| Cervical cancer is a serious female gynaecological oncology disease and is the fourth most common cancer among women worldwide.Over500,000 new cases of cervical cancer are diagnosed each year,resulting in over 300,000 deaths.Despite significant advances in diagnosis and treatment,cervical cancer is still one of the leading causes of cancer deaths.Hence,investigating the oncogenic mechanism of cervical cancer can be more beneficial to the clinical treatment of cervical cancer.TGR5 is a transmembrane protein receptor that affects metabolic homeostasis,belongs to the G protein-coupled receptor superfamily,and is widely expressed in various tissues.The role of TGR5 in metabolism-related processes has been a hot topic of research;meanwhile,TGR5 has also been shown to be involved in regulating physiopathological processes such as inflammation,gastric cancer and liver cancer,but the role of TGR5 in cervical cancer has never been reported.Therefore,investigating the mechanism of TGR5 in the process of cervical cancer may provide new ideas for the clinical treatment of cervical cancer.Long non-coding RNAs(lnc RNAs)are a class of non-coding RNAs that are more than 200 nt in length and are widely expressed in all types of organizations.Although lnc RNAs do not have the ability to encode proteins,they can participate in regulating various physiological processes by interacting with proteins,mi RNAs and other bioactive molecules.Meanwhile,a large number of studies have shown that lnc RNAs also play an important role in the development of cancer,and researches on the role of lnc RNAs in cancer may provide new potential targets for cancer treatment.This paper mainly explores the role and regulation molecular mechanism of TGR5 in cervical cancer.The role of TGR5 in the process of cervical inflammatory response was used as an entry point to verify the inhibitory effect of TGR5 activation on cervical inflammation as well as cervical cancer.Subsequently lnc RNA high-throughput sequencing technology was used to screen the differentially expressed lnc RNA after TGR5 activation,and revealed the molecular mechanism by which TGR5 regulated its expression.The specific molecular pathways underlying the role of the TGR5-HCP5 signaling axis in cervical cancer were then further explored.The main content and conclusions of the paper are as follows.Through analyzing the expression level of TGR5 in cervical cancer tissue and its relationship with the prognosis of patients,the antagonistic effect of TGR5 on cervical cancer was preliminarily determined.Subsequent animal experiments showed that the expression level of inflammatory genes in cervical tissues of TGR5 knockout mice(TGR5 KO)was higher than that in wild-type(WT)mice.And TGR5 KO mice were more sensitive to bacterial lipopolysaccharide(LPS)-induced inflammation,exhibiting severe inflammation reaction in cervix.At the same time,after feeding WT mice with TGR5 agonist to activate TGR5 in vivo,LPS-induced cervical inflammation in mice was significantly inhibited.The results of mechanism experiments showed that TGR5 activation can inhibit the activation of STAT3 pathway to antagonize the development of cervical inflammation in mice.In vitro experiments,TGR5 activation could inhibit the proliferation and cell clone formation of cervical cancer cells and induce apoptosis and can also inhibit the growth of cervical cancer cells in vivo.In summary,TGR5 activation antagonizes the development of cervical inflammation and cancer,suggesting its potential as a target for the treatment of cervical-related diseases.In order to further explore the downstream molecular mechanism of TGR5 antagonizing cervical cancer,lnc RNA HCP5 was screened out as a downstream lnc RNA through lnc RNA sequencing.The results of cell proliferation assay and cell apoptosis experiments confirmed that HCP5 participated in the process of TGR5 antagonizing cervical cancer.Then,the results of luciferase reporter gene assay(luciferase)and chromatin immunoprecipitation(Ch IP)assay suggested that TGR5 activation can inhibit the nuclear translocation of p65 protein in the cytoplasm and weaken the induction of p65 on HCP5 transcriptional activity,thereby downregulating the expression of HCP5.In addition,HCP5 is highly expressed in cervical cancer tissues and its expression is positively correlated with poor prognosis of patients.The results of in vivo and in vitro experiments confirmed that down-regulating of HCP5 can inhibit the cell proliferation,colony formation,migration and tumor growth of cervical cancer cells in nude mice.The above results indicate that HCP5 plays a pro-cancer role in cervical cancer;they also suggest that TGR5 can play a role in inhibiting cervical cancer by down-regulating the expression of HCP5.In order to further explore the potential downstream molecules and pathways by which HCP5 promoting cervical cancer progression,the molecular mechanism of HCP5 regulation of cervical cancer have been fully investigated.Via analyzing the subcellular localization of HCP5,it was proved that HCP5 has a significant distribution in the cytoplasm of cervical cancer cells,indicating that HCP5 may regulate cervical cancer through the mechanism of competing endogenous RNAs(ce RNA).The downstream mi RNA regulated by HCP5—mi R-139-5p was screened via using bioinformatics analysis tools,and the mutual regulation relationship between HCP5 and mi R-139-5p was confirmed by q RT-PCR.At the same time,the results of luciferase experiments proved the interaction between HCP5 and mi R-139-5p,and RNA co-immunoprecipitation(RIP)further confirmed the binding interaction between HCP5 and mi R-139-5p.Next,according to the mechanism of mi RNA’s biological function,DNA damage-induced transcript 4(DDIT4)was predicted as the target gene of mi R-139-5p through using the bioinformatics analysis tools.Through the analysis of cervical cancer-related data sets in TCGA and GEO databases,it was determined that DDIT4 was upregulated in cervical cancer tissues and was significantly correlated with poor prognosis of patients.The inhibitory effect of mi R-139-5p on DDIT4 was confirmed by q RT-PCR,western blot and luciferase experiment.Next,we explored the mutual regulatory relationship among HCP5,mi R-139-5p,and DDIT4 by setting up complementary experiments in different groups.expression levels,thereby promoting the development of cervical cancer.To sum up,the study of this topic firstly reveals that TGR5 acts as a negative regulator to inhibit the development of cervical inflammation and cancer,and also specifically elucidated that TGR5 activation could exert tumor suppressive role via regulating the HCP5/mi R-139-5p/DDIT4 signaling axis.These results not only enrich the biological function of TGR5,but also provide new potential targets for the treatment of cervical cancer. |
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