Research On The Mechanism Of LncRNA UCA1 Promoting Paclitaxel Resistance In Colorectal Cancer

Background and Objective:Colorectal cancer is one of the most common malignancies,with the third incidence in the world.Surgery is the effective method of treatment,but the survival rate after excision of the primary tumour remains low.Systemic therapy such as chemotherapy,radiotherapy,targeted therapy and immunotherapy are necessary treatments for distant spread of colorectal cancer cells.Paclitaxel is a powerful and effective anti-cancer drug.However,like some conventional chemotherapeutic agents,patients show resistance to paclitaxel in the course of treatment,which limits the clinical efficacy to a certain extent,and the specific mechanism is not completely clear,it is of great significance to clarify the mechanism of paclitaxel tolerance in colorectal cancer.Extensive evidence shows that non-coding RNA is an indispensable regulatory factor in a variety of biological processes and plays an important role in regulating the occurrence and development of tumor,and long non-coding RNA(lncRNAs)is an important part of it.lncRNAs can mediate the interaction between DNA and protein and adsorbing microRNA.Some studies have shown that lncRNAs inhibits glycolysis and apoptosis resistance of tumor cells.It is suggested that IncRNAs has an important effect on tumor metabolism and emphasizes the great potential of lncRNA in tumor therapy.The purpose of this study was to explore the role of Long non-coding RNA urothelial carcinoma associated 1(UCA1)in regulating paclitaxel resistance in human colorectal cancer cells from tumor metabolism.Methods:1.The expression of ucal in colorectal tumor and adjacent normal tissue was analyzed by TCGA database,and the result was verified in tissue and cell levels by detecting the expression of UCA1 in tissue samples from colorectal cancer patients,between normal colorectal cell lines and colorectal cancer cell lines were detected by qRT-PCR.2.To explore the interaction between UCA1 and paclitaxel in colorectal cancer,we then overexpressed UCA1 in colorectal cells,the survival rate of overexpressed and control group treated with different concentrations of paclitaxel and the expression of UCA1 in colorectal cancer cells treated with different concentrations of paclitaxel were detected.3.Established a Taxol-resistant colorectal cancer cell line(LoVo TR),the expression of UCA1 in LoVo TR and normal LoVo cell were detected by qRT-PCR.4.For exploring the metabolic characteristics of two groups of cells,the levels of glucose consumption,lactate production,extracellular acidification rates(ECAR)were detected.5.The protein and mRNA expression levels of the key glycolytic enzymes HK2 and LDHA between Taxol-sensitive and Taxol-resistant colorectal cancer cells were detected by qRT-PCR or Western blot.6.To explore the interaction between glycolysis and the sensitive to Taxol of colorectal cancer cells,we detected the survival rate of LoVo Taxol-sensitive and Taxol-resistant cells,which were treated with glycolysis inhibitors,3-BrPA or Oxamate.7.To study whether UCA1 could regulate glycolysis,we transfected LoVo cells containing the UCA1 overexpression vector or UCA1-specific siRNA,and the protein and mRNA expression levels of the key glycolytic enzymes HK2 and LDHA between these cells and control cells were detected by qRT-PCR or Western blot,meanwhile,glucose metabolism,lactate production,and ECAR were measured.8.To determine whether the sensitization of colorectal cancer cells to Taxol by UCA1 is through the regulation of glycolysis,the survival rates of siUCAl and control cells under treated with different concentrations of paclitaxel were determined,then glycolysis rescue experiments were carried out:(1)We co-transfected the overexpression vector of HK2 and siUCAl to rescue glycolysis;(2)control vectors or UCA1 overexpression vectors were transfected into colorectal cancer cells respectively,and then the cell survival rate was determined after glycolysis inhibitor 3-BrPA or PBS treatment.Results:1.The analysis based on the TCGA database showed that the expression of ucal in colorectal tumor tissue was significantly up-regulated compared with the normal tissue,and the expression of ucal in tissue samples from colorectal cancer patients also verified this result.Consistently,the expression levels of UCA1 were higher in six colorectal cancer cell lines than in two normal colorectal cell lines.2.The survival rate in colorectal cells with UCA1 overexpression were higher compared with that in the control group,the difference increased with the increased in Taxol concentration.And the expression level of UCA1 was found to be increased with increase in the Taxol concentration.3.The survival rate of resistant cells was significantly higher than that of sensitive cells after Taxol treatment,when the Taxol concentration was increased to 16 nM,the difference was much more apparent.Besides,the expression level of UCA1 was significantly higher in Taxol-resistant cells than in Taxol-sensitive cells4.The analysis of the glucose metabolism demonstrated that glucose consumption,lactate production,extracellular acidification rates(ECAR),and glucose metabolism level in Taxol-resistant cells were significantly higher than those in the Taxol-sensitive cells.5.Compared with Taxol-sensitive colorectal cancer cells,the protein and mRNA expression levels of the key glycolytic enzymes HK2and LDHA were upregulated in Taxol-resistant cells.6.The relative survival rates of Taxol-resistant cells and Taxol-sensitive cells were decreased,while the survival rate of Taxol-resistant cells was significantly decreased after 3-BrPA and Oxamate treatment.7.Glycolysis key enzymes HK2 and LDHA were regulated by the overexpression or downregulation of UCA1 at both protein and mRNA levels.Consistently,overexpression of UCA1 significantly upregulated glucose consumption,lactate production,and ECAR.In contrast,knockdown of UCA1 downregulated glucose metabolism.8.The result of rescue experiments shows that the survival rates of siUCAl were lower and seems to easier killed by Taxol.Restoration of glycolysis in co-transfected cells rendered the cells with resistance to Taxol treatments.;exogenous UCA1 increased Taxol resistance in colorectal cancer cells,treatment with a glycolysis inhibitor could help the cells overcame the resistance and colorectal cells could be effectively damaged.Conclusions:1.UCA1 can regulate the sensitivity of colorectal cancer cells to paclitaxel,and its overexpression can promote paclitaxel resistance in colorectal cancer cells.2.Glycolysis is involved in resistance to paclitaxel in colorectal cancer cells.3.In colorectal cancer cells,overexpression of UCA1 can promote the glycolysis process by regulating the key enzymes of glycolytic enzymes HK2 and LDHA,and further enhance the drug resistance of colorectal cancer cells to paclitaxel.