| Background:Colorectal cancer(CRC)has risen to the third highest incidence of malignant tumors in the world,with poor prognosis and high mortality.The occurrence and development of CRC involves a variety of mechanisms,including intestinal flora disorder,heredity,gene mutation and intestinal inflammation,etc,accompanied by a large number of proto-oncogene activation and inhibitory gene inactivation.At present,the most effective way to treat CRC is radical surgery combined with radiotherapy and chemotherapy,molecular targeted therapy,and immunotherapy.Although adjuvant drugs significantly improve the prognosis of CRC patients in late stages,only 15%-25%of the patients are responsive to the treatments.In addition,among patients who can undergo radical surgery,up to 20%of patients have local or distant recurrence within 5 years after surgery.Therefore,searching for oncogenes related to the development of CRC is essential to further regulate the treatment of CRC and improve the prognosis of CRC patients.Mex3 RNA-binding protein family member A(Mex3a)was originally found in C.elegans as a translation-regulating protein that helps to maintain the totipotency of the reproductive line.In humans,the Mex3 family has four homologous proteins Mex3a,b,c,and d.Previous studies have shown that Mex3a plays a key carcinogenic effect in lung cancer,gastric cancer,breast cancer,liver cancer,bladder urothelial cancer,Wilm's tumor and glioblastoma multiforme.In the intestinal tract,multiple studies have shown that Mex3a mainly affects intestinal differentiation,polarity and stemness,and helps to maintain intestinal homeostasis.This prompted us to study the role and regulatory mechanism of Mex3a in CRC.MicroRNAs(miRNA)are short-chain non-coding RNAs consisting of 19-23 nucleotides.miRNAs inhibit target gene expression by sequence-specific interaction with the 3'-UTR,leading to mRNA degradation and inhibiting protein translation miRNAs play important roles in the processes of cancer development,such as differentiation,invasion,proliferation,and apoptosis.An increasing number of miRNAs are related to the occurrence of CRC and are considered as markers of prognosis and CRC treatment.For instance,miR-214 was shown to inhibit the growth and liver metastasis of CRC by regulating fibroblast growth factor receptor 1(FGFR1).Therefore,we speculate that a certain miRNA may play a key role for the regulation of Mex3a in CRC.This study aims to explore the expression and role of Mex3a in colorectal cancer,and further explore its upstream and downstream mechanisms,and provide a new perspective on the pathogenesis of colorectal cancer.This study first detected the expression of Mex3a in CRC clinical samples,and then verified the expression of Mex3a in public databases,and analyzed its relationship with CRC clinicopathological parameters.In vivo and in vitro functional experiments are conducted to explore the effects of Mex3a on CRC cell proliferation,invasion and other abilities,and to provide a new perspective for the pathogenesis of CRC by looking for miRNA directly bound to Mex3 A and its downstream regulated pathways.Methods:1.Immunohistochemical(IHC)found that the expression level of Mex3a protein in CRC tissue was significantly up-regulated compared to adjacent tissues.Similarly,Western blot also confirmed the expression of this protein in cancer and adjacent tissues.Subsequently,gene expression omnibus(GEO)and The Cancer Genome Atlas(TCGA)databases were used to verify the expression of Mex3a in cancer and para-cancerous tissues.The H score was determined according to the intensity of IHC staining,and the relationship between Mex3a expression level and clinicopathological characteristics of CRC patients was analyzed.The follow-up data of CRC patients was sorted and analyzed by Kaplan-Meier method to draw the relationship between Mex3a expression and survival time of CRC patients.2.The expression of Mex3a in CRC cell lines was detected by qRT-PCR and Western blot,and we screen out endogenous cell lines with low expression of Mex3a and high expression of Mex3a,selected cell lines with high Mex3a expression SW620 and SW480 to transfect Mex3a small interference,the cell line HCT116 with low Mex3a expression to transfect the Mex3a overexpression plasmid.EDU assay was performed to observe the effect of Mex3a on CRC cell proliferation,tube formation assay was used to observe the effect of Mex3a on angiogenesis,transwell experiment was used to observe the effect of Mex3a on CRC cell invasion and migration,Drug sensitivity assay was used to observe the effect of Mex3A on the anti-chemotherapeutic ability of CRC cells.3.The SW620 and SW480 cell lines with stable knockdown of Mex3a,HCT116 cell line with stable overexpression of Mex3a were constructed by transfection with lentivirus.After puromycin screening,the CRC cell lines with stable knockdown or overexpression of Mex3a and control cell lines were constructed.Then it was subcutaneously injected into nude mice to observe the effect of Mex3a on the growth and metastasis of CRC cells under in vivo conditions.4.Through transcriptomics sequencing and KEGG analysis,we screened the downstream signaling pathways of Mex3a that affect the development of CRC:RAP1GAP/MEK/ERK/HIF-1?,and verified the influence of Mex3a on the RAPIGAP/MEK/ERK/HIF-la signaling pathway by Western blot.MEK inhibitor(U0126)verified that Mex3a affect the proliferation,invasion and migration of CRC cells through the MEK/ERK/HIF-la signaling pathway.Western blot verified that U0126 could reversed the effect of Mex3a on RAP1GAP/MEK/ERK/HIF-1? pathway.5.Using Co-immunoprecipitation(Co-IP)experiment to verify the interaction between Mex3a and RAP1GAP.Using clinical samples to verify the correlation between Mex3a and RAP 1 GAP.Using TCGA database and clinical samples to verify the expression changes of RAP1GAP in CRC tissues and adjacent tissues.Using Kaplan-Meier methods to analyze the relationship between the expression of RAP1GAP and the survival time of CRC patients in the TCGA database.Western blot proved that RAP1GAP could reversed the effect of Mex3a on the RAP1GAP/MEK/ERK/HIF-1?pathway.6.We predicted the miRNA that regulates Mex3a by TargetScan(www.targetscan.org)and miRDB(mirdb.org),and the binding site and mutation site sequence of hsa-miR-6887-3p on Mex3a mRNA 3'-UTR were obtained.Through luciferase experiments we verified that hsa-miR-6887-3p was a possible upstream target of Mex3a.The TCGA database verified the expression of hsa-miR-6887-3p in CRC tissues and adjacent tissues.The relationship between the expression of hsa-miR-6887-3p in the TCGA database and the survival time of CRC patients was analyzed by Kaplan-Meier method.The expression of hsa-miR-6887-3p in CRC cell lines was detected by qRT-PCR,and we screen out endogenous cell lines with low expression of hsa-miR-6887-3p and high expression of hsa-miR-6887-3p.Selected with relatively high hsa-miR-6887-3p expression CRC cell lines HCT116 to transfect hsa-miR-6887-3p inhibitor,with low hsa-miR-6887-3p expression CRC cell lines SW620 to transfect the hsa-miR-6887-3p mimics,then the effect of hsa-miR-6887-3p mRNA expression levels was verified by qRT-PCR.The effect of hsa-miR-6887-3p on Mex3a mRNA and protein expression levels was detected by qRT-PCR and Western blot.7.Western blot was used to detect the transfection of Mex3a small interference,overexpression of hsa-miR-6887-3p and Mex3a in the SW620 cell line.Effect on Mex3a expression in SW620 cell line.Through functional recovery experiments,the effect of hsa-miR-6887-3p on the proliferation and invasion of CRC cells in vitro was explored.8.The SW620 cell line stable overexpression of hsa-miR-6887-3p was constructed by transfection with lentivirus.After puromycin screening,the CRC cell lines with stable overexpression of hsa-miR-6887-3p and control cell lines were constructed.Then it was subcutaneously injected into nude mice to observe the effect of hsa-miR-6887-3p on the subcutaneous tumorigenesis ability of CRC cells in nude mice under in vivo conditions.Result:1.Mex3a is highly expressed in CRC and correlates with poor prognosisThe protein levels of Mex3a in 101 CRC tissues and 79 normal adjacent tissues were analyzed by immunohistochemical staining.The results showed that Mex3a was highly expressed in CRC tissues,but almost not expressed in adjacent tissues.Through GEO(GSE39582)and TCGA database,we found that the expression level of Mex3a mRNA in CRC was significantly higher than in normal adjacent tissues.Correlation analysis of clinicopathological parameters of 101 CRC patients showed that the high expression of Mex3a in CRC tissues was positively correlated with T stage(P=0.025),pathological grade(P=0.035),patient survival status(P=0.012)and others clinicopathological parameters,but there was no significant correlation with age(P=0.134),gender(P=0.392),N stage(P=0.919),M stage(P=1)and TNM grade(P=0.325).Kaplan-Meier survival curve analysis was performed on 101 CRC patients.The results showed that the overall survival rate(OS)of patients with Mex3a high expression group was significantly shorter than that with Mex3a low expression group(P=0.003).Secondly,the survival analysis of the GSE39582 dataset showed that compared with patients with Mex3a low expression group,the OS of the patients with Mex3a high expression group was significantly shorter(P=0.022),and the recurrence-free survival rate(RFS)was also significantly reduced(P=0.048).2.High expression of Mex3a promotes tumorigenic properties of CRC cellsThe results of qRT-PCR and Western blot showed that Mex3a had the relatively high expression level in SW620 and SW480 CRC cell lines,and the relatively low expression level in HCT116 cell line.Western blot results showed that compared with the control group,the small interference of Mex3a(Mex3a siRNA)and the overexpression plasmid(Mex3a-flag)could effectively reduced and increased the expression of Mex3a in CRC cells,respectively.The EDU experiment showed that the proliferation ability of CRC cells was significantly weakened after interference the expression of Mex3a,while the proliferation ability of CRC cells was significantly enhanced after overexpression of Mex3a.The angiogenesis experiment showed that ability of CRC cells to promote tube formation was significantly weakened after interference the expression of Mex3a,while the ability of CRC cells to promote tube formation was enhanced after overexpression of Mex3a.Transwell results showed that interference the expression of Mex3a inhibited the invasion and migration ability of CRC cells,while overexpression of Mex3a could significantly promoted the invasion and migration ability of CRC cells.Drug sensitivity experiments showed that the anti-chemotherapeutic drug ability of CRC cells was significantly weakened after interference the expression of Mex3a,and the anti-chemotherapeutic drug ability of CRC cells was enhanced after overexpression of Mex3a.3.Mex3a enhances oncogenesis of CRC cells in vivoConstructed Mex3a stable knockdown(Mex3a KD)and control(Control KD)CRC cell lines,Mex3a stable overexpression(OE Mex3a)and control(OE Control)CRC cell lines.The subcutaneous transplanted tumor model in nude mice showed that the tumor weight and tumor volume of Mex3a KD group was significantly smaller than Control KD group,Ki-67 staining showed that the Mex3a KD group had a significantly lower staining density than the Control KD group.However,the tumor volume,tumor weight,and Ki-67 staining in the OE Mex3a group had opposite results.4.Mex3a acts as a co-activator of RAP1GAP/MEK/ERK/HIF-1? signaling in CRC cellsTranscriptome sequencing was performed on the stable SW480 Mex3a KD and SW480 Control KD CRC cell lines to find the downstream target genes and related signal pathways of Mex3a.The results showed that the low expression of Mex3a caused changes in many signal pathways including MAPK and Rap1 signal pathway.Considering the differentially expressed genes involved in this pathway,we speculated that Mex3a may affected the malignant biological behavior of CRC cells through the RAPIGAP/MEK/ERX/HIF-1? pathway.Western blot results showed that after knock down of Mex3a in SW620 and SW480 cell lines,the protein expression level of RAP 1 GAP was increased,and the protein expression level of p-MEK1/2,p-ERK1/2 and HIF-1? were decreased.Functional rescue experiment showed that MEK inhibitor U0126 can reversed the proliferation,invasion and migration effects of Mex3a on CRC cells.Western blot results showed that U0126 can reversed the effect of Mex3a on the RAP1GAP/MEK/ERK/HIF-1? pathway.5.RAP1GAP is the downstream target gene of Mex3a in CRC cellsCo-immunoprecipitation(Co-IP)results showed that Mex3a in CRC cells can interact with RAP1GAP.The protein expression levels of Mex3a and RAP1GAP were analyzed by immunohistochemical staining of 100 cases of CRC tissues and 6 cases of adjacent tissues.The results showed that Mex3a was highly expressed in CRC tissues,RAP1GAP was lowly expressed in CRC tissues,and RAP1GAP was negatively correlated with Mex3a.Western blot results showed that RAP1GAP can reverse the effect of Mex3a on the RAP1GAP/MEK/ERK/HIF-1? pathway.The TCGA database found that the expression of RAP 1 GAP mRNA in CRC tissues was significantly lower than that in adjacent tissues,and Kaplan-Meier survival curve analysis found that compared with patients in the RAP 1 GAP high expression group,the OS of the patients in the RAP 1 GAP low expression group was significantly shorter(P=0.032).6.hsa-miR-6887-3p is the upstream target of Mex3a in CRC cellsUsing TargetScan and miRDB to predicted the miRNA that regulates Mex3a,and the binding site and mutation site sequence of hsa-miR-6887-3p on Mex3a mRNA 3'-UTR were obtained.Luciferase experiments showed that overexpression of hsa-miR-6887-3p can significantly reduce the luciferase activity of Mex3a wild-type 3'-UTR.Through Starbase(starbase.sysu.edu.cn)analysis,we found that hsa-miR-6887-3p was under-expressed in CRC tissues compared with normal tissues.Kaplan-Meier survival analysis results showed that the survival time of patients in the hsa-miR-6887-3p low expression group was significantly shorter than that in the hsa-miR-6887-3p high expression group(P=0.047).The qRT-PCR results showed that hsa-miR-6887-3p had the lowest expression in the CRC cell line SW620 and relatively high expression in the HCT116 cell line.The qRT-PCR results showed that compared with the control group,hsa-miR-6887-3p mimics and hsa-miR-6887-3p inhibitor could effectively increase and decrease the expression of hsa-miR-6887-3p in CRC cells,respectively.The results of qRT-PCR and Western blot showed that overexpression or inhibition of hsa-miR-6887-3p expression could significantly inhibited or promoted the mRNA and protein expression levels of Mex3a.7.hsa-miR-6887-3p represses Mex3a to inhibit CRC growthWestern blot results showed that overexpression of Mex3a could reversed the inhibitory effect of hsa-miR-6887-3p on Mex3a.EDU and Transwell experiments showed that similar to the knock-down of Mex3a expression group,the overexpression of hsa-miR-6887-3p group could inhibited the proliferation and invasion of CRC cells,while overexpression of Mex3a could reversed the proliferation and invasion effects of overexpression of hsa-miR-6887-3p on CRC cells,and vice versa.8.hsa-miR-6887-3p suppresses CRC cell growth in vivoThe hsa-miR-6887-3p stable overexpression(LV-miR-6887-3p)and control(LV-miR-NC)CRC cell lines were constructed.The subcutaneous transplanted tumor model in nude mice showed that the tumor volume and tumor weight in the LV-miR-6887-3p group was significantly smaller than the LV-miR-NC group.Ki-67 staining showed that the staining density of LV-miR-6887-3p cell group was significantly lower than that of LV-miR-NC group,and the difference was statistically significant.Conclusion:1.The expression of Mex3a is increased in CRC and is related to poor clinical prognosis.2.In vitro and in vivo experiments shows that Mex3a enhanced the malignant biological behavior of CRC cells3.RAP1GAP is a downstream target molecule of Mex3a,which can reverse the effect of Mex3a on the activity of MEK/ERK/HIF-1? signaling pathway.4.hsa-miR-6887-3p inhibits the malignant biological behavior of CRC cells by directly regulating the expression of Mex3a. |
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