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. 2021 Dec 29;23(1):373.
doi: 10.3390/ijms23010373.

High-Grade Cervical Intraepithelial Neoplasia (CIN) Associates with Increased Proliferation and Attenuated Immune Signaling

Affiliations

High-Grade Cervical Intraepithelial Neoplasia (CIN) Associates with Increased Proliferation and Attenuated Immune Signaling

Irene Tveiterås Øvestad et al. Int J Mol Sci. .

Abstract

Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

Keywords: CIN progression; cervical cancer screening; differential gene expression; transcriptomic analysis.

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Conflict of interest statement

The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Overview of the index biopsies and additional cervical samples, before and after the index biopsies, from the women included in the study.
Figure 2
Figure 2
Gene expression from the 27 cervical biopsies (14 normal and 13 CIN3/AIS biopsies) were analyzed using Oncomine™ Immune Response Research Assay. (A) Distribution of p-values (−log10) as a function of fold change (log2) between CIN3/AIS and normal biopsies. Differentially expressed genes (DEGs, fold change > |2| and p < 0.05) showing downregulation in CIN3/AIS are depicted in green, whereas genes upregulated in CIN3/AIS are depicted in orange. (B) Hierarchical clustering based on the 27 significant DEGs with correlation clustering distance and average linkage. The HPV status is included; HPV16 (green), HPV18 (orange), HPV52 (purple) and HPV negative (pink). (C) Principal component analysis (PCA) of the first two principal components of the gene expression data. Each point represents a biopsy, normal (green), and CIN3/AIS biopsies (orange).
Figure 3
Figure 3
Protein expression of NCAM1. (A) Boxplots illustrating the distribution of NCAM1 positive cells/1.0 mm2 of normal and CIN3/AIS biopsies: in total (orange), in the epithelium (green) and in the stroma (olive green). Immunohistochemical staining of NCAM1 positive lymphocytes in (B) a normal biopsy and (C) a CIN3 biopsy (40× magnification).
Figure 4
Figure 4
Ingenuity Pathway analysis (IPA) illustrates the association between the transcription factors FOXM1, JUN, MXD1 and MYC and differentially expressed genes in CIN3/AIS and normal biopsies, limited to z score, ≥2 (activation) and ≤−2 (inhibition) and overlay of KIAA0101.

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