A Study On Mechanisms Of LncRNA HOTTIP Facilitating The Stemness Of Breast Cancer Stem Cells

Objective:Currently,malignant tumors are the second leading cause of death in humans.According to the World Health Organization,approximately 9.6 million people died of tumors in 2018 worldwide.Among them,breast cancer is one of the most common malignant tumors in women,and it is also the most common cause of tumor-related deaths in women,which seriously threatens women’s life and health.More and more evidence indicates that the occurrence and maintenance of breast cancer may be regulated by a small number of cells in the tumor,which are called cancer stem cells（CSCs）.Cancer stem cells have become a major obstacle to breast treatment due to their strong tumorigenicity,self-renewal ability,metastatic ability,and resistance to radiation and chemotherapy.Therefore,it is of great significance to explore effective tumor molecular markers and key regulatory mechanisms in CSCs for the treatment of breast cancer.Long-chain non-coding RNA（lncRNA）is defined as RNA that is greater than200 nt in length and lack of the ability to encode proteins.However,in recent years,more and more studies have confirmed that lncRNA is related to the occurrence and maintenance of various tumors.Therefore,exploring the role of lncRNAs in tumorigenesis and development and its molecular mechanism has become a hot spot in cancer research.This study was aimed to investigate the role of long-chain non-coding RNA HOTTIP（HOXA transcript at the distal tip）in the maintenance of breast cancer stem cells and its molecular regulatory mechanism,and to provide new targets for the diagnosis and treatment of breast cancer.Methods:1.ISH was used to detect the expression of HOTTIP in breast cancer tissues.The relationship between HOTTIP expression and tumor prognosis was analyzed using Kaplan-Meier Plotter（KM Plotter）database.2.The serum-free suspension culture method was used to culture MCF7 and T47D cells to enrich BCSCs and identify the stemness of the enriched BCSCs.QRT-PCR were used to detect the expression level of HOTTIP in normal breast epithelial cells MCF10A,breast cancer parental cells,and spheres.3.Over-expression HOTTIP cell model and HOTTIP-silencing cell model were constructed by infecting with lentiviruses carrying HOTTIP-cDNA and HOTTIP shRNA plasmid.Flow cytometry was used to detect the effects of HOTTIP on the percentage of CD44⁺/CD24^-BCSCs;Soft agar colony formation assay and mammosphere formation assay were performed to detect the self-renewal of BCSCs;western blot was used to detect the expression of OCT4,SOX2,CK14 and CK18.And ten of 6-week BALB/C nude mice were purchased from Beijing Weitong Lihua Company and randomly divided into two groups for BCSC transplantation.This xenograft tumor model was used to investigate the effect of HOTTIP on the tumorigenicity of BCSCs.4、ISH was used to detect the expression of mi R-148a-3p in breast cancer tissues.QRT-PCR were used to detect the expression level of miR-148a-3p in breast cancer parental cells,and spheres.Flow cytometry,Soft agar colony formation assay,mammosphere formation assay and western blot were conducted to detect the role of miR-148a-3p in maintaining the stemness of BCSCs.5、DIANA was used to explore the binding between HOTTIP and miR-148a-3p.According to predicted binding site between HOTTIP and miR-148a-3p,we designed luciferase plasmid,and conducted dual-luciferase reporter assays to determine whether miR-148a-3p directly binds to HOTTIP.Results:1.HOTTIP expression is increased in human breast cancer tissues compared with paracancerous tissue.And its expression level is associated with poor prognosis.（1）ISH results indicated that breast cancer tissue had a hinger expression of HOTTIP.QRT-PCR results showed that compared with normal breast epithelial cells（MCF10A）,the expression of HOTTIP in breast cancer cells was significantly increased.KM ploter results showed that high expression of HOTTIP suggested poor prognosis.（2）HOTTIP is specifically overexpressed in CD44⁺/CD24^-low breast cancer tissues.（3）QRT-PCR results showed the expression of HOTTIP is significantly increased in BCSCs.2.HOTTP regulates the self-renewal ability of BCSCs and maintains the stemness of BCSCs.（1）Depletion of HOTTIP decreased the CD44+/CD24-population in BCSCs,reduced the self-renewal capacity of BCSCs,decreased the expressions of stemness-related proteins OCT4 and SOX2,and increased the expression of differentiation markers CK14 and CK18.And Knockdown of HOTTIP inhibited the growth of tumor in vivo.（2）Over-expression of HOTTIP increased the colony formation ability,upregulated the expression of OCT4 and SOX2,and reduced CK14 and CK18 levels.3.MiR-148a-3p is down-regulated in breast cancer and regulates the stemness of BCSCs through WNT1.（1）MiR-148a-3p is significantly down-regulated in breast cancer and low expression of mir-148a indicates poor prognosis.（2）MiR-148a-3p expression is lower in the CD44+/CD24-low cells.（3）WNT1 level negatively correlates with miR-148a-3p in breast cancer tissues.（4）QRT-PCR results showed that miR-148a-3p is down-regulated in BCSCs and functional studies verified that miR-148a-3p could inhibits the stemness of BCSCs by targeting WNT1.4.HOTTIP maintains the stemness of BCSCs through the miR-148a-3p/WNT1pathway.（1）HOTTIP expression is negatively correlated with miR-148a expression in breast cancer tissue.（2）HOTTIP is positively correlated with WNT1 expression in breast cancer tissues.（3）Dual-luciferase reporter assay results showed that HOTTIP could directly bind to miR-148a-3p.（4）HOTTIP could regulates the stemness of BCSCs through the miR-148a-3p/WNT1 pathway.Conclusions:1.HOTTIP is highly expressed in breast cancer tissues and associated with poor prognosis.2.HOTTIP is highly expressed in BCSCs.HOTTIP maintains the stemness of BCSCs,and promotes the tumorigenicity in vivo.3.MiR-148a-3p is down regulated in BCSCs and regulates BCSCs stemness by inhibiting WNT1.4.HOTTIP is negatively correlated with miR-148a expression and positively correlated with WNT1 expression.HOTTIP may maintain the stemness of BCSCs through the miR-148a-3p/WNT1 pathway.