Dataset Information

Reprogrammed CRISPR-Cas13a targeting the HPV16/18 E6 gene inhibits proliferation and induces apoptosis in E6-transformed keratinocytes.

ABSTRACT: The long-lasting infection of high-risk type (type 16 or type 18) human papillomavirus (HPV) is the main cause of gynecological and urinary malignancies. Given the high mortality rate after surgery, the development of a new molecular therapy would be of value to clinicians. The aim of the present study was to achieve targeted inactivation of the viral E6 gene in keratinocytes using the reprogrammed CRISPR-Cas13a system. To accomplish this, a reprogrammed CRISPR-Cas13a system, targeting both the HPV16/18 E6 genes was constructed using a guide RNA expressing vector. The expression levels of E6 protein were measured using western blot analysis after transfection of the vector into E6-transformed keratin oocytes. Cell proliferation was analyzed using CCK-8 assays and cell apoptosis was evaluated using Hoechst 33258 staining and ELISAs examining caspase-3 levels. The results indicated that both the HPV16/18 E6 genes can be inactivated using the CRISPR-Cas13a system. Furthermore, silencing E6 inhibited cell proliferation (14±1.8% on average) and induced apoptosis (80.2±3.2% on average) in E6-transformed keratinocytes but not in normal keratinocytes. In conclusion, results of the present study demonstrate that the reprogrammed CRISPR-Cas13a system has the potential for inactivating HPV E6 gene functions.

SUBMITTER: Li C

PROVIDER: S-EPMC7185167 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Reprogrammed CRISPR-Cas13a targeting the HPV16/18 E6 gene inhibits proliferation and induces apoptosis in E6-transformed keratinocytes.

Li Chunjing C Guo Liwen L Liu Guoqing G Guo Mingjuan M Wei Huiling H Yang Qiqiong Q Wang Jianfeng J Chen Huihua H

Experimental and therapeutic medicine 20200331 6

The long-lasting infection of high-risk type (type 16 or type 18) human papillomavirus (HPV) is the main cause of gynecological and urinary malignancies. Given the high mortality rate after surgery, the development of a new molecular therapy would be of value to clinicians. The aim of the present study was to achieve targeted inactivation of the viral E6 gene in keratinocytes using the reprogrammed CRISPR-Cas13a system. To accomplish this, a reprogrammed CRISPR-Cas13a system, targeting both the ...[more]

PMID: 32346451

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Project description:Keratinocyte infection with high-risk human papillomavirus genotypes has been linked to cancer development. In cervix, the alpha HPV16 and HPV18 have been reported as the mayor causative agents of cervical cancer. Oncogenic progression and chronic inflammation are closely related processes, with IL-6 as one of the main pro-inflammatory cytokines involved. However, there are limited studies about the regulation of IL-6 by low and high risk HPVs and the HPV proteins implicated in this modulation. In this work, we report the overexpression of IL-6 in HPV infected cervical cancer derived cell lines (HeLa and SiHa) compared to non-tumorigenic keratinocytes (HaCaT), and in Cervical Intraepithelial Neoplasia grade 1 HPV16 and HPV18 positive cervical samples compared to HPV negative samples without lesions. Moreover, we generated HaCaT keratinocytes that express E5, E6, and E7 from high risk (16 or 18) or low risk (62 and 84) HPVs. E5 proteins do not modify IL-6 expression, while E7 modestly increase it. Interestingly, E6 proteins in HaCaT cells upregulate IL-6 mRNA expression and protein secretion. Indeed, in HaCaT cells that express high risk HPV16E6 or HPV18E6 proteins, only the truncated E6* isoforms were expressed, showing the stronger IL-6 overexpression, while in HaCaT cells that express low risk HPV62 and HPV84 full length E6 proteins, IL-6 was also upregulated but not so drastically. Since HaCaT cells have a mutated p53 form that is not degraded by the introduction of E6 or E6/E7, it seems that E6/E7 regulate IL-6 by an additional mechanism independent of p53. In addition, basal keratinocytes showed a strong expression of IL-6R using immunohistochemistry, suggesting an autocrine mechanism over proliferative cells. Altogether, IL-6 cytokine expression in keratinocytes is upregulated by E6 and E7 proteins from HPVs 16, 18, 62, and 84, especially by high risk HPV16 and HPV18 E6*, which may contribute to promote a pro-inflammatory and highly proliferative microenvironment that can persist over time and lead to cervical tumorigenesis.

Project description:Different types of cells infected with Epstein-Barr virus (EBV) can release exosomes containing viral components that functionally affect neighboring cells. Previously, we found that EBV was localized mostly in infiltrating lymphocytes within the stromal layer of cervical lesions. In this study, we aimed to determine effects of exosome-transferred EBV-encoded RNAs (EBERs) on keratinocytes expressing human papillomavirus (HPV) 16 E6/E7 (DonorI-HPV16 HFKs). Lipid transfection of in vitro-transcribed EBER1 molecules (ivt EBER1) into DonorI-HPV16 HFKs caused strong induction of interferon (IFN)-related genes and interleukin 6 (IL-6). To gain insights into the physiological situation, monocyte-derived dendritic cells (moDCs), low passage DonorI-HPV16 HFKs and primary keratinocytes were used as recipient cells for internalization of exosomes from wild-type EBV (wt EBV) or B95-8 EBV-infected lymphoblastoid cell lines (LCLs). qRT-PCR was used to determine the expression of EBER1, HPV16 E6/E7, IFN-related genes and IL-6 in recipient cells. The secretion of inflammatory cytokines was investigated using cytometric bead array. Wt EBV-modified exosomes induced both IFN-related genes and IL-6 upon uptake into moDCs, while exosomes from B95-8 EBV LCLs induced only IL-6 in moDCs. Internalization of EBV-modified exosomes was demonstrated in DonorI-HPV16 HFKs, yielding only EBER1 but not EBER2. However, EBER1 transferred by exosomes did not induce IFN-related genes or IL-6 expression and inflammatory cytokine secretion in DonorI-HPV16 HFKs and primary keratinocytes. EBER1 copy numbers in exosomes from wt EBV-infected LCLs were 10-fold higher than in exosomes from B95-8 LCLs (equal cell equivalent), whereas ivt EBER1 was used at approximately 100-fold higher concentration than in exosomes. These results demonstrated that the induction of IFN-related genes and IL-6 by EBER1 depends on quantity of EBER1 and type of recipient cells. High levels of EBER1 in cervical cells or infiltrating dendritic cells may play a role in the inflammation-to-oncogenesis transition of HPV-associated cervical cancer through modulation of innate immune signals.

Dataset Information

Reprogrammed CRISPR-Cas13a targeting the HPV16/18 E6 gene inhibits proliferation and induces apoptosis in E6-transformed keratinocytes.

Publications

Reprogrammed CRISPR-Cas13a targeting the HPV16/18 E6 gene inhibits proliferation and induces apoptosis in E6-transformed keratinocytes.

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