Mir-22-3p Suppresses Colorectal Cancer Proliferation And Migration By Targeting KDM3A

Background:Colorectal cancer refers to tumors occurring in the colon or rectum,and its incidence is increasing,seriously threatening people’s health.Under the current level of diagnosis and treatment,comprehensive treatment mainly through surgery for patients initially diagnosed with early-stage colorectal cancer often achieves better therapeutic effects,while those initially diagnosed with advanced colorectal cancer often have a poor prognosis.Therefore,it is of great significance to find effective interventions for colorectal cancer through new biological target molecules.The biological process of colorectal cancer cell proliferation,migration and invasion is one of the most important biological processes leading to the progression of colorectal cancer from early to late stages.It is very important to study this biological process and explore related biological markers for intervention in the treatment of colorectal cancer.In addition,while studying colorectal cancer cells,changes in the tumor microenvironment are also key factors affecting the treatment of colorectal cancer.Tumor-associated macrophages are increasingly being paid attention to in the tumor microenvironment.Due to the influence of tumor cells,the original function of macrophages around tumor cells changes from inhibiting and killing colorectal cancer cells to promoting the growth or metastasis of tumor cells.Therefore,in addition to focusing on the changes in the cell functions of colorectal cancer cells themselves,the changes of tumor-associated macrophages in the colorectal cancer tumor microenvironment also need to be focused on.The main cause of the functional changes of colorectal cancer cells and macrophages is the abnormal expression of tumor-related genes.Post-transcriptional regulation plays an important role in the regulation of gene abnormal expression.Post-transcriptional regulation refers to the process in which mRNA translation and expression are regulated after mRNA transcription.It is an important gene expression regulation mechanism that can regulate gene expression level and thus affect cell growth,differentiation and function.Among transcriptional regulatory molecules,microRNAs(miRNAs)play an important role.There are a large number of miRNAs in human body,which regulate gene expression by degrading target genes through binding to 3’UTR.miR-22-3p is a kind of miRNA,which participates in many biological processes,including cell growth,differentiation and apoptosis.In recent years,it has been found to play an important role in many tumors,but it has been studied less in colorectal cancer.In our previous work,we found that miR-22-3p may target the KDM3A molecule.KDM3A is a histone lysine demethylase involved in epigenetic regulation of gene expression.It is known that KDM3A participates in many biological processes,including cell cycle regulation,stem cell differentiation and cancer progression.It has been found to be up-regulated in many cancers and associated with macrophage polarization,suggesting that it may play a role in tumorigenesis.Therefore,we intend to explore the role of miR-22-3p/KDM3A on the proliferation,migration and invasion functions of colorectal cancer cells and the effect on tumor-related macrophages.Methods:(1)Analysis of miR-22-3p Expression and Clinical Prognostic Value in Colorectal Cancer1.Expression of miR-22-3p in Colorectal Cancer:Differential expression of mir-22 in colorectal cancer tissues and normal mucosal tissues from TCGA data by bioinformatics analysis.Differential expression of miR-22-3p in colorectal cancer cell lines(CaCo2,DLD-1,SW480,SW620)and normal mucosal epithelial cell line NCM460 by qRT-PCR detection.2.Study of miR-22-3p and Prognosis of Colorectal Cancer:Bioinformatics analysis of the relationship between mir-22 expression in colorectal cancer patients and their prognosis.(2)Exploring the effects of changes in miR-22-3p expression on the proliferation,migration and invasion functions of colorectal cancer cells.1.Establishing colorectal cancer cell lines with overexpression of miR-22-3p in colorectal cancer cells:Transfecting colorectal cancer cells with miR-22-3p mimic/NC-mimic to construct colorectal cancer cell lines with overexpression of miR-22-3p and control group cells.Evaluating transfection efficiency by qRT-PCR detection of miR-22-3p expression.2.Establishing colorectal cancer cell lines with low expression of miR-22-3p:Transfecting colorectal cancer cells with miR-22-3p inhibitor/NC-inhibitor to construct colorectal cancer cell lines with low expression of miR-22-3p and control group cells.Evaluating transfection efficiency by qRT-PCR detection of miR-22-3p expression.3.Comparing the changes in proliferation,migration and invasion abilities of colorectal cancer cells with different miR-22-3p expression levels:Detecting the changes in proliferation ability of different treatment groups of colorectal cancer cells by CCK-8 assay.Detecting the changes in migration ability of different treatment groups of colorectal cancer cells by Transwell migration assay.Detecting the changes in invasion ability of different treatment groups of colorectal cancer cells by Transwell invasion assay.Detecting the changes in expression of proliferation,migration and invasion related marker proteins by Western Blot.(3)Exploring the possible mechanisms of miR-22-3p inhibiting the proliferation,migration and invasion abilities of colorectal cancer cells.1.Verification of the binding between miR-22-3p and KDM3A:Reliability of miR-22-3p targeting KDM3 A binding and potential binding sites were analyzed by bioinformatics.KDM3A 3’UTR wild type and mutant fluorescent reporter gene plasmids were constructed according to the predicted binding sites,and the binding between miR-22-3p and KDM3A was verified by dual fluorescent reporter gene assay.The expression of KDM3A mRNA and protein was detected by qRT-PCR and Western Blot respectively in colorectal cancer cell lines after overexpression of miR-22-3p,to verify the effect of miR-22-3p on KDM3A expression in colorectal cancer cells.2.Exploration of the role of KDM3A in colorectal cancer:Bioinformatics analysis of KDM3A expression and prognosis in colorectal cancer.Colorectal cancer cells overexpressing KDM3A were constructed by KDM3A lentivirus transfection in colorectal cancer cells.The role of KDM3A in colorectal cancer was explored by CCK-8,Transwell migration and invasion assays.3.Exploration of the role of KDM3A in the inhibition of colorectal cancer cell proliferation,migration and invasion by miR-22-3p:Simultaneous transfection of miR-22-3p mimics and overexpression of KDM3A in colorectal cancer cells was performed to observe the effect of KDM3 A overexpression on the anticancer effect of miR-22-3p.4.Exploration of the effect of miR-22-3p/KDM3A axis on the expression of HIPPO pathway key marker YAP1:miR-22-3p mimics were transfected into colorectal cancer cells,and the expression of HIPPO pathway marker protein YAP1 was detected by Western Blot to observe the effect of miR-22-3p on HIPPO pathway.The expression of HIPPO pathway marker protein YAP1 was detected by Western Blot when KDM3A molecule was overexpressed in colorectal cancer cells transfected with miR-22-3p mimics,to explore whether miR-22-3p regulates HIPPO pathway by affecting KDM3A molecule.(4)Exploring the Effects of miR-22-3p on Tumor-Associated Macrophages in the Microenvironment of Colorectal Cancer1.Constructing tumor-associated macrophages related to colorectal cancer:M0 macrophages were constructed by stimulating THP1 cells with PM A.Tumor-associated macrophages related to colorectal cancer were constructed by indirect culture of M0 macrophages in colorectal cancer cell SW480 culture medium.Control group macrophages were constructed by indirect culture of M0 macrophages in normal intestinal mucosal epithelial cell NCM460 culture medium.The mRNA of tumor-associated macrophages and control group macrophages were extracted and the molecular markers CD86 and CD 163 of macrophages were compared by qRT-PCR to verify whether tumor-associated macrophages were successfully constructed.2.Cell functional verification of constructed tumor-associated macrophages:The culture medium of tumor macrophages and control group macrophages were collected,and colorectal cancer cells SW480 and DLD-1 cells were stimulated to observe the changes in their proliferation,migration and invasion abilities,and to verify whether tumor-associated macrophages were successfully constructed in cell functional verification.3.Exploring the expression differences of miR-22-3p and KDM3A in tumor-associated macrophages:The mRNA of tumor-associated macrophages and control group macrophages were extracted,and the expression differences of miR-22-3p and KDM3A in two groups of macrophages were compared by qRT-PCR.4.Exploring the role of miR-22-3p in tumor-associated macrophages:The tumor-associated macrophages were transfected with miR-22-3p mimic or NC-mimic to construct tumor-associated macrophages with high expression of miR-22-3p and NC tumor-associated macrophages,respectively,named as miR-22-3p macrophage group and NC macrophage group.qRT-PCR was used to detect the changes of molecular markers CD86 and CD163 in two groups of cells.The effect of miR-22-3p on the tumor-associated macrophages’ pro-cancer biological function was explored by collecting the culture medium of two groups of cells to indirectly culture tumor cells.Results:(1)The Expression and Clinical Prognostic Value of miR-22-3p in Colorectal Cancer1.Bioinformatics analysis showed that the expression of miR-22 in normal colonic tissues was higher than that in colorectal cancer tissues.The results of miR-22-3p detection in different cell lines showed that the expression of miR-22-3p in normal intestinal mucosal epithelial cells was higher than that in colorectal cancer cell lines(CaCo2,DLD-1,SW480,SW620).2.Bioinformatics prognostic analysis showed that high expression of miR-22 was associated with good prognosis of colorectal cancer patients.(2)The Impact of miR-22-3p Expression Changes on the Proliferation,Migration and Invasion Functions of Colorectal Cancer Cells1.qRT-PCR detection of miR-22-3p expression in colorectal cancer cells transfected with miR-22-3p mimic and NC-mimic showed that the expression of miR-22-3p in colorectal cancer cells transfected with miR-22-3p mimic was significantly increased,indicating the successful construction of miR-22-3p overexpression colorectal cancer cells and control cells.2.qRT-PCR detection of miR-22-3p expression in colorectal cancer cells transfected with miR-22-3p inhibitor/NC-inhibitor showed that the expression of miR-22-3p in colorectal cancer cells transfected with miR-22-3p inhibitor was significantly decreased,indicating the successful construction of miR-22-3p low expression colorectal cancer cells and control cells.3.CCK-8 results showed that the proliferation,migration and invasion ability of colorectal cancer cells overexpressing miR-22-3p were lower than those of the control group.The proliferation,migration and invasion ability of colorectal cancer cells with low expression of miR-22-3p were higher than those of the control group.WesternBlot results showed that compared with the control group,the expression of proliferation marker PCNA decreased in the miR-22-3p overexpression group,and the expression of migration and invasion marker E-cadherin increased.In the miR-22-3p low expression group,compared with the control group,its PCNA expression increased,and the expression of migration and invasion marker E-cadherin decreased.(3)The Mechanism of miR-22-3p Inhibiting the Proliferation,Migration and Invasion Ability of Colorectal Cancer Cells1.Bioinformatics prediction showed that miR-22-3p could target and regulate KDM3A molecule.Dual-luciferase reporter gene assay confirmed that miR-22-3p could bind to the 3’UTR of KDM3 A mRNA.After mutating the predicted binding sites of KDM3A 3’UTR,dual-luciferase reporter gene assay showed that the inhibitory fluorescence effect of miR-22-3p disappeared.After transfecting miR-22-3p mimic into colorectal cancer cell lines,the expression of KDM3A mRNA and protein in colorectal cancer cells decreased.2.Bioinformatics analysis showed that KDM3A was highly expressed in colorectal cancer compared with normal tissues.The high expression of KDM3A was associated with poor prognosis of colorectal cancer patients.Overexpression of KDM3A in colorectal cancer cells enhanced the proliferation,migration and invasion ability of colorectal cancer cells.3.Overexpression of KDM3A weakened the inhibitory effect of miR-22-3p on the proliferation,migration and invasion of colorectal cancer.4.Overexpression of miR-22-3p in colorectal cancer cells reduced the expression of YAP1 protein.Overexpression of KDM3A could weaken the inhibitory effect of miR-22-3p on the expression of YAP1 protein.(4)The Effect of miR-22-3p on Tumor-Associated Macrophages in the Microenvironment of Colorectal Cancer1.qRT-PCR results showed that the CD86 molecule marker of the constructed tumor-associated macrophages decreased and the CD 163 molecule marker increased,indicating that the macrophages constructed by us successfully differentiated into tumor-associated macrophages.2.Compared with the control group,the proliferation,migration and invasion ability of colorectal cancer cells indirectly cultured in the culture medium of tumor-associated macrophages were significantly increased.3.qRT-PCR results showed that the expression of miR-22-3p in tumor-associated macrophages decreased and the expression of KDM3A increased.4.After miR-22-3p treatment,compared with the control,the expression of CD86 in miR-22-3p macrophages increased and the expression of CD163 decreased.The ability of miR-22-3p macrophages to promote the proliferation,migration and invasion of colorectal cancer cells was weakened.Conclusion:1.miR-22-3p is a differentially expressed molecule in colorectal cancer,and its overexpression in colorectal cancer is associated with a good prognosis for colorectal cancer patients.2.miR-22-3p can inhibit the proliferation,migration and invasion of colorectal cancer cells.3.miR-22-3p can inhibit the proliferation,migration and invasion of colorectal cancer by targeting KDM3 A to regulate the HIPPO pathway.4.miR-22-3p is differentially expressed in tumor-associated macrophages,which can affect the differentiation of tumor-associated macrophages and weaken their pro-tumorigenic effects.