Silencing long non‐coding RNA DLX6‐AS1 or restoring microRNA‐193b‐3p enhances thyroid carcinoma cell autophagy and apoptosis via depressing HOXA1

Abstract Long non‐coding RNA DLX6 antisense RNA 1 (DLX6‐AS1) lists a critical position in thyroid carcinoma (TC) development. However, the overall comprehension about DLX6‐AS1, microRNA (miR)‐193b‐3p and homeobox A1 (HOXA1) in TC is not thoroughly enough. Concerning to this, this work is pivoted on DLX6‐AS1/miR‐193b‐3p/HOXA1 axis in TC cell growth and autophagy. TC tissues and adjacent normal thyroid tissues were collected, in which expression of DLX6‐AS1, miR‐193b‐3p and HOXA1 was tested, together with their interactions. TC cells were transfected with DLX6‐AS1/miR‐193b‐3p‐related oligonucleotides or plasmids to test cell growth and autophagy. Tumorigenesis in nude mice was observed. DLX6‐AS1 and HOXA1 were up‐regulated, and miR‐193b‐3p was down‐regulated in TC. Depleted DLX6‐AS1 or restored miR‐193b‐3p disturbed cell growth and promoted autophagy. DLX6‐AS1 targeted miR‐193b‐3p and positively regulated HOXA1. miR‐193b‐3p inhibition mitigated the impaired tumorigenesis induced by down‐regulated DLX6‐AS1. Tumorigenesis in nude mice was consistent with that in cells. It is clear that DLX6‐AS1 depletion hinders TC cell growth and promotes autophagy via up‐regulating miR‐193b‐3p and down‐regulating HOXA1.

NEAT1 enables itself to impede ATC cell migration and invasion under hypoxic conditions. 5 Independently, an observational study has elucidated that lncRNA DLX6 antisense RNA 1 (DLX6-AS1) is repressive for TC cell migration and invasion. 6 Surprisingly, miR-193b-3p is investigated to tie up with PTC invasiveness. 7 However, the integrated performance of DLX6-AS1 and targeted miR-193b-3p in TC has seldom been navigated. Homeobox A (HOXA) group has been suggested to take part in tumorigenesis of follicular TC, such as HOXA9. 8 Recently, HOXA9 has been discovered to connect with invasion and migration in PTC. 9 Specifically, it has been demonstrated that there is a significant discrepancy in methylation status of HOXA1 between benign thyroid lesions and PTC of formalinfixed, paraffin-embedded tissues. 10 Briefly, how DLX6-AS1 and miR-193b-3p cooperate in the process of TC is decoded in this work from the perspective of HOXA1.

| Ethics statement
This study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University, and the written informed consent of each participant was obtained. All animal plans were approved by the Animal Ethics Committee of Beijing Tongren Hospital, Capital Medical University.

| Clinical specimen collection
Patients (n = 108) who were diagnosed with TC in Beijing Tongren Hospital, Capital Medical University, during 2017-2019 were recruited, whose cancer tissues were collected while the adjacent normal thyroid tissues were taken as controls. None of patients had received treatment (including radiotherapy and chemotherapy) before operation.

| Cell culture
Human TC cell lines K1, BCPAP, IHH4 and TPC1 and human nor-

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA from tissues and cells was obtained by Trizol (Invitrogen), and the concentration and purity of RNA were evaluated by a  Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were considered as loading controls for genes, whose expression levels were calculated by 2 −ΔΔCt method. 13

| Western blot assay
Pre-lysed in radio-immunoprecipitation assay cell lysis buffer (Gibco), proteins from cells and tissues were extracted by centrifu-

| Tumour xenografts in nude mice
Nude mice (4-6 weeks old, female) were originated from HFK (Beijing, China) and raised under pathogen-free conditions. Stably transfected K1 cells (2 × 10 6 cells) were injected subcutaneously in nude mice. Xenografted tumours were measured every 5 days: volume = (a × b 2 )/2 (a was the longest diameter and b was the shortest diameter of the xenografted tumour). Mice were euthanized 25 days later to obtain the tumours, which were photographed and weighed.

| Transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) staining
Tumour tissues were fixed in 10% paraformaldehyde and embedded in paraffin. Then, the tissues were reacted with TUNEL mixture by following the guidance of the in situ cell apoptosis detection kit (POD, Roche Diagnostics GmbH, Mannheim, Germany). The stained slides were detected with an Olympus IX51 fluorescence microscope (Olympus), and the apoptosis rate was calculated.

| Statistical analysis
All data were analysed by SPSS21.0 (IBM, NY, USA) statistical software. Measurement data were expressed as mean ± standard deviation. Discrepancy between two groups was assessed by t test while that among multiple groups by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. Pearson analysis was applied in correlation assessment. At p < 0.05, statistical significance was set.

| Depleted DLX6-AS1 disturbs cell progression and promotes autophagy in TC
K1 cells were screened out to explore the functional performance of  108). Measurement data were expressed as mean ± standard deviation, N = 3. *p < 0.05 compared with Nthyori3-1 cells. Discrepancy between two groups was assessed by t test while that among multiple groups by one-way ANOVA, followed by Tukey's post hoc test

| DLX6-AS1 targets miR-193b-3p
The regulatory mechanism of DLX6-AS1 and miR-193b-3p was firstly tested by RNA22 to predict the binding site between the two ( Figure 3A) and then by dual-luciferase reporter gene assay to verify the binding of miR-193b-3p a DLX6-AS1 ( Figure 3B). It was outlined that miR-193b-3p-mimic caused an impairment in fluorescence activity of DLX6-AS1-WT while had nothing to do with fluorescence activity of DLX6-AS1-MUT. RNA pull-down assay depicted that ( Figure 3C) miR-193b-3p was enriched in Bio-DLX6-AS1-WT of K1 cells while its enrichment was not changed in Bio-DLX6-AS1-MUT.

| DLX6-AS1 positively regulates HOXA1 in TC
It has been surveyed that the oncogene HOXA1 was overexpressed in tumour tissues 24 and cells. 25 In this work, it was implied that miR-193b-3p could bind to HOXA1 3′-untranslated region and regulate HOXA1 expression in K1 cells ( Figure 5A). Additionally, RT-qPCR and Western blot assay depicted that silencing DLX6-AS1 or restoring miR-

| Silencing DLX6-AS1 or restoring miR-193b-3p impedes tumour formation in mice with TC
To verify whether the regulatory function of DLX6-AS1 in TC oc-

| DISCUSS ION
Rarely occurred TC is a heterogeneous disease, accounting for less than 1% of all tumours. 26 LncRNAs and miRNAs are elaborated to expression, thereby to encourage TC cell progression and suppress autophagy ( Figure S1).
Importantly, this work has delved out DLX6-AS1 is expressed Also, increased DLX6-AS1 is presented in colorectal cancer tissues and depleting DLX6-AS1 restricts metastasis of colorectal cancer cells and arrests cell phase progression. 28 Besides, oesophageal squamous cell carcinoma specimens express elevated DLX6-AS1 and DLX6-AS1 down-regulation is attributed to depressed cell proliferation and metastasis. 29 Crucially, the network between DLX6-AS1 and its regulatory gene miR-193b-3p has been deciphered and the results imply that DLX6-AS1 is negatively connected with and targeted to miR-193b-3p. Then, focused on miR-193b-3p, we have investigated that it is expressed at a low level in TC, and its restoration attributes to inhibited TC cell progression and reinforced autophagy. In fact, the targeting relationship between DLX6-AS1 and miR-193b-3p has not been verified yet in previous researches, which requires further validation. Experimentally, a study has made it clear that miR-193b-3p expression is decreased in metastatic TC by comparison with non-invasive TC tissues. 7 Mechanistically, miR-193b-3p expression trends to down-regulate in osteosarcoma and miR-193b-3p depletion would result in enhanced cell proliferation, reduced G0/G1 phase and impaired apoptosis. 30 Another study also stresses out that miR-193b-3p expression is reduced in lung cancer and miR-193b-3p elevation restrains migration, invasion and colony formation activities of cells. 31 For validation, tumorigenesis To proceed, the mechanism of miR-193b-3p/DLX6-AS1 with HOXA1 is deeply explored to reveal a negative connection between miR-193b-3p and HOXA1 and the positive one between DLX6-AS1

ACK N OWLED G EM ENT
We would like to acknowledge the reviewers for their helpful comments on this paper.

CO N FLI C T S O F I NTE R E S T
The authors declare that they 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.