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Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 295-298

Expression of microRNAs-106b in nonsmall cell lung cancer

1 Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science, Tianjin 300192, China
2 Department of Inner Medicine, Shandong Cancer Hospital and Institute, Jinan 250117, Shandong Province, China
3 The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, Guangxi Province, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Xiao-Chun Wang
Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science, Tianjin 300192
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.235344

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 > Abstract 

Aim: To explore the expression of microRNA-106b (miRNA-106b) in nonsmall cell lung cancer (NSCLC).
Settings and Design: miRNAs are short regulatory RNAs that negatively modulate gene expression at the posttranscriptional level, and are deeply involved in the pathogenesis of several types of cancer. miRNA-106b has been shown to play an oncogenic role in tumor progression. The expression of miRNA-106b is detected in this study.
Subjects and Methods: Quantitative reverse transcription polymerase chain reaction and Northern blotting were used to detect the expression level of miRNA-106b in 200 NSCLC samples.
Statistical Analysis Used: All statistical analyses were performed using SPSS 16.0 software. Results were statistically evaluated using the Kruskal–Wallis test and Mann–Whitney U-test. Survival curves were estimated by the Kaplan–Meier method and P < 0.05 was considered to be statistically significant.
Results: miRNA-106b expression is increased in NSCLC tissues. Statistical analysis showed that overexpression of miRNA-106b was strongly associated with lymph node metastasis, stage of tumor node metastasis classification, and poor prognosis. Moreover, there was a significant difference in the miRNA-106b expression levels between smoking and nonsmoking patients. Multivariate Cox regression analysis showed that miRNA-106b was an independent prognostic factor for NSCLC patients.
Conclusions: These data suggest that aberrantly expressed miRNA-106b may contribute to the development of NSCLC.

Keywords: MicroRNAs-106b, nonsmall cell lung cancer, overexpression, prognosis

How to cite this article:
Li Y, Tian J, Guo ZJ, Zhang ZB, Xiao CY, Wang XC. Expression of microRNAs-106b in nonsmall cell lung cancer. J Can Res Ther 2018;14, Suppl S2:295-8

How to cite this URL:
Li Y, Tian J, Guo ZJ, Zhang ZB, Xiao CY, Wang XC. Expression of microRNAs-106b in nonsmall cell lung cancer. J Can Res Ther [serial online] 2018 [cited 2020 Feb 25];14:295-8. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/295/235344

 > Introduction Top

It is a well-known fact that lung cancer ranks as the single biggest cause of cancer deaths in developed countries. The number of new lung cancer cases and the number of deaths from lung cancer are very similar, and the cure rate is regarded to be about 15% in advanced countries and 7–8% in developing countries. Nonsmall cell lung cancer (NSCLC) accounts for approximately 80% of all the cases.[1] Accumulating evidence suggests that microRNAs (miRNAs) may be involved in controlling lung cancer development and play critical roles in its pathogenesis.[2]

miRNAs are a species of small noncoding single-stranded RNAs of about 21 to 23 nucleotides that function by binding to target mRNAs, resulting in their degradation or translational inhibition based upon the degree of complementary with their target mRNA. The primary transcripts of miRNAs are generated by RNA polymerase II, after which they are sequentially processed by RNase III class enzymes, Drosha, and Dicer, to first produce ~70 nt long intermediate hairpin structures (pre-miRNAs) and finally the mature miRNAs.[3] Human miRNAs have been reported and a number of these have been shown to play normal physiologic roles in cell proliferation, apoptosis, and differentiation.[4] In addition, studies have showed that miRNAs contribute to oncogenesis by promoting the expression of oncogenes or by inhibiting tumor suppressor genes. As such, some miRNAs may be markers for cancer diagnosis and prognosis.[5] In this study, the expression of miRNA-106b was investigated in NSCLC and analyzed for significance. The results indicated that miRNA-106b was overexpressed and was associated with lymph node metastasis and poor prognosis, suggesting that miRNA-106b may be a potential diagnosis and prognosis marker for NSCLC.

 > Subjects and Methods Top

Tissue specimens

Fresh tissue samples, containing NSCLC and adjacent histologically normal tissue, were procured from surgical resection specimens collected by the Department of Tumor Medicine, xxx Hospital from 2001 to 2007. Primary tumor regions and corresponding histologically normal tissues from the same patients were separated by experienced pathologists, and immediately stored at −70°C until use. All the patients received no treatment before surgery and signed informed consent forms for sample collection. Use of patient samples comprising tumor and adjacent histologically normal tissues had been approved by our Institutional Ethics Committee.

RNA extraction

Total RNA was extracted from NSCLC tissue and its corresponding normal tissue using the absolutely RNA reverse transcription-polymerase chain reaction (RT-PCR) Miniprep kit (stratagene), according to the manufacturer's instructions and quantification was done with the DUVR 800 ultraviolet/visible spectrophotometer (Beckman Coulter, Fullerton, CA, USA).

Quantitative reverse transcription-polymerase chain reaction

TaqMan miRNA assays (ABI PRISM) used the stem-loop method to detect the expression levels of mature miRNA-106b. For RT reactions, 10 ng total RNA was used in each reaction and mixed with RT primer. RT reactions were carried out at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min, and then maintained at 4°C. Following RT reactions, 1.5 μl cDNA was used for a PCR along with TaqMan primers (2 μl). PCR was conducted at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and at 60°C for 60 s in the ABI 7500 real-time PCR system. Real-time PCR results were analyzed and expressed as relative miRNA expression of the quantification cycle values. RT and PCR primers for miR-106b were purchased from ABI PRISM. U6B was used for normalization. Relative expression levels among treatments were then calculated using the following equation: Relative gene expression = 2ΔΔCt, −ΔΔCt = (Ct gene of interestCt internal control gene)Treated− (Ct gene of interestCt internal control gene)Untreated.[6]

MicroRNAs Northern blots

For miRNA Northern blots, 15 μg total RNA were separated on 15% denaturing polyacrylamide gels, electrotransferred onto GeneScreen Plus membranes (PerkinElmer), and hybridized using UltraHyb-Oligo buffer (Ambion). Oligonucleotides complementary to mature miRNA-106b were end-labeled with T4 Kinase (Invitrogen) and used as probes. Hybridization was performed at 42°C overnight, and membranes were washed twice in 0.1 × subacute sclerosing panencephalitis and 0.1% sodium dodecyl sulfate at 42°C for 15 min each. Membranes were then exposed to a storage phosphor screen (GE Healthcare Bio-Sciences) for 8 h and imaged using a Typhoon 9410 Variable Mode Imager (GE Healthcare Bio-Sciences). Saved images were cropped using Photoshop 6.0 (Adobe Systems).

Statistical analysis

All statistical analyses were performed using SPSS 16.0 software (IBM, NY, USA). Results were statistically evaluated using the Kruskal–Wallis test and Mann–Whitney U-test. Survival curves were estimated by the Kaplan–Meier method and P < 0.05 was considered to be statistically significant.

 > Results Top

Expression of microRNAs-106b in nonsmall cell lung cancer tissue

In this study, expression of miRNA-106b was detected in 200 NSCLC samples and adjacent histologically normal tissue using RT-quantitative PCR (RT-qPCR), and its expression was normalized to that of the control U6B small nuclear RNA gene. Results showed miRNA-106b expression levels were significantly higher in NSCLC tissues than in corresponding noncancerous tissues. These results were confirmed by agarose gel electrophoresis of RT-qPCR products [Figure 1]a and [Figure 1]b.
Figure 1: Expression of microRNAs-106b in nonsmall cell lung cancer. (a) Representative quantitative polymerase chain reaction results of microRNAs-106b expression in nonsmall cell lung cancer tissue and adjacent noncancerous tissue. microRNAs-106b expression levels were significantly higher in nonsmall cell lung cancer tissue than in corresponding noncancerous tissue. Triplicate assays were performed for each sample, (b) agarose gel electrophoresis results of quantitative polymerase chain reaction, (c) Northern blots show that expression of microRNAs-106b in nonsmall cell lung cancer tissue was increased compared with corresponding noncancerous tissue, which was in accordance with quantitative polymerase chain reaction results. N = Normal tissue, T = Tumor tissue

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To further confirm the RT-qPCR results, Northern blot analysis for miRNA-106b was performed in the same four pair samples that were examined using RT-qPCR. Increased miRNA-106b expression was observed in all NSCLC tissues [Figure 1]c.

Correlation between microRNAs-106b expression and clinicopathological features in nonsmall cell lung cancer

Statistical analysis showed that overexpression of miRNA-106b was associated with lymph node metastasis, stage of tumor node metastasis (TNM) classification, and smoking [Table 1]. To determine the association between miRNA-106b expression and prognosis, we plotted Kaplan–Meier curves for overall survival. Significant differences were observed in NSCLC patient survival according to miRNA-106b expression in tumor tissues. The survival rate of patients with a low miRNA-106b expression (60 months) was higher than that of patients with high miRNA-106b expression (37 months, P = 0.008) [Figure 2]. Multivariate Cox regression analysis showed that miRNA-106b expression (P = 0.017), regional lymph node metastasis (P = 0.021), and smoking (P = 0.019) were independent prognostic factors for NSCLC patients [Table 2].
Table 1: Relationship between miRNA-106b expression and tumor clinicopathologic features

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Figure 2: Kaplan–Meier survival curve of nonsmall cell lung cancer patients sub-grouped as microRNAs-106b-low or high expression. The prognosis of microRNAs-106b-positive cases was significantly shorter than that of microRNAs-106b-negative cases (P = 0.024)

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Table 2: Multivariate analysis of prognostic factors by the Cox proportional hazards model in NSCLC patients

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 > Discussion Top

Increasing numbers of reports have revealed aberrant expression of certain miRNAs in tumors. Not only is the spectrum of miRNAs expressed in malignant cells significantly different from that of normal counterpart cells, but also miRNA expression profiles can better classify poorly differentiated tumors compared with the mRNA (EST)-based classifier.[7] A potential role for miRNAs in cancer has been suggested by the location of genes encoding many miRNAs in, or close to, minimal regions of amplicons, loss of heterozygosity, or breakpoint cluster regions.[8] Amplification or overexpression of an oncogenic miRNA could eliminate the expression of a miRNA-target tumor suppressor gene, and result in cancer progression. Overexpression of miR-106b has been observed in a variety of human tumors, including colorectal cancer,[9] gastric cancer,[10] hepatocellular carcinoma,[11] glioma tumor,[12] renal cell carcinoma,[13] and head and neck squamous cell carcinomas.[5] Phenotypic analyses revealed that miR-106b family promoted exit from G1 and entry into S phase. Prasad and Katiyar revealed that down-regulation of miRNA-106b inhibits the growth of melanoma cells by promoting G1-phase cell cycle arrest and reactivation of p21/WAF1/Cip1 protein.[14] The oncogenic properties of the miR-106b family of miRNAs may stem from combined positive regulation of the cell cycle and additional functions.[15] However, the expression status of miRNA-106b in NSCLC has not been reported until recently. In this study, expression of miRNA-106b was detected for the first time in NSCLC samples, and the results showed miRNA-106b level was increased.

Studies have shown that miRNA expression fingerprints correlate with clinical and biological characteristics of tumors, including tissue type, differentiation, aggression, response to therapy, and prognosis.[16] A large amount of diagnostic information was encoded in a relatively small number of miRNAs. For lung cancer, it has been shown that miRNA-let-7 expression was frequently reduced both in vivo and in vitro and that this was significantly associated with shortened postoperative survival, independent of disease stage.[17] High mRNA-155 and a low miRNA-let-7a-2 expression levels were shown to correlate with poor survival.[18] Markou et al. found overexpression of mature miRNA-21 was an independent negative prognostic factor for overall survival in NSCLC patients.[19] Given these results, it was decided to investigate whether miRNA-106b expression also correlated with the clinicopathological features and prognosis of NSCLC patients. So, the relationship between miRNA-106b expression and patient clinicopathological factors was analyzed using 200 NSCLC samples. Statistical analysis showed that overexpression of miRNA-106b was significantly positively associated with lymph node metastasis and stage of TNM classification. An interesting result was that miRNA-106b expression was significantly associated with the smoking status of NSCLC patients. It is well-reported that epidemic studies have shown smoking as a high risk factor for lung cancer. The exact effects of smoking upon miRNA-106b expression in NSCLC patients require further study.

 > Conclusions Top

Our results revealed for the first time that miRNA-106b was overexpressed in NSCLC tumors. Increased expression of miRNA-106b was strongly associated with lymph node metastasis and poor prognosis. Moreover, miRNA-106b expression was an independent prognostic factor for NSCLC patients. Our findings provide a new role of miRNA-106b in NSCLC, and it may be considered as a potential novel prognostic marker and a target for future development of specific therapeutic interventions in NSCLC.

Financial support and sponsorship

This study was supported by the National Natural Science Foundation of China (81272511), Natural Science Foundation of Tianjin (14JCZCJC13700), Youth Research Foundation of Peking Union Medical College(33320140126), the Development Foundation of Institute of Radiation of Medicine (SF1414) and the Research Foundation of Institute of Radiation of Medicine (ST1557).

Conflicts of interest

There are no conflicts of interest.

 > References Top

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Prasad R, Katiyar SK. Down-regulation of miRNA-106b inhibits growth of melanoma cells by promoting G1-phase cell cycle arrest and reactivation of p21/WAF1/Cip1 protein. Oncotarget 2014;5:10636-49.  Back to cited text no. 14
Ivanovska I, Ball AS, Diaz RL, Magnus JF, Kibukawa M, Schelter JM, et al. MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression. Mol Cell Biol 2008;28:2167-74.  Back to cited text no. 15
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Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 2004;64:3753-6.  Back to cited text no. 17
Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006;9:189-98.  Back to cited text no. 18
Markou A, Tsaroucha EG, Kaklamanis L, Fotinou M, Georgoulias V, Lianidou ES. Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR. Clin Chem 2008;54:1696-704.  Back to cited text no. 19


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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