Long noncoding RNA ZMIZ1‐AS1 promotes gefitinib resistance via binding to hnRNPA1

To gain an insight into the molecular mechanisms of gefitinib resistance in nonsmall cell lung cancer, we screened out the long noncoding RNA related to gefitinib resistance through microarray data in gefitinib‐sensitive and resistant cells. lncRNA ZMIZ1‐AS1 was significantly upregulated in HCC827/GR cells by screening the microarray data. Further real‐time‐qPCR results were consistent with the microarray data. Cell viability assay and flow cytometry showed that ZMIZ1‐AS1 influenced the sensitivity of HCC827/GR cells to gefitinib. RNA pull‐down assay demonstrated that hnRNPA1 was a specific binding protein for ZMIZ1‐AS1. Our results help to reveal the role and mechanism of lncRNA in the secondary resistance of gefitinib and provide a new therapeutic target for gefitinib therapy.


| INTRODUCTION
The incidence and mortality of lung cancer rank first in the world, and more than 80% of patients are nonsmall cell lung cancer (NSCLC). 1 More than 50% of NSCLC patients are in the middle or advanced stage when they are first diagnosed, and they have lost the opportunity for surgical treatment. The traditional treatments usually focus on radiotherapy and chemotherapy, and the results are often unsatisfactory. The appearance of small molecule tyrosine kinase inhibitors (TKIs) that target EGFR, such as gefitinib, is a major advance in NSCLC treatment in recent years, and they become the first line treatment of NSCLC patients with sensitive mutations of EGFR. However, these patients relapse after 9-13 months of continuous use of the drug, that is, secondary drug resistance. 2 Although the application of the irreversible TKI inhibitors afatinib and AZD9291, which specifically target the EGFR T790M mutation, can partially alleviate resistance, it can still produce new site resistance mutations. Therefore, research on the mechanism of EGFR TKI resistance is in the ascendant, and there is a long way to go to find further targets related to EGFR TKIs resistance.
Long noncoding RNA (long noncoding RNA) is a type of noncoding RNA with a length greater than 200 nt, and its relationship with tumor chemotherapy resistance has become a research hotspot in recent years. Many studies have confirmed that lncRNA is closely related to drug sensitivity. For example, lncARSR could up-regulate AXL and c-MET by competitively binding miR-34/miR-449 to induce sunitinib resistance in renal cancer cells. At the same time, lncARSR also transmitted drug-resistant phenotypes through exosomes. The drug-resistant phenotype could be transmitted through exosomes which may become a potential therapeutic target for sunitinib resistance. 3 The miR-100 and miR-125b produced by lncRNA MIR100HG synergistically inhibit five Wnt/β-catenin negative regulators, activate the Wnt signaling pathway, and thereby mediate cetuximab resistance. 4 These studies suggest that the interaction between noncoding RNAs may play an important role in tumor resistance. Therefore, further searching for new targets for gefitinib resistance from the perspective of lncRNA will help provide new strategies for the treatment of lung cancer resistance.
We continuously induced the gefitinib-sensitive cell line HCC827 and successfully established the secondary drug-resistant cell line HCC827/GR. 5 In this study, we screened out the lncRNAs related to gefitinib resistance through microarray data. This study will help reveal the role and mechanism of lncRNA in the secondary resistance of gefitinib and provide a new therapeutic target for reversing the secondary resistance of EGFR-TKI.

| Cell transfection
The small interfering RNAs (siRNAs) that specifically target human lncRNA ZMIZ1-AS1 were purchased from RiboBio Co., Ltd. For transfection, the cells were placed in six-well or 96-well plates. Twenty-four hours later, they were transfected with lncRNA ZMIZ1-AS1 silencer or negative control (NC) by Lipofectamine ® RNAiMAX reagent (cat. no. 13778075) and Gibco ® Opti-MEM ® (cat. no. 31985062) according to the manufacturer's instructions. The cells were treated with or without different concentrations of gefitinib for 48 h.

| Cell viability assay
The CCK8 assay was used to detect cell viability. The cells were placed at approximately 5 Â 10 3 cells per well in a 96-well plate and stimulated with different concentrations of gefitinib after 24 h of cell attachment. CCK8 reagents were added into the wells after 48 h and the OD values (absorbance) were measured at 450 nm using a Microplate Reader (BioTek ELx800; BioTek Instruments, Winooski, Vermont).

| Cell apoptosis analysis
The cells were plated at 1 Â 10 6 cells/well in a six-well plate followed by gefitinib treatment. Twenty-four hours later, the cells were stained using a fluorescein isothiocyanate/Annexin V apoptosis detection kit (Cat.no. 556547; BD Biosciences, San Diego, California). The samples were then loaded onto a flow cytometer (C6; Becton Dickinson, San Diego, California).

| Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen). And then, the cDNA was synthesized from 100 ng extracted total RNA using the

| Statistical analysis
All the figures were prepared using GraphPad Prism 8.0.2 software (GraphPad Inc., La Jolla, California). SPSS software (version 20.0; IBM Corp., Armonk, New York) was performed for statistical analysis. The comparison of cell viability and apoptosis between multiple groups was performed using two-way analysis of variance (ANOVA). The differences between two groups were analyzed using student's t test.
Differences with p < .05 were considered statistically significant.

| LncRNA ZMIZ1-AS1 was increased in gefitinib resistance cells
Cell viability was determined by the CCK-8 assay to assess the resistance index of the gefitinib resistance cells. As shown in Figure 1A, the IC50 value of HCC827 for gefitinib was 0.05 μM. However, the IC50 of HCC827/ GR became >10 μM. The resistance index was more than 200-fold higher. The microarray experiment in gefitinib-sensitive HCC827 and gefitinib-resistant HCC827/GR cells in pairs was performed as described previously. 6 The microarray data have been submitted to the Gene Expression Omnibus (accession number, GSE74575). We found that ZMIZ1-AS1 was significantly upregulated in HCC827/GR cells by screening the microarray data. Further RT-qPCR results were consistent with the microarray data ( Figure 1B).  Figure 2B). Cells were analyzed using flow cytometry and the results of the apoptosis assay were similar to those of the CCK-8 assay.

| ZMIZ1-AS1 can bind to hnRNPA1
To explore the potential binding proteins of ZMIZ1-AS1in HCC827/ GR cells, we performed a biotin-labeled RNA pull-down assay followed by silver staining ( Figure 3A). A protein band specifically presented in lncRNA-HGBC was located at approximately >35 kD and then was subjected to sequence analysis via mass spectrometry.
With great interest, we paid particular attention to an RNA-binding candidate hnRNPA1 that has high confidence score. Western blot assay confirmed that hnRNPA1 was a specific binding protein for ZMIZ1-AS1 ( Figure 3B).

| DISCUSSION
Gefitinib is a first-line treatment drug used to treat lung cancer with sensitive mutations, which can significantly improve their overall survival rate. However, acquired gefitinib-resistance is still a Growing evidence has pointed to the notion that many lncRNAs can function to regulate some target gene expression through direct interaction with proteins. hnRNPA1 is the most abundant and ubiquitously expressed member of heterogeneous nuclear ribonucleoproteins (hnRNPs). It is a RNA-binding protein associated with complex and diverse biological processes such as processing heterogeneous nuclear RNAs into mature mRNAs, RNA splicing, transactivation of gene expression, and modulation of protein translation. 11-13 hnRNPA1 has been reported to participate in multiple molecular events in cancer transformation. [14][15][16] Here, we reported that ZMIZ1-AS1 binded to hnRNPA1 by RNA pull-down assay and mass spectrometry. Our study has limitations because we did not reveal how ZMIZ1-AS1 promoted gefitinib resistance by combining with hnRNPA1. The specific mechanism needs further study.
In summary, the present study showed that ZMIZ1-AS1 promoted gefitinib resistance by binding with hnRNPA1. These findings enhanced our understanding of ZMIZ1-AS1in NSCLC gefitinib resistance. So far, there has been no report on the involvement of ZMIZ1-AS1 in the process and molecular mechanism of gefitinib resistance in NSCLC, so this study is original and innovative.

This work was supported by scientific research project of Jiangsu
Commission of Health (M2020032).