ERGIC3, Which Is Regulated by Mir-203A, Is a Potential Biomarker for Non-Small Cell Lung Cancer

ERGIC3, which is regulated by miR-203a, is a potential biomarker for non-small cell lung cancer

Qing-Hai Lin1, Kui-Dong Zhang1, He-Xian Duan1, Wan-Li Wei2, Yi Cao1*

1Laboratory of Molecular and Experimental Pathology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China

2Department of Pathology, Third Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China

*Address for correspondence:

Prof. Dr. Yi Cao, Laboratory of Molecular and Experimental Pathology, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan 650223, China. E-mail:

Abstract

In the previous study, through screening libraries of differentially expressed genes, we found that ERGIC3 was a novel lung cancer-related gene. In this study,we developed a new murine monoclonal antibody (mAb)against ERGIC3. This avid antibody (6-C4) is well suited for immunohistochemistry, immunoblotting, and solid-phase immunoassays. Furthermore, we systematically investigated expressions of ERGIC3 ina broad variety of normal human tissues and various types of tumors by immunohistochemistry. In normal human tissues, 6-C4 reacted only some epithelial cells such as hepatocytes, gastrointestinalepithelium, ducts and acini of pancreas, proximal and distal tubules of kidney, mammaryepithelial cells etc, but the most normal human tissues were not stained.Moreover, almost all carcinomas that originate from epithelial cells, were positive for 6-C4, whileall sarcomas were negative. Notably, 6-C4 stained strongly NSCLC cells, but it did not react with normal lung tissues. The ERGIC3mAb might be used in histopathological diagnosis and in the cytopathological test for early detection of NSCLC. Additionally, we also studied mechanisms of ERGIC3regulationin vitro and in vivoby means of bioinformatics analysis, miRNA expression profiling, miRNA transfectionetc, and found miR-203adown-regulation induced ERGIC3 over-expression in NSCLC cells.

Key words:non-small cell lung cancer,ERGIC3, biomarker, monoclonal antibody, miR-203a.

Introduction

Cancers have become a major cause of morbidity and mortality around the world, leadingto approximately 14 million new cases and 8 million cancer-related deaths in 2012 worldwide.(1) Among all, lung cancer that was estimated for 13% of all cancers and 20% of all cancer deaths, is the leading cause of cancer-related deaths. (2) Since much diversity in histological and biological characteristics, lung cancer could be divided into small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), and the latter which accounts more than 80% of all lung cancer rates, including adenocarcinoma (AC), squamous cell carcinoma (SCC) and large-cell carcinoma. (3)The overall 5 years survival rate for lung cancer has merely improved from 12% to 16% in the past 3 decades, largely due to that 80% of patients have been diagnosed with metastatic disease and more than half of patients have distant metastases. (4)Sputum cytology and detectionof several blood biomarkers such as circulating DNA and RNA, exosomal microRNA, circulating tumor cells and various lung cancer specific antigens have received encouraging results. (5)However, no faultless biomarkers for the early detection of lung cancer have been approved sofar.Though some biomarkers like carcinoembryonic antigen and cytokeratin 19 fragment antigen 21–1 have been used for screening lung cancer, specificity and sensitivity were not satisfied.(6,7)Therefore, it makes a lot of sense to find new tumor biomarkers and develop detection methods. (8)In the previous study, we used NSCLC and normal lung tissues to construct libraries of differentially expressed genes by the suppression subtractive hybridization.Among these differentially expressed genes, we found that ERGIC3 (endoplasmic reticulum-Golgi intermediate compartment protein 3) was strongly over-expressed in NSCLCs and the over-expressionpromoted the proliferation and migration of NSCLCcells (9).

ERGIC3, also labeled as Erv46 and ERp43, is located in the cis face of the Golgi apparatus and vesicular tubular structures between the transitional endoplasmic reticulum(ER) and cis-Golgi. (10)ERGIC3had significant effect upon cell growth and ER stress-induced cell death in HEK-293 cell line. (11)Moreover,ERGIC3 was involved in the cellular growth, migration, and the invasion as well as metastasis in hepatocellular carcinomas (HCCs).(12)Considering the high sensitivity and specificity of ERGIC3 expression in lung cancer as well as its roles in the development and progression of cancers,we thought that ERGIC3 may be a potential biomarker of NSCLC. (13)In the study, we developed a new murine monoclonal antibody (mAb)against ERGIC3. The avid antibody is well suited for immunohistochemistry, immunoblotting, and ELISA. Furthermore, we systematicallyinvestigated expressions of ERGIC3 ina broad variety of normal human tissues and various types of human tumors.Additionally, we also studied mechanisms of ERGIC3 regulation and found the ERGIC3 over-expressionresulting from the down-regulation of microRNA-203 (miR-203a)in NSCLCs.

Materials and Methods

Results

A new mAb toERGIC3 was developed

After the cell fusion, we obtained 15 positive clones including 6 strongly positive clones (Fig. S1A). A monoclonal hybridoma was established from 06-C4 after 3 rounds sub-cloning by limiting dilution(Fig. S1B). The mAb secreted by the monoclonal hybridoma was named as "6-C4". The isotype of the mAb 6-C4was IgG2b (, kappa light chain).

6-C4 reacted with the ERGIC3 peptide and the native protein extracted from cultured NSCLC cells,but did not reacted withBSA,as well as plasma, saliva and urine samplesfrom 3 normal adult peopleby ELISA analysis (Fig. 1B). Furthermore, a clear single band was detected at about 50KD by6-C4throughwestern-blot analysis using the native protein, which was consistent with the preliminary result usingthe mouse serums ofimmunized mice(Fig. 1C). The positiveimmunofluorescence staining of6-C4 was localized around the Golgi apparatus and ER(Fig. 1D), same asourprevious result using the anti-ERGIC3 serum purchased from Abcam (Cambridge, UK).(9)Inimmunohistochemistry, NSCLC and HCC tissues were strongly stained by 6-C4 like theprevious studies(Fig. S1D).(9, 12)Theseresults indicated that 6-C4 recognized specifically ERGIC3 protein.

The expression of ERGIC3 was determined in normal adult human tissues

There are few studies on ERGIC3 expression in normal human tissues. Therefore, we examinedexpressions of ERGIC3in abroad variety of humannormaltissuesusing 6-C4. The results are showed in Table 1 and Fig. 2. Mostof normal tissues were not stained by 6-C4. However, the positive staining was observed in the cytoplasm of some epithelial cells.We would like to emphasize that all non-malignant lung tissues were negative for 6-C4 staining.

The expression of ERGIC3 was determinedin varioustumortissues

Expression of ERGIC3was examinedin 15 types of human tumorsby immunohistochemistry using 6-C4. The results are demonstrated in Table 2 and Fig. 3. ERGIC3 was strongly expressed in all carcinomas originating from epithelial cells.In contrast, all sarcomas were negative for 6-C4 in this study.

We also examined expression of ERGIC3protein incultured cells by western-blotusing 6-C4.All NSCLC lines expressed higher levels of ERGIC3 than 16HBE (Fig. 4A and 4B). The tendency of ERGIC3 protein expression was consistent with that ofERGIC3 mRNA(Fig. 4D).

The ERGIC3-related miRNAswere identified in NSCLC cells

Many cancer-related genes are regulated bymiRNAs.(18)Throughbioinformatics analysis we predicted 398miRNAsthat may bind to the3′-untranslated region (3′-UTR) of ERGIC3.In addition, we found 87 consensus differentially expressedmiRNAthrough comparing miRNA profilesof NSCLC cells (A549, 801D, and EPLC-32M1) with that of 16HBE.Integrating the candidate miRNAs predicted by bioinformatics analysisand differentially expressed miRNAsdetectedby the miRNA expression profiling, both miR-140-3p and miR-203a may target ERGIC3 and were differentially expressed in NSCLC cells. Therefore, the two miRNAswere selectedfor further research.Since miR-490-3p was abnormally expressed in HCC and participated in ERGIC3 regulation,(12)miR-490-3p was also investigated in the study.

Q-RT-PCR analysis was performed to validate expressions of the three candidate miRNAs (miR-140-3p, miR-203a and miR-490-3p)in cultured cell lines. The expression of miR-140-3p was higher in NSCLC cellscompared with 16HBE (Fig. S2A), while NSCLC cellsexpressed much lower levels of miR-203athan16HBE (Fig. 4C). Theq-RT-PCR results of miR-140-3p and miR-203awere consistent with that of the miRNA expression profiling.No significantdifference of the miR-490-3p expression was found between NSCLC cells and 16HBE (Fig. S2C). Interestingly, levels of miR-203awere negativelycorrelated with ERGIC3 expression (Fig. 4C; Fig. 4D).

The values of ERGIC3 were negatively correlated with miR-203a in NSCLCs

MiR-203a was down-expressed in NSCLC tissues compared with their adjacent nonmalignant tissues(Fig. 4F), whileERGIC3 was up-regulated in NSCLC tissues(Fig. 4E). The expressions of ERGIC3 and miR-203awere negatively correlated in patient tissueslike the findings in cultured cells.

The expression of ERGIC3 was regulated by miR-203ain NSCLC cells

MiRNA could down-regulated gene expression via either translation inhibition or mRNA degradation through binding 3’-UTR of mRNA(18). In bioinformatic analysis, the sequence alignment of the miR-203a binding site was exactly conservative among primates, even across mammals (Fig. 5A).To test whether ERGIC3 over-expression was associated with miR-203adown-expression, we performed interference experiments in vitro. Expression ofmiR-203a was increasedbymiR-203amimictreatment(Fig. 5B).Importantly, the expression of ERGIC3 was significantly decreased at mRNA and protein levels after miR-203a mimic treatmentcompared with the control(Fig. 5C, D, E).However,ERGIC3 expression was increasedat mRNA and protein levels after miR-203a inhibitortreatment(Fig. 5F, G, H).

We also performed interference experiments to investigate relationships between ERGIC3 as well as miR-140-3p and miR-490-3p. Unfortunately, ERGIC3 expressions were not significantly changed aftermiR-140-3p and miR-490-3pmimicstreatments(Fig. S2B and Fig. S2D).

The expression of miR-203aaffected the cellularmorphologyandproliferation

Considering the novel discovery of miR-203ain NSCLC, we further investigated possible functions of miR-203a. XLA-07 (with the low level of endogenous miR-203a) and 16HBE (with the high level of endogenous miR-203a) were treated with miR-203a mimic or inhibitor, respectively. The miR-203a mimic treatment led to an obviously morphological change (from the spindle-like appearance to the round shape,Fig. 6A) and significantly reduced the cell proliferationin XLA-07 cells(Fig. 6C). Notably, the changes that were induced by miR-203a mimic treatment, were similar as thealterations caused by the ERGIC3 RNA interferencein NSCLC cells.(9)Interestingly, significant alterations in the cellular morphology and proliferation were not observed in 16HBE cells after miR-203a inhibitortreatment (Fig. 6A, B).

Discussion

Cancer is a disease caused by the accumulation of genetic and epigenetic alterations. Studying expressionsand functions of cancer-related genes is crucial to understanding carcinogenesis and identifying novel biomarkers for cancer. In previous study, we found that ERGIC3 was a novel lung cancer-related gene, and over-expressed ERGIC3 promoted the cellular proliferation and migration. (9)ERGIC3could be coupled with Erv41p to form an integral membrane protein complex as the components of COPII vesicles playing an important role in protein transportation through the secretory pathway. (19-21) Abnormal expression of ERGIC3 could affect cell growth and ER stress-induced cell death, and contributed to epithelial to mesenchymal transition (EMT).(11, 12)Thus, ERGIC3 maybe a potential biomarker for cancer. In the study, we developed a new mAb (6-C4) against ERGIC3. The avid antibody is well suited for immunohistochemistry, immunoblotting, and ELISA. To our knowledge, 6-C4 is the first murine mAb towards ERGIC3.6-C4 could be used for studying the expressionand functions of ERGIC3.

Furthermore, we systematicallystudied expressions of ERGIC3 in normal human tissues and tumors by immunohistochemistry using 6-C4. The staining was not observed in the most normal human tissues. However, 6-C4 reacted with some epithelial cells such as hepatocytes, gastrointestinalepithelium, ducts and acini of pancreas, proximal and distal tubules of kidney, mammaryepithelial cells etc.The expression of ERGIC3 intheseepithelial cells may be closely related with its functions. ERGIC3participatesto form vesiclesof the protein transport and secretion.(20, 21)In fact, these epithelial cells stained by the ERGIC3 mAbare very active in protein synthesisand secretion.Interestingly, we found that almost all carcinomas that originate from epithelial cells, were positively stained by6-C4, but all sarcomas were negative for 6-C4. Therefore, we supposed that ERGIC3 may be a useful biomarker for distinguishing between carcinomas and sarcomasin histopathological diagnosis. However, a larger number of cases must be investigated to permit final conclusions. More importantly, we noticed that NSCLCcells were strongly stained by 6-C4, but all cells (including bronchial epithelial cells and alveolar cells) in nonmalignant lung tissues were negative for 6-C4. These results weresimilar as our previous observation using the anti-ERGIC3 serum purchased from Abcam. (9)It is tempting to speculate that the ERGIC3mAb might be used in the sputum test for early detection of lung cancer. The early diagnosis andtreatmenthas a decisiveinfluence on theprognosis of lung cancer. Thus, it is of great value to develop new methods for early detection of lung cancer. Thiswork has beencarried outin our laboratory.

ERGIC3 over-expression has been observed in NSCLCs. However, molecular mechanisms underlying ERGIC3 alteration remain elusive. MiRNAs are a family of small non-coding RNAs, approximately 21 to 25 nucleotides in length,(22, 23)that target mRNAs at complementary sites in the 3′-UTR to regulate gene expression at the post-transcriptional level. (24) In the study, we identified that miR-203a is a regulator of ERGIC3 in NSCLCs. MiR-203a(also named as miR-203) thatwas firstly assigned as skin-related miRNA, (25, 26)was involved in the development and progression of cancers by targeting different genes.(27-30)Here we found that the reduced expression of miR-203a was one of mechanisms underlying ERGIC3 over-expression in NSCLCs. Our finding was supported by the following evidence:1) MiR-203acan bind the 3’-UTR of ERGIC3 mRNA through bioinformatic prediction; 2) ERGIC3 expressions were negatively correlated with miR-203a in cultured cells andtissues obtained from NSCLC patients; 3) ERGIC3 was down-regulated at mRNA and protein levels by the elevated miR-203a expression inNSCLC cells; 4) ERGIC3 was up-regulated at mRNA and protein levels by the reduced miR-203a expression inhuman bronchial epithelial cells; 5)The forced expression of miR-203a could lead to changes of cellular morphologyandproliferation in cultured NSCLC cells, which were of like the effects induced by the decreased expression of ERGIC3 (9).In a previous study,unlike most miRNA-mRNA interactions, miR-490-3p increased ERGIC3 mRNA and protein levels by binding the 3’-UTR of ERGIC3 in HCC cells.(12) However, we did not observe that miR-490-3p significantly affectedERGIC3 expression in NSCLC cells. It is seem that mechanisms of ERGIC3regulationare different in various cancers.

In conclusion, we developed a new murine mAb against ERGIC3 (6-C4). The avid antibody is well suited for immunohistochemistry, immunoblotting, and ELISA. 6-C4 stained strongly NSCLC cells, but did not react with normal lung tissues. The ERGIC3mAb might be used in the histopathological diagnosisand cytopathological test for early detection of lung cancer. Additionally, we found that miR-203adown-regulation induced ERGIC3 over-expression in NSCLC cells.

Figure legends

Figure 1. Generation and identification of the mAb to ERGIC3. A,the tertiary structure of ERGIC3 protein and the sequence of the synthetic peptide used as antigen. B, the reactivity of 6-C4 in solid-phase immunoassays. C, 6-C4 recognized protein extracted from cultured cellsat band about 50KD inimmunoblot. D, positive staining of 6-C4around Golgi apparatus and ER of NSCLC cells in immunofluorescence. SP2/0 cell supernatant used as thenegative control.

Figure 2. Immunohistochemical analysis of ERGIC3 innormal human tissues using 6-C4. A, brain. B, cerebellum. C, heart. D, lung. E, gallbladder. F, esophagus. G, urinary bladder. H, testis. I, prostate. J, uterus. K, thyroid gland. L, spleen. M, thymus. N, muscle. O, liver. P, stomach. Q, intestine. R, colon. S, kidney. T,breast.

Figure 3. Immunohistochemical analysis of ERGIC3 invarious tumor tissues using 6-C4. A, NSCLC. B, pancreatic carcinoma. C, HCC. D, esophagus carcinoma. E, gastric carcinoma. F, colon carcinoma. G, renal cell carcinoma. H, bladder carcinoma. I, mammarycarcinoma. J, cervical carcinoma. K, prostate carcinoma. L, thyroid carcinoma. M, osteosarcoma. N, chondrosarcoma. O,fibrosarcoma.

Figure 4.Expressions of ERGIC3 and miR-203a in cultured cells and tissues. Expression of ERGIC3 protein (A and B)in cultured cells by western blot analysis. Expressionsof miR-203a(C) and ERGIC3 mRNA (D) in cultured cells by q-RT-PCR. Expressionsof ERGIC3 mRNA(E) and miR-203a(F) in 7 pairs of NSCLC (LCT) and their adjacent lung tissues (LNT)by q-RT-PCR.

Figure 5. The expression of ERGIC3 was regulated by miR-203a.A, the potential binding site of miR-203aatthe 3’-UTR of ERGIC3 mRNA predicted by RNAhybrid (v2.2)and comparison of the sequence alignment among several species. B, the level of miR-203a was significantly elevated in EPLC-32M1 after miR-203a mimic treatment. C-E,ERGIC3 expression was reducedat mRNA (C)and protein (D-E) levels in NSCLC cells by miR-203a mimic treatment. F-H, ERGIC3 expressions were increased at mRNA (F) and protein (G and H) levels in16HBE by miR-203a inhibitor treatment.Student’s t-test, *: p<0.05; **: p<0.01.ncRNA:negative control miRNA.

Figure 6.MiR-203aexpression affected the cellular morphologyand proliferation. The changesin the morphology (A, top) and proliferation (B) could be seen in XLA-07 after miR-203a mimic treatment. However, miR-203a inhibitor treatment did not significantly affectthe cellular morphology (A, bottom) and proliferation(C) in 16HBE.Student’s t-test, p>0.05. Control:negative control miRNA.

A list of Supporting Information

Table S1. The primers used in this study.

Fig.S1. The establishment and identification of monoclonal antibodies (mAbs) to ERGIC3.

Fig. S2.Effects of miR-140-3p and miR-490-3p on the ERGIC3 expressionin NSCLC cells.

Supplementary data

Table S1.The primers used in this study.

Fig.S1. The establishment and identification of monoclonal antibodies (mAbs) to ERGIC3. ELISA analysis demonstrated positive hybridomas of anti-ERGIC3 mAbs after cell fusion (A) and the positive rate of about 95% after rounds of sub-cloning by limiting dilution (B). The serums of mice immunized with the ERGIC3 peptiderecognized protein extracted from cultured cells at about 50KD in immunoblot (C). 6-C4 stained strongly non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC) tissues in immunohistochemistry, and SP2/0 supernatant was used as the negative control (D).

Fig. S2.Effects of miR-140-3p and miR-490-3p on the ERGIC3 expressionin NSCLC cells. A, miR-140-3p was highly expressed in cultured NSCLC cells. B, miR-140-3p mimic treatment did not significantly affect ERGIC3 expression in 16HBE (B). C, miR-490-3p was expressed with varying degreesin NSCLC cells.D, miR-490-3p mimic treatment tended to reduce ERGIC3 expression in A549,but there was not significant difference betweenmiR-490-3p mimictreatment and the negative control (Student’s t-test, p>0.05). ncRNA:negative control miRNA.

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