In vitro and in vivo growth inhibition of human cervical cancer cells via human papillomavirus E6/E7 mRNAs' cleavage by CRISPR/Cas13a system.

Sustained infection of high-risk human papillomavirus (HR-HPVs), especially HPV16 and HPV18, is a major cause of cervical cancer. E6 and E7 oncoproteins, encoded by the HPV genome, are critical for transformation and maintenance of malignant phenotypes of cervical cancer. Here, we used an emerging programmable clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13a system to cleave HPV 16/18 E6/E7 messenger RNAs (mRNAs). The results showed that customized CRISPR/Cas13a system effectively and specifically knocked down HPV 16/18 E6/E7 mRNAs, inducing growth inhibition and apoptosis in HPV16-positive SiHa and HPV18-positive HeLa Cell lines, but not in HPV-negative C33A cell line. Simultaneously, we detected downregulation of E6/E7 oncoproteins and upregulation of tumor suppressor P53 and RB proteins. In addition, we used subcutaneous xenograft tumor growth assays to find that the weight and volume of tumors in the SiHa-16E6CR1 group knocked down by the CRISPR/Cas13a system were significantly lower than those in the SiHa-VECTOR group lacking crRNA. Our study demonstrated that targeting HPV E6/E7 mRNAs by the CRISPR/Cas13a system may be a candidate therapeutic strategy for HPV-related cervical cancer.

Chen Y ，Jiang H ，Wang T ，He D ，Tian R ，Cui Z ，Tian X ，Gao Q ，Ma X ，Yang J ，Wu J ，Tan S ，Xu H ，Tang X ，Wang Y ，Yu Z ，Han H ，Das BC ，Severinov K ，Hitzeroth II ，Debata PR ，Xu W ，Fan W ，Jin Z ，Cao C ，Yu M ，Xie W ，Huang Z ，Hu Z ，You Z ... -  《-》

Honeybee venom possesses anticancer and antiviral effects by differential inhibition of HPV E6 and E7 expression on cervical cancer cell line.

Kim YW ，Chaturvedi PK ，Chun SN ，Lee YG ，Ahn WS ... -  《-》

Gene Targeting of HPV18 E6 and E7 Synchronously by Nonviral Transfection of CRISPR/Cas9 System in Cervical Cancer.

Ling K ，Yang L ，Yang N ，Chen M ，Wang Y ，Liang S ，Li Y ，Jiang L ，Yan P ，Liang Z ... -  《-》

HPV E6/E7 promotes aerobic glycolysis in cervical cancer by regulating IGF2BP2 to stabilize m(6)A-MYC expression.

Enhanced aerobic glycolysis constitutes an additional source of energy for tumor proliferation and metastasis. Human papillomavirus (HPV) infection is the main cause of cervical cancer (CC); however, the associated molecular mechanisms remain poorly defined, as does the relationship between CC and aerobic glycolysis. To investigate whether HPV 16/18 E6/E7 can enhance aerobic glycolysis in CC, E6/E7 expression was knocked down in SiHa and HeLa cells using small interfering RNA (siRNA). Then, glucose uptake, lactate production, ATP levels, reactive oxygen species (ROS) content, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were evaluated. RNA-seq was used to probe the molecular mechanism involved in E6/E7-driven aerobic glycolysis, and identified IGF2BP2 as a target of E6/E7. The regulatory effect of IGF2BP2 was confirmed by qRT-PCR, western blot, and RIP assay. The biological roles and mechanisms underlying how HPV E6/E7 and IGF2BP2 promote CC progression were confirmed in vitro and in vivo. Human CC tissue microarrays were used to analyze IGF2BP2 expression in CC. The knockdown of E6/E7 and IGF2BP2 attenuated the aerobic glycolytic capacity and growth of CC cells, while IGF2BP2 overexpression rescued this effect in vitro and in vivo. IGF2BP2 expression was higher in CC tissues than in adjacent tissues and was positively correlated with tumor stage. Mechanistically, E6/E7 proteins promoted aerobic glycolysis, proliferation, and metastasis in CC cells by regulating MYC mRNA m6A modifications through IGF2BP2. We found that E6/E7 promote CC by regulating MYC methylation sites via activating IGF2BP2 and established a link between E6/E7 and the promotion of aerobic glycolysis and CC progression. Blocking the HPV E6/E7-related metabolic pathway represents a potential strategy for the treatment of CC.

Hu C ，Liu T ，Han C ，Xuan Y ，Jiang D ，Sun Y ，Zhang X ，Zhang W ，Xu Y ，Liu Y ，Pan J ，Wang J ，Fan J ，Che Y ，Huang Y ，Zhang J ，Ding J ，Yang S ，Yang K ... -  《International Journal of Biological Sciences》

Transcriptional gene silencing of HPV16 E6/E7 induces growth inhibition via apoptosis in vitro and in vivo.

Transcriptional silencing of HPV oncogenes using short interfering RNA (siRNA) blocks E6/E7 expression. Our objective was to estimate the effective value of E6/E7 specific siRNA-induced transcriptional gene silencing as a potential therapeutic strategy for cervical cancer. In vitro studies were performed by employing two categories of siRNA targeting promoter of E6/E7 gene and E7 transcript, respectively, and inhibitory effect of both siRNAs was further observed in vitro and on xenograft in BALB/c mice that were inoculated with siRNA transfected SiHa cells and parental SiHa cells followed by siRNA intratumoral injection in vivo. Tumor volume and growth curves were assessed. Furthermore, cellular proliferation and apoptosis of inoculated tumors were determined by immunohistochemistry staining and TUNEL assay. The two most active siRNA sequences specifically knockdown E6/E7 expressions at mRNA level in HPV16 positive Siha cells, increased p53 and decreased p16 expressions at protein level, inhibited cell proliferation, and induced cell apoptosis in vitro. Furthermore, both siRNAs effectively inhibited tumor formation and growth no matter in mice with siRNA transfected cells in vitro or with siRNA intratumoral injection in vivo. TUNEL staining and FCM assay consistently showed that tumor retardation was through induction of cellular apoptosis. RNAi targeting the promoter of HPV16 E6/E7 acts effectively in vitro and in vivo, especially through intratumoral delivery, and may be a candidate therapeutic strategy for cervical cancer.

Zhou J ，Peng C ，Li B ，Wang F ，Zhou C ，Hong D ，Ye F ，Cheng X ，Lü W ，Xie X ... -  《-》