| BackgroundColorectal cancer(CRC),also known as colon cancer,is one of the most common gastrointestinal malignancies,ranking second in incidence and third in mortality worldwide.About 50%of CRC patients will appear colorectal liver metastases(CRLM)in the course of disease,which is the main cause of death of patients.Therefore,prevention and control of CRLM is the top priority in overcoming CRC and extending the survival time of patients.A large number of studies have confirmed that microRNAs play an important regulatory role in the occurrence,development,invasion and metastasis of a variety of tumors,which is also the focus of CRC research at present.Microrna(miRNAs)is a kind of small non-coding RNA that exists in a variety of cells.Once microRNAs interact with relevant target genes,they will further regulate the expression of downstream gene proteins.Currently,miRNAs have been found to regulate the expression of more than 70%of human genes.MiRNAs are specific in different tissues and organs and are increasingly identified as new biomarkers or therapeutic targets.Mir-708 is a small subset of several cancer-related micrornas,but has shown unique molecular biology in many cancer studies.The expression level of mir-708 in tumor cells is closely related to cell proliferation,migration,invasion,epithelial-mesenchymal transformation(EMT)and chemical drug sensitivity,which is why miR-708 has attracted more attention.Zinc finger e-box binding homeobox 1(ZEB1),a transcription factor,is an important member of the e-box binding protein family and can promote tumor invasion and metastasis by inducing epithelial-mesenchymal transformation(EMT)of cancer cells.According to previous research found that ZEB1 may be direct target of miR-708,but it has not been researched in the CRC,our research on this subject that aims to explore the miR-708 and ZEB1 expression and its significance in the CRC and explore relationships may exist between the target and the relevant mechanisms,so as to seek for CRC,especially CRLM new targets,new treatment strategies,and provides the theory basis.Part One Expression of miR-708 in colorectal cancer tissues and its effect and mechanism on proliferation,cycle,apoptosis,migration and invasion of colorectal cells.Materials and methodsColorectal cancer tissues and adjacent tissues were from 10 colorectal cancer patients who had signed the informed consent.After the serum was treated by centrifugation,RNA was extracted by Trizol method for backup use.First,qRT-PCR was used to detect the difference in mir-708 expression between cancer tissues and adjacent tissues,CRC cell line(HCT-116)and normal liver epithelial cell line(ECT-1).In HCT-116,Cell models of the high expression miR-708 group(miR-708 mimics group)and the low expression miR-708 inhibitor group were established with transfection technology,and cell proliferation was detected with the Cell counting kit-8(CCK-8)kit.Flow cytometry was used to detect cell cycle and apoptosis.Differences in cell migration and invasion were detected by Transwell assay.The difference in mRNA and protein levels of apoptosis-related Bax and Bcl-2 in HCT-116 cells and EMT-related E-cadherin and N-cadherin were detected by qRT-PCR and western blot.ResultsFirst,the expression of mir-708 in colorectal cancer tissues was significantly lower than that in paracancer tissues.The detection showed that the expression of miR-708 in CRC cell line(HCT-116)was significantly lower than that in normal liver epithelial cell line(ECT-1).Then CCK-8 kit was used to detect that the number of cell proliferation in the miR-708 mimics group was significantly reduced compared with that in the miR-708 inhibitor group.Then the results of qRT-PCR,flow cytometry and Transwell chamber showed that compared with the corresponding negative control group,CRC cells with high expression of miR-708 could significantly inhibit cell proliferation,prolong cell cycle,increase apoptosis and decrease the ability of cell migration and invasion.After that western blot and qRT-PCR were used to detect the expressions of apoptosis-related Bax and Bcl-2.The results showed that miR-708 mimics group could significantly increase the expression of pro-apoptotic gene Bax and reduce the expression of anti-apoptotic gene Bcl-2,while the miR-708 inhibitor group had the opposite results.Finally,EMT-related E-cadherin and N-cadherin expression were detected.The results showed that miR-708 mimics group could increase E-cadherin expression and decrease E-cadherin expression,while miR-708 inhibitor group showed the opposite results in the terms of protein and mRiA levels.ConclusionThe expression of miR-708 in colorectal cancer tissues was significantly decreased,and the high expression of miR-708 in CRC cell lines could reduce the proliferation,migration and invasion of tumor cells,and significantly increase the number of apoptosis.The expression level of miR-708 in CRC cell lines is directly negatively correlated with the degree of tumor malignancy.In addition,miR-708 could promote the apoptosis of CRC cells by up-regulating the expression of pro-apoptotic protein Bax and down-regulating the anti-apoptotic protein Bcl-2.At the same time,miR-708 could also inhibit EMT in tumor cells by increasing the expression of E-cadherin and reducing the expression of N-cadherin,thereby reduce the migration and invasion ability of CRC cells.These results further highlight the role of miR-708 in promoting the apoptosis of tumor cells and inhibiting the proliferation,migration and invasion of CRC cells.Part Two Expression of miR-708 in colorectal cancer tissues and its effect and mechanism on proliferation,cycle,apoptosis,migration and invasion of colorectal cells.In the first part of the study,we found that the expression level of miR-708 in CRC cell lines was negatively correlated with the degree of tumor malignancy,especially in the process of regulating EMT in CRC cells.And ZEB1 is an important driving factor of EMT,occurrence of EMT by inducing tumor cells,and promote the tumor invasion and metastasis.This part of the research mainly through further exploration of ZEB1 which is closely related to the EMT in CRC tissues and the influence of the differences between the different expression of the cells of CRC.And discusses the effect and possible mechanism on cell apoptosis,migration and invasion by the different expression of ZEB1 in CRC cells.Materials and methodsColorectal cancer tissues and adjacent tissues were from 10 colorectal cancer patients who had signed the informed consent.After the serum was treated by centrifugation,RNA was extracted by Trizol method for backup use.First,the difference of ZEB1 expression in cancer tissues and paracancer tissues was detected by immunohistochemical staining,and then the difference of ZEB1 protein and gene expression in cancer tissues and paracancer tissues,CRC cell line(HCT-116)and normal liver epithelial cell line(ECT-1)was detected by qRT-PCR and western blot.CRC cell line models of ZEB1 silenced group(ZEB1 siRNA group)and over-expressed group(ZEB1 OE group)were constructed by plasmid transfection technology.Differences in cell proliferation were detected by CCK-8 kit.The cell cycle and apoptosis were examined by flow cytometry.Differences in cell migration and invasion were detected by Transwell assay.Finally,qRT-PCR and western blot were used to detect the difference in mRNA and protein expressions of apoptosis-related Bax and Bcl-2 and EMT-related E-cadherin and N-cadherin in ZEB1 siRNA group and ZEB1 OE group.ResultsFirst of all,immunohistochemical results showed that ZEB1 expression in colon cancer tissues was significantly increased compared with that in adjacent tissues,and this result was verified in protein level and gene level by qRT-PCR and western blot.In addition,the expression of ZEB1 in CRC cell line(HCT-116)was significantly increased than that in normal liver epithelial cell line(ECT-1)by qRT-PCR and western blot.After the successful construction of ZEB1 siRNA group and ZEB1 OE group by plasmid transfection,CCK-8 kit was used to detect that the number of cell proliferation.In ZEB1 siRNA group the number was significantly lower than that in the control group.The cell cycle of ZEB1 siRNA group was examined by flow cytometry and found that the G1 phase of the cell cycle was significantly prolonged and tumor cell apoptosis was increased in the ZEB1 siRNA group.Transwell assay results showed that the cell migration and invasion ability of ZEB1 siRNA group was significantly lower than that of ZEB1 OE group.The expression of Bax and Bcl-2 related to apoptosis was detected by western blot and qRT-PCR.Compared with the control group,the expression of pro-apoptotic gene Bax was significantly increased and the expression of anti-apoptotic gene Bcl-2 was significantly decreased in the ZEB1 siRNA group.The ZEB1 OE group had the opposite result.Finally,EMT-related E-cadherin and N-cadherin expressions were detected.The results showed that the expression of E-cadherin was significantly increased in CRC cells of ZEB1 siRNA group,while the expression of Ncadherin was significantly decreased And the opposite results were found in ZEB1 OE group.ConclusionZEB1 expression is significantly increased in colorectal cancer tissues.Silencing ZEB1 expression in CRC cell lines can reduce the proliferation,migration and invasion ability of tumor cells,prolong cell cycle and increase the number of apoptosis of tumor cells.The level of ZEB1 expression in CRC cell line is positively correlated with the degree of tumor malignancy.In addition,ZEB1 could inhibit the apoptosis of tumor cells by reducing the expression of pro-apoptotic gene Bax and increasing the anti-apoptotic protein Bcl-2.Meanwhile,ZEB1 could also promote EMT in CRC cells by reducing the expression of E-cadherin and increasing the expression of N-cadherin,thus promoting the migration and invasion ability of CRC cells.These results further highlight the role of ZEB1 in promoting the proliferation,migration and invasion of CRC cells and inhibiting the apoptosis of tumor cells.Part Three Mir-708 directly targets ZEB1 and promotes apoptosis of colorectal cancer cells by inhibiting the AKT/mTOR signaling pathwayAfter the studies in the previous two parts,we found that miR-708 inhibited the proliferation,migration and invasion of CRC cells and promoted the apoptosis of tumor cells,while ZEB1 had the opposite effect on CRC cells,so whether if there was a certain functional relationship between the miR-708 and ZEB1 has attracted the attention of our team.If there is a directly target relationship,how do they function in CRC cells?Based on the above research basis and experimental hypothesis,we designed the third part of the experiment,hoping to further explore the relationship between miR-708 and ZEB1,as well as the potential mechanism of action or influence on the AKT/mTOR signaling pathway by verifying the targeting relationship between miR-708 and ZEB1.Materials and methodsUsing database software analysis of miR-708 and whether if there is a possible target relation between ZEB1,then adopts double luciferase report gene targeting the relation between experiment:The wild plasmid vector of ZEB1 gene 3'-UTR luciferase was constructed and transferred into HCT-116 cells at the same time as miR-708 mimics to detect the change of relative fluorescence expression of ZEB1 in HCT-116 cells.The changes in ZEB1 mRNA and protein levels in the CRC model miR-708 mimics group and miR-708 inhibitor group were detected by western blot and qRT-PCR.In order to illustrate the effects of mir-708 and ZEB1 on AKT/mTOR signaling pathway,western blotting was used to detect the differences in p-AKT and p-mTOR protein expressions in mir-708 mimics and mir-708 inhibitor groups,which were closely related to AKT/mTOR signaling pathway.The same method was used to detect the difference of p-akt and p-mtor protein expression in ZEB1 siRNA and ZEB1 OE cells.ResultsFirst,the analysis results of TargetScan7.0 and miRBase biological database showed that ZEB1 was one of the potential functional targets of miR-708,and predicted the binding site of ZEB1 mRNA 3'-UTR with miR-708.In order to further verify the correlation between ZEB1 and miR-708,the dual-luciferase reporter gene test was used.The results showed that co-transfection of miR-708 mimics and ZEB1 luciferase wild plasmid vector could significantly decrease the relative fluorescence expression of ZEB1 in HCT-116 cells compared with the negative control group.Then,the ZEB1 expression in the miR-708 mimics group was significantly decreased by western blotting,while the ZEB1 expression in the miR-708 inhibitor group was significantly increased.Finally,the expression of p-AKT and p-mTOR proteins closely related to the AKT/mTOR signaling pathway was detected by western blot.The results showed that p-AKT and p-mtTOR protein expressions were significantly decreased in the miR-708 mimics group,while p-AKT and p-mTOR protein expressions were significantly increased in the mir-708 inhibitor group.The same method showed that the expression of p-AKT and p-mTOR protein in ZEB1 siRNA group cells was significantly decreased,while the expression of p-AKT and p-mTOR protein in ZEB1 OE group cells was significantly increased.ConclusionZEB1 may be one of the direct targets of miR-708 in CRC cells,and the expressions of miR-708 and ZEB1 are negatively correlated.Finally,we found that both high expression of miR-708 and low expression of ZEB1 could reduced the expression of p-AKT and p-mTOR proteins in CRC cells,thus inhibiting the AKT/mTOR signaling pathway and ultimately inducing apoptosis of CRC cells.Therefore,we can speculate a new potential anti-colorectal cancer signaling pathway,namely the miR-708--ZEB1--AKT/mTOR signaling pathway.SummaryCompared with para-colorectal cancer tissues,mir-708 expression in cancer tissues was low,while ZEB1 expression was high.Mir-708 inhibits the proliferation,migration and invasion of CRC cells and promotes the apoptosis of tumor cells.On the contrary,ZEB1 promotes the proliferation,migration and invasion of CRC cells and inhibits the apoptosis of tumor cells.ZEB1 may be one of the direct targets of miR-708 in CRC cells.We found that the expressions of miR-708 and ZEB1 are directly negatively correlated.Mir-708 has directly target by ZEB1 which could promote CRC cell apoptosis and inhibit the invasion and metastasis of CRC cellsinvasion by inhibiting the AKT/mTORr signaling pathway,namely,miR-708-ZEB1-AKT/mTOR anti-colorectal cancer signaling pathway. |
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