Mechanism Study Of P53/miR-29c-3p Circuit Regulation Of Colorectal Cancer Cell Proliferation And Metastasis

BackgroundRecurrence and metastasis of colorectal cancer(CRC)are the main challenges in clinic,In addition to surgery and radiotherapy,chemotherapy is a major option for this disease.However,currently available chemotherapies of colorectal cancer are not ideal,as patients often fail the treatments due to drug resistance.Previous studies showed that p53 mutations or dysfunction are associated with reduced susceptibility of tumor cells to chemotherapy.The latest experimental results show that p53 can be used as the intermediate link between different stress and cellular responses,and play an important role in tumorigenesis and drug response,through a variety of feedback signal stimulation.Previous study confirmed that p53 is the main substrate of SIRT1(Sirtuin 1,silent mating type information regulation 2 homolog),which is an NAD+ dependent histone deacetylase.It was found that 90% of p53 protein acetylation could be deacetylated by SIRT1,which reduces p53 activity,and increases the risk of cancer.Meanwhile,p53,as transcriptional factor,is also involved in various biological processes of tumor cells by regulating the expression of its downstream target genes.Therefore,it is of great interest to further understand the functions of p53 and its related pathways in tumor biology by exploring the upstream and the downstream regulatory mechanisms of p53.In recent years,great attention has been attracted to identifying non-coding RNA molecule micro RNA(miRNA)that can be directly targeted by p53 for negatively regulating gene expression at post transcription level.Growing evidences have proved that alteration in miRNA expression could contribute significantly to cancerous phenotypes of human tumors,including CRC.As one such important miRNA,miR-29 c has been found down-regulated in multiple cancer types,such as chronic lymphocytic leukemia,lung cancer,gastric cancer,malignant lymphoma.Although the experimental results showed that miR-29 c may be p53 target gene,and participate in the p53 tumor signaling pathway.In addition,study indicated that miR-29 c may participate in the expression of post-translational modified genes,such as the SIRT family.It is suggested that the regulation of tumor suppressor gene p53 may have some circuits,However,little is known about the mechanism of these ciruits regulation in tumor.Previous studies have conducted a large number of experiments using miRNA,which have enriched the understanding of p53.For example,some people think that there is a close relationship between p53 and PI3 K.Because the mechanism of tumor regulation is extremely complex,it is difficult to obtain a clear explanation using traditional experimental methods.In order to further study the pathway of p53/miRNA,we need to combine the cell biological exprements.In summary,by using computer software and cell biological exprements,we are interested in addressing the following questions:(1)whether mir-29 c is regulated by p53 to participate in the antitumor effects toward CRC;(2)whether miR-29 c forms p53 positive or negative feedback by regulating other tumor related genes;and(3)identification of novel mir-29 c target gene that is responsible for CRC phenotypes.ObjectiveTo evaluate the functional relationship between miR-29c-3p and p53 in human colorectal cancer cell lines HCT116 p53+/+ and HCT116 p53-/-.To identify novel target gene of miR-29c-3p and to explore its biological effects on CRC.Methods(1)Effect of miR-29c-3p on cell proliferation and apoptosis were assessed by 5-FU,Act D,CCK-8 and apoptosis kit in HCT116 p53+/+ and HCT116 p53-/-cells.(2)Effects of transfection of Flag-PHLDB2 and miR-29c-3p mimics,and cotransfection on cell invasion were assessed by Trans Well invasion assay using HCT116 p53-/-cells.and effects of transfection of Flag-PHLDB2 and miR-29c-3p mimics,and co-transfection on cell migration were assessed by wound healing assay using HCT116 p53-/-cells.(3)Expression of miR-29c-3p in HCT116 p53+/+ and HCT116 p53-/-cells,effect of ectopic FLAG-p53 on miR-29c-3p expression in HCT116 p53-/-cells,and miR-29c-3p expression following transfection of miR-29c-3p mimics and anti-miR-29c-3p in two cell lines were assessed by RT-q PCR.(4)Protein expression of p53 and target genes,such as SIRT1 and PHLDB2,were tested by western blot.(5)miR-29 c gene sequence was analyzed by p53 MH software to identify potential p53 responsive element(RE).Online analysis was employed to screen for potential miR-29c-3p target and PHLDB3 was selected for further validation.(6)Luciferase report gene plasmid were constructed.Dual luciferase reporter assay following co-transfection of these plasmids was performed in HCT116 p53-/-cells.The regulation of RE by p53 and PHLDB2 3'-UTR by miR-29c-3p were detected by Dual luciferase reporter assay.Results(1)5-FU and Act D significantly increased expression of p53 and its target gene MDM2,accompanied by elevated expression of miR-29c-3p,in HCT116 p53+/+,but not HCT116 p53-/-cells.These results indicate that the expression of miR-29c-3p is inducible by p53 activating drugs.(2)Ectopic expression of FLAG-p53 significantly up-regulated miR-29c-3p expression in HCT116 p53-/-cells.(3)p53 MH software analysis showed that the upstream of miR-29c-3p gene contains two highly conservative p53 responsive elements(RE1,RE2),and p GL3-RE1 and p GL3-RE2 were constructed successfully.Dual luciferase reporter assay showed that p GL3-RE1,but not p GL3-RE2,could be regulated by p53 in a dose-dependent manner,suggesting that miR-29c-3p is a direct target gene of p53.(4)RT-q PCR confirmed that transfection of miR-29c-3p mimics in HCT116 p53+/+ and HCT116 p53-/-cells could increase the expression of miR-29c-3p,while anti-miR-29c-3p decreased miR-29c-3p expression.(5)CCK-8 proliferation experiment showed that miR-29c-3p mimics significantly inhibited cell proliferation of HCT116 p53+/+ one day after transfection,while similar inhibition on HCT116 p53-/-cells occurred four days after transfection.anti-miR-29c-3p promoted proliferation of HCT116 p53+/+ cells two days after transfection,but no significant effect was observed in HCT116 p53-/-cells,further supporting that the effect of miR-29c-3p on the proliferation of CRC was p53 dependent.The apoptosis experiment showed that miR-29c-3p mimics could induce cell apoptosis by ~14 times compared with the control group in HCT116 p53+/+ cells.In HCT116 p53-/-cells,only ~ 4 times.This result suggests that induction of cell X apoptosis by miR-29c-3p is more significant in p53 positive cells than that in p53 negative cells.CCK-8 proliferation experiment showed that Act D inhibition of HCT116 p53+/+ cell proliferation was significantly lower in the presence of antimiR-29c-3p than that in the control group.This result indicates that the inhibitory effect of Act D on HCT116 p53+/+ cell proliferation is partially mir-29c-3p dependent.(6)WB showed that transfection of miR-29c-3p mimics in HCT116 p53+/+ cells could significantly reduce the expression of SIRT1 and increase expression of p53 and p21,confirming that miR-29c-3p overexpression could down-regulate SIRT1 and in turn activate p53.SIRT1 expression was decreased in response to SIRT1 si RNA transfection in HCT116 p53+/+ cells.While in the presence of miR-29c-3p overexpression,compared with SIRT1 si RNA alone,p53 acetylation was not further induced by SIRT1 knockdown,and p53 and p21 expression were not increased.These findings indicate that miR-29c-3p activates p53 through targeting SIRT1.Then WB showed that in HCT116 p53+/+ cells,5n M Act D could activate p53 pathway,accompanied by decrease of SIRT1 expression.However,in the presence of antimiR-29c-3p,SIRT1 was not changed.This observation suggests that p53 could suppress SIRT1 through induction of miR-29c-3p.(7)The online software found that the 3'-UTR of PHLDB2 m RNA contains a putative miR-29c-3p target sequence.It suggests that miR-29c-3p has the potential to regulate PHLDB2.(8)RT-q PCR showed that PHLDB2 m RNA level was not significant changed in HCT116 p53-/-cell after transfection of miR-29c-3p mimics or anti-miR-29c-3p.WB showed that miR-29c-3p mimics could reduce,while anti-miR-29c-3p could increase,the expression of PHLDB2 protein.This result indicates that miR-29c-3p could indeed regulate PHLDB2 expression.(9)p MIR-PHLDB2(WT)and p MIR-PHLDB2(MU)were constructed successfully,and Dual luciferase reporter assay showed that miR-29c-3p mimics wild-type,but not mutant,miR-29c-3p binding site of the 3'-UTR of PHLDB2 m RNA.(10)Analysis of human cancer databases available from PROGgene V2 showed that higher expression of PHLDB2 is associated with shorter overall survival and metastasis-free survival of colon cancer patients.It was concluded that the PHLDB2 expression level was negatively correlated with the prognosis of colorectal cancer.(11)Mammalian FLAG-PHLDB2 expression vector was generated successfully.(12)Trans Well experiment showed that suppression of colon cancer cell invasion by miR-29c-3p was significantly attenuated in the presence of ectopic PHLDB2.Wound healing experiment showed that suppression of colon cancer cell migration by miR-29c-3p was significantly attenuated in the presence of ectopic PHLDB2.Conclusions(1)We found that the upstream of miR-29c-3p gene contains a functional p53 consensus responsive element that is driven by p53 transcriptional factor activity,suggesting miR-29c-3p as a direct p53 target gene.(2)miR-29c-3p exerts significant anti-tumor effects,such as inhibition of cell proliferation,induction of apoptosis,in a partially p53-dependent manner,highly likely through suppression of SIRT1,a known miR-29c-3p target and a deacetylase for p53.(3)Through online software prediction and in vivo validation,we demonstrated that Pleckstrin Homology Like Domain Family Member 2(PHLDB2)is a valid miR-29c-3p target gene.Analysis of human cancer databases available from PROGgene V2 showed that higher expression of PHLDB2 is associated with shorter overall survival and metastasis-free survival of colon cancer patients.Further,suppression of colon cancer cell invasion and migration by miR-29c-3p was significantly attenuated in the presence of ectopic PHLDB2,indicating that PHLDB2 is a critical downstream target of miR-29c-3p.Collectively,our findings present the first to elucidate that miR-29c-3p is a direct p53 target gene,it can form positive feedback regulation of p53 through SIRT1,and also identify PHLDB2 as an important miR-29c-3p target gene involved in colorectal cancer metastasis.It confirms that p53/miR-29c-3p circuit has regulation effect on proliferation and metastasis of colorectal cancer.The further study may clarify the role of PHLDB2 in the regulation of p53 network.