NR2F2-AS1 Suppressed Trastuzumab Effects on Esophageal Cancer By Inhibiting miR-4429 and miR-425-5p Expression Through Targeting IGF1R

Aim:In this manuscript, we aimed to investigate the involvement of non-coding RNAs in mediating trastuzumab effects in EAC. Background: Scarce evidences supported that targeted drugs, like Trastuzumab, can be applied to esophageal adenocarcinoma patients (EAC). Objective: Evaluating the role and mechanism of NR2F2-AS1 in regulating Trastuzumab effects in EAC patients. Method: RNA sequencing to screen IGF1R related lncRNAs. qRT-PCR and western blot were used to evaluate the expression level of genes. CCK-8 was used to test the cell proliferation ability. Dual luciferase reporter gene assay and RNA pull-down were used for crosstalk evaluation. Results: NR2F2-AS1 was identied to be associated with HER2 expression by RNA sequencing and its expression related to worse prognosis and advanced T and N stage.NR2F2-AS1 expression induced by Trastuzumab through mediating H3K27ac. Furtherly, miR-4429 and miR-425-5p, which were predicted and proved to interact with NR2F2-AS1 and IGF1R, expressed lowly in esophageal cancer both in vivo and in vitro and suppressed cell viability. Most importantly, miR-4429 and miR-425-5p overexpression could increase trastuzumab’s inhibitory effect on cell viability. Conclusion: Trastuzumab has mainly of miR-4429 and miR-425-5p overexpression targeting HER2. However, Trastuzumab induces exosomal NR2F2-AS1 expression, which binds to miR-4429 and miR-425-5p to suppress their expression, resulting in the failure of trastuzumab treatment. Therefore, targeting exosomes might be a novel way to develop auxiliary drugs for trastuzumab in EAC. IGF1R positive and IGF1R negative esophageal cancer patients. F miR-4429 expression in esophageal cancer patients stratied by T stage. G miR-4429 expression in esophageal cancer patients stratied by N stage. H miR-425-5p expression in esophageal cancer patients stratied by T stage. I miR-425-5p expression in esophageal cancer patients stratied by N stage.


Introduction
HER2(Epidermal Growth Factor Receptor 2) , usually activated by HER1, 3 and 4 by forming dimers, is proved to mediate malignant behaviors, for example, promoting cell proliferation, migration, invasion and angiogenesis [1] . Therefore, HER2 is a feasible therapeutic target and widely applicated in treating breast cancer [1] . Numerous studies have elucidated the distinguished effect of Trastuzumab in prolonging HER2 positive breast cancer by suppressing the HER2 activation [2] . Accumulating studies have shown that HER2 overexpressing esophageal cancer patients have shorter overall survival months than HER2 negative patients with more than 70% HER2 positive rate [3] . Besides, around 30% esophageal cancer patients had overexpressed HER2, indicating the possibility of applying Trastuzumab [4] . A phase II study showed that Trastuzumab did not increase the toxicity and side effects, suggesting the accepted safety [5] . Under this circumstance, successfully applying trastuzumab to esophageal cancer will signi cantly improve the prognosis [6,7] . However, a substantial proportion of HER2 positive EAC patients did not well respond to trastuzumab, partially due to acquired resistance [8,9] .Loss of PTEN is one of the most important mechanisms that contributed to the trastuzumab resistance [10,11] . Apart from PTEN, IGF1R (insulin-like growth factor-I receptor) activation induces HER2 phosphorylation and impairs the HER2 activation in trastuzumab resistant cell lines [12] . IGF1R knock down will block the heterodimerization with HER2 and improve cancer cells' response to trastuzumab [13] . Here, we have assumed that NR2F2-AS1 might regulate trastuzumab sensitivity by targeting IGF1R.
Increasing evidence have shown the neglectable impacts of non-coding RNAs in the process of developing trastuzumab resistance [14] . For example, linc-ATB functioned as a microRNA sponge to suppress the expression of miR-200c to up-regulate the ZEB1 and ZNF-27 to activate EMT process, resulting in the trastuzumab resistance [15] . In this manuscript, lncRNA NR2F2-AS1 was identi ed using RNA-sequencing in vivo as candidate trastuzumab resistance associated genes. lncRNA NR2F2-AS1 is involved in the cancer cell proliferation, apoptosis, cell cycle arrest, cancer stemness and etc. as an oncogene [16][17][18][19] . Lin et al provided valuable evidence that NR2F2-AS1 enhanced Rac1 expression to increase cancer stemness in clear cell renal cell carcinoma; therefore, it can be inferred that NR2F2-AS1 might be engaged in developing drug resistance [16] . Current studies reported that NR2F2-AS1 mainly promotes tumorigenesis by means of acting as a microRNA sponge. For instance, NR2F2-AS1 sponges miR-230b and miR-4429 to regulate their regulation of downstream genes [16,20] . Therefore, we assume that NR2F2-AS1 might regulate IGF1R expression through sponging potential microRNAs. In this manuscript, we predicted that miR-4429 and miR-425-5p could interact with both NR2F2-AS1 and IGF1R.

Esophageal adenocarcinoma patients
In total 201 esophageal cancer patients (Her2 positive) were enrolled in rst a liated hospital of Xi`an Jiaotong University between March 2011 and March 2013. Every enrolled patient has received and signed the written consent. Protein and RNA were extracted from esophageal cancer and corresponding normal tissue samples obtained from each patient immediately; and remnant samples were para n-embedded.
The main inclusion criteria were as follows: 1. Pathologically diagnosed as esophageal adenocarcinoma; 2. Receive surgery plus chemotherapy; 3. Did not receive any other treatment related to EAC elsewhere; 4.
Older than 18-year-old and ability to take care of herself/himself. The main exclusion criteria were: 1. Diagnosed with other primary malignancies; 2. IV stage (based on 7th edition of UICC tumor staging system); 3. Refuse to sing the informed consent. Two employees were in charge of documenting clinical and follow-up records of each patient. Besides, the corresponding author quality controlled every data and validated the data integrity. Our study has gained approval from the Ethics Committee of rst a liated hospital of Xi`an Jiaotong University. This study was performed in accordance with the Declaration of Helsinki. qRT-PCR Total RNA was extracted from cell lines, EAC patients' cancer tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Cytoplasmic & Nuclear RNA Puri cation Kit (Norgen, Canada) was used to isolate cytoplasmic and nuclear RNA. First-strand cDNA synthesis kit (Tiangen Biotech, Beijing, China) was used for reverse transcription to generate cDNA. SYBR® Premix Dimer Eraser kit (Takara Shiga, Japan) was used to perform qRT-PCR and test the expression of NR2F2-AS1 and IGF1R. β-actin was used as internal control. miScript microRNA RT PCR kit (Qiagen, Toronto, ON, Canada) was used for cDNA synthesis and qRT-PCR process for miR-4429 and miR-425-5p expression. U6 was used as the internal control. ABI 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) was used to perform the qRT-PCR process. The expression level was calculated by 2 -ΔΔCt method.

Western blot
Total protein was extracted from cells using RIPA buffer (Sigma-Aldrich, Darmstadt, Germany) and was quanti ed by BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were transferred to PVDF after separated by SDS-PAGE. 5% skim milk was used for transferred PVDF membranes block for 1 hour at room temperature. IGF1Rβ (1:1000, CST, Shanghai, China) was incubated overnight at 4 •C, following secondary antibody(1:10000, Beyotime, Shanghai, China) Enhanced chemiluminescence (ECL, Beyotime, Shanghai, China) was used to quantify the protein expression level.
Dual luciferase reporter gene assay Plasmids of pGL3-NR2F2-AS1 wild type (WT) or NR2F2-AS1 mutant type were co-transfected with the miR-425-5p/NC or miR-4429/NC mimic into SK-GT-4 cells for 48 hours with the assistance of Lipofectamine 2000 (Beyotime, Shanghai, China). Plasmids of pGL3-IGF1R wild type (WT) or IGF1R mutant type were co-transfected with the miR-425-5p/NC or miR-4429/NC mimic into SK-GT-4 cells as well. Then Dual-luciferase reporter system (Promega, Madison, WI, USA) was used to measure the re y luciferase activity, which was normalized to renilla luciferase activity. . Trizol was then used to purify the bound RNA and qRT-PCR was used to test the NR2F2-AS1 enrichment.

Statistics
GraphPad Prism 8.2 was used for plotting statistical graphs. R 3.3.1 was used for statistic analysis: WGCNA and Limma package were used for RNA sequencing analysis. t-test was used for two groups comparison; one-way ANOVA for multiple groups comparison and repeated measurement ANOVA for CCK-8 results analysis. P < 0.05 was indicative of statistically signi cant difference.

Results
1. RNA sequencing to identify the potential involvement of NR2F2-AS1 in regulating IGF1R expression RNA sequencing was used to identify differential expressed lncRNAs between IGF1R positive and IGF1R negative esophageal adenocarcinoma patients (4 Vs 4): we have plotted heatmap to visualized the top 30 differentially expressed lncRNAs ( Figure 1A). Further in vivo experiments showed that NR2F2-AS1 was highly expressed in 201 Her2 positive esophageal squamous cell carcinoma patients rather than that in normal adjacent esophageal tissues ( Figure 1B). Moreover, we noticed that NR2F2-AS1 expression was related to advanced T ( Figure 1C) and N stages ( Figure 1D); and NR2F2-AS1 expressed higher in IGF1R positive patients ( Figure 1E).

NR2F2-AS1 promotes migration and inhibits apoptosis of EAC in vitro by regulating IGF1R
We found that NR2F2-AS1 expressed higher in OE-33 and SK-GT-4 cell line (Figure 2A), besides IGF1R was aberrantly up regulated in OE-33 and SK-GT-4 cell line both in mRNA and protein level ( Figure 2B and 2C). Then we found that NR2F2-AS1 resided in the cytoplasm of OE-33 and SK-GT-4( Figure 2D and 2E).

miR-4429 and miR-425-5p expression pro le in esophageal cancer patients
Starbase V3.0 was used to predict the potential crosstalk microRNAs for NR2F2-AS1 and IGF1R; miR-4429 and miR-425-5p were identi ed to be both interacted with NR2F2 and IGF1R ( Figure 4A). Further in vivo experiments showed that miR-4429 and miR-425-5p were both suppressed in Her2 positive esophageal adenocarcinoma patients ( Figure 4B and 4C). Besides, miR-4429 and miR-425-5p expressed higher in IGF1R positive patients ( Figure 4D and 4E). miR-4429 was negatively associated with advanced T stage and N stages ( Figure 4F and 4G). However, miR-425-5p seems not to be relevant with T or N stage ( Figure 4H and 4I).
Moreover, we found that miR-4429 knock down could reverse the inhibitory effect of NR2F2-AS1 knock down on the IGF1R expression ( Figure 6H to 6I). Same results were detected for miR-425-5p knock down ( Figure 6J to 6K). In conclusion, NR2F2-AS1 binds to miR-4429 and miR-425-5p to regulate IGF1R expression to exert biological functions.

Discussion
Trastuzumab contained regimens have made great progress in improving the prognosis of breast cancer [10] ; recent clinical trials implies the possibility of applying trastuzumab in esophageal cancer [2,5] .
There is a growing consensus on this issue that HER2 overexpression EAC patients are eligible for receiving trastuzumab [2,5] . However, lessons from breast cancer application show that a large proportion of patients would experience acquired trastuzumab resistance even though they are well responsive to the initial therapy [10] . IGF1R signaling pathway activation mainly accounts for trastuzumab resistance by interacting with both HER2 and EGFR to suppress the activation of downstream pathways [13] . Therefore, we have utilized RNA sequencing to identify that NR2F2-AS1 that was associated with IGF1R.
NR2F2-AS1 has been identi ed to be oncogenic in various cancers. In this manuscript, we further investigated the role of NR2F2-AS1 in trastuzumab treatment. We found that trastuzumab can signi cantly induce the expression of NR2F2-AS1, especially exosomal NR2F2-AS1. Further experiments indicate the unneglectable role of NR2F2-AS1 in promoting proliferation of esophageal cancer cell. Then, our result showed that exosomal NR2F2-AS1 induced by trastuzumab played an important role in promoting migration and inhibiting apoptosis of EAC cell lines. Most importantly, exosomal NR2F2-AS1 could suppress the anti-cancer function of trastuzumab treatment. Therefore, we assume that trastuzumab could induce the exosomal NR2F2-AS1 expression and in verse inhibit trastuzumab's anticancer effects.
IGF1R regulated receptor tyrosine kinase signaling, which might play signi cant role in trastuzumab resistance; and its overexpression is associated with trastuzumab resistance in breast cancer cells [13] .
With the assistance of online bioinformatics tools, we have noticed that NR2F2-AS1 might sponge miR-4429 and miR-425-5p to regulate IGF1R expression, resulting in impaired trastuzumab effects. Dual luciferase reporter gene assay and RNA pull down further con rmed that NR2F2-AS1 could interact with miR-4429 and miR-425-5p; and miR-4429 and miR-425-5p could bind to the 3`UTR of IGF1R to suppress its expression.
In conclusion, we have identi ed the key regulator NR2F2-AS1 for IGF1R based on RNA sequencing analysis. Furthermore, exosomal NR2F2-AS1, induced by trastuzumab treatment, could inhibit the anticancer effect of trastuzumab by regulating IGF1R through sponging miR-4429 and miR-425-5p.

Declarations
The data that support the ndings of this study are available from the corresponding author on request.

Competing interests
The authors declare no con icts of interest.

Funding information
This work was supported by the Chinese National Science Foundation Projects (NSFC 30740022).

Consents
All patients involved in this study has signed the written informed consent to participate in our study. Our study was approved to be published by Ethics Committee of the First A liated Hospital of Xi`an Jiaotong University.