| Background:Colorectal cancer is a common digestive system malignancy,Its incidence rate is third and second respectively in male and female cancer patients.Mortality rate is only inferior to lung cancer and liver cancer,which seriously endanger human health and life.Although the incidence and mortality of colorectal cancer have declined in recent years,the incidence rate of colorectal cancer is still rising in China.At present,the treatment of colorectal cancer is mainly radical resection,For the patients in the advanced stage,they usually receive the comprehensive treatment of radiotherapy and chemotherapy after operation.However,it is regrettable that a large data from multiple centers around the world shows that,despite the standardized comprehensive anti-tumor treatment,hundreds of thousands of people still die of colorectal cancer every year.The reason is that colorectal cancer has a wide range of drug resistance to a variety of chemotherapy drugs.At the same time,the side effects of chemotherapy on patients seriously affect the quality of life of patients and waste a lot of medical resources.Therefore,it is urgent to select effective and low toxic side effects of anti-tumor drugs and find effective treatment targets.Micro RNA(mi RNA)is a kind of small,regulatory,endogenous non coding single stranded small molecular RNA composed of 19-25 nucleotides.Mi RNA gene expression has the following characteristics: high stability,tumor correlation and tissue specificity.These characteristics make mi RNA become a potential biomarker.Mi RNA plays an important role in cell differentiation,metabolism and tumor formation.Abnormal mi RNA expression can directly promote or inhibit the transcription of downstream target genes,play the role of oncogenes or tumor suppressor genes,and thus affect the progression of colorectal cancer.At present,the main difficulty of mi RNA research is the prediction of target gene and the evaluation of biological function.How mi RNA play a regulatory role in the occurrence and development of colorectal cancer needs to be further explored.It has become a research hotspot of tumor therapy to select the appropriate tumor related gene as the target and carry out specific treatment for tumor.According to the literature,mi R-491 has been found to play a role in regulating the proliferation,apoptosis,invasion and metastasis of cancer cells and drug resistance in a variety of tumors,such as glioma,oral squamous cell carcinoma,breast cancer,lung cancer,gastric cancer,adrenal cancer,cervical cancer,etc.In addition,the abnormal increase of mi R-491 is related to the invasion and metastasis of colorectal cancer.Down regulating the expression of mi R-491 through specific inhibitors can increase the sensitivity of chemotherapy drugs to animal models.Although the current study can show that mi R-491 is closely related to the occurrence and prognosis of colorectal cancer,the regulatory mechanism of mi R-491 is still unclear.PEG10 is a paternal imprinted gene,which has been proved to be a cancer gene,which can regulate cell growth and differentiation,and promote cell growth and proliferation.Foreign scholars have found that IGF-2(imprinted gene insulin-like growth factor 2)is overexpressed by methylation in most colorectal cancer,which leads to the proliferation and differentiation of tumor cells,thus promoting the occurrence and development of tumor.Therefore,we speculate that PEG10,a paternal expression gene homologous with IGF-2,is also overexpressed in colorectal cancer.Wnt gene is highly conserved in all kinds of organisms.There is no Wnt gene in normal mature cells.Among them,Wnt1 may be the initiating factor leading to the activation of classical Wnt signaling pathway.?-catenin is a new proto oncogene product,which is highly expressed in colorectal cancer,but there is no free ?-catenin in normal colorectal epithelial cytoplasm.The results showed that Wnt / ?-catenin could not be degraded in colorectal cancer cells due to its mutation and abnormal activation.As a result,a large number of Wnt /?-catenin accumulated in the cytoplasm and translocated into the nucleus,combined with the corresponding transcription factors(such as TCF4 / LEF)to promote the expression of some downstream proto oncogenes,make the cells over proliferate,differentiate,inhibit cell apoptosis and lead to tumorigenesis.Traditional Chinese medicine,as a unique medical means in China,is an important part of comprehensive anti-tumor treatment,with its unique advantages.It can repair the side effects of chemotherapy drugs in time,improve the normal function of patients,improve the quality of life of patients,and increase the sensitivity of tumor to chemotherapy drugs.Its anti-tumor effect has been widely used in clinical.Curcumin,as a traditional Chinese medicine,can regulate many signal transduction pathways.It has been proved to play an important role in chemoprevention and chemotherapy of colorectal cancer in vitro cell model and animal model.At present,curcumin has been listed as the third generation of chemopreventive drugs by the National Cancer Research Institute of the United States.However,up to now,the molecular mechanism of its anticancer effect has not been fully understood.Objective:This study is based on mi RNA level to explore the mechanism of curcumin on colorectal cancer.The effects of curcumin on mi R-491,PEG10 and Wnt1/?-catenin signaling pathway were analyzed in HCT-116 cells to determine the level changes of curcumin-mi R-491-PEG10-Wnt1/?-catenin regulatory axis in the development of colorectal cancer.This paper expounds the regulatory mechanism of curcumin on the occurrence and development of colorectal cancer,and provides scientific basis for further diagnosis and prognosis of colorectal cancer.It is of great significance to study the mechanism of traditional Chinese medicine(TCM)on the level of mi RNA in the prevention and treatment of colorectal cancer,which can provide a new molecular target for TCM in the prevention and treatment of colorectal cancer.The combination of TCM and Western medicine is expected to improve the five-year survival rate of colorectal cancer patients.Method:Experiment 1:From June 2016 to January 2017,we collected specimens of colorectal cancer tissues and corresponding adjacent tissues(? 5cm from tumor tissues)which were removed by colorectal and anal surgery in the first hospital of Jilin University.The expression of mi R-491,PEG10,Wnt1 and ?-catenin m RNA was detected at tissue level by q PCR.Western blot was used to detect the expression of PEG10,Wnt1 and ?-catenin in colorectal cancer and adjacent tissues.Experiment 2:The expression of mi R-491 in HCT116,Caco2 and HT29 colorectal cancer cells was detected by q PCR.The construction and sequencing of mi R-491 vector were completed by Shanghai Gene pharma company.The target relationship between mi R-491 and PEG10 was verified by double Luciferase reporter gene detection system.The expression levels of mi R-491,PEG10,Wnt1 and ?-catenin m RNA in HCT116 cells transfected with mi R-491 mimics,mi R-491 inhibitor and mi R-NC were detected by q PCR.Western blot was used to detect the expression of PEG10,Wnt1 and ?-catenin in HCT116 cells after transfection of mi R-491 mimics,mi R-491 inhibitor and mi R-NC,respectively.Experiment 3:MTT method was used to detect the effect of curcumin and PEG10 overexpression on the proliferation of HCT116 cells.Flow cytometry was used to detect the effect of curcumin and PEG10 overexpression on the apoptosis of HCT116 cells.The m RNA levels of mi R-491,PEG10,Wnt1 and ?-catenin in curcumin group and PEG10 overexpression group were detected by q PCR.The expression levels of PEG10,Wnt1 and ?-catenin in curcumin group,PEG10 overexpression group and negative control group were detected by Western blot.In order to observe the inhibitory effect of curcumin on tumor growth in vivo,the xenograft model of immunodeficient mice was established by subcutaneous inoculation of HCT116 cells.Curcumin was injected intraperitoneally for 5 weeks.MTT method was used to detect the effect of curcumin on the proliferation of HCT116 cells.The effect of curcumin on apoptosis of HCT116 cells was detected by flow cytometry.The expression of PEG10,Wnt1 and ?-catenin m RNA in HCT116 cells transfected with mi R-491 inhibitor was detected by q PCR compared with that in mi R-491 inhibitor + curcumin and negative control group.Western blot was used to detect the expression level of PEG10,Wnt1 and ?-catenin in HCT116 cells transfected with mi R-491 inhibitor compared with mi R-491 inhibitor + curcumin and negative control group.Result:Experiment 1:The results of q PCR showed that the expression level of mi R-491 in colorectal cancer was significantly lower than that in adjacent tissues,while the expression levels of PEG10,Wnt1 and ?-catenin m RNA were significantly higher than that in adjacent tissues(**P<0.01).The expression of PEG10,Wnt1 and ?-catenin in colorectal cancer was significantly higher than that in adjacent tissues.Experiment 2:The results showed that the expression level of mi R-491 in HCT116 cells was lower than that in other colorectal cancer cells,and it could be used for further experimental study in vitro.Using bioinformatics websites such as mi RBase and Targetscan to predict that PEG10 is a potential downstream target gene of mi R-491.The detection results of double Luciferase reporter gene indicated that mi R-491 could bind through the binding site of 3'UTR region of PEG10,thus inhibiting the expression of PEG10,which showed the decrease of Luciferase activity,further confirmed that PEG10 was the downstream target gene of mi R-491.The expression of mi R-491 in HCT116 cells transfected with mi R-491 mimics was significantly higher than that of mi R-491 inhibitor or mi R-sh NC.The expression of PEG10 m RNA decreased significantly after mi R-491 mimics was transfected,and mi R-491 inhibitor significantly increased the expression level of PEG10 m RNA.In mi R-491 mice group,Wnt1 and ?-catenin m RNA expression level was down regulated,while mi R-491 inhibitor significantly increased Wnt1 and ?-catenin m RNA expression level.Western blot was used to detect the comparison of mi R-491 mimics transfected in HCT116 cells with mi R-491 inhibitor or mi R-sh NC transfected in HCT116 cells,PEG10,Wnt1 and ?-catenin protein decreased significantly in mi R-491 mimics group,while mi R-491 inhibitor could significantly increase the expression levels of PEG10,Wnt1 and ?-catenin protein.Experiment 3:MTT results showed that curcumin can significantly reduce the activity of HCT116 cells,and PEG10 overexpression can significantly increase the activity of HCT116 cells.The results of flow cytometry showed that curcumin could induce apoptosis of HCT116 cells,while PEG10 over expression could significantly reduce the apoptosis rate of HCT116 cells.Compared with the control group,the tumor volume and weight decreased,the difference was statistically significant.The results showed that curcumin can inhibit tumor growth in mice.The results of q PCR showed that curcumin could up regulate the expression of mi R-491 and down regulate the expression of mi R-491,PEG10,Wnt1 and ?-catenin m RNA.Overexpression of PEG10 significantly increased the m RNA expression of Wnt1 and ?-catenin.Western blot analysis showed that PEG10 overexpression increased the expression of PEG10,Wnt1 and ?-catenin,while curcumin decreased the expression of PEG10,Wnt1 and ?-catenin.MTT results showed that curcumin could significantly reduce the activity of HCT116 cells after mi R-491 was inhibited,which indicated that curcumin could reduce the proliferation of HCT116 cells.Flow cytometry showed that curcumin could induce apoptosis of HCT116 cells after mi R-491 was inhibited.The expression of PEG10,Wnt1 and ?-catenin m RNA in HCT116 cells transfected with mi R-491 inhibitor was significantly higher than that in mi R-491 inhibitor + curcumin and negative control groups.The expression level of PEG10,Wnt1 and ?-catenin m RNA in HCT116 cells transfected with mi R-491 inhibitor + curcumin was significantly lower than that in mi R-491 inhibitor.Western blot was used to detect the expression levels of PEG10,Wnt1 and ?-catenin in HCT116 cells transfected with mi R-491 inhibitor,while the expression levels of PEG10,Wnt1 and ?-catenin in HCT116 cells transfected with mi R-491 inhibitor + curcumin were significantly lower than that of mi R-491 inhibitor.Conclusion:Experiment 1:(1)In this study,real-time fluorescent quantitative PCR was used to detect the expression of mi R-491 in paracancerous tissues,which was significantly higher than that in colorectal cancer tissues.It was confirmed that mi R-491 played a role of "tumor suppressor gene" in the occurrence and development of colorectal cancer.(2)The expression of PEG10,Wnt1 and ?-catenin in colorectal cancer was significantly higher than that in adjacent tissues,both at RNA level or protein level,indicating that PEG10,Wnt1 and ?-catenin played a role in promoting the occurrence and development of colorectal cancer.(3)We also found that the expression of mi R-491 was negatively correlated with PEG10,Wnt1 and ?-catenin m RNA.Experiment two:(1)Compared with other colorectal cancer cells,mir-491 decreased most significantly in HCT116 cells.(2)In this experiment,PEG10 was predicted to be the target gene of mi R-491 by gene prediction program,and the interaction between mi R-491 and PEG10 was verified by double Luciferase Report System,and PEG10 was the downstream target gene of mi R-491.(3)It was confirmed by overexpression and silencing mi R-491 that mi R-491 could inhibit the expression of PEG10,Wnt1 and ?-catenin m RNA and protein after overexpression,while silencing mi R-491 could promote the expression of PEG10,Wnt1 and ?-catenin m RNA and protein,further indicating the negative regulatory effect of mi R-491 on PEG10,Wnt1 and ?-catenin.Experiment three:(1)From the results of MTT colorimetry and flow cytometry,curcumin can inhibit the proliferation of HCT116 cells in vitro and promote the apoptosis of HCT116 cells;curcumin can inhibit tumor growth in mice;moreover,curcumin can still produce the above effects and promote the expression of mi R-491 after the silence of mi R-491;after the overexpression of PEG10,it can promote the proliferation of HCT116 cells and inhibit the apoptosis of HCT116 cells.(2)Wnt1 and ?-catenin are downstream signal molecules of PEG10.The anti-tumor effect of mi R-491 on colorectal cancer is achieved by regulating PEG10 and Wnt1 / ?-catenin signaling pathway.(3)Curcumin can effectively promote the expression of miR-491 and inhibit the expression of PEG10,Wnt1 and ?-catenin m RNA and protein.Therefore,the antitumor effect of curcumin on colorectal cancer is realized by up regulating the expression of mi R-491,thereby reducing the expression of PEG10,and then inhibiting the Wnt1 / ?-catenin signaling pathway.(4)This study is to research the anti-tumor mechanism of curcumin at mi RNA level,and to report the regulatory effect of curcumin on mi R-491 and PEG10 for the first time,which is expected to become a new target of anti-colorectal cancer treatment,and to provide new ideas and directions for the research and development of anti-colorectal cancer drugs. |
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