| Objective: In recent years,with the widespread use of early screening strategies,the incidence and mortality of cervical cancer have declined significantly.However,cervical cancer remains one of the major malignancies leading to morbidity and death among women,especially in developing countries.There were about 600,000 new cases of cervical cancer worldwide in 2020,and more than 340,000 deaths.According to the statistics released by the National Cancer Center in 2019,the incidence of cervical cancer in China ranked sixth among female malignancies,and the mortality ranked eighth among female malignancies.The incidence and death of cervical cancer have a tendency to be "younger".When cervical cancer metastases or recurs,the 5-year overall survival rate of patients is only 17%,which seriously affects the health of women.Therefore,it is still necessary to explore the mechanism of the occurrence and development of cervical cancer and to seek new therapeutic targets.HPV is unenveloped DNA virus with circular double-stranded genome,which is the first virus to be identified as the cause of cancer.Persistent high risk HPV serotype infection,especially HPV16 and 18 types,is an important risk factor for cervical cancer.HPV16 is responsible for more than 50% of cervical cancers,while HPV16/18 are responsible for more than 70% of cervical cancers worldwide.Cervical squamous cell carcinoma is mainly associated with HPV16,while cervical adenocarcinoma is mainly associated with HPV18.The HPV genome can encode viral proteins,which are involved in viral genome replication and transcription,viral assembly and infection,promotion of the integration of virus genome with the host chromosomes,and disruption of the molecular regulatory network and genomic stability of host cells at genomic level,transcriptional level and protein level,resulting in the malignant transformation of host cells.In particular,the E6 and E7 proteins encoded by the high risk HPV genome play an important role in the initiation and progression of cervical cancer.Therefore,most of the current studies on cervical cancer focused on the effects of high risk HPV infection on the host cells,especially the effects of the viral oncoproteins E6/E7 and the downstream molecules and pathways of E6/E7 translated from the high risk HPV genome on the host cells.These studies have not only led to a better understanding of the molecular pathways disrupted by the virus,but also led to some breakthroughs in targeted therapy of cervical cancer.Cervical squamous cell carcinoma is the most common pathological type of cervical cancer.This study sought to explore the role of HCN2 in cervical squamous cell carcinoma and its upstream and downstream mechanisms.Methods: 1.Kaplan-Meier plotter and GEPIA websites were used to observe the prognostic information of HCN2 in cervical cancer.The expression level of candidate research target HCN2 in normal cervical tissues(n=29)and cervical cancer tissues(n=113,including 87 cases of cervical squamous cell carcinoma)was detected by RT-q PCR,and the correlation between its expression level and the clinicopathological features of cervical cancer was analyzed.By means of si RNA interference,the expression of HCN2 was silenced in the cervical squamous cell carcinoma cell lines Si Ha and Ca Ski.MTT assay and EDU assay were used to detect the effect of HCN2 interference on the proliferation of cervical squamous cell carcinoma cell lines.The effect of HCN2 interference on the migration of cervical squamous cell carcinoma cell lines was detected by cell scratch assay.The effect of HCN2 interference on the invasion of cervical squamous cell carcinoma cell lines was detected by Transwell assay.The effect of HCN2 interference on the apoptosis of cervical squamous cell carcinoma cell lines was detected by apoptosis assay.Western blot was used to detect the effect of HCN2 knockdown on the cervical cancer metastasis-and anti-apoptosis-related molecules Bcl2 and survivin.2.Total cell proteins of si-scramble transfection group and si-HCN2 transfection group were collected,and hybridized with antibody chips containing 55 targets of Akt,JAK/STAT,MAPK,NFk B and TGF-beta pathways to screen out proteins that might be regulated by HCN2.The screened differential proteins were enriched by KEGG pathway analysis.The affected key protein and key pathway were verified by RT-q PCR and Western blot.3.The expression of HPV16 E6 was interfered in HPV16 positive cervical squamous cell carcinama cell lines Si Ha and Ca Ski,and the regulation of HCN2 by HPV16 E6 was detected by Western blot.Target Scan Human and ENCORI bioinformatics websites were used to predict micro RNAs that might bind with HCN2 m RNA 3'UTR.The binding of related micro RNA to the 3'UTR of HCN2 m RNA was verified by duel luciferase reporter gene assay.Western blot was used to verify the mutual regulation between HPV16 E6 downstream protein and HCN2.Results:1.According to the information on the Kaplan-Meier plotter and GEPIA websites,the high expression of HCN2 was associated with poor prognosis of cervical cancer.RT-q PCR results showed that in cervical cancer tissues(n = 113)or just in cervical squamous cell carcinoma tissues(n = 87),the expression level of HCN2 was significantly higher than that of normal cervical tissues(n = 29).MTT and Ed U assay showed that HCN2 interference inhibited the proliferation ability of cervical squamous cell carcinoma cell lines.Cell scratch assay showed that HCN2 interference inhibited the migration ability of cervical squamous cell carcinoma cell lines.Transwell assay showed that HCN2 interference inhibited the invasion ability of cervical squamous cell carcinoma cell lines.Apoptosis assay showed that HCN2 interference promoted apoptosis of cervical squamous cell carcinoma cell lines.Western blot analysis showed that HCN2 knockdown inhibited the expression of Bcl2 and survivin.2.Antibody chips results showed that HCN2 interference significantly reduced the activation of p38,which was confirmed again by Western blot.KEGG pathway enrichment analysis showed that PD-L1 expression and PD-1 checkpoint pathway in cancer might be the key pathway that was affected by HCN2 in cervical squamous cell carcinama cell lines.RT-q PCR and Western blot results showed that HCN2 knockdown resulted in the decrease of PD-L1 in both protein and RNA levels.Western blot results showed that HCN2 knockdown resulted in the decrease of MYC protein,which regulated PD-L1 m RNA transcription.3.The expression of HPV16 E6 was interfered in HPV16 positive cervical squamous cell carcinama cell lines Si Ha and Ca Ski,and found the expression of HCN2 was down-regulated by Western blot.Bioinformatics websites Target Scan Human and ENCORI both predicted that there were two miR-137-3p binding sites in HCN2 m RNA3'UTR.Dual luciferase assay confirmed that miR-137-3p could bind to HCN2 m RNA3'UTR.Western blot results showed that the interference of EZH2 that was the downstream protein of HPV16 E6 and which m RNA 3'UTR was also the target of miR-137-3p,decreased the expression of HCN2.On the basis of EZH2 interference,miR-137-3p inhibitors treatment reversed the expression of HCN2.Western blot also showed that HCN2 knockdown reduced the expression of EZH2.On the basis of HCN2 interference,miR-137-3p inhibitors treatment also reversed the expression of EZH2.Conclusion: 1.Kaplan-Meier plotter and GEPIA websites showed that the high expression of HCN2 is a factor of poor prognosis of cervical cancer.Histological detection showed that HCN2 expression in cervical squamous cell carcinoma was higher than that in normal cervical tissues.HCN2 interference reduced the proliferation,migration and invasion of cervical squamous cell carcinoma cell lines and promoted cell apoptosis.2.HCN2 promoted the activation of p38 in cervical squamous cell carcinama cell lines and regulated the expression of PD-L1 through MYC.3.HCN2 was regulated by EZH2 that was the downstream protein of HPV16 E6 through competitive endogenous RNA pathways.At the same time,HCN2 also regulated the expression of EZH2 by sponging miR-137-3p. |
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