Effect Of LncRNA XLOC₀01659 On The Proliferation And Invasion Of Esophageal Squamous Carcinoma Cells And Its Molecular Mechanism

Background and objectiveEsophageal Cancer（EC）is a common malignant tumor of the digestive tract,ranking second in the world as a cause of death due to cancer.EC is a tumor with high incidence,poor prognosis,and strong invasion.EC includes two important pathological types:Esophageal Adenocarcinoma（EAC）and Esophageal Squamous Cell Carcinoma（ESCC）.Esophageal adenocarcinoma is more common in Western countries,and esophageal squamous cell carcinoma is the main pathological type in developing countries.Because of the rising incidence and poor prognosis of EC,it is necessary to find novel early diagnosis parameters and methods of EC.Therefore,a better understanding of the molecular mechanisms of EC carcinogenesis is essential for improving the diagnosis,personal treatment and prognosis of EC.Although many oncogenes and tumor suppressor genes associated with EC have been reported,the biological functions and underlying mechanisms of EC tumorigenesis have not been fully elucidated.Long non-coding RNA（lncRNA）is a type of non-coding RNA with 200 nucleotides.It has no obvious protein coding potential.However,recent studies have shown that IncRNA is ubiquitous in the genome and plays a key role in many diseases,especially related to tumorigenesis.lncRNA is specifically expressed in some cancers,so lncRNA is considered to be a novel potential biomarker and cancer treatment target.Previous research by our research group showed that lncRNA XLOC 001659 is highly expressed in esophageal squamous cell carcinoma.There are very few reports about lncRNA XLOC₀01659 in the literature at home and abroad.Our study first comprehensively analyzed its expression and biological role in esophageal squamous cell carcinoma.miRNAs are a class of small non-coding RNAs found in eukaryotes.They consist of 22-24 nucleotides.Studies have found that miRNAs are involved in the regulation of many tumors and act as tumor suppressor genes or oncogenes.miR-490-5p has inhibitory effects on a variety of tumors,including breast cancer,but its role in esophageal cancer has not yet been clarified.It is well known that lncRNA and miRNA can interact and participate in the regulation of different cancers.Tumor-related miRNAs and lnc RNAs may be developed as specific cancer biomarkers for the diagnosis and prognosis of cancer.In this study,we explored the effects of lncRNA XLOC 001659 and miR-490-5p on esophageal squamous cell carcinoma,and explored their potential molecular mechanisms.Methods1.Collect normal esophageal epithelial cells HET-1A and esophageal squamous cell carcinoma cells EC9706,EC-1,and extract RNA for reverse transcription.2.Use qRT-PCR to detect the expression of lncRNA XLOC₀01659 in HET-1A,EC9706 and EC-1.3.Synthesize knockdown lncRNA XLOC₀01659 siRNA and unrelated sequence control NC,and transfect EC9706 and EC-1 cells to obtain EC9706 and EC-1 cells that down-regulate the expression of lncRNA XLOC₀01659.4.Use qRT-PCR to verify the expression of lncRNA XLOC₀01659 in EC9706 and EC-1 cells after transfection of siRNA and NC.5.CCK-8 and plate clone formation experiments were performed to detect the effects of down-regulated expression of lncRNA XLOC₀01659 on the proliferation ability of EC9706 and EC-1 cells.6.Transwell assay was used to detect the effect of down-regulated expression of lncRNA XLOC₀01659 on the invasion ability of EC9706 and EC-1 cells.7.The cell scratching assay detected the migration ability of EC9706 and EC-1 cells after down-regulating the expression of lncRNA XLOC₀01659.8.Hoechst 33342 staining was used to detect the effect of down-regulated expression of lncRNA XLOC₀01659 on the apoptosis of EC9706 and EC-1 cells.9.Use bioinformatics to analyze miRNAs that interact with lncRNA XLOC₀01659.10.According to the results of bioinformatics analysis,qRT-PCR was used todetect the effect of down-regulating the expression of lncRNA XLOC 001659 in EC9706 and EC-1 on the expression of miR-490-5p.11.Construct lncRNA XLOC 001659 wild-type and mutant vectors,and use double luciferase reporting experiments to further verify the interaction between lncRNA XLOC₀01659 and miR-490-5p.12.Transfect miR-490-5p mimics into EC9706 and EC-1 cells to prepare miR-490-5p overexpressing EC9706 and EC-1 cells.Transwell and plate cloning experiments were performed to detect the upregulation of miR-490-5p Effects of Esophageal Squamous Carcinoma Cell Line Invasion and Proliferation13.Using bioinformatics to analyze target genes that interact with miR-490-5p.14.Construct PIK3CA wild-type and mutant vectors,and verify that PIK3CA is a target gene of miR-490-5p using a double luciferase report.15.Western-blot was used to detect the expression level of PIK3CA after up-regulating the expression of miR-490-5p.16.The effect of overexpression PIK3CA on the growth and invasion of esophageal squamous cell carcinoma cell lines was tested by response experiments.Results1.qRT-PCR results showed that the expression of lncRNA XLOC 001659 in esophageal squamous carcinoma cell lines EC9706 and EC-1 was significantly higher than that in normal esophageal cells HET-1 A.2.The qRT-PCR results showed that the expression of lncRNA XLOC 001659 in EC9706 and EC-1 cells transfected with siRNA was significantly lower than that in the control group compared with the unrelated sequence control group.3.The results of CCK-8 experiment showed that compared with NC control group,EC9706 and EC-1 cells transfected with siRNA were significantly inhibited in growth after 24 hours,and became more and more significant with time.4.The results of plate clone formation experiments showed that compared with the irrelevant sequence control group,the number of EC9706 and EC-1 cells transfected with siRNA was significantly lower than that of the NC control group.5.The results of the Transwell invasion experiment showed that the cell invasion number of the transfected siRNA group was significantly different from that of the unrelated sequence control group.6.The results of the cell scratch test showed that the migration ability of the cells transfected with the siRNA group was not significantly different from that of the irrelevant sequence control group at 0 h,and the cell migration ability was weakened at 24 and 48 hours.7.Hoechst 33342 staining results showed that there was no significant difference between the cells transfected with the siRNA group and the cells transfected with the unrelated sequence control group（P>0.05）.8.The results of bioinformatics analysis showed that there is a specific binding site in the seed region that interacts with lncRNA XLOC 001659 and miR-490-5p.Located at chr2:126754156-126754172.9.qRT-PCR results showed that the expression of lncRNA XLOC₀01659 was down-regulated in EC9706 and EC-1 cells,and the expression of miR-490-5p was significantly increased.10.The results of the double luciferase report showed that co-transfection of miR-490-5p mimics and wild-type lncRNA XLOC 001659 recombinant reporter vector,esophageal squamous carcinoma cell line luciferase activity was significantly reduced,and co-transfection of miR-490-5p mimics and mutations There was no significant difference in the luciferase activity of the type lncRNA XLOC 001659 recombinant reporter vector in esophageal squamous carcinoma cell lines.11.Plate colony formation experiments showed that the number of colony formation in esophageal squamous carcinoma cell lines transfected with miR-490-5p mimics was significantly reduced,compared with the control group.12.After overexpression of miR-490-5p in esophageal squamous cell carcinoma cell lines,Transwell results showed that the number of esophageal squamous cell carcinoma cell lines invaded was significantly less.13.Bioinformatics analysis showed that miR-490-5p and PIK3CA had an interacting seed region binding site.14.The results of the double luciferase report showed that co-transfection of miR-490-5p mimics and wild-type PIK3CA recombinant reporter vector,esophageal squamous carcinoma cell line luciferase activity was significantly reduced,and co-transfection of miR-490-5p mimics and mutants There was no significant difference in the luciferase activity of PIK3KA recombinant reporter vector,and cells transfected with miR-490-5p NC and recombinant vector.15.Western-blot results showed that compared with the unrelated sequence control group,overexpression of miR-490-5p significantly inhibited PIK3CA protein expression.16.After over-expressing PIK3CA,the results of the Transwell experiment showed a significant increase in the number of invasion,and the results of the plate colony formation experiment showed that the number of colony formation in the esophageal squamous carcinoma cell line increased significantly.Conclusions1.Compared with normal esophageal epithelial cells HET-1A,the expression of lncRNA XLOC₀01659 in esophageal squamous cell carcinoma EC9706 and EC-1 cells was significantly increased.2.Down-regulating the expression of lncRNA XLOC₀01659 in esophageal squamous cell carcinoma EC9706 and EC-1 cells significantly inhibited the proliferation and invasion of esophageal squamous carcinoma cell lines.3.PIK3CA is one of the downstream targets of miR-490-5p.The mechanism of lncRNA XLOC₀01659 on proliferation and invasion in esophageal squamous cell carcinoma may be due in part to its regulation of miR-490-5p expression,which in turn affects PIK3CA To play its biological role.