| Introduction: Cervical cancer is one of the most common malignant tumors in women.In 2020,there are more than 600,000 new cases of cervical cancer and more than340,000 patients dead worldwide.Globally,cervical cancer occupies both fourth place in the incidence and mortality of female cancer.Due to effective primary(HPV vaccine)and secondary(screening)preventive measures,cervical cancer is considered preventable cancer.However,these measures have not been widely implemented worldwide.Compared to the developed countries,the incidence of cervical cancer in the developing countries is still 7-10 times higher,and the mortality rate gap can reach up to 18 times.Therefore,studying the molecular mechanism of cervical cancer,looking for opportunities to treat invasive tumors,and discovering molecular targets for early diagnosis and treatment have important theoretical significance for reducing the heavy burden of cervical cancer worldwide and improving the inequality of cervical cancer treatment.MYCT1(Myc target 1)gene is related to laryngeal cancer discovered by our research group using electronic hybridization technology.It is located on chromosome6q25 and encodes 235 amino acids.It is the direct target gene of the MYC gene and has tissue specificity and regulatory complexity in different tumors.In laryngeal,gastric,and liver cancer,MYCT1 is low expressed,exerting tumor suppressor function.Its role in hematological tumors is controversial,and it can play a role as a tumor suppressor or oncogene.Moreover,its related research in cervical cancer has not been reported yet.micro RNA is a small non-coding RNA that exists widely in various species.It regulates the expression of target genes by binding to m RNA 3’UTR and is an important molecular target for disease diagnosis and treatment.Among them,miR-106 b is the focus of current research.miR-106 b plays a vital role in various tumors.miR-106 b is highly expressed in cervical cancer.Inhibition of miR-106 b expression can significantly repress cervical cancer cells’ migration and invasion.In cervical cancer cells,HPV oncoprotein upregulates the miR-106 b gene expression,suggesting that miR-106 b presents a vital role in the occurrence and development of cervical cancer,especially HPV-positive cervical cancer,but the specific regulatory mechanism is still unclear.Through bioinformatics prediction,we have found that miR-106 b is negatively correlated with MYCT1 gene expression,and there is a miR-106 b binding site in the3’UTR region of MYCT1 suggesting that MYCT1 may be a potential target gene of miR-106 b.The interferon regulatory factor 1(IRF-1)gene encodes a transcription inhibitor response to viruses and bacteria and plays a vital role in multiple biological processes.Acts as a tumor suppressor,IRF-1 not only inhibits the growth of tumor cells but also stimulates the immune response against tumor cells.By inhibiting the expression of P21 and P53,HPV16 E6 in cervical cancer can inhibit IRF-1-induced apoptosis and promote cell growth.In addition,HPV16 E7 interferes with its transactivation ability by recruiting histone deacetylase to the IRF-1 promoter.The MCM7 is the miR-106 b host gene.We have also found that its promoter region has been predicted to have an IRF-1 binding site,suggesting that IRF-1 may regulate the expression of miR-106 b through the IRF-1binding site of MCM7.In summary,we speculate that there may be an IRF-1/miR-106/MYCT1 signal axis in the human body,and MYCT1 may be regulated by this signal axis and play an important role in cervical squamous cell carcinoma.Therefore,we will first clarify the essential role of MYCT1 in cervical squamous cell carcinoma and then conduct an in-depth and comprehensive study of its upstream regulatory mechanism to further verify the biological role of this signal axis in cervical cancer in this study.The results will enrich the mechanism of MYCT1 in tumorigenesis and provide an essential clue for finding meaningfully molecular targets related to the axis in cervical cancer diagnosis and treatment.Materials and Methods: 1.Materials: Caski and Siha cell lines,human squamous cell carcinoma tissue specimens,normal cervical tissue specimens.2.Methods: immunohistochemistry technology,cell culture,gene transient transfection,RT-q PCR,Western Blot,CCK-8 experiment,transwell experiment,Ch IP,dual-luciferase reporter gene activity detection.Results: 1.RT-q PCR result showed that in cervical squamous cell carcinoma tissue,the expression level of MYCT1 m RNA was significantly lower(P<0.001).Immunohistochemistry results displayed that the expression level of MYCT1 protein in cervical squamous cell carcinoma tissue was significantly lower(P<0.001).Statistical analysis of clinical data showed that,in cervical squamous cell carcinoma,MYCT1 expression level was significantly negatively correlated with tumor diameter(R=-0.523,P<0.01),significantly negatively correlated with clinical stage(R=-0.614,P<0.01)and significantly negatively correlated with distant metastasis of cervical cancer(R=-0.243,P<0.05).2.CCK-8 result showed that overexpressed MYCT1 Caski and Siha cells’ proliferation ability was significantly lower(P<0.05).On the contrary,knocked down MYCT1,the proliferation ability was significantly increased(P<0.05).Transwell results revealed that overexpressed MYCT1 Caski and Siha cells’ migration and invasion abilities were significantly lower(P<0.05).On the contrary,both the migration and invasion abilities of Caski and Siha cells knocking down MYCT1 were significantly increased(P<0.05).3.RT-q PCR and Western Blot results showed,in transfected with miR-106 b mimic Caski and Siha cells,MYCT1 was significantly downregulated at m RNA and protein levels,respectively(P<0.01).Whereas transfected with miR-106 b inhibitor,MYCT1 was significantly upregulated both at m RNA and protein levels,respectively(P<0.01).The bioinformatics prediction results displayed that there are potential binding sites for miR-106 b in the 3’UTR region of MYCT1.In cervical squamous cell carcinoma Caski cells,the luciferase activity of wild-type MYCT1 3’UTR and miR-106 b mimic co-transfected group was significantly lower than that of the control group(P<0.01),and mutant MYCT1 3’UTR and miR-106 b mimic co-transfected group had no significant change compared with the control group(P>0.05).4.RT-q PCR and Western Blot results showed that in IRF-1 knocked-down Caski and Siha cells,IRF-1 m RNA and protein levels were significantly lower(P<0.01).MCM7 is the host gene of miR-106 b.Bioinformatics prediction result revealed that,in the promoter region of MCM7,there were two adjacent IRF-1 potential binding sites.RT-q PCR results showed that the expression of miR-106 b and MCM7 both significantly increased in Caski and Siha cells knocked down IRF-1(P<0.01).In addition,both Ch IP and dual-luciferase reporters showed that IRF-1 could directly bind the MCM7’s promoter in vivo and in vitro.5.RT-q PCR and Western Blot results showed that MYCT1 m RNA and protein levels in Caski and Siha cells of cervical squamous cell carcinoma with IRF-1 knockdown were significantly lower than that in the control group(P<0.01),while co-transfected with si IRF-1 and miR-106 b inhibitor,MYCT1 m RNA and protein levels were significantly higher than that of the knockdown group(P<0.05),suggesting that IRF-1 promotes the expression of MYCT1 through miR-106 b.6.CCK-8 result showed that,in Caski and Siha cells,miR-106 b inhibitor significantly suppressed the proliferation.si MYCT1 could significantly restore the effect(P< 0.05).Transwell results showed that,in Caski and Siha cells,miR-106 b inhibitor significantly inhibited migration and invasion.si MYCT1 could significantly restore the impact(P<0.05).7.CCK-8 results displayed that Caski and Siha cells knocked down IRF-1 had significantly higher proliferation capacities than those in the control group,respectively.miR-106 b inhibitor or MYCT1 could restore the effects of si IRF-1 on the proliferation abilities of Caski and Siha cells,respectively(P<0.05).Transwell results showed that,in knocked down IRF-1 Caski and Siha cells,the migration and invasion abilities were significantly higher than those of the control groups.miR-106 b inhibitor or MYCT1 can restore the effects of si IRF-1 on Caski and Siha cells’ migration and invasion ability(P<0.01),respectively.8.TCGA database analysis results showed that the levels of IRF-1 and miR-106 b were negatively correlated(R=-0.1342,P<0.05);miR-106 b and MYCT1 levels were negatively correlated(R=-0.2532,P<0.01).The lower level of IRF-1,the higher the tumor size,local invasion,lymph node,and distant metastasis(P<0.01);the higher expression level of miR-106 b,the higher the tumor size,local invasion,and distant metastasis rate(P<0.01);the lower expression level of MYCT1,the larger the tumor size,local invasion and distant metastasis rate(P<0.01).Conclusion: 1.MYCT1 downregulated in cervical squamous cell carcinoma.In cervical squamous cell carcinoma cells,MYCT1 inhibits proliferation,migration,and invasion.Thus,MYCT1 plays a tumor suppressor role in cervical squamous cell carcinoma.2.IRF-1/miR-106b/MYCT1 signaling pathway exists in cervical squamous cell carcinoma.miR-106 b represses MYCT1 expression by directly targeting its 3’UTR region;IRF-1 suppresses miR-106 b transcription directly binding to the MCM7’s promoter region.3.In cervical squamous carcinoma cells,IRF-1 inhibits the proliferation,migration,and invasion through the miR-106b/MYCT1 signal axis. |
PDF Full Text Request