Detection of miR-155-5p and imaging lung cancer for early diagnosis: in vitro and in vivo study

Abstract

Purpose: Currently, the routine screening program has insufficient capacity for the early diagnosis of lung cancer. Therefore, a type of chitosan-molecular beacon (CS-MB) probe was developed to recognize the miR-155-5p and image the lung cancer cells for the early diagnosis.

Methods: Based on the molecular beacon (MB) technology and nanotechnology, the CS-MB probe was synthesized self-assembly. There are four types of cells-three kinds of animal models and one type of histopathological sections of human lung cancer were utilized as models, including A549, SPC-A1, H446 lung cancer cells, tumor-initiating cells (TICs), subcutaneous and lung xenografts mice, and lox-stop-lox(LSL) K-ras G12D transgenic mice. The transgenic mice dynamically displayed the process from normal lung tissues to atypical hyperplasia, adenoma, carcinoma in situ, and adenocarcinoma. The different miR-155-5p expression levels in these cells and models were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The CS-MB probe was used to recognize the miR-155-5p and image the lung cancer cells by confocal microscopy in vitro and by living imaging system in vivo.

Results: The CS-MB probe could be used to recognize the miR-155-5p and image the lung cancer cells significantly in these cells and models. The fluorescence intensity trends detected by the CS-MB probe were similar to the expression levels trends of miR-155 tested by qRT-PCR. Moreover, the fluorescence intensity showed an increasing trend with the tumor progression in the transgenic mice model, and the occurrence and development of lung cancer were dynamically monitored by the differen fluorescence intensity. In addition, the miR-155-5p in human lung cancer tissues could be detected by the miR-155-5p MB.

Conclusion: Both in vivo and in vitro experiments demonstrated that the CS-MB probe could be utilized to recognize the miR-155-5p and image the lung cancer cells. It provided a novel experimental and theoretical basis for the early diagnosis of the disease. Also, the histopathological sections of human lung cancer research laid the foundation for subsequent preclinical studies. In addition, different MBs could be designed to detect other miRNAs for the early diagnosis of other tumors.

Keywords: Chitosan; Lung cancer; MicroRNA; Molecular beacon; Molecular imaging; Tumor-initiating cell.

Fig. 1

Schematic showing the delivery of miR-155-5p MB into the cells via CS nanoparticles for the detection and imaging of miRNA

Fig. 2

Fluorescence imaging and detection in viable cancer cells. a Confocal microscopy imaging of the four cells after delivery of the miR-155-5p MB (red) by CS nanoparticles. RS MB was used as a negative control. The cell nucleuses were stained by Hoechst33342 (blue). Scale bar = 25 μm. b Fluorescence intensity of Cy5 was measured after imaging (n = 6) (*p < 0.05). c Relative miR-155-5p expression was detected in A549, SPC-A1, H446 cells, and TICs by qRT-PCR

Fig. 3

Establishment of animal models and detection of miR-155-5p expression. a HE staining in A549 and H446 lung xenograft(LX)models (× 200). b HE staining at 4, 6, 8, and 12 weeks in transgenic mice models after instillation of adenovirus (× 400). c, d miR-155-5p expression in the subcutaneous xenografts (SX) and lung xenografts (LX) of nude mice models (n = 8). e miR-155-5p expression in transgenic mice at different disease stages (n = 8) (*p < 0.05)

Fig. 4

In vivo identification of miR-155-5p and fluorescence imaging of cancer cells in xenografts models. a Imaging the subcutaneous and lung xenografts after injection of CS-MB via the tail veins. Subcutaneous xenografts model, a: A549 treated with CS-RS MB, b:A549 treated with CS-miR-155-5p MB, c: H446 treated with CS-miR-155-5p MB. Lung xenografts model, d: A549 treated with CS-RS MB, e:A549 treated with CS-miR-155-5p MB, f: H446 treated with CS-miR-155-5p MB. Groups a and d were used as the negative controls. b Imaging the xenografts after removal. c Fluorescence intensity was analyzed after injection (n = 8) (*p < 0.05). d Confocal microscopy imaging of the xenografts tissues after transfection with CS-miR155-5p MB or CS-RS MB (red). Cell nuclei were stained by DAPI (blue). Scale bar 25, 50 μm. Arrow: planting nodule and cancer cells

Fig. 5

In vivo identification of miR-155-5p and fluorescence imaging of cancer cells in transgenic mice at different stages of the disease. a Imaging the lung after injection of CS-miR-155-5p MB nanoparticles via the tail vein. Mice without intranasal inhalation of the adenovirus were used as the control group. b Imaging the lungs after removal. c Fluorescence intensity was analyzed after injection (n = 8) (*p < 0.05). d Confocal microscopy imaging of the different pathological changes after transfection with CS-miR-155-5p MB. Cell nuclei were stained by DAPI (blue). Scale bar 50 μm. Arrow ①: atypical hyperplasia. Arrow ②: adenoma. Arrow③: carcinoma in situ. Arrow ④: adenocarcinoma

Fig. 6

Identification of miR-155-5p and fluorescence imaging of the cancer cells in human lung squamous carcinoma and adenocarcinoma tissues. RS MB was used as a negative control. Scale bar 50 μm