The Effect Of LncRNA LINC00941 On Biological Behavior Of Colon Cancer Cells And Its Molecular Mechanism

As a common malignant tumors of digestive tract in clinic,colon cancer remains continuing high morbidity and mortality.The occurrence of colon cancer is complex,which is associated with various factors such as environment,lifestyle,diet,heredity.The early diagnosis and treatment of colon cancer has made some progress in recent years along with molecular biology and clinical diagnosis technology developing,however,the overall survival rate of patients has not been significantly improved.About 20-25% of colon cancer patients have distant metastasis and poor prognosis.In addition,the molecular mechanism about colon cancer progression remains unclear.Therefore,the potential mechanism of colon cancer progression become current research hotspot,in order to find the key molecules involved in tumor growth and metastasis and improve the therapeutic effect and prognosis of colon cancer.The studies have shown that,in addition to protein and micro RNA(miRNA),long non-coding RNAs(lnc RNAs)is also involved in the regulation of the occurrence and development of colon cancer.In the process of tumor progression,the imbalance of lnc RNAs expression is a common phenomenon,and the abnormality of lnc RNAs expression profile is closely related to the occurrence of various malignant tumors.LINC00941 is called MSC upregulation factor,which is a novel lnc RNA located in 12p11.21 region of human genome.It had been demonstrated in previous studies that LINC00941 is involved in the growth and metastasis of multiple tumors including gastric cancer,lung cancer.Because of the ambiguous biological effectiveness and possible molecular mechanism of LINC00941 in colon cancer,it has been particularly necessary to investigate its function in colon cancer for discovering new targets of genetic therapy of colon cancer.In this study,the expression,biological function and possible mechanism of LINC00941 in colon cancer were investigated in vitro and in vivo.The results show that LINC00941 regulates the expression of miR-205-5p through sponging miRNA,and then positively regulates the expression of MYC,which accelerates colon cancer progression.The experiment furnishes a novel basis and idea for the application of lnc RNAs in the diagnosis and treatment of colon cancer.Part Ⅰ the expression of LINC00941 in colon cancer tissues and cells Methods1.42 cases of colon cancer tissues and matched normal colon tissues were collected from patients undergoing surgery at the Affiliated Tumor Hospital of Zhengzhou University.All tissue specimens were confirmed by histopathology.2.The normal colon epithelial NCM460 cell line and seven diverse colon cancer cell lines(LS174T,HCT116,CT26,HCT8,HCT29,SW480,Lo Vo)were cultured.3.qRT-PCR method was used to detect the difference of LINC00941 expression between colon cancer and adjacent normal tissues.The same is true for colon cells and colon cancer cells.4.Statistical analysis: all the experimental datas were expressed as mean±standard deviation.T-test was used for comparison between the two groups,single-factor or multi-factor analysis of variance was used for comparison between groups in the SPSS 20.0 software.P < 0.05 was considered to be statistically significant.Results1.In contrast with normal colon tissues,the level of LINC00941 expression in colon cancer tissues was markedly elevated.2.In contrast with NCM460 cell line,the level of LINC00941 expression in seven colon cancer cell lines(especially HCT116 and Lo Vo cell lines)was markedly elevated.ConclusionThere was expressed LINC00941 in both colon cancer tissues and cells,and the level of LINC00941 expression was markedly elevated by comparsion with normal colon tissue and colon epithelial cells.Part Ⅱ the effect on the biological behavior of LINC00941 in colon cancer cells Methods1.HCT116 and Lo Vo cells were cultured and transfected with the plasmid overexpressing LINC00941 and empty vector respectively,in order to construct stable cell lines overexpressing LINC00941 and negative control cell lines.2.LINC00941 expression interference by siRNA : colon cancer cell lines with LINC00941 silencing was established by transfecting siLINC00941-1,siLINC00941-2 and negative control si-NC into HCT116 and Lo Vo cells respectively.3.After stable transfection,the LINC00941 expression in colon cancer cells was tested using qRT-PCR;the ability of colon cancer cells proliferating,migrating and invading was test using CCK-8 assay and Transwell assay.4.Establishment of subcutaneous xenograft tumor model in nude mice: observe and measure the volume changes of the transplanted tumor and the weight of the exfoliated tumor.Establishment of the lung metastasis model of colon cancer in nude mice: counte the number of lung metastatic nodules after killing the nude mice.5.Statistical analysis: all the experimental datas were expressed as mean±standard deviation.T-test was used for comparison between the two groups,single-factor or multi-factor analysis of variance was used for comparison between groups in the SPSS 20.0 software.P < 0.05 was considered to be statistically significant.Results1.The results of qRT-PCR showed that after stable overexpression and silencing transfection in HCT116 and Lo Vo cells,the level of LINC00941 expression in cells with LINC00941 plasmid was remarkably up-regulated by comparison with cells with empty vector as the negative control.On the contrary,the level of LINC00941 expression in cells with siLINC00941-1 and siLINC00941-2 remarkably down-regulated by comparison with cells with si-NC as the negative control.2.The results of CCK-8 assay reflected that after stable overexpression and silencing transfection,HCT116 and Lo Vo cells with LINC00941 plasmid showed a higher proliferation ability than cells with empty vector as the negative control.On the contrary,the proliferative activity of cells with siLINC00941-1 and siLINC00941-2 was evidently decreased by comparison with cells with si-NC as the negative control.3.The results of Transwell assay showed that after stable overexpression and silencing transfection in HCT116 and Lo Vo cells,the activity of cells migrating and invading with LINC00941 plasmid was evidently increased by comparison with cells with empty vector as the negative control.On the contrary,the activity of cells migrating and invading with siLINC00941-1 and siLINC00941-2 was evidently decreased by comparison with cells with si-NC as the negative control.4.The vivo experimental results in nude mice showed that: in the model of transplanted tumor in nude mice,comparing sh LINC00941 group with sh NC group,the tumor growth rate,the tumor volume and tumor weight in sh LINC00941 group was obviously decreased.Besides,in the model of lung metastasis of colon cancer in nude mice,knocking down LINC00941 can significantly inhibit the formation of metastatic pulmonary nodules.ConclusionOverexpressing LINC00941 can accelerate the ability of colon cancer cells proliferating,migrating and invading both in vitro and in vivo.On the contrary,silencing LINC00941 can restrain the ability of colon cancer cells proliferating,migrating and invading.Part Ⅲ Molecular mechanism of the effect of LINC00941 on the biological behavior of colon cancer cells Methods1.LINC00941 localization in colon cancer cells was detected using RNA-FISH assay.2.The pmir GLO vector was inserted with the 3’UTR sequence binding to miR-205-5p and the corresponding mutant sequence of LINC00941,for constructing the vector of LINC00941-wt(wild type)and LINC00941-mut(mutant).LINC00941-wt or LINC00941-mut were cotransfected into colon cancer cells with miR-205-5p mimics or miR-205-5p inhibitors,and NC mimics or NC inhibitors were used as negative controls.Double luciferase report assay was used to detect luciferase activity,in order to verify whether there is a targeted binding between LINC00941 and miR-205-5p,RIP test was used to further verify the relationship between LINC00941 and miR-205-5p.3.Si NC,siLINC00941,siNC+miR-205-5p inhibitor and siLINC00941+miR-205-5p inhibitor were transfected into Lo Vo cells respectively.Taking four groups of colon cancer cells as the object,detected miR-205-5p expression by qRT-PCR,and the ability of cells proliferating,migrating and invading by CCK-8assay and Transwell assay.4.The 3’UTR sequence and mutant sequence of MYC binding to miR-205-5p were inserted into pmir GLO carrier to construct MYC-wt(wild type)vector and MYC-mut(mutant)vector.MYC-wt or MYC-mut were cotransfected into Lo Vo cells with NC mimics,miR-205-5p mimics,miR-205-5p mimics+empty vector and miR-205-5p mimics+LINC00941 plasmid vector,respectively.Luciferase activity was detected by double luciferase report analysis to verify the direct binding between LINC00941 and miR-205-5p.5.The transfected cells were divided into groups: NC,miR-205-5p,miR-205-5p+vector,miR-205-5p+LINC00941,si-NC and si-LINC00941.The MYC m RNA expression was detected by qRT-PCR with protein detected by Western blot.6.Statistical analysis: all the experimental datas were expressed as mean±standard deviation.T-test was used for comparison between the two groups,single-factor or multi-factor analysis of variance was used for comparison between groups in the SPSS 20.0 software.P < 0.05 was considered to be statistically significant.Results1.The results of RNA-FISH experiment demonstrated that LINC00941 was mainly located in the cytoplasm of colon cancer cells.2.The results of double luciferase report experiment reflected that the luciferase activity of cells transfected with LINC00941-wt declined when miR-205-5p was overexpressed,while increased when the miR-205-5p expression was knocked down.However,the luciferase activity of cells transfected with LINC00941-mut had no significant change.At the same time,Luciferase activity was remarkably inhibited in the cells co-transfected with MYC-wt and miR-205-5p mimics,but not in the cells co-transfected with MYC-mut and miR-205-5p mimics.In addition,the inhibition of luciferase activity induced by miR-205-5p mimics was reversed in the colon cancer cells co-transfected with MYC-wt and miR-205-5p mimics + LINC00941.3.The results of RIP assay revealed that expression of LINC00941 and miR-205-5p could be detected in Ag O2 immunoprecipitate at the same time.However,after down-regulating the expression of miR-205-5p,the expression of LINC00941 detected in Ag O2 immunoprecipitate was also significantly down-regulated.4.It can be seen from the results of qRT-PCR that miR-205-5p expression of colon cancer cells with siLINC00941 distinctly increased,which was reversed by adding miR-205-5p inhibitor.5.The results of CCK-8 and Transwell experiments demonstrated that LINC00941 silencing inhibited the ability of colon cancer cells proliferating,migrating and invading,however partial reversal of the inhibition was achieved by silencing miR-205-5p.6.The results of qRT-PCR and Western-blot experiments showed that the MYC m RNA expression in both miR-205-5p group and si-LINC00941 group declined remarkably in contrast to the control group.In the meantime,comparing miR-205-5p+LINC00941 group with miR-205-5p+vector group,MYC m RNA showed a higher expression in miR-205-5p+LINC00941 group.The same is MYC protein expression.ConclusionLINC00941 acting as a molecular sponge of miR-205-5p elevates MYC expression by ceRNA mechanism,which promotes colon cancer progression by acting on the miR-205-5p/MYC axis ultimately.