Analysis of microRNA Expression in Liquid-Based Cytology Samples may be Useful for Primary Lung Cancer Diagnosis

Yusuke Araki, MD; Koji Arihiro, MD, PhD; Kakuhiro Yamaguchi, MD, PhD; Shinjiro Sakamoto, MD, PhD; Yasushi Horimasu, MD, PhD; Takeshi Masuda, MD, PhD; Shintaro Miyamoto, MD, PhD; Taku Nakashima, MD, PhD; Hiroshi Iwamoto, MD, PhD; Kazunori Fujitaka, MD, PhD; Hironobu Hamada, MD, PhD; Noboru Hattori, MD, PhD


Am J Clin Pathol. 2021;156(4):644-652. 

In This Article

Materials and Methods

Tissue Samples

We collected 18 patient samples of primary lung cancer tissues and paired adjacent normal tissue surgically resected at Hiroshima University Hospital and kept in our lung cancer file. Characteristics of the 18 lung cancer patients are shown in Table 1. Normal and malignant tissue samples were acquired during routine patient therapeutic surgery. We collected tissue samples of approximately 100 mg, which were immediately placed in RNAlater (Ambion) and stored in a –20°C freezer until use.

Cytologic Samples

When we performed bronchoscopy, we submitted bronchial lavage fluid and the washed device fluid as cytologic specimens. We assembled 136 bronchial cytologic samples kept in our lung cytology file. Patient characteristics are shown in Table 2. Each case was followed until a final diagnosis of cancer was or was not made. Submitted specimens were centrifuged at 600g to obtain cell pellets, which were then fixed in Cellprep (Roche Diagnostics) to prepare for cytologic specimens. Fixed cytologic specimens were stored at 26°C. After preparation, approximately half of the residual samples were used for this study within 24 hours.

Patient Selection of Cytologic Samples

A total of 136 bronchial cytologic samples were collected. We examined the expression of miRs according to the selection criteria Figure 1. Before performing reverse transcription–quantitative polymerase chain reaction (RT-qPCR), 10 samples were excluded. Cytologic examination of the samples was successfully performed in 134 cases; 2 samples were judged as inappropriate because few cells were present. Four samples involved metastatic cancer, and 5 samples were not used because the total amount of RNA recovered was too low for the pres-ent analysis. Finally, 125 samples that could be detected were examined by RT-qPCR. The relationship of examined cases involving histologic diagnosis and cytologic classification is shown in Table 3. The number of samples that yielded positive amplification results was different for each miR; miR-21 was detected in all samples, but other miRs were not detected in certain samples. In 29 samples, cycle threshold (Ct) values for 1 to 3 of the 4 miRs were undetectable within 45 cycles; in 96 samples, Ct values for all 4 miRs were detected. Expression of the 4 miRs was compared with pathologic diagnosis. Histologic examination of surgically resected lung tumors and biopsy specimens was performed in 31 cases (37.3%) and 50 cases (60.2%), respectively. If a case was diagnosed as cancer based on pathologic examination, including histologic and/or cytologic examinations, it was classified as being in the tumor group. If a case was diagnosed as having a benign lesion, such as a granuloma or infectious nodule based on pathologic diagnosis or on radiologic examination showing that a tumor had become smaller or on bronchoalveolar lavage testing showing an interstitial pneumonia pattern, it was classified as being in the nontumor group.

Figure 1.

Selection protocol for liquid-based cytologic specimens. Cancer was diagnosed histologically. miR, microRNA; N, cases diagnosed as noncancerous; T, cases diagnosed as cancerous.

RNA Extraction and RT-qPCR

Total RNA was extracted from tissues using the mirVANA Isolation Kit (Thermo Fisher Scientific), according to the manufacturer's protocol. Total RNA was extracted from LBC samples using the Magcore Compact Automated Nucleic Acid Extractor (RBC Bioscience) and Magcore Total RNA Cultured Cells Kit (RBC Bioscience), according to the manufacturer's protocol. RNA concentration was measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific). Extracted RNA samples were stored at –80°C until use.

Total RNA was diluted to 2 ng/μL with nuclease free water, and 10 ng of total RNA was reverse transcribed to complementary DNA using a TaqMan miR reverse transcription kit and TaqMan miR assays (Thermo Fisher Scientific). According to the manufacturer's protocol, the reverse transcriptase reaction was performed in a total volume of 15 μL per reaction and under the following conditions: 16°C for 30 minutes, 42°C for 30 minutes, 85°C for 5 minutes, and then held at 4°C. The following primers were used: RNU6B (U6 small nuclear RNA; assay 001093), miR-21 (assay 000397), miR-31 (assay 002279), miR-182 (assay 002334), and miR-183 (assay 002269).

RT-qPCR was performed using an Illumina Eco Real-Time PCR System. Real-time PCR was performed in a total volume of 20 μL per reaction. After the reverse transcriptase reaction, 1.33 μL of the reverse transcriptase product was mixed with 10 μL of TaqMan Universal Mater Mix II (Thermo Fisher Scientific), 1 μL of specific probes of TaqMan miR assays (Thermo Fisher Scientific), and nuclease-free water and subjected to real-time PCR. RNU6B was used as an endogenous control for the normalization of expression levels. The relative expression levels of target miRs were calculated by the ΔCt method (Ctsample – CtRNU6B sample) and compared between samples obtained from lung cancer and normal tissues. All experiments were repeated 3 times.

Statistical Analysis

The data were analyzed using JMP Pro version 14.1.0 (SAS Institute). The Mann-Whitney test was used to analyze the differences in miR expression levels between patients. Receiver operating characteristic curves and area under the curve analyses were used to determine the sensitivity and specificity of each miR. P < .05 was considered statistically significant.