MicroRNA-326 Negatively Regulates CD155 Expression in Lung Adenocarcinoma

doi:10.21203/rs.3.rs-2229871/v1

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MicroRNA-326 Negatively Regulates CD155 Expression in Lung Adenocarcinoma

https://doi.org/10.21203/rs.3.rs-2229871/v1

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Treatment with immune checkpoint inhibitors induces a durable response in some patients with non–small cell lung cancer, but eventually gives rise to drug resistance. Upregulation of CD155 expression is implicated as one mechanism of resistance to PD-1/PD-L1 inhibitors, and it is therefore important to characterize the mechanisms underlying regulation of CD155 expression in tumor cells. The aim of this study was to identify microRNAs (miRNAs) that might regulate CD155 expression at the posttranscriptional level in lung cancer. Comprehensive miRNA screening with target prediction programs and a dual-luciferase reporter assay identified miR-346, miR-328-3p, miR-326, and miR-330-5p as miRNAs that bind to the 3′ untranslated region of CD155 mRNA. Forced expression of these miRNAs suppressed CD155 expression in lung cancer cell lines. Immunohistochemical staining of CD155 in tissue specimens from 57 patients with lung adenocarcinoma revealed the median tumor proportion score for CD155 to be 68%. The abundance of miR-326 in these specimens with a low level of CD155 expression was significantly greater than that in those with a high level (P < 0.005). Our results thus suggest that miR-326 negatively regulates CD155 expression in lung adenocarcinoma and might therefore play a role in the development of resistance to PD-1/PD-L1 inhibitors.

microRNA

miR-326

CD155

lung adenocarcinoma

poliovirus receptor (PVR)

nectin-like molecule 5 (Necl5)

Cancer cells evade immune surveillance by expressing multiple immune checkpoint molecules [1]. Immune checkpoint inhibitors (ICIs) that target inhibitory signaling in T cells mediated by the interaction of programmed cell death–1 (PD-1) with its ligand PD-L1 have shown unprecedented clinical activity for the treatment of non–small cell lung cancer (NSCLC) without targetable mutations of EGFR or ALK genes, having become a standard therapy for this disease [2–6]. Expression of PD-L1 in tumor cells has been found to be related to the response to PD-1/PD-L1 inhibitors and has been approved as a biomarker for such treatment in individuals with NSCLC. However, not all PD-L1–positive patients respond to these agents, with the identification of new drug resistance biomarkers and development of new therapies being clinical priorities [7, 8].

CD155, also known as the poliovirus receptor (PVR) and nectin-like molecule 5 (Necl5), is often upregulated in tumor cells and endows them with a growth advantage and increased migratory capacity [9, 10]. It also binds with high affinity to the immune checkpoint molecule TIGIT (T cell immunoreceptor with Ig and ITIM domains) and thereby inhibits T cell activation (11), and recent evidence suggests that it may mediate resistance to ICIs in melanoma and NSCLC [12–15]. A phase 2 study provided preliminary evidence for the efficacy and safety of combination therapy with antibodies to TIGIT and to PD-L1 for advanced NSCLC [16], with such therapy offering the potential to overcome resistance to PD-1/PD-L1 inhibitors. Multiple pathways may contribute to the expression and function of CD155 related to immune suppression, with a detailed understanding of the upregulation of CD155 in tumor cells having been lacking.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that comprise 19 to 25 nucleotides and mediate posttranscriptional regulation of gene expression by binding to the 3′ untranslated region (3′-UTR) of protein-coding mRNAs with imperfect complementarity, with such binding resulting in inhibition of translation or in transcript cleavage [17]. Several miRNAs expressed in immune cells or cancer cells have been found to play key roles in cancer-related immune responses by targeting immunosuppressive or immunostimulatory factors [18]. Moreover, miRNAs regulate the expression of genes for immune checkpoint ligands such as PD-L1 [19–21].

Given the growing evidence implicating upregulation of CD155 in tumor cells in immune evasion, we have now investigated the possible role of miRNAs in the posttranscriptional regulation of CD155 expression in lung adenocarcinoma. Comprehensive miRNA screening with sequence-based target prediction algorithms and the use of a luciferase reporter assay allowed us to identify several miRNAs as potential regulators of CD155 expression in lung adenocarcinoma.

miRNA target prediction

Seven target prediction programs—including TargetScan (https://www.targetscan.org/vert_80), miRMap (http://mirmap.ezlab.org), RNA22 (https://cm.jefferson.edu/rna22), miRWalk (http://zmf.umm.uni-heidelberg.de), miRanda (www.microrna.org), microT-CDS (https://dianalab.e-ce.uth.gr/html/dianauniverse/index.php?r=microT_CDS), and miRDB (http://www.mirdb.org)—were applied to screen for miRNAs that potentially bind to the 3′-UTR of CD155 mRNA.

Cell Culture

The human lung cancer cell lines A549, H322, and H23 as well as the human embryonic kidney cell line HEK293T were obtained from American Type Culture Collection (Manassas, VA). The human lung cancer cell line EBC-1 was obtained from RIKEN BioResource Research Center (Ibaraki, Japan). Cells were cultured under 5% CO₂ at 37°C in Dulbecco’s modified Eagle’s medium for A439 and HEK293T, RPMI 1640 medium for H322 and H23, or Eagle’s minimum essential medium for EBC-1 (Thermo Fisher Scientific), each supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific).

Mirna Transfection

Cells were transiently transfected during the exponential growth phase with 50 nM miScript miR-346 mimic (ID:MSY0000773), miScript miR-330-5p mimic (ID:MSY0004693), miScript miR-326 mimic (ID:MSY0000756), miScript miR-423-5p mimic (ID:MSY0004748), miRCURY LNA miR-328-3p mimic (ID:YM00473147), miRCURY LNA miR-6884-5p mimic (ID:YM00472866), miRCURY LNA miR-485-5p mimic (ID:YM00473278), miRCURY LNA miR-1306-5p mimic (ID:YM00473249), miRCURY LNA miR-423-5p mimic (ID:MSY0004748), miRCURY LNA miR-324-5p mimic (ID:YM00473205), mirVana miR-3184-5p mimic (ID:PM18582), or AllStars negative control (ID: SI03650318), all obtained from Qiagen. Transfection was performed for 48 h with the use of the Lipofectamine RNAiMAX or Lipofectamine 3000 reagents (Thermo Fisher Scientific), and the cells were then subjected to experiments.

Construction Of Reporter Plasmids

Complementary DNA corresponding to the full-length 3′-UTR of CD155 mRNA was amplified by PCR from total cDNA of A549 cells with PrimeSTAR GXL DNA Polymerase (Takara) and the primers 5′-TAATTCTAGTTGTTTAAACTAGCGGCCGCCAGCGTCGGGACTGAG-3′ (forward) and 5′-GCATGCCTGCAGGTCGACTCTAGATTTTGCATTATTAAAAGAAGAGTCAAGG-3′ (reverse). Complementary 18- to 22-bp DNA oligonucleotides containing putative miR-346, miR-328-3p, miR-326, or miR-330-5p binding sites of the 3′-UTR of CD155 mRNA, or corresponding mutant sites, were synthesized. The sense and antisense strands were annealed by incubation of 2000 ng of each oligonucleotide in 46 µl of Oligo Annealing Buffer (Promega) first for 3 min at 90°C and then for 1 h at 37°C. The PCR amplicon for the full-length 3′-UTR of CD155 mRNA and the annealed oligonucleotides were separately cloned between the PmeI and XbaI sites of the pmirGLO Dual-Luciferase vector (Promega) with the use of an In-Fusion HD Cloning Kit (Takara). All plasmids used in this study were verified by Sanger sequencing.

Luciferase Reporter Assay

Each reporter plasmid (100 ng) was introduced together with miRNA mimics (50 nM) or a negative control miRNA into HEK293T cells in 96-well plates by transfection for 48 h with the use of Lipofectamine 3000 reagents (Thermo Fisher Scientific). Luciferase activity in cell lysates was then measured with the use of a Dual-Luciferase Assay System (Promega), and firefly luciferase activity was normalized by Renilla luciferase activity.

Rna Extraction And Rt-qpcr Analysis

Total RNA was isolated from cultured cells with the use of an RNeasy Mini Kit or an miRNeasy Mini Kit (Qiagen). Total RNA from tissue samples was extracted with the use of an AllPrep DNA/RNA FFPE Kit (Qiagen). For analysis of the abundance of CD155 mRNA, total RNA (400 ng) was subjected to reverse transcription (RT) with PrimeScript RT Master Mix (Takara) and the resulting cDNA was subjected to quantitative PCR (qPCR) analysis in triplicate with TB Green Premix Ex Taq II (Takara) in a Thermal Cycler Dice Real Time System (Takara). The qPCR primers (forward and reverse, respectively) were 5′-GGCAACTACACCTGCCTGTT-3′ and 5′-AATCACCTGGCACTCAGACC-3′ for CD155 mRNA and 5′-ACTCAACACGGGAAACCTCA-3′ and 5′-AACCAGACAAATCGCTCCAC-3′ for 18S rRNA (endogenous normalization control). For analysis of mature miRNAs, total RNA (10 ng) was subjected to RT with a miRCURY LNA RT Kit (Qiagen) and the resulting cDNA was subjected to qPCR analysis in triplicate with a miRCURY LNA SYBR Green PCR Kit (Qiagen) in a Thermal Cycler Dice Real Time System (Takara). The small nuclear RNAs U6 and SNORD38B were used as endogenous controls for lung cancer cell lines and tumor samples, respectively (Supplementary Table S1). Relative mRNA or miRNA abundance was determined with SDS software by the 2^–ΔΔCt method.

Flow Cytometry

For analysis of CD155 expression at the cell surface, cultured cells were harvested, suspended in ice-cold PBS supplemented with 1% FBS, and incubated for 30 min on ice in the dark with phycoerythrin-conjugated mouse monoclonal antibodies to human CD155 (diluted 1:100; #337610, BioLegend) or an immunoglobulin G1 κ isotype control (diluted 1:100; #400114, BioLegend). The cells were then subjected to flow cytometric analysis with a FACSVerse instrument (BD Biosciences) and Flowjo software (Tree Star). The relative median fluorescence intensity (MFI) ratio was calculated as CD155 MFI divided by isotype-control MFI.

Immunoblot Analysis

Immunoblot analysis

Cultured cells were lysed in RIPA buffer (Thermo Fisher Scientific), the lysates were fractionated by SDS-PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated overnight at 4°C with antibodies to CD155 (diluted 1:1000; clone D8A5G, #81254, Cell Signaling Technology), and immune complexes were then detected with horseradish peroxidase–conjugated goat antibodies to rabbit immunoglobulin G (GE Healthcare UK), Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific), and a ChemiDoc Touch MP system (Bio-Rad). β-actin was detected with corresponding primary antibodies (diluted 1:1000; clone 13E5, #4970, Cell Signaling Technology) and horseradish peroxidase–conjugated goat antibodies to rabbit immunoglobulin G (GE Healthcare UK) as a loading control.

Patients And Tissue Samples

Formalin-fixed, paraffin-embedded tissue samples were collected retrospectively from 57 individuals who had undergone surgical resection of lung adenocarcinoma at Kyushu University Hospital between February and December 2020. Diagnosis of the 57 patients was based on the WHO Classification of Lung Tumors. All 57 samples were subjected to immunohistochemistry and RT-qPCR analysis. The characteristics of the patients were obtained from their medical records. This aspect of the study was performed in accordance with the Declaration of Helsinki (as revised in 2013) and was approved by the Ethics Committee of Kyushu University and Kyushu University Hospital (ethics approval ID: 2021 − 289).

Immunohistochemistry

All tumor samples were subjected to immunohistochemical analysis with rabbit monoclonal antibodies to CD155 (clone D8A5G, #81254, Cell Signaling Technology) at a dilution of 1:200. Fixed sections were incubated with the primary antibodies overnight at 4°C, after which immune complexes were detected with the use of a Histofine Simple Stain Max-PO Kit (Nichirei Bioscience) and the peroxidase substrate 3,3′-diaminobenzidine. All staining was evaluated by two investigators including a pathologist, and the tumor proportion score (TPS) for CD155 expression in tumor cells was determined as the percentage of viable tumor cells showing staining at any intensity in a section containing at least 100 viable tumor cells.

Statistical analysis

Differences in miRNA abundance or luciferase reporter activity were assessed with the Student’s t test or Mann-Whitney U test, as appropriate. All statistical analysis was performed with GraphPad Prism 8 (GraphPad Software). A P value of < 0.05 was considered statistically significant.

Data Availability

The data that support the findings of this study are available from the corresponding author, YY, upon reasonable request.

Identification of miRNAs as putative regulators of CD155 expression

To explore the possible regulation of CD155 expression by miRNAs, we identified miRNAs predicted to bind to the 3′-UTR of CD155 mRNA with the use of the TargetScan online prediction tool. A total of 48 miRNAs was predicted to bind to sites that are broadly conserved in the 3′-UTR of mammalian CD155 mRNA sequences (Fig. 1A). We selected the 10 miRNAs with the highest TargetScan Total Context + + scores (Fig. 1B) and tested them with a panel of six additional algorithms (miRMap, RNA22, miRWalk, miRanda, microT-CDS, and miRDB). All of the selected miRNAs were predicted to bind to the 3′-UTR of CD155 mRNA by at least three of these algorithms in addition to TargetScan (Fig. 1C).

Evaluation Of Mirna Binding To Cd155 Mrna

To further evaluate miRNA binding to CD155 mRNA, we performed a dual-luciferase reporter assay with the 10 predicted miRNAs. The coding sequence for the 4.3-kb full-length 3′-UTR of human CD155 mRNA was cloned downstream of the firefly luciferase gene in the pmirGLO Dual-Luciferase vector to yield pmirGLO-CD155 (Fig. 2A), which was then introduced together with each miRNA mimic or a negative control miRNA into HEK293T cells by transfection. Six miRNAs (miR-346, miR-328-3p, miR-326, miR-330-5p, miR-6884-5p, and miR-324-5p) were found to attenuate normalized firefly luciferase activity by at least 20% and were therefore determined to bind directly to the 3′-UTR of CD155 mRNA (Fig. 2B).

Regulation Of Cd155 Expression By Mirnas In Nsclc Cells

We next evaluated whether the six miRNAs found to bind to the 3′-UTR of CD155 mRNA regulate CD155 mRNA abundance in NSCLC cells. Transfection of A549 cells with these miRNA mimics (Supplementary Fig. S1) revealed that overexpression of miR-346, miR-328-3p, miR-326, and miR-330-5p each significantly reduced the abundance of CD155 mRNA, whereas overexpression of miR-6884-5p or miR-324-5p had no such effect (Fig. 3A). Overexpression of miR-346, miR-328-3p, miR-326, or miR-330-5p also significantly attenuated the expression of CD155 at the cell surface as determined by flow cytometry (Fig. 3B) as well as reduced the total abundance of CD155 in cell lysates as determined by immunoblot analysis (Fig. 3C). Similar inhibitory effects of these four miRNAs on cell surface expression of CD155 were apparent in the NSCLC cell lines H322, H23, and EBC-1, with the exception of miR-346 and miR-328-3p in H23 cells (Supplementary Fig. S2). These results thus indicated that miR-346, miR-328-3p, miR-326, and miR-330-5p regulate CD155 expression in NSCLC cells.

Validation Of Mirna Binding Sites In The 3′-utr Of Cd155 Mrna

Our results indicated that miR-346, miR-328-3p, miRNA-326, and miR-330-5p bind directly to the 3′-UTR of CD155 mRNA and suppress cell surface and total protein expression of CD155 in NSCLC cells. TargetScan analysis predicted three binding sites for each of these miRNAs in the 3′-UTR of CD155 mRNA. To confirm binding of each miRNA to these sites, we constructed dual-luciferase reporter plasmids containing each putative binding site with either the wild-type or a mutated sequence (Supplementary Fig. S3). Transfection of HEK293T cells with these plasmids and the corresponding miRNA mimics revealed that each miRNA significantly inhibited the luciferase reporter activity for one of the three wild-type sequences, and that such inhibition was abolished by mutation of these sequences (Fig. 4A–D). These results indicated that miR-346, miR-328-3p, miR-326, and miR-330-5p each directly binds to at least one of the corresponding predicted sites in the 3′-UTR of CD155 mRNA and thereby suppresses CD155 expression.

Association Of Mir-326 Abundance And Cd155 Expression In Lung Adenocarcinoma Patients

To clarify further the role of miRNAs in the regulation of CD155 expression in lung cancer, we determined the abundance of miR-346, miR-328-3p, miRNA-326, and miR-330-5p in CD155-low and CD155-high lung adenocarcinoma tissue. We collected tissue specimens from 57 patients with lung adenocarcinoma who underwent surgical resection. The tissue was subjected to immunohistochemical staining of CD155, and the CD155 TPS was determined (Fig. 5A). The median TPS for CD155 expression was 68% and was adopted as a cutoff for classification of the lung adenocarcinoma specimens as low or high for CD155 expression. The clinical characteristics of the study patients according to CD155 expression level are shown in Table 1. The proportions of men, former smokers, and tumors with higher pathological and TN stages tended to be greater for patients with CD155-high lung adenocarcinoma. RT-qPCR analysis showed that the abundance of miR-326 was significantly lower in CD155-high tumors than in CD155-low tumors (P < 0.005), whereas there was no significant difference in the expression levels of miR-346, miR-328-3p, or miR-330-5p between CD155-low and CD155-high tumors (Fig. 5B). These results indicated that the abundance of miR-326 is negatively associated with CD155 expression in lung adenocarcinoma.

Table 1

Baseline characteristics of the study patients according to CD155 expression level.
Characteristic	CD155 expression		Total
Characteristic	Low (n = 29)	High (n = 28)	Total
Age (years)
<70	15 (51.7)	14 (50.0)	29 (50.9)
≥70	14 (48.3)	14 (50.0)	28 (49.1)
Sex
Male	12 (41.4)	16 (57.1)	28 (49.1)
Female	17 (58.6)	12 (42.9)	29 (50.9)
Smoking history
Former smoker	14 (48.3)	19 (67.9)	33 (57.9)
Never smoker	15 (51.7)	9 (32.1)	24 (42.1)
Pathological stage
Stage I	27 (93.1)	18 (64.3)	45 (78.9)
Stage II/III	2 (6.9)	10 (35.7)	12 (21.1)
T status
T1	26 (89.7)	21 (75.0)	47 (82.5)
T2/T3/T4	3 (10.3)	7 (25.0)	10 (17.5)
N status
N0	28 (96.6)	20 (71.4)	48 (84.2)
N1/N2/N3	1 (3.4)	8 (28.6)	9 (15.8)
All values are number (%).

We set out to identify miRNAs that might regulate CD155 expression at the posttranscriptional level in lung cancer. We first applied target prediction algorithms and a luciferase reporter assay to discover functional miRNA binding sites in the 3′-UTR of CD155 mRNA. We then found that overexpression of four of the identified miRNAs (miR-346, miR-328-3p, miR-326, and miR-330-5p) suppressed CD155 expression as examined both at the cell surface and in cell lysates of lung cancer cell lines. Analysis of clinical samples revealed that miR-326 abundance was negatively associated with CD155 expression in patients with lung adenocarcinoma. Our results have thus revealed a previously unrecognized mechanism for posttranscriptional regulation of CD155 expression in lung adenocarcinoma.

CD155 is a member (Necl5) of the nectin-like molecule family and serves as the receptor for poliovirus (PVR) [22]. Upregulation of CD155 has been detected in many cancer types including lung adenocarcinoma [15, 23], and its overexpression has been associated with tumor progression and poor prognosis in retrospective studies of various types of solid cancer [24]. In addition, CD155 overexpression promotes tumor cell invasion and migration as well as proliferation [25]. The upregulation of CD155 expression in tumors is likely due to a variety of mechanisms. Reactive oxygen species and reactive nitrogen species were shown to increase CD155 expression by inducing a DNA damage response in multiple myeloma cells [26]. Furthermore, IFN-γ has been shown to increase CD155 expression in melanoma cells [27]. However, the mechanisms underlying regulation of CD155 expression in lung cancer have remained unclear.

We here identified miR-326 as an miRNA that negatively regulates CD155 expression in lung adenocarcinoma. Among four miRNAs found to regulate CD155 expression in lung cancer cells, forced expression of miR-326 or miR-330-5p significantly suppressed CD155 expression in all four lung cancer cell lines examined (A549, H322, H23, and EBC-1). Expression of miR-330-5p was lower than that of miR-326 in lung adenocarcinoma tissue samples (Supplementary Fig. S4), possibly explaining why only miR-326 abundance was found to be associated with CD155 expression in these specimens.

Previous studies have found that miR-326 acts as a tumor suppressor in glioma and osteosarcoma [28–30]. A search of the miRBase database (https://www.mirbase.org) and TargetScan identified > 300 potential target genes of miR-326, including that for NSBP1 (nucleosome binding protein 1), which promotes cell proliferation and invasion and whose expression is suppressed by miR-326 in NSCLC cells [31]. Recent studies have also indicated that miR-326 directly binds to the mRNA for and suppresses the expression of FSCN1 (fascin actin-bundling protein 1), which is implicated in the progression of lung cancer [32, 33]. In the present study, we now show that miR-326 directly binds to the mRNA for and suppresses the expression of CD155, which also contributes to tumor progression, consistent with the proposed role for miR-326 as a tumor suppressor.

Treatment with ICIs induces a durable response in some patients with NSCLC, but existing cancer immunotherapies are associated with both innate and acquired resistance [34, 35]. Upregulation of CD155 is implicated as one mechanism of resistance to PD-1/PD-L1 inhibitors [14, 15], and it will therefore be important to identify the mechanisms underlying regulation of CD155 expression in tumors. However, repeated lung biopsy for the detection of resistance markers is highly invasive in patients with advanced lung cancer, with the development of biomarkers that can be measured with minimally invasive approaches being highly desirable. MicroRNAs detected in serum are thought to mediate intercellular communication by regulating gene expression at the posttranscriptional level in target cells [36–38]. Both intracellular and cell-free circulating miRNAs have been associated with the diagnosis and prognosis of lung cancer [39, 40]. Our results now indicate that miR-326 in tumor cells or the tumor microenvironment suppresses CD155 expression in lung adenocarcinoma, suggesting that miR-326 present in serum might negatively regulate the expression of CD155 in lung cancer cells and that the serum level of miR-326 might be a resistance marker for PD-1/PD-L1 inhibitor treatment.

There are several limitations to the present study. First, the clinical samples were obtained from a relatively small patient population and at early disease stages, given that patients at an inoperable stage could not be included. Second, the clinical samples were limited to tumor tissue, with no corresponding serum samples being available. Third, the study explored miRNA-mediated regulation of CD155 expression largely in lung adenocarcinoma, with further investigation being necessary to assess the potential role of miR-326 in tumor or serum samples as a resistance biomarker for PD-1/PD-L1 inhibitors in patients with various cancer types.

In conclusion, our study has shown that miR-326 negatively regulates CD155 expression in lung adenocarcinoma. Our findings suggest that downregulation of miR-326 results in upregulation of CD155 expression and might therefore promote tumor progression and the development of resistance to PD-1/PD-L1 inhibitors.

Financial Support: This work was supported by Japan Society for the Promotion of Science KAKENHI grant number 21K15553.

Corresponding Author: Yasuto Yoneshima, MD, PhD, Department of Respiratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: +81-92-642-5378. Fax: +81-92-642-5382.

Email: [email protected]

Authors’ Disclosures: The authors declare no competing interests.

Acknowledgments

We thank Cell Innovator Co. Ltd. and The Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences, for technical assistance.

Authors’ Contributions

T. Nakanishi: Methodology, formal analysis, data curation, investigation, writing–original draft. Y. Yoneshima: Conceptualization, methodology, formal analysis, data curation, investigation, funding acquisition, project administration, writing–original draft, writing–review and editing. K. Okamura: Methodology, writing–review and editing. M. Hashisako: Methodology, writing–review and editing. N. Haratake: Resources, writing–review and editing. K. Ota: Methodology, writing–review and editing. E. Iwama: Methodology, writing–review and editing. T. Takenaka: Resources, writing–review and editing. K. Tanaka: Methodology, writing–review and editing. T. Yoshizumi: Writing–review and editing. Y. Oda: Writing–review and editing. I. Okamoto: Supervision, writing–review and editing.

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MicroRNA-326 Negatively Regulates CD155 Expression in Lung Adenocarcinoma

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Abstract

Figures

Introduction

Materials And Methods

miRNA target prediction

Cell Culture

Mirna Transfection

Construction Of Reporter Plasmids

Luciferase Reporter Assay

Rna Extraction And Rt-qpcr Analysis

Flow Cytometry

Immunoblot Analysis

Patients And Tissue Samples

Immunohistochemistry

Statistical analysis

Data Availability

Results

Identification of miRNAs as putative regulators of CD155 expression

Evaluation Of Mirna Binding To Cd155 Mrna

Regulation Of Cd155 Expression By Mirnas In Nsclc Cells

Validation Of Mirna Binding Sites In The 3′-utr Of Cd155 Mrna

Association Of Mir-326 Abundance And Cd155 Expression In Lung Adenocarcinoma Patients

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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