| Breast cancer have become a common lethal disease,70%of these patients with estrogen receptor-positive（ER+）.Tamoxifen（TAM）is the gold standard for endocrine therapy of breast cancer patients with ER+,which has greatly improved the survival rate of patients.Although TAM showed fine anti-cancer effects,approximately 40%of patients still relapsed after they received TAM adjuvant therapy.Postmenopausal patients with early breast cancer initially respond well to TAM therapy.Recurrent tumors that occur in the breast and endometrium will become resistant to the drug over time,which greatly limits the application of TAM.In addition,some patients may also develop primary drug resistance,making the TAM initial treatment appears ineffective.Therefore,it has always been a hot topic for scholars to find effective methods to reverse drug resistance,prevent the development of drug resistance,and thus improve the endocrine therapy efficacy for breast cancer and the survival rate of patients.Long non-coding RNAs（lnc RNAs）and micro RNAs（mi RNAs）are the most widely studied nc RNAs.More and more evidences show that they are related to tumorigenesis,tumor progression and drug resistance.Studies have confirmed that lnc RNAs and mi RNAs can function as members of ce RNAs.They can regulate each other at the post-transcriptional level through competing for shared mi RNAs,thereby influencing a series of biological processes and link protein-coding m RNAs to the function of nc RNAs.The term RNA sponge was coined to describe this phenomenon.These currently reported ce RNA-related molecules are only a small part of the complex ce RNA network,continuous screening and validation is still needed to discover more reliable biomarkers so that more effective regulatory pathways can be found to elucidate the mechanism of tumorigenesis,development and drug resistance.Tumor research is a topic that scholars have discussed and difficult to overcome for many years.A suitable tumor model is not only conducive to the discovery of pathogenic mechanism,but also helpful to the development of effective antitumor drugs.Tumor cell lines are obtained from the patients by extraction and purification,they are the most widely studied disease model,which have the advantages of practicality,convenient operation and repeatability.As the life partner of human beings,dogs have similarities with humans in terms of living habits,environmental conditions and dietary structures.Canine mammary gland tumor（CMGT）is also a common disease in female dogs,its incidence is about three times that of humans,more than 50%of the cases are diagnosed as malignant tumors,causing serious harm to the health of dogs.The effects of surgical treatment alone are not satisfactory,and adjuvant treatment is often required.Using canine mammary tumors as the research object,screening differentially expressed lnc RNAs and mi RNAs in TAM-resistant and non-resistant cells,and exploring the mechanism involved in TAM resistance,can not only promote the research progress of veterinary clinical endocrinotherapy,but also contribute to discover and screen out more reliable molecular markers.The study protocol is shown below:Firstly,two TAM resistant canine mammary gland tumor（CMGT）cell lines,CHMpTAM and CHMmTAM respectively,were established by concentration gradient and drug maintenance method.Secondly,lnc RNAs and mi RNAs differentially expressed in CHMmTAM and CHMm were screened by RNA sequencing.The interaction between lnc RNA-mi RNA was analyzed.Five up/down-regulated lnc RNAs and mi RNAs were randomly selected and verified by q RT-PCR for the accuracy of sequencing results.Finally,the study explored the mechanisms of lnc RNAs and mi RNAs involvement in drug resistance.The experimental results are as follows:（1）In this study,two TAM-drug-resistant cell lines were cultured,named CHMpTAM and CHMmTAM respectively,they were collectively called CMGT/TR.Functional verification results showed that drug-resistant cells exhibited enhanced abilities of cell proliferation,migration,invasion and clone formation.The anti-apoptotic ability,EMT and CSCs character of drug-resistant cells were also enhanced.（2）Sequencing results showed that a total of 162 differentially expressed lnc RNAs were screened out in CHMmTAM and CHMM cells,and 66 were down-regulated and 96 were up-regulated in drug-resistant cells;29 mi RNAs were differentially expressed,of which 15 were down-regulated and 14 were up-regulated.The intersection of the two algorithms（Miranda（Score>150,Energy<-20）and RNAhybrid（Energy<-25））was used as the final result to draw multiple lnc RNA-mi RNA interaction diagrams,one mi RNA is regulated by multiple lnc RNAs,while one lnc RNA can also regulate multiple mi RNAs.The results of GO and KEGG enrichment analysis showed that the generation of drug resistance was mainly related to Hedgehog pathway,P53 pathway,Hippo pathway,MAPK pathway,Wnt pathway and other pathways.Ten randomly selected lnc RNAs and mi RNAs genes were validated by q RT-PCR in drug-resistant and parental cells,and lnc-45594,lnc-42060 and mi R-10b were found to be significantly up-regulated,and lnc-33364 and mi R-204-5p were significantly down-regulated.Combined with the lnc RNA-mi RNA interaction map,it was determined that lnc-42060 and mi R-204-5p might be involved in TAM resistance in breast tumors.（3）The results of q RT-PCR showed that lnc-42060 was up-regulated and mi R-204-5p was down-regulated in tumor tissues compared to the paracancerous,indicating that lnc-42060 and mi R-204-5p were not only involved in drug resistance generation but also in tumor development.The nucleus and cytoplasm separation experiments showed that lnc-42060 was mainly located in the cytoplasm suggesting that lnc-42060 may function as ce RNA.Bioinformatics,dual luciferase reporter gene and RIP experiments confirmed lnc-42060 could specifically bind to mi R-204-5p.Finally,rescue experiments showed that lnc-42060 as an oncogene could enhance cell proliferation,migration,cloning and microspheres formation ability and TAM chemical resistance,while mi R-204-5p as a cancer suppressor gene could inhibit generation and development of tumor.And the overexpression/silencing of mi R-204-5p and lnc-42060 at the same time counteracted each other’s effects.The above results suggest that lnc-42060 regulates breast tumor development and TAM drug resistance through mi R-204-5p.（4）Bioinformatics prediction and dual-luciferase assay confirmed that SOX4 is one of the target genes of mi R-204-5p.Rescue experiments showed that SOX4 expression was positively correlated with lnc-42060 and negatively correlated with mi R-204-5p.And then functional assays confirmed that SOX4 silencing resulted in reduction of proliferation,migration and clone formation in drug-resistant cells.The above results suggest that lnc-42060 can target SOX4 by acting as a mi R-204-5p molecular"sponge",which regulates the malignant phenotype and TAM chemical resistance of CMGT cells.（5）The in vivo nude mouse transplantation tumor experiment illustrated that silence lnc-42060 expression inhibited the tumor formation ability of CHMmTAM and reduce tumor volume and weight.In summary,differentially expressed lnc RNAs and mi RNAs were screened in breast tumor parents and drug-resistant cells.As a novel marker,lnc-42060 can play a role in promoting cancer through mi R-204-5p/SOX4 molecular axis and participate in the regulation of TAM sensitive type.Clinical histological examination also confirmed that high expression of lnc-42060 and SOX4occured in ER+CMGT tissues,while mi R-204-5p expressed at a low level.Therefore,lnc-42060mi R-204-5p and SOX4 may serve as important biomarkers for diagnosis and treatment in the future,they are also expected to be applied in practice as new molecular markers to predict TAM resistance. |
