Exploring the Differential Gene of CHOP-Related Factors in Hepatocellular Injury Caused by Endoplasmic Reticulum Stress

Objective: CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP/DDIT3) is a protein activated by endoplasmic reticulum stress (ERS). However, the downstream genes of CHOP on liver damage caused by ER-stress have been unclear. Herein, we investigated the potential downstream related factors of CHOP in L-02 cells. Methods and Material: Tunicamycin (TM) was used to induce ER-stress. Short hairpin RNA (shRNA) was used to knocked down CHOP, and the functions of differentially expressed genes (DEGs) were obtained from functional annotations. qRT-PCR was employed to validate the expression levels of candidate DEGs. Results: 633 genes were differentially expressed between ERS L-02 cells and normal L-02 cells(cid:0)and 131 genes were differentially expressed between shRNA-CHOP and shRNA-NC in ERS L-02 cells. By analyze these results, we luckily found 8 genes including Interferon a-inducible protein 27 (IFI27), Lipocalin 2 (LCN2), Chromosome 11 Open Reading Frame 86 (C11orf86), Calmegin (CLGN), Kelch domain-containing 7B (KLHDC7B), Niban Apoptosis Regulator 1 (Niban), T-Cell Receptor Gamma- Chain Constant Region (TARP), Lysosome associated membrane protein 3 (LAMP3) were intimately related to chop. Conclusion: Our study might contribute to better understand how CHOP functions during ER-stress, and these results can expand databases of CHOP in GenBank or others.


Introduction
The liver is an essential metabolic organ in the body, and it acts a crucial role in antioxidants, storing glycogen, synthesizing secreted proteins and so on [1]. In recent years, the incidence of hepatic diseases trend to raise year by year in the world. The liver is rich in endoplasmic reticulum (ER). There is a conspicuous correlation between the hepatic diseases and dysfunction of the ER. ER is a main organelle for protein synthesis and folding to form the correct three-dimensional conformation of the proteins. Various factors would lead to the malfunctions of ER, such as oxygen deprivation, calcium metabolic disorders, oxidative stress and so on [2].
Endoplasmic reticulum stress (ERS) would be induced if the perturbation of ER is serious or persistent.
The unfolded protein response (UPR) is a part of the global Endoplasmic reticulum stress (ERS) response. the unfolded and misfolded proteins accumulate in ER would result in UPR [3]. The UPR protects cell from damage by enhancing the protein folding ability, stagnating the translation of most proteins and accelerating protein degradation. Protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositolrequiring enzyme-1 (IRE-1) and activating transcription factor 6 (ATF6) are important ER-localized transmembrane proteins in the three classical signaling pathways of UPR [4]. PERK/eIF2P/ATF4 pathway is necessary to regulate CHOP expression in ERS. The increasing translation of activating transcription factor 4 (ATF4) directly induces the expression of downstream gene CHOP, and then accelerates the apoptosis of cells, which suggesting that CHOP plays an important role in the apoptosis process induced by ERS [5].
CHOP, also known as GADDl53/ DDIT3, is a multifunctional transcription factor that is critical in the ERSinduced apoptotic programs. It is expressed at a very low level in physiological state and is signi cantly up-regulated in response to a variety of cellular stresses and induces cell cycle arrest and apoptosis [6]. It also plays a dual role both as an inhibitor of CCAAT/enhancer binding protein (C/EBP) function and as an activator of other genes. CHOP plays a considerable part in ER stress-induced apoptosis, which is the main upregulated pro-apoptotic player. There are a lot of researches which focus on upstream regulatory pathway of CHOP. And few of downstream genes are associated with CHOP in public databases at present, so it is signi cant to nd the downstream genes regulated by CHOP [7].
Nowadays, the next-generation of sequencing technology has been widely used, and it has become a powerful tool for simultaneous sequencing of millions of genomic features and identi cation of speci c genes in sample tissues. High-throughput RNA sequencing is superior to expression pro les from the microarray technology. The high sensitivity of RNA-Seq data could provide a qualitative and quantitative description of gene expression [8]. In this study, we used RNA-seq to obtain mRNA expression data from L-02 cells, and to identify Differentially expressed genes between ERS L-02 cells and normal L-02 cells and between shRNA-CHOP and shRNA-NC in ERS L-02 cells. Our aim was to discover the potential downstream related factors for CHOP.

Cell culture
L-02 cells were from Procell Life Science & Technology Co., Ltd (Wuhan, China). L-02 cells were cultured in 1640 medium containing 10% fetal bovine serum, 100 units/ml penicillin G and 100 µg/ml streptomycin sulfate. The culture medium was kept replacing every 2 days, and cells were maintained in the logarithmic growth phase.

Knockdown of CHOP by shRNA and treatment
Knockdown of CHOP was performed using short hairpin RNA (shRNA) in L-02 cells. Brie y, The L-02 cells were divided into four groups ( A: The normal L-02 cells, B: The L-02 cells were treated with TM for 24 h in a dose of 60 µg/mL, C: The L-02 cells were rstly transfected shRNA-CHOP for 24 h, then were treated with TM for 24 h in a dose of 60 µg/mL, D: The L-02 cells were rstly transfected shRNA-NC for 24 h, then were treated with TM for 24 h in a dose of 60 µg/mL). Western blotting was used to identify the knockdown e ciency. The most e cient shRNA sequences were as following: shRNA 1: 5′-GCGCATG AAGGAG-AAAGAACAGG; shRNA2: 5′-CCGAGCTCTGATTGACCGAATGG[9].

Extraction and construction of RNA-seq library
Total RNA of each group was extracted by using TRIzol reagent (Invitrogen, USA). The library preparation followed the standard procedures of Baerveldt glaucoma implant (BGI). Oligo (dT) magnetic beads are used to select mRNA with polyA tail from total RNA, and the mRNA was cleaved into short fragments, and reverse transcribed into double-strand cDNA (dscDNA) by N6 random primer. The ends of the dscDNA were lled in, then the 5' end was phosphorylated, and a sticky end protruding from A was formed at the 3' end, followed by a bubbling junction with a protruding 'T' at the 3' end. After polymerase chain reaction (PCR) ampli cation with two speci c primers, the enriched cDNA libraries were sequenced with a BGISEQ-500 (BGI, Inc., Wu Han, China).

Differentially expressed genes analysis
Differentially expressed genes were identi ed by using the Noise Sensitivity Questionnaire (NOISeq) method, which could screen differentially expressed genes between two groups, showing a good performance compared with other differential expression methods [10]. we screen differentially expressed genes according to the following default criteria: fold change ≥ 2 and diverge probability ≥ 0.8.

Functional annotation of differentially expressed genes.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict the biological functions of DEGs. GO is an international standard gene functional classi cation system. It rstly mapped all DEGs to GO terms in the database (http://www.geneontology.org/), calculating gene numbers for every term, then using the hypergeometric test to nd signi cantly enriched GO terms in the input list of DEGs. KEGG was used to perform pathway enrichment analysis of DEGs. This analysis identi ed by signi cantly enriched metabolic pathways or signal transduction pathways in DEGs comparing with the whole genome [11]. P-value ≤ 0.05 were set as the threshold for selecting signi cantly enriched functional GO terms and KEGG pathway enrichment.
2.6. Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain (qRT-PCR) analysis was used to validate the DGEs selected from the RNA-seq analysis. According to the manufacturer's instructions (Invitrogen, USA), total RNA was extracted using TRIzol reagent. The primers pairs designed using Primer Premier 5.0 were listed in Table 1. The HiScript® 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China) was used to synthesize the cDNA. AceQ® qPCR SYBR® Green Master Mix (Vazyme Biotech, Nanjing, China) was used to perform qRT-PCR reactions on a Bio-Rad CFX96 Real-Time PCR machine (Bio-Rad, USA). These reactions were used the following cycle parameters: 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds, and then 72°C for 30 seconds. The relative expression of target genes was calculated by the 2 −ΔΔCT equation [12]. The GraphPad Prism version 5.01 software was used to output gures.

E cient CHOP knockdown and RNA-seq analysis
Western blotting showed that CHOP was knocked down in L-02 cells by short hairpin RNA (shRNA), as shown in Fig. 1. These results identi ed an e cient decrease of the CHOP protein level in sh-RNA-3, which was used to subsequent experiments [13].

RNA-seq was performed on the L-02 samples of 8 subjects (normal L-02 cells, ERS L-02 cells groups, shRNA-CHOP in ERS L-02 cells and shRNA-NC in ERS L-02 cells, each group has two biological repeats).
Approximately, averagely generating 24137291 raw sequencing reads and then 23980654 clean reads after ltering low quality. All of the clean reads were aligned to the UCSC human reference genome (hg.19) using HISAT/ Bowtie2 tool. The average mapping ratio with reference gene is 80.77% and the average genome mapping ratio is 95.87%. Speci c information of sequencing data for each sample is shown in Table 2.   (Fig. 2). We used cluster and Java TreeView software to perform clustering analysis of DEGs, the result as the following Fig. 3A and Fig. 3B. We were interested in the DEGs which were highly changed between the two compared groups. According to the data of DEGs, we found eight genes were signi cantly altered, Calmegin (CLGN), Lipocalin 2 (LCN2), The family of lysosome-associated membrane protein 3 (LAMP3), Kelch domain-containing 7B (KLHDC7B) and family with sequence similarity 129 member A (FAM129A) were up-regulated when ERS occurred and were down-regulated when CHOP was knockdown. Conversely, Interferon a-inducible protein 27 (IFI27), T-cell receptor gamma chain alternate reading frame protein (TARP) and Chromosome 11 Open Reading Frame 86 (C11orf86) were down regulated when ERS occurred and were up-regulated when CHOP was knockdown. The speci c information of DEGs we selected were listed in Table 3.  shown.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs
KEGG is a knowledge database for the systematic functional analysis of genes to connect genomic information with higher functional information by computerizing current knowledge on cellular processes and standardizing gene annotations. Thus, KEGG analyses were performed to assign pathways and functionally classify genes. FDR < 0.05 was de ned as signi cant KEGG pathway enrichment. As shown in Table 4, pathways in protein processing, mTOR signaling, oxidative phosphorylation, PI3K-Akt signaling pathway, Rap1 signaling pathway, MAPK signaling pathway and others were signi cantly enriched KEGG pathways between ERS L-02 cells and normal L-02 cells. The signaling pathways of DEGs between shRNA-CHOP and shRNA-NC in ERS L-02 cells were not available in the KEGG database.

Veri cation of the expression level of DEGs The expression levels of DEGs between ERS L-02 cells groups and normal L-02 cells groups and between
shRNA-CHOP in ERS L-02 cells groups and shRNA-NC in ERS L-02 cells groups were quanti ed by qRT-PCR. As shown in Fig. 3, DDIT3, CLGN, LCN2, LAMP3, KLHDC7B and FAM129A were signi cantly upregulated in ERS L-02 cells compared with normal controls (P < 0.05), and they were down-regulated after we knocked down DDIT3. In Fig. 6, IFI27, TARP and C11orf86 shown the exact opposite situations, they were signi cantly down-regulated in ERS L-02 cells compared with normal controls (P < 0.05), and they were up-regulated after we knocked down CHOP (P < 0.05).The qRT-PCR results were all in accordance with our RNA-seq data.

Discussion
There are numerous researches on the expression CHOP involved in ER-stress at present. CHOP plays a considerable role in many biological processes such as autophagy and apoptosis. In this study, we used RNA-seq to analyze these potential genes located downstream of CHOP gene, which showed that knockdown of CHOP resulted in an enormous impact on other genes whose role remains unknown in ERstress. These results indicated that CHOP contributed to important functions in ER-stress signaling pathways and others. After contrasting with cell-to-cell variation in both L-02 cells and normal L-02 cells, and that change between shRNA-CHOP and shRNA-NC in ERS L-02 cells, we identi ed that considerable genes with obvious changes after CHOP knockdown. To our knowledge, this study is the rst attempt to investigate the gene regulatory network after CHOP knockdown. Our study advances the understanding of CHOP function in ER-stress and more importantly, initially clari ed the relevant factors and pathways of CHOP. Interestingly, we identi ed eight genes showing a close relationship with CHOP gene which have been rarely reported. We further noticed that a kind of unknown regulatory mechanism may exist between these genes and CHOP gene [14].
These genes are further explained in detail as follows: LCN2 belongs to the lipocalin family. The protein encoded by this gene is a neutrophil gelatinase-associated lipocalin (NGAL) and participates actively in a lot of physiological activities, such as innate immunity, in ammation, apoptosis, proliferation and differentiation of cells and so on [15,16]. A number of studies discovered that up-regulation of LCN2 is associated with the occurrence and development of many liver diseases, suppression or knockdown of LCN2 could alleviate various liver diseases, such as liver brosis [17], alcoholic fatty liver[18], liver failure [19], in ammation and infection [20]. In addition, Thyroid hormone (T3)/thyroid hormone receptor (TR) could regulate LCN2 via the Met/FAK cascade, which suggested that LCN2 may be effectively utilized as a novel marker and therapeutic target in hepatocellular carcinoma (HCC) [21]. What's more, the urinary neutrophil gelatinase-associated lipocalin (NGAL) may be a sensitive biomarker of acute kidney injury (AKI) in serious complication after liver transplantation [22]. In this study, we found that LCN2 was up-regulation in ERS L-02 cells and down-regulation when we knockdown CHOP, suggested that it had an intimate connection with ERS [23].
The family of lysosome-associated membrane proteins (LAMPs) included three members, LAMP1, LAMP2 and LAMP3. The function of LAMP1 and LAMP2 is mainly to maintain the stability of the lysosomal membrane, while LAMP3 is closely related to all kinds of pathological processes, such as ovarian cancer [24], breast cancer [25], oral squamous cell carcinoma[26], uterine cervical cancer [27] et al.
Several studies have reported that LAMP3 could be induced up-regulated by PERK-elF2α-ATF4 signaling pathways and promote the invasion and metastasis of tumors [28]. In patients with esophageal squamous cell carcinoma, the high expression of LAMP3 may serve as a novel biomarker of poor prognosis [29]. It is also a downstream target gene of tumor suppressor TP53 [30]. In addition, The coding for LAMP3 is considered to be a risk factor for Parkinson's disease (PD) [31]. CHOP is a downstream target gene of PERK-elF2α-ATF4 signaling pathways. The results of our dates showed that LAMP3 could be regulated by CHOP. The mechanism needs further studies.
FAM129A, also known as Niban, is highly expressed in several cancer cells, and it is closely related to changes in mitochondrial function during preneoplastic and neoplastic processes [32]. Previous research has shown that FAM129A mRNA and protein were induced up-regulated by tunicamycin, which was in conformance with our results, and it could modulate cell death signaling by regulating translation, such as the phosphorylation of eIF2α and S6 subunit kinase (S6K) 1/4E-binding protein (4E-BP) 1 [33]. It could also promote the interaction of MDM2-p53 and subsequent p53 degradation, thereby providing an antiapoptotic effect [34]. What's more, overexpression of zinc-nger protein 777 (ZNF777) inhibits proliferation at low cell density through down-regulation of FAM129A [35]. There are only 32 literature reports on this factor at present. Our study rst identi ed a regulatory mechanism between FAM129A and chop, which provided a new ideas to explore this factor.
CLGN, a homologue of endoplasmic reticulum (ER) residing lectin chaperone calnexin, is required for the generation of normal spermatozoa [36]. Previous studies have shown that CLGN was transcriptionally regulated by histone deacetylase (HDAC) and the cytosine-phosphate-guanine (CpG) methyltransferase [37]. In APAs, CLGN served as an ER-associated gene, which was highly expressed and got involved in aldosterone production by translational regulation of CYP11B2 [38]. So far, almost no literature has reported the relationship between CLGN and the liver. In this study, we found that CLGN had a close relationship with CHOP in L-02 cells. The further regulative mechanism needs a more in-depth research.
IFI27, a highly inducible proteins, has been suggested to be a putative cell proliferation marker, IFI27 knockdown resulted in S-phase arrest that was found to be associated with increased Tyrosine-15 (Tyr15) phosphorylation of cyclin-dependent kinase 1 (CDK1), reduced cell division cycle 25B (CDC25B) and reduced formation of cyclin A/CDK1 complex [39]. It is also supposed to be biological markers of variety of diseases. In respiratory infection, it could be an immune biomarker to discriminate in uenza and bacterial infections in patients [40]. It could serve as a noninvasive diagnostic marker for chronic glomerulon-ephritis using peripheral blood [41]. It was also supposed to be a novel marker of epithelial stromal tumor [42]. Our research found for the rst time that IFI27 is expressed in l-02 cells. In addition, it may be a stress-induced genes which lies downstream of CHOP.
TARP is the rst mitochondrial membrane protein found in human prostate [43]. Several studies have found that TARP could severe as a new target for immunotherapy [44]. It could act as a molecular prognostic marker since it is highly expressed in prostate cancer, breast cancer, endometrial carcinomas and so on [45]. Surprisingly, we found that TARP may play a role in liver injury caused by ER-stress in this experiment, and its regulatory mechanism of this process may be related to CHOP.
There are almost no reports about KLHDC7B and C11orf86. In this study, the up-regulated of CHOP would cause the increase of KLHDC7B and the decrease of C11orf86, and they both had the opposite changes when CHOP was knockdown. Therefore, we predict that they may be regulated by CHOP, but this conclusion remains to be further studied. Exploring the upstream and downstream genes of CHOP in ERstress will help us better understand the regulatory mechanism of ER-stress and provide a new perspective and strategy for the prevention and treatment of diseases related to liver injury.

Conclusion
We totally identi ed 633 DEGs between ERS L-02 cells and normal L-02 cells, 131 DEGs between shRNA-CHOP and shRNA-NC in ERS L-02 cells. GO and KEGG enrichment were used to analysis the potential functions of dysregulated genes. We found 9 genes were both changed between ERS L-02 cells and normal L-02 cells and between shRNA-CHOP and shRNA-NC in ERS L-02 cells. They were DDIT3, CLGN, LCN2, LAMP3, KLHDC7B, FAM129A, IFI27, TARP and C11orf86. Their expression levels were also detected through qRT-PCR. At present, the relationship between these DEGs and DDIT3 was rarely reported and in our future work, the biological functions of these DEGs will be further researches. Our ndings might provide a foundation for the intense research of biological functions CHOP. In addition, according to these genes may provide new intervention targets for the treatment of liver diseases, and contribute to new design ideas for molecular targeted drugs used to treat liver injury.

Con icts of interest
The authors declare that they have no con icts of interest concerning this article.    General analyses of DEGs in model vs control, EJ vs model groups. Note:X axis means number of DEGs (the number is presented by its square root value). Y axis represents GO terms. All GO terms are grouped in to three ontologies: blue is for biological process, green is for cellular component and red is for molecular function. The GO Terms enrichment of DEGs between shRNA-CHOP and shRNA-NC in ERS L-02 cells. Note:X axis means number of DEGs (the number is presented by its square root value). Y axis represents GO terms.
All GO terms are grouped in to three ontologies: blue is for biological process, green is for cellular component and red is for molecular function. Relative mRNA levels of representative DEGs. Note: A: control group B: model group C: sh-CHOP group D: sh-NC group Relative mRNA levels of representative DEGs (DDIT3, CLGN, LCN2, LAMP3, KLHDC7B, FAM129A, C11orf86, IFI27, TARP) were quanti ed using q-PCR. Compared with control group:*p < 0.05, **p < 0.01. Compared with model group: ##p < 0.01.