The Experimental Study On The Effect Of Long Non-coding RNA LINC00460/miR-503-5p Axis On Proliferation And Apoptosis Of Cervical Cancer

Objective: Cervical cancer is the second largest gynecological tumor in the world.The incidence of cervical cancer has been rising rapidly in Asia in recent years,especially in developing countries.It has been recognized that the occurrence of cervical cancer is related to the continuous infection of HPV.However,some studies believe that the abnormal expression of oncogenes can also affect the occurrence and development of cervical cancer as an independent factor.At present,early cervical cancer can be controlled,but the prognosis of recurrent and metastatic cervical cancer is not ideal.Therefore,in-depth study of the pathogenesis of cervical cancer is of great significance for the prevention and treatment of cervical cancer.Long non-coding RNAs(Lnc RNAs)are a class of non-coding RNAs with a length of more than 200 nucleotides,which can participate in the regulation of a variety of biological processes in cells.Long non-coding RNAs are associated with the pathogenesis of cancers.Through analysis on the GEPIA website(http://gepia.cancer-pku.cn/),we found that the high expression of long non-coding RNA LINC00460 is related to the poor prognosis of cervical cancer.LINC00460 is a newly discovered long intergenic non-protein coding RNA,and its cancer-promoting effect in gastric cancer,meningioma,ovarian cancer,lung cancer,etc.has been relatively clear.However,its effect on cervical cancer has not been reported yet.So we speculated that it could also promote the development of cervical cancer.Since Salmena proposed the hypothesis of competing endogenous RNA(ce RNA)in 2011,people have gained a new understanding of the regulatory mechanisms of micro RNA(miRNA)and lnc RNAs.We predict the binding sites of miR-503-5p on LINC00460 though online bioinformatics tools Star Base(http://starbase.sysu.edu.cn/).As an anti-tumor miRNA,miR-503-5p can inhibit the progression of bladder cancer,lung cancer and ovarian cancer,etc.More importantly,it has been reported that miR-503-5p is associated with poor prognosis of cervical cancer and can inhibit the proliferation of cervical cancer cells through the regulation of its downstream target genes.Therefore,we speculated that LINC00460 could affect the occurrence and development of cervical cancer by regulating miR-503-5p.This study’s aims were to investigate the expression level of LINC00460 in cervical cancer tissues and paracancerous tissues and to analyze its correlation with clinicopathological parameters and ultrasound results of cervical cancer patients.Furthermore,the effect of the expression of LINC00460 on the proliferation and apoptosis of cervical cancer cells was investigated.The molecular regulation mechanism of LINC00460 promoting the proliferation and apoptosis of cervical cancer was explored based on the theory of competitive endogenous RNA targeting absorption of miRNA.Methods: Through analysis of GEPIA website,we found that the high expression of LINC00460 is related to the poor prognosis of cervical cancer patients.Thirty pairs of cervical cancer tissues and para-cancerous tissues were collected from patients who had been taken surgical resection at the gynecology department of Shenjing Hospital of China Medical University(Shenyang,China).All the tissue samples were pathologically confirmed,and they were immediately frozen by liquid nitrogen and then stored at-80°C.The clinicopathological data of all the patients were collected.All patients underwent three-dimensional energy Doppler ultrasonography before surgery.Vascular classification and the blood flow parameters were assessed.The expression level of LINC00460 in the tissue samples were detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR),and the correlation between the expression level and the clinicopathological characteristics of patients and the results of ultrasonic examination was analyzed.2.The basic expression of LINC00460 in four cervical cancer cell lines(Siha,C-33 a,Hela and Caski)was detected by RT-qPCR,and two highly expressed cell lines(Siha and Hela cells)were screened out.The LINC00460 interference vector and negative control interference vector were constructed and transfected into Siha and Hela cells,respectively.The experimental groups were as follows: control group,NC shRNA group,LINC00460 shRNA1 group,LINC00460 shRNA2 group.The transfection efficiency of LINC00460 in Siha and Hela cells was verified by RT-qPCR after transfection.Flow cytometry was used to detect cell cycle distribution in each group,methyl thiazolyl tetrazolium(MTT)assay was used to detect cell viability,and Western blot was used to detect the expression of Ki67 and cyclin D1 to verify the effect of silencing LINC00460 on proliferation of cervical cancer cells in vitro.3.Flow cytometry was used to detect cell apoptosis,Hoechst staining was used to observe cell apoptosis,and Western blot was used to detect the expression of Cleaved-caspase-3 and Cleaved-PARP,so as to verify the effect of silencing LINC00460 on apoptosis of cervical cancer cells.4.By predicting miR-503-5p as the downstream miRNA of LINC00460 through Star Base database,wild-type LINC00460 vector containing miR-503-5p binding site(WT-LINC00460)and LINC00460 vector containing miR-503-5p binding site mutation(MUT-LINC00460)were constructed and co-transfected into a tool cell line(HEK-293T)with NC mimic or miR-503-5p mimic,respectively.The experiment was divided into four groups: WT-LINC00460+NC mimic group,MUT-LINC00460+NC mimic group,WT-LINC00460+miR-503-5p mimic group,MUT-LINC00460+miR-503-5p mimic group.The interaction between LINC00460 and miR-503-5p was analyzed by using dual luciferase reporter assay.RT-qPCR was used to detect the expression levels of miR-503-5p in Siha and Hela cells in each group,and Western blot was used to detect the protein expressions of miR-503-5p target genes AKT2,HMGA2 and SHOX2,so as to confirm the regulatory effect of LINC00460 on miR-503-5p and its downstream target genes.5.Rescue experiments were designed,and Siha cells were co-transfected with LINC00460 shRNA1 and miR-503-5p inhibitor or NC inhibitor.The experimental groups were as follows: LINC00460 shRNA1+NC inhibitor group and LINC00460 shRNA1+miR-503-5p inhibitor group.Cell proliferation was detected by MTT,cell cycle distribution was detected by flow cytometry,cell apoptosis was detected by flow cytometry,and the expression of AKT2,HMGA2,SHOX2,Cyclin D1 and Cleaved caspase-3 was detected by Western blot.6.Tumor formation experiments in nude mice were conducted to verify the effect of silencing expression of LINC00460 on tumor-formation ability of nude mice.The expressions of LINC000460 and miR-503-5p in tumor tissues were detected by RT-qPCR,the expression of Ki67 protein in tumor tissues was detected by immunohistochemistry(IHC),the apoptosis of cells was detected by TUNEL assay,and the protein expressions of AKT2,HMGA2 and SHOX2 were detected by Western blot.The effect of LINC00460 silencing on miR-503-5p and its downstream target genes was further verified in vivo,as well as the effect on the proliferation and apoptosis of cervical cancer cells in vivo.Results: 1.LINC00460 levels in cervical cancer tissues were higher than para-cancerous tissues(P<0.01).The expression level of LINC00460 in cervical cancer tissues is correlated with the degree of differentiation of cervical cancer tissues,and the expression level is positively correlated with lymph node metastasis of patients,but not with the patient’s age,tumor size,clinical FIGO stage and pathological type.The expression level of LINC00460 in cervical cancer tissues was correlated with the ultrasound results of vascular classification.2.LINC00460 silencing significantly suppressed the proliferation of cervical cancer cells as evidenced by decreased cell viability.Flow cytometry showed that LINC00460 silencing promoted the arrest of cervical cancer cell cycle in G1 phase and inhibited the G1/S transition of cervical cancer cell cycle.Silencing of LINC00460 inhibited the expression of Ki67 and Cyclin D1,suggesting that silencing of LINC00460 inhibited the proliferation of cervical cancer(P<0.05).3.Flow cytometry and Hoechst staining showed that LINC00460 silencing significantly increased apoptotic cells and Hoechst-positive cells,and enhanced the expressions of apoptosis-related protein Cleaved-caspase-3 and Cleaved-PARP.These suggested that LINC00460 silencing could promote the apoptosis of cervical cancer cells(P<0.05).4.It was predicted by Star Base database that LINC00460 and miR-503-5p had targeted binding sites.The dual luciferase reporter assay showed that the luciferase activity of 293 T cells transfected with WT-LINC00460 was significantly reduced by miR-503-5p mimic(P<0.01),while the luciferase activity of 293 T cells transfected with MUT-LINC00460 was not significantly affected.RT-qPCR showed that LINC00460 silencing could significantly up-regulate the expression level of miR-503-5p in Siha and Hela cells(P<0.01).And Western blot showed that LINC00460 silencing could reduce the expression levels of miR-503-5p target genes AKT2,HMGA2 and SHOX2 in cervical cancer cells(P<0.01).LINC00460 could bind to miR-503-5p and LINC00460 silencing enhanced miR-503-5p expression and inhibited its target gene expressions in cervical cancer cells.5.The rescue experiments showed that compared with the LINC00460 shRNA1+NC inhibitor group,the cell proliferation ability of the LINC00460 shRNA1+miR-503-5p inhibitor group was significantly improved,the G1/S transition of cervical cancer cells was improved,the proportion of apoptotic cells was significantly reduced,the expression of cyclin D1 was significantly up-regulated,the expression of Cleaved caspase-3 was significantly down-regulated,and the expressions of AKT2,HMGA2,and SHOX2 were significantly up-regulated.It was verified that miR-503-5p inhibition reversed LINC00460silencing-caused inhibition of cell proliferation,miR-503-5p target gene expressions and promotion of cell apoptosis.6.Tumor formation experiments in nude mice showed that the growth rate,volume and weight of the transplanted tumor in the LINC00460 shRNA group were significantly lower than those in the NC shRNA group(P<0.01).RT-qPCR results showed that the expression level of LINC00460 in the LINC00460 shRNA group was significantly lower than that in the NC shRNA group,while the expression level of miR-503-5p was significantly higher than that in the NC shRNA group.IHC results showed that the expression level of Ki67 in the LINC00460 shRNA group was significantly lower than that in the NC shRNA group(P<0.01).TUNEL assay showed that there were more apoptotic cells in the LINC00460 shRNA group.Western blot results showed that the protein expression levels of AKT2,HMGA2 and SHOX2 in the LINC00460 shRNA group were significantly lower than those in the NC shRNA group.LINC00460 silencing also attenuated tumor growth,promoted miR-503-5p levels and cell apoptosis,and inhibited cell proliferation and miR-503-5p target gene expressions in tumor tissues.Conclusion: 1.This study confirmed the high expression level of LINC00460 in cervical cancer tissues and cell lines,suggesting that LINC00460 is related to the occurrence and development of cervical cancer.2.Silencing of LINC00460 expression can inhibit the proliferation ability of cervical cancer cells in vitro.3.Silencing of LINC00460 expression can also enhance the apoptotic ability of cervical cancer cells in vitro.4.LINC00460 targets miR-503-5p and regulates the downstream target genes AKT2,HMGA2 and SHOX2 of miR-503-5p in cervical cancer cells.5.LINC00460 affected cell proliferation and apoptosis via sponging miR-503-5p.6.LINC00460 regulates the level of miR-503-5p in vivo,and silencing of LINC00460 can inhibit the growth ability of cervical cancer tumor and the proliferation of cervical cancer cells in nude mice.It also can promote the apoptosis of cervical cancer cells.It is suggested that LINC00460 may play a role as an oncogene in the pathogenesis of cervical cancer,and it is expected to be a novel therapeutic target for cervical cancer.