LncRNA SNHG5 Affects Cell Proliferation,Metastasis And Migration Of Colorectal Cancer Through Regulating MiR-132-3p/CREB5

Background and PurposeAll over the world,colorectal cancer(CRC)is one of the most common digestive tumors.Substantial development in the tools to prevent,diagnose and treat CRC has been gained,and as a result the survival of CRC patients at early stage has improved in the last decades.However,the overall five-year survival rate of patients with CRC remains low because of the fact that the molecular nosogenesis of this carcinoma remains unclear.Therefore,to clarify the molecular mechanism of colorectal cancer proliferation and metastasis is the keypoint to guide early diagnosis of CRC and improve the prognosis.Long non-coding RNAs(lncRNAs)are 200-10’000 nucleotides in length.They lack protein coding ability.Plenty of studies have shown that lncRNAs are abnormally expressed in a variety of tumors,such as colorectal cancer,breast cancer,gastric cancer,esophageal cancer,bladder cancer and so on.SNHG5 is a member of the host gene family of non-coding nucleolus small molecule RNA(snoRNA),with a length of 524 base pairs,and is located at the breaking point of 6q15 chromosome translocation.Previous studies have shown that SNHG5 has a certain correlation with B-cell lymphoma.SNHG5 encodes nucleolar small RNA U50 and U500 of internal factor 4 and 5,respectively.U50 mutation in both somatic and germ cell lines has been reported in breast cancer and prostate cancer.However,the effects of SNHG5 have not been characterized in CRC.MicroRNA(miRNA)is a class of endogenous non-coding single-stranded small RNA with a length of 19-25 nucleotides,which potentially mediate protein expressions accounting for 20-30%.In recent years,many miRNAs have been proved to play an important role in the pathogenesis of tumors via regulating mRNA expression.A large number of previous studies have confirmed that miR-132 is located on chromosome 17p13.3.Previous studies on glioma,CRC,breast cancer,prostate cancer and other malignant tumors all suggested the correlation between miRNA-132 and SNHG5.Some studies suggested that miRNA-132 miR-132 could promote proliferative capacity of cancer cells and may be a potential target for SNHG5.Additionally,miR-132 has also been described in a number of other fields,such as inflammation and cell transformation..CAMP-responsive element binding protein 5(CREB5),the product of which belongs to the CRE(cAMP response element)-binding protein family,which contain zinc-finger and bZIP DNA-binding domains.A previous research revealed that higher expressions of CREB5,predicted favorable disease free survival in patients with pulmonary carcinoid tumors.The biological functions of CREB5 in CRC have not been characterized in previous studies.Based on the above related studies,we established the subject of "the effect of LncRNA SNHG5 on the proliferation,metastasis and migration of colorectal cancer through mi-132-3p/CREB5".The purpose of our study was to observe the biological functions of LncRNA SNHG5 in colorectal cancer and explore its mechanisms,so as to guide the treatment of colorectal cancer.Methods1.GSE18105 microarray from GPL96 platform was analyzed using the R project for statistical computing.LncRNAs with abnormal expression were screened,and in combination with literatures,SNHG5 was selected as the research target.By qRT-PCR,we detected and compared the expression levels of SNHG5 in tumor tissues and adjacent tissues.The expressions of SNHG5 in CRC cell lines RKO,SW480,LoVo and normal colorectal mucosa cell FHC were also detected by qRT-PCR.Finally,CRC cell line with the highest level of SNHG5 expression was selected for subsequent experiments.2.By using the Starbase database,we found that miR-132-3p,miR-155,miR-205 and miR-150 could interact with SNHG5.The expression levels of the above related miRNAs were detected by qRT-PCR,among which the up-regulation level of miR-132-3p was the highest.Therefore,we selected miR-132-3p for the follow-up experiments.Luciferase reporter gene and qRT-PCR were used to detect the expression levels of miR-132-3p and SNHG5 during the knockout and enhancement of each other,so as to clarify the interaction between miR-132-3p and SNHG5.3.To determine the effects of SNHG5 on tumorigenesis,we divided CRC cells into five groups:miR-132-3p mimics group,miR-132-3p inhibitor group,si-SNHG5 group,si-SNHG5+miR-132-3p inhibitor group and NC group.The effects of SNHG5 and miR-132-3p on the proliferation,metastasis and migration of CRC cells were evaluated by qRT-PCR,CCK-8 assay,transwell migration assay and wound healing assay.4.The apoptosis rates of CRC cells in the above five groups were detected by flow cytometry to determine the effects of SNHG5 on the apoptosis of CRC cells.5.The mRNA levels in tumor and normal tissues were analyzed,and mRNAs with the fold change value greater than 2 were used to draw the heat map.CREB5 was selected as the research object,and the expression of CREB5 in tumor and adjacent tissues was confirmed by Western blotting and qRT-PCR.6.Relationship between miR-132-3p and CREB5:The bioinformatics software(Targetscan 7.1)predicted miR-132-3p could target to CREB5.Luciferase activity analysis:the addition of miR-132-3p mimics was able to inhibit luciferase activity of cells transfected with CREB5-wt,but not that of cells transfected with CREB5-mut,indicating that miR-132-3p could directly bind to the 3’UTR region of CREB5 and inhibit its expression.In order to further demonstrate that miR-1 32-3p could inhibit the expression of CREB5,we detected the mRNA and protein expression levels of CREB5 by qRT-PCR and Western blot in LoVo cells which were transfected with miR-132-3p inhibitor or miR-132-3p mimics.7.To evaluate the effects of miR-132-3p on proliferation,metastasis,migration and apoptosis of CRC cells by targeting CREB5:we divided the cells into four groups:CREB5 group,si-CREB5 group,CREB5+miR-132-3p mimics group and NC group.The expression of CREB5 was detected by qRT-PCR.CCK8,transwell assay and wound healing assay were used to detect the proliferation ability of LoVo cells.Flow cytometry exhibited the apoptosis rate.8.In vivo experiments verified the effect of lncrna SNHG5 on CRC:The experiments include two groups:si-SNHG5 group and NC group.Nude mice were sacrificed after 25 days of feeding,and the size and weight of subcutaneous tumors were measured.The expression levels of CREB5 in two groups were detected by Western blot.9.Effects of IncRNA SNHG5 or miR-132-3p on CREB family protein in CRC cells:Western blot experiments were used to verified the effects of IncRNA SNHG5 and miR-132-3p on the changes of CREB family protein expression levels.While after treated with miR-132-3p inhibitor or miR-132-3p mimics,the variation trends of CREB 1 and CREB3 proteins level were evaluated。Results1.Lncrna SNHG5 was overexpressed in CRC tissues and cells.Microarray analysis indicated that the expression level of SNHG5 in tumor increased to 2.79 times.The expressions of SNHG5 in tumor tissues and adjacent tissues were detected by qRT-PCR and the difference between them were statistically significant.The expressions of SNHG5 in RKO,SW480 and LoVo colorectal cancer cell lines and normal colorectal cells FHC showed that:the expression of SNHG5 was up-regulated in CRC cells;among which,it was highest in LoVo cell lines.2.Relationship between miR-132-3p and SNHG5:The expression of miR-132-3p in CRC cells was significantly lower than that in adjacent tissues.The addition of miR-132-3p mimics was able to inhibit luciferase activity of cells transfected ith SNHG5-wt,but not that transfected with SNHG5-mut.The results indicated that SNHG5 could directly bind to the 3’UTR region of miR-132-3p and inhibit its expression.At the transcriptional level,SNHG5 expression in miR-132-3p mimics group didn’t change remarkably compared with NC group,indicating that this was a unidirectional adjustment.MiR-132-3p was negatively correlated with SNHG5 expression.In a word,we reached a conclusion that mik-132-3p was a downstream target of SNHG5 in CRC cells.3.Effect of SNHG5 on proliferation,metastasis and migration of CRC cells by regulating miR-132-3p:CCK-8 assay displayed that the multiplication capacity of LoVo cells transfected with miR-132-3p inhibitor was dramatically higher than that of NC group,whereas the multiplication capacity of LoVo cells transfected with miR-132-3p mimics or si-SNHG5 was decreased in comparison to that of NC group.The metastasis and migration ability of LoVo cells were detected by transwell assay and wound healing assay respectively.Compared with NC group,the metastasis and migration ability of LoVo cells transfected with miR-132-3p inhibitor were enhanced,the metastasis and migration ability of LoVo cells transfected with miR-132-3p mimics or si-SNHG5 were inhibited.Our results indicated that SNHG5 regulated miR-132-3p to promote cell proliferation,metastasis and migration in CRC cells.4.Effects of SNHG5 on apoptosis of CRC cells by regulating miR-132-3p:Flow cytometry was employed and its results displayed the apoptosis rate of miR-132-3p mimics group and si-SNHG5 group were dramatically increased,the apoptosis rate of miR-132-3p inhibitor group was significantly inhibited while the apoptosis rate of si-SNHG5+miR-132-3p inhibitor group was comparable to that of NC group.Our results indicated that SNHG5 regulated miR-132-3p to reduce apoptosis in CRC cells.5.CREB5 was differentially expressed in CRC tissues and cells:Statistical analysis proved that CREB5 was up-regulated in human CRC cell.QRT-PCR results confirmed the expression of CREB5 in CRC tissues was dramatically higher than that in adjacent tissues.Results of Western blot were consistent with the results of qRT-PCR.6.Relationship between miR-132-3p and CREB5:The addition of miR-132-3p mimics was able to inhibit luciferase activity of cells transfected with CREB5-wt,but not that of cells transfected with CREB5-mut,indicating that miR-132-3p could directly bind to the 3’UTR region of CREB5 and inhibit its expression.In LoVo cell lines,the expression levels of mRNA and protein related to CREB5 were detected by qRT-PCR and Western blot.MiR-132-3p was negatively correlated with CREB5 expression.7.Effects of miR-132-3p on proliferation,metastasis,migration and apoptosis of CRC cells by targeting CREB5:CCK-8,transwell migration assay and Wound healing assay results exhibited that compared with NC group,transfection of CREB5 promoted the proliferation ability of LoVo cells,and transfection of si-CREB5 inhibited the proliferation ability of LoVo cells,while the proliferation ability of cells in CREB5+miR-132-3p mimics group was similar to that of NC group.These results illustrated that miR-132-3p inhibited cell proliferation,metastasis and migration,and promoted apoptosis of CRC cells by targeting CREB5.8.In vivo experiments verified the effect of lncrna SNHG5 on CRC:Compared with NC group,si-SNHG5 group could observably inhibit the growth of subcutaneous tumor in nude mice.Western blot exhibited that si-SNHG5 group significantly inhibited the expression of CREB5.These results exhibited that in vivo SNHG5 could affect the multiplication capacity of CRC by regulating CREB5.9.Effects of lncRNA SNHG5 or miR-132-3p on CREB family protein in CRC cells:Western blot experiments displayed that CREB1 and CREB3 proteins were decreased in si-SNHG5 group compared with NC group.While after treated with miR-132-3p inhibitor or miR-132-3p mimics,the variation trends of CREB1 and CREB3 proteins level were similar to CREB5.Conclusions1.Lncrna SNHG5 was overexpressed in CRC tissues and cells2.LncRNA SNHG5 promoted the proliferation,metastasis and migration of CRC cells and inhibited the apoptosis of CRC cells by a unidirectional adjustment of the downstream target miR-132-3p.3.The expression of miR-132-3p in CRC tissues and cells was down-regulated,and CREB5 expression in CRC tissues and cells was up-regulated.4.MiR-132-3p inhibited the proliferation,metastasis and migration of CRC cells,and promoted the apoptosis of CRC cells by regulating CREB5.