|Year : 2018 | Volume
| Issue : 4 | Page : 860-863
Expression level of long noncoding RNA H19 in plasma of patients with nonsmall cell lung cancer and its clinical significance
Jianhua Luo1, Qiaoqiao Li1, Junsu Pan1, Longxiang Li1, Liangjie Fang2, Ying Zhang1
1 Department of Respiratory, Taizhou Municipal Hospital, Taizhou, Zhejiang, PR China
2 Department of Respiratory, The First Affiliated Hospital, Zhejiang University, Hangzhou, PR China
|Date of Web Publication||27-Jun-2018|
Department of Respiratory, Taizhou Municipal Hospital, Taizhou, Zhejiang, 318000
Source of Support: None, Conflict of Interest: None
Objective: This study was aimed to explore the expression level of long noncoding RNA H19 in the plasma of patients with nonsmall cell lung cancer (NSCLC) and its clinical significance.
Methods: A total of 66 NSCLC patients (case group) and 31 patients with benign lung disease (control group) admitted from February 2015 to February 2017 were included in this study. Real-time polymerase chain reaction assay was applied to examine the relative expression level of long noncoding RNA H19 in the plasma of the two groups. The relationship between H19 expression and clinical, pathological features was explored. Receiver-operating characteristic (ROC) curve was applied to evaluate the clinical value of plasma H19 as a tumor marker in the auxiliary diagnosis of NSCLC.
Results: The relative expression levels of plasma H19 inpatients from NSCLC group and benign lung disease group were 5.62 ± 2.02 (ΔCt) and 7.74 ± 2.75 (ΔCt), respectively. The NSCLC group presented with significantly higher levels than that of the benign disease group (P < 0.05). According to the median of relative expression level of 5.54, the plasma H19 of NSCLC patients was classified into low expression group ≥5.54 (n = 34) and high expression group <5.52 (n = 32). The relationship between the patients' clinical, pathological features, and the expression level of H19 was analyzed. The expression of H19 was not significantly correlated with the gender, age, clinical staging, tumor diameter, and pathological type of the patients (Pall > 0.05). With the serum H19 as a diagnosis reference, the diagnostic sensitivity of NSCLC was 67.74%, and the specificity was 63.08%. The area under the ROC curve was 0.73, and the diagnostic cutoff value was 6.62.
Conclusion: Plasma level of H19 in NSCLC patients was significantly increased, which could be applied as a serological marker for the auxiliary diagnosis of NSCLC.
Keywords: H19, long noncoding RNA, nonsmall cell lung cancer, plasma
|How to cite this article:|
Luo J, Li Q, Pan J, Li L, Fang L, Zhang Y. Expression level of long noncoding RNA H19 in plasma of patients with nonsmall cell lung cancer and its clinical significance. J Can Res Ther 2018;14:860-3
|How to cite this URL:|
Luo J, Li Q, Pan J, Li L, Fang L, Zhang Y. Expression level of long noncoding RNA H19 in plasma of patients with nonsmall cell lung cancer and its clinical significance. J Can Res Ther [serial online] 2018 [cited 2019 Nov 14];14:860-3. Available from: http://www.cancerjournal.net/text.asp?2018/14/4/860/235096
| > Introduction|| |
Lung cancer was one of the most commonly diagnosed malignant tumors with the highest morbidity and mortality in the world, especially in China., At present, it was lack of large-scale epidemiological data on lung cancer in China. However, regional epidemiological studies recently showed that lung cancer had become a malignant tumor for males in China. Meanwhile, lung cancer contributed to the second morbidity rate and the first mortality rate for females in China. Therefore, lung cancer has become a major public health problem threatening human health. However, the pathogenesis of lung cancer has not yet been fully elucidated, neither the molecular mechanism of the pathogenesis. Thus, the molecular mechanism on lung cancer genesis, development, invasion, and metastasis has been becoming the focus of current research.
In recent years, long noncoding RNA has been confirmed to play an important role in biological functions, including tumor genesis,, development, invasion, and metastasis. The mechanisms that regulated the proliferation, invasion, and metastasis , of tumor cells have been elucidated. Meanwhile, some studies showed that the expression of long noncoding RNA in the peripheral blood of patients with lung cancer was different from that of normal population., This finding revealed that long noncoding RNA might be a serological marker for the auxiliary diagnosis of lung cancer. In this paper, 66 patients with nonsmall cell lung cancer (NSCLC) were included and investigated. Real-time polymerase chain reaction (RT-PCR) assay was applied to examine the expression of long noncoding RNA H19 in the serum of patients. It aimed to explore the relationship between the expression of H19 and the patients' clinical, pathological features and the feasibility of expression to become a serological marker for NSCLC patients.
| > Methods|| |
A total of 66 NSCLC patients (case group) and 31 patients with benign lung disease (control group) admitted from February 2015 to February 2017 in our hospital were included. Informed consent has been obtained from all individuals included in this study. The research related to the application of human tissues has been complied with all the relevant national regulations, institutional policies, and the tenets of the Helsinki Declaration. The study has been approved by our institutional review board or equivalent committee. The inclusion criteria for involved NSCLC patients were as follows: (1) who were confirmed as NSCLC with pathology or cytology; (2) who did not receive any prior treatment, including radiotherapy, chemotherapy, and biotherapy, before collecting peripheral blood; and (3) without other systemic malignancies. Meanwhile, the exclusion criteria were: (1) with small cell lung cancer; (2) who were treated with radiotherapy, chemotherapy, or immunotherapy; (3) who were pregnant and lactating women; (4) with other systematic malignancies; and (5) with pulmonary metastasis. For the control group, no history of malignancy was observed in all patients with benign lung diseases. The patients signed an informed consent before their specimens were obtained. There were a total of 40 males and 26 females in the 66 NSCLC patients. A total of 23 patients were aged ≤60 years old, and 43 patients were >60. In addition, 22 patients were in clinical Stage I/II and 44 patients were in Stage III/IV.
Five milliliters peripheral venous blood was extracted from the patients after undergoing fasting at early morning. After all the blood samples were collected, plasma centrifugation collection and cryopreservation were performed within 1 h. Plasma was initially centrifuged for 10 min at 4500 r/min and 4°C. Then, the supernatant was transferred to a new Eppendorf tube for 10 min and centrifuged at 13000 r/min and 4°C. Subsequently, plasma samples were packaged (400 μL/tube) and kept at 80°C for further extraction of total RNA.
Serum H19 expression detection
Exactly 400 μL of plasma was taken and ablated with ice for later use. Plasma total RNA was extracted strictly according to the instruction of mirVana PARIS Kit and stored at −80°C for later use. Then, 5 μL RNA eluent with fixed volume was taken to perform reverse transcription as cDNA by Prime Script™ RT reagent Kit with gDNA Eraser Kit. Afterward, 2 μL cDNA was applied as the template. SYBR green (Takara Japan) method was adopted to detect the expression of Inc., RNA H19 with ABI7900PCR meter. Each sample was determined in the triplet. The specificity of primer reaction was determined according to dissolution curve. Glyceraldehyde 3-phosphate dehydrogenase was selected as the reference gene.
The SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied to deal with the data. The data were expressed with x¯ ± s and the comparison between groups was performed based on the t-test of the sample mean. The enumeration data was expressed with n (%). The comparison of sample rate was processed by Chi-square tests two-tailed P < 0.05 indicated statistical different.
| > Results|| |
Plasma H19 level
The relative expression levels of the plasma H19 of patients in NSCLC group and benign lung disease group were 5.62 ± 2.02 (ΔCt) and 7.74 ± 2.75 (ΔCt), respectively. Significantly higher levels were observed in the NSCLC group than that of in the benign disease group (P< 0.01) [Figure 1].
|Figure 1: Plasma H19 relative expression in patients with nonsmall cell lung cancer and benign lung disease (**P < 0.01)|
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Correlation between plasma H19 level and patients' clinical, pathological features
According to a median of 5.54 for the relative expression levels, NSCLC patients were classified into low expression group with the plasma H19 ≥5.54 (n = 34) and high expression group with the plasma H19 <5.52 (n = 32). The relationship between the patients' clinical, pathological features, and the expression level of H19 was analyzed. The expression of H19 was not significantly correlated with the gender, age, clinical staging, the diameter of tumor and pathological type of the patients (P > 0.05) [Table 1].
|Table 1: Correlation between plasma H19 level and patients' clinical pathological features|
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Clinical value of plasma H19 for diagnosis of nonsmall cell lung cancer
The plasma H19 was applied as the diagnosis reference. The diagnostic sensitivity for NSCLC was 67.74%, and the specificity was 63.08%. The area under the receiver-operating characteristic (ROC) curve was 0.73, and the diagnostic cutoff value was 6.62 [Figure 2].
|Figure 2: The receiver-operating characteristic curve for plasma H19 in diagnosis of nonsmall cell lung cancer|
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| > Discussion|| |
H19 has been a typical imprint-related long non-coding RNA, with the length of 2.3 kb. Its maternal allelic expression and paternal imprinting represented evolutionary conservation in mammals. Furthermore, H19 has been one of the first identified imprinted genes. H19/Igf2 imprinted genes belonged to a genomic imprinting group located at human chromosome 11P15.5, indicating a highly evolutionary conservation. H19 gene was a maternal imprinted gene, whereas Igf2 gene was a paternal imprinted gene. Both of these genes were 90 kb apart. They were regulated by differentially methylated region at 4 kb in the upstream of H19 gene or imprinting control region. The high abundance of H19 gene was expressed during embryonic development, which was mainly expressed in endoderm- and mesoderm-derived tissues. However, the expression of postnatal H19 was decreased and found only in myocardium and skeletal muscles. The full-length 2.5 kb of H19 gene contains five exons and four introns. The mature H19 generated from H19 gene products was 2.3 kb in full length. Due to the lack of distinct open reading frame, H19 gene was called noncoding RNA. H19RNA molecule can be detected in the cytoplasm and nucleus but mainly existed in the cytoplasm, which played its role in the mode of regulatory RNAs or ribose regulators.
Recent studies found that H19 was abnormally expressed in various tumor tissues. The findings revealed that H19 might be served as a potential biomarker for cancer. However, the expression of H19 in the plasma of tumor patients and its relationship with the expression in tissues has been rarely reported.
Zhang et al. applied RT-PCR to detect the expression of H19 in tumor tissues and plasma of patients with breast cancer. The study showed that the expression of H19 in breast cancer tissues was significantly higher than that of in adjacent tissues, and the level of H19 in the plasma of breast cancer group was significantly higher than that of in benign disease group. The area under the ROC curve of the plasma H19 for the solitary diagnosis of breast cancer was 0.83, and the sensitivity and specificity were 57.7% and 86.4%, respectively. Thus, the diagnostic efficacy for breast cancer was high. Studies showed that highly expressed H19 in plasma might be a potential biomarker for the diagnosis of breast cancer.
The present study suggested that there was a significantly different expression of H19 in the plasma between NSCLC group and benign disease group. Therefore, H19 could be applied as a serological marker for the auxiliary diagnosis of NSCLC, which was consistent with the conclusion on breast cancer by Zhang et al. However, the expression of H19 was not related to the clinical, pathological features of the subjects.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Zheng R, Zeng H, Zuo T, Zhang S, Qiao Y, Zhou Q, et al.
Lung cancer incidence and mortality in China, 2011. Thorac Cancer 2016;7:94-9.
Zheng R, Zeng H, Zhang S, Fan Y, Qiao Y, Zhou Q, et al.
Lung cancer incidence and mortality in China, 2010. Thorac Cancer 2014;5:330-6.
Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al
. Cancer statistics in China, 2015. CA Cancer J Clin 2016;66:115-32.
Kolenda T, Guglas K, Ryś M, Bogaczyńska M, Teresiak A, Bliźniak R, et al.
Biological role of long non-coding RNA in head and neck cancers. Rep Pract Oncol Radiother 2017;22:378-88.
Palmieri G, Paliogiannis P, Sini MC, Manca A, Palomba G, Doneddu V, et al.
Long non-coding RNA CASC2 in human cancer. Crit Rev Oncol Hematol 2017;111:31-8.
Chen T, Yang P, He ZY. Long non-coding RNA H19 can predict a poor prognosis and lymph node metastasis: A meta-analysis in human cancer. Minerva Med 2016;107:251-8.
Zhang J, Zhang P, Wang L, Piao HL, Ma L. Long non-coding RNA HOTAIR in carcinogenesis and metastasis. Acta Biochim Biophys Sin (Shanghai) 2014;46:1-5.
Zhang R, Xia Y, Wang Z, Zheng J, Chen Y, Li X, et al.
Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer. Biochem Biophys Res Commun 2017;490:406-14.
Tantai J, Hu D, Yang Y, Geng J. Combined identification of long non-coding RNA XIST and HIF1A-AS1 in serum as an effective screening for non-small cell lung cancer. Int J Clin Exp Pathol 2015;8:7887-95.
Gabory A, Ripoche MA, Yoshimizu T, Dandolo L. The H19 gene: Regulation and function of a non-coding RNA. Cytogenet Genome Res 2006;113:188-93.
Nordin M, Bergman D, Halje M, Engström W, Ward A. Epigenetic regulation of the igf2/H19 gene cluster. Cell Prolif 2014;47:189-99.
Gabory A, Jammes H, Dandolo L. The H19 locus: Role of an imprinted non-coding RNA in growth and development. Bioessays 2010;32:473-80.
Chen SW, Zhu J, Ma J, Zhang JL, Zuo S, Chen GW, et al.
Overexpression of long non-coding RNA H19 is associated with unfavorable prognosis in patients with colorectal cancer and increased proliferation and migration in colon cancer cells. Oncol Lett 2017;14:2446-52.
Zhang Y, Luo ZL, Zhang KJ, Wu LC, Zhang L, Liu JB. The potential diagnostic value of long non coding RNA H19 expression in plasma for breast cancer. Chin J Clin Lab Sci 2016;34:264-7.
[Figure 1], [Figure 2]