cells transfected with oelncRNA TNRC6C-AS1 and shlncRNA TNRC6C-AS1, respectively, compared with the NC group. F, Western blot analysis was performed to assess the expression of apoptosis (Casepase-3) and autophagy-related (Beclin1 and LC3-II/I) factors in SW579 cells transfected with oelncRNA TNRC6C-AS1 and shlncRNA TNRC6C-AS1, respectively, normalized to GAPDH. G, The representative pictures, weight and volume of tumours in nude mice (n = 14). *P < .05, **P < .01, ***P < .001 vs the NC group. The comparison among multiple groups was analysed by one-way or repeated measures ANOVA (mean ± standard deviation). Values were obtained from 3 independent experiments in triplicate

| LncRNA TNRC6C-AS1 silencing promotes apoptosis and autophagy of TC cells through STK4/ Hippo axis
RT-qPCR and Western blot analysis were carried out to determine effect of lncRNA TNRC6C-AS1 on the apoptosis and autophagy of TC cells through STK4/Hippo axis. When compared with the blank group and the NC group, mRNA and protein levels of Beclin1 decreased, the protein levels of LC3-II/I and Casepase-3 were reduced significantly ( Figure 5A-C), the number of autophagic vesicles decreased significantly and the proliferation ability was enhanced obviously, the ability of apoptosis and autophagy was declined F I G U R E 3 lncRNA TNRC6C-AS1 promotes STK4 methylation A, lncATLAS website was applied to predict the subcellular localization of lncRNA TNRC6C-AS1. B, FISH was used to detect lncRNA TNRC6C-AS1 localization. C, Results of Blast comparison between lncRNA TNRC6C-AS1 and STK4. D, ChIP was used to assess expression of lncRNA TNRC6C-AS1 (**P < .01 vs the IgG group). E, RIP was used to detect the binding between lncRNA TNRC6C-AS1 and methyltransferase. F, The distribution of CpG islands in promoter region of STK4 analysed by bioinformatics. G, MSP was used to detect methylation of STK4 (U: Unmethylation, Non-methylation; M: Methylation). **P < .01, ***P < .001 vs the blank group. The comparison between two groups was analysed by t test while comparison among multiple groups should be statistically analysed by one-way ANOVA (mean ± standard deviation). Values were obtained from 3 independent experiments in triplicate

| D ISCUSS I ON
TC is the most common endocrine malignancy, and the incidence has increased in both developed and developing countries. 20 TC is a major health threat to human beings all over the world. 21 Meanwhile, a new study has showed the influence of lncRNA TNRC6C-AS1 on proliferation, apoptosis and invasion of papillary TC cells. 8 In this study, we explored the role of the lncRNA TNRC6C-AS1, STK4 and Hippo signalling pathway in TC cells. Consequently, this study In this study, we found that lncRNA TNRC6C-AS1 expression in TC tissues was significantly increased and STK4 expression was significantly decreased. A study has approached that lncRNA TNRC6C-AS1 is up-regulated in TC tissues and cells, 9 which is consistent with our results. STK4 is the key kinase participated in the Hippo signalling pathway. 22 A study has showed that the protein level of STK4 decreases in the progression of prostate cancer. 23 Based on RIP assay in this study, the expression of STK4 was apoptotic and autophagic ability. LATS1 is an upstream protein of YAP and adjusts YAP by removing it from the nuclear chamber. 28 A study has showed that LATS proteins are notably down-expressed in breast cancer and non-small cell lung cancer. 29 Dephosphorylated YAP allows its entry into nucleus where it activates the transcription of proproliferative and anti-apoptotic targets. 25 YAP can be phosphorylated and made inactive by alive LATS1. 30 A study has showed that downregulated lncRNA taurine-up-regulated gene 1 (TUG1) occurs with decreased YAP levels. 31 A new study has noted that Beclin1 works in autophagy via complexes like the Beclin1-PI3KC3 or Beclin1-Bcl-2 complex. 32 LC3II was considered as an autophagy-linked marker in cell tissues. 33 YAP plays a role in the processes of autophagy and apoptosis 34 and silencing YAP increased the ratio of LC3-I and LC3-II, accompanied by the autophagy-related protein Beclin1. 15 Caspase-3 plays an important role in the caspase cascade reaction and represents a major enzyme and promoter of apoptosis in cancer cells. 35 A new study has determined a point that multiple myeloma cell apoptosis is accelerated by the improvement of Caspase-3 activity. 36 This study has supported that lncRNA TNRC6C-AS1 silencing inhibits STK4 promoter methylation, up-regulates the expression of STK4 and inactivates the Hippo signalling pathway, thus suppressing the proliferation and promoting cell apoptosis and autophagy of TC cells ( Figure 7). However, due to the limited simple size and experimental conditions, clinical analysis should be conducted in the future in order to validate the results of this study and excavate the predictive values of lncRNA TNRC6C-AS1 in diagnosis and prognosis of TC. We speculate that lncRNA TNRC6C-AS1 or STK4 is a promising F I G U R E 5 LncRNA TNRC6C-AS1 silencing promotes the apoptosis and autophagy of TC cells through STK4/Hippo. A, The mRNA expression of Beclin1 in transfected SW579 cells normalized to GAPDH was determined by RT-qPCR. B, Western blot analysis was used to determine the protein levels of Beclin1, LC3-II/I and Casepase-3 in SW579 cells normalized to GAPDH after transfection. C, The proliferation ability of SW579 cells in each group was tested by EdU assay. D, TUNEL method was used to detect the apoptosis rate of SW579 cells. E, MDC method was used to detect the number of autophagic vesicles of SW579 cells in each group. *P < .05 vs the blank group, #P < .05 vs the shSTK4 group. The comparison among multiple groups was analysed by one-way ANOVA (mean ± standard deviation). Values were obtained from 3 independent experiments in triplicate F I G U R E 6 LncRNA DLX6-AS1 silencing inhibits tumorigenesis and tumour growth of TC cells in vivo through STK4/Hippo. A, Representative pictures of tumours of nude mice with different cells after transfection. B, Tumour weight in nude mice. C, Tumour volume in nude mice. *P < .05 vs the blank group, # P < .05 vs the shSTK4 group. The comparison among multiple groups was analysed by one-way ANOVA. The comparison of data at different time points was analysed by repeated measures ANOVA (mean ± standard deviation). Values were obtained from 3 independent experiments in triplicate

ACK N OWLED G EM ENTS
We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.

CO N FLI C T O F I NTE R E S T
We declare that we have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Xinzhi Peng
https://orcid.org/0000-0001-9436-5550 F I G U R E 7 LncRNA TNRC6C-AS1 silencing inhibits proliferation and promotes apoptosis and autophagy of TC cells. LncRNA TNRC6C-AS1 is upregulated in TC cells, by binding DNMT to STK4 promoter region, which further promotes the methylation of STK4 promoter, thereby down-regulating STK4 and activating Hippo signalling pathway. This further promotes the proliferation of TC cells and inhibits the apoptosis and autophagy of TC cells