Expression of Ciz1b in patients with non small cell lung cancer

Primary lung cancer is one of the most common malignant diseases. The expression of Ciz1b appears to be limited to tumor cells, suggesting that this variant can be used as a marker for the cancer. This study is to evaluate the expression and diagnostic role of Ciz1b in lung cancer patients.

EXPRESSION OF Ciz1b IN PATIENTS WITH

NON-SMALL CELL LUNG CANCER

Ho Van Son 1,2 ; Ngo Tat Trung 1,3 ; Nguyen Linh Toan 1

SUMMARY

Introduction: Primary lung cancer is one of the most common malignant diseases The expression

of Ciz1b appears to be limited to tumor cells, suggesting that this variant can be used as a marker for the cancer This study is to evaluate the expression and diagnostic role of Ciz1b in lung cancer patients Subjects and methods: 100 patients with non-small cell lung cancer and

51 healthy controls were recruited for this study Relative expression of Ciz1b mRNA was determined by realtime PCR Results: Increased Ci1zb mRNA expression was observed

in patients with non-small cell lung cancer compared to healthy individuals (p = 0.008) The expression of Ciz1b mRNA was negatively correlated with the ALT enzyme levels (Spearman's r = -0.22, p = 0.045) and with the AST levels (Spearman' r = -0.2, p = 0.068) The level of mRNA expression of the Ci1zb gene was significantly associated with tumor cell differentiation in patients with non-small cell lung cancer Ciz1b mRNA expression in serum was capable of distinguishing lung cancer patients from healthy control (AUC = 0.602, p = 0.05) Conclusion: The expression of Ciz1b is regulated by the occurrence of lung cancer and Ci1zb plays an important role in lung cancer development, as well as has the potential as a biomarker for diagnosis and prognosis of lung cancer

* Keywords: Lung cancer; Non-small-cell lung carcinoma; Ciz1b; mRNA expression

INTRODUCTION

Primary lung cancer is one of the most

common malignant diseases and also is

one of the leading causes of death from

cancers Lung cancer accounts for about

12% of all types of cancers and 18% of

cancer-related deaths [1, 2] Of these,

non-small-cell lung carcinoma (NSCLC)

accounts for more than 80% of lung

cancer In Vietnam, lung cancer has

increased rapidly due to smoking, air

pollution, dust emissions and industrial

waste According to the Ministry of Health,

there are about 20,000 to 30,000 new

cases of lung cancer annually and about 90% of primary lung cancer patients die in the first five year [1]

Ciz1 (Cip1-interacting zinc finger protein) enhances the activation of DNA replication

in mammals by interacting with cyclin-dependent E and A protein kinases

It interacts directly with E and A cyclins, CDK2, and p21 cyclin-dependent kinase inhibitors It also plays an important role in

DNA replication by regulating the expression

of genes, including cyclin D, which influence cell proliferation [3] Normally, Ciz1 is attached

to the salt- and nuclease-resistant protein

1 175 Military Hospital

2 Vietnam Military Medical University

3 108 Military Central Hospital

Corresponding author: Nguyen Linh Toan (toanntl@gmail.com)

Date received: 02/08/2018

Date accepted: 20/09/2018

component of the nucleus called a

"nuclear matrix" Ciz1 is in the spatial

structure of DNA replication Some recent

studies described a Ciz1 variant “missing

the C-terminal region associated with the

nuclear matrix attachment and the

expression of this stable variant appears

to be limited to tumor cells, suggesting

that this variant can be used as a marker

for cancer” Recent studies showed that

determining this variant of Cip1 can be

used for early diagnosis of lung cancer [3]

A previous study measured the

expression of Ciz1 using Western blot

and found that Ciz1 was present in the

lung tumor, but was absent in the nearby

tissues The study also found that the

Ciz1 variant accurately identified patients

with stage I lung cancer [4] The data

showed that the Ciz1 variant was an

important candidate for a specific marker

of cancer, which can detect early stage

lung cancer in the risky groups [5]

However, there are no reports of the

expression of this gene at diagnosis or

prognosis for NSCLC in Vietnam This study

is to Evaluate the expression and diagnostic

role of Ciz1 in lung cancer patients

SUBJECTS AND METHODS

1 Subjects

The study was conducted in a

cross-sectional model, described in combination

with clinical examination and laboratory

analysis 100 patients with NSCLC were

diagnosed by histopathology or cytology

Patients were grouped according to the

stages of the disease according to TNM

classification criteria [6] The control

group consisted of 51 healthy individuals who had the corresponding age as those with lung cancer The group of healthy people was examined for health and were confirmed to be healthy and agreed to participate in the study

2 Methods

* Preclinical and clinical examination:

All patients and the control group were examined clinically and laboratory tests such as hematology, biochemistry, imaging (chest X-ray, chest CT) were performed The overall condition of the participants was assessed using KPI (KPI: Karnofsky Performance Index from 0 - 100 points) [7] Patients were classified according to the classification criteria [6] The procedure for sampling and storing samples was conducted at 175 Military Hospital 5 mL

of blood from each participant, both the patients and healthy people was stored at -20°C until use

* Expression of Ciz1b mRNA:

The expression level of Ciz1b in the serum of patients with lung cancer and control group was determined by realtime PCR Total RNA was extracted from plasma samples of patients and healthy individuals using an RNA extraction kit following the manufacturer's instructions cDNA don’t need this were synthesized from RNA using the Thermo Scientific cDNA kit You have already said this at the beginning of the paragraph Each realtime PCR reactions include Master Mix SYBR Maxima 2X, Primers 5 pm (Ciz1b and ABL), Water and cDNA template The total volume of each reaction is 12 μL

The thermal cycle for the realtime PCR

reaction for ABL and Ciz1b was 50°C for

2 mins, 95°C for 10 mins, 95°C for 15s,

and repeat for 45 cycles

* Statistical analysis:

Data were analyzer using SPSS V.19

The diference between groups was analyzed

using Mann-Whitnay U test for

non-parameter variables Sperman correlation

coefficient (RS) was used to investigate the

relationship between continuous variables

p < 0.05 was considered statistical

significance

RESULTS

1 Baseline characteristics of the

study subjects

Lung cancer patients had an average

age of 60 years, while the control group

had an average age of 53.88 years

No significant difference in age between

patients with lung cancer and the healthy

controls was observed (p = 0.158)

In terms of gender distribution among the

patients, 73 males and 27 females There

were 35 men, and 16 women in the

control group No significant difference in

gender distribution between the two groups

was found (p = 0.225)

2 Clinical characteristics of lung

cancer patients

On admission, the common clinical

symptoms were coughing, chest pain,

difficulty breathing and weight loss

49 patients (49%) had coughing, 43 patients

(43%) had chest pain, 12 patients (12%)

had shortness of breath, 7 patients (7%)

had weight loss and 24 patients (24%)

had other clinical symptoms Of the

100 patients with lung cancer, 34 had tumors in the upper lobes of the right lung, 26 had lobular tumors in the left lung, 18 had lobular tumors in the right lung, 13 had tumors in the lower lobes of the left lung and 9 patients with tumors in the right lobe of the lung

According to the TNM classification system [6], patients were classified into stages IA, IIA, IIIA, IIIB and IV The majority

of patients were in stage IV (76%), 15% in stage IIIB, 5% of patients was in stage IIA, and only 2% of patients were in stages

IA and IIIA In terms of tumor cell differentiation, 56% of patients had grade 2, 35% of patients had grade 3, and 9% of patients had grade 1 These results indicated that the majority of patients were at the late stages

General ultrasound was performed for 82 patients and we found 38 patients (46%) had abnormal images Bone morphogenesis was performed for 63 patients, 34 of whom accounted for 54%

of abnormal images Radiography revealed that 49/51 patients (96.1%) had abnormal images CT images showed 82/93 patients (88%) with tumors, 18/93 patients (19%) with pleural effusion, and 8/93 patients (8.6%) with thickened lung membranes PET-CT was performed for 26 patients and 100% of patients had abnormal images, of which 16/26 patients (61.5%) had tumors, 7/26 patients (26.92%) had pulmonary effusion, 13/26 patients (50%) had malignant lesions, and 17/26 patients (65.4%) had lymph nodes

3 Expression of Ciz1b in patients with lung cancer and healthy individuals

Figure 1: Expression levels of Cizb1 mRNA in patients and controls

The relative expression of the Ciz1b gene in the plasma of patients with lung and healthy cancers is calculated by the ΔCt method with the expression of ALB gene as the reference The analysis showed that the expression of the Ciz1b gene in patients with lung cancer (10.02 ± 4.83) was significantly higher than that in the controls (7.75 ± 2.63) (p = 0.008)

4 Correlation between Ciz1b expression and clinical parameters

Table 1: Association between Ciz1b expression and differentiation of tumor

(n = 63)

Differentiation

p

Ciz1b relative

Tumor differentiation reflects the degree of malignancy of cancer cells To assess the association of Ciz1b expression with tumor development, we compared the expression level of Ciz1b with degrees of differentiation including grade 1, grade 2, and grade 3 The results showed that Ciz1b mRNA expression was the lowest in the group with grade 1, increased in the group with grade 2 and the highest in the group with grade 3 (p < 0.05) These results suggested that the expression levels of Ciz1b increased with the degree of differentiation of the tumor and was closely related to the development

and differentiation of lung cancer cells

Figure 2: Correlation between Ciz1b mRNA expression and liver enzyme levels

We also analyzed the association of Ciz1b expression with other clinical parameters Results showed that Ciz1b mRNA expression was negatively correlated with ALT enzyme levels (Spearman's r = -0.22, p = 0.045) and with the AST levels (Spearman' r = -0.2, p = 0.068) However, there was no association between Ciz1b mRNA levels with analyzed clinical parameters (p > 0.05) In addition, Ciz1b mRNA expression was not correlated with the level of hemoglobin, hematocrit, red blood cells, lymphocytes, platelet counts and monocytes (p > 0.05) Ciz1b mRNA expression was also not correlated with the ion index tests such as Na+, K+ and Cl- concentrations, kidney function tests such as urea and creatinine, and with cancer markers such as CEA and CyFra 21.1 (p > 0.05)

5 Potential diagnostic function of Ciz1b expression for lung cancer

Figure 3: Diagnostic potential of Ciz1b mRNA expression in lung cancer

Ciz1b mRNA expression was significantly

different between lung cancer patients

and controls, and was associated with the

differentiation of lung cancer tumor

Therefore, Ciz1b mRNA expression may

have a potential for screening and

prognosis of lung cancer We analyzed

the effectiveness of Ciz1b expression for

identification of lung cancer from the

control group The results showed that

Ciz1b mRNA expression in serum was

capable of distinguishing lung cancer

patients from healthy control However,

the diagnostic efficacy was not high, and

the AUC was only 0.602 (p = 0.05)

DISCUSSION

Primary lung cancer is one of the most

common malignant diseases and also is

one of the leading causes of death from

cancers Ciz1 plays an important role in

triggering DNA replication by regulating

the function of cyclin E and A-dependent

protein kinases Ciz1 also interacts

directly with cyclins E, cyclins A, CDK2

(cyclin-dependent kinase 2) and with the

cyclin-dependent p21 kinase inhibitor to

regulate DNA replication and the expression

of genes involved in the cell cycle

including cyclin D [8] However, only a few

studies have addressed the role of the

Ciz1b gene in lung cancer

The level of Ciz1 expression has been

shown in early cell differentiation Some

variants of Ciz1 that are involved in

cancer development are deficient in exon

4 and the expression of different Ciz1

variants is closely related to the expression

of the cyclin A1 protein This is a vital protein

in the development and differentiation of

cells [9] A previous study showed that the Ciz1b had the potential as a biomarker that was usually found in patients with lung cancer but not in healthy individuals [3] In that study, the authors used the Western blot method for protein detection, which was quite complex and was not optimized to be a method used for diagnostic purposes Therefore, theELISA method of quantifying serum protein levels has been established [3] In our study, specific primers were designed to amplify and quantify the expression of Ciz1b mRNA in the serum by realtime PCR, which provides a rapid and accurate result regarding the expression of Ciz1b mRNA in the serum

Another recent study has shown that Ciz1b was sensitive enough to identify patients with stage I cancer in high-risk groups, including patients with benign pulmonary lung disease, pneumonia, asthma, chronic obstructive pulmonary disease (COPD) and smokers [3] This study indicated that cancer patients had significantly higher levels of Ciz1b compared

to those in controls and the Ciz1b levels also increase with age in patients with NSCLC The study also found that Ciz1b levels in patients with stage I lung cancer were significantly higher compared to smokers, and Ciz1b levels were the highest

in patients with basal cell carcinoma followed by adenocarcinoma, pneumothorax and patients with benign neoplasia In our study, the level of mRNA expression of the Ciz1b gene in the serum of patients with NSCLC was significantly higher than that of controls This result confirms Ciz1b gene expression is affected by the

disease status, suggesting that Ciz1b plays

an important role in the development of

lung cancer The results also suggest that

Ciz1b gene may be used as a biomarker

for diagnosis and prognosis of lung

cancer [5]

The study showed that Ciz1b levels

help diagnose cancer patients with an

accuracy threshold of 98% for patients

with NSCLC and controls (AUC = 0.96)

When comparing patients with NSCLC in

stage I with asymptomatic smokers, or those

with benign pulmonary lymph nodes, the

AUC reached 0.913, and 0.905, respectively

In this study, we also analyzed the

diagnostic potential of Ciz1b for patients

with NSCLC and the results showed that

Ciz1b was capable of detecting patients

with NSCLC (AUC=0.602) Therefore,

determining the presence of Ciz1b in the

serum of high-risk groups is an effective

non-invasive method for diagnosing,

screening and predicting lung cancer

CONCLUSION

Increased Ci1zb mRNA expression in

patients with NSCLC was observed

compared to healthy individuals Ci1zb

mRNA expression levels were associated

with tumor cell differentiation and

correlated with liver enzyme levels Ci1zb

plays an important role in lung cancer

development and has the potential as a

biomarker for diagnosis and prognosis of

lung cancer

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