| Colorectal cancer is a very common malignant tumor disease in the digestive system in China.Incidence of colorectal cancer has obviously increased in the past years,especially in some large cities.According to statistics released on 2015 Chinese Cancer Registry Annual Report,the incidence of colorectal cancer has overrun that of liver cancer,which makes it ranked the third among malignant tumor diseases,behind lung cancer and gastric cancer.Its mortality is on the fifth place,next only to that of lung cancer,liver cancer,gastric cancer and esophageal cancer.In China,the early diagnosis of the cancer is still in low rate,of which 60% patients are found to be in the mid and late stage.In addition,colorectal cancer has such characteristics as the large number of patients and low 5-year survival rate,which makes colorectal cancer the main cause of death for most cancer patients.Surgical treatment is considered to be the only way that can help treat the cancer.However,many patients may confront with relapse or even cancerous metastasis after receiving radical treatment for the cancer.Risks of relapse and cancerous metastasis may be reduced if adjuvant radiochemotherapy in treatment is adopted after surgery.For patients with colorectal cancer of late stage,chemotherapy also plays a crucial role in the synthetic treatment.However,drug resistance becomes an unavoidable issue for colorectal cancer patients during chemotherapy session.Thus,it can be seen that the sooner standardized treatment is adopted for colorectal cancer patients,the higher survival rate can be achieved.Therefore,it is of great significance to explore molecular bio-markers of intense sensitivity,find out molecular targets and implement prognostic assessment.Mi RNA is an important non coding small molecule RNA with length of about 18-25 nucleotides,which is endogenously expressed in human body.In human genome,the proportion of mi RNA molecules only accounts for 1%,but it has vital regulating effect on the gene expression,modification,translation and transcription of 1/3 or even more genes.Mi RNA may be regarded as a molecular marker with outstanding value,providing new reference for clinical treatment,early diagnosis of malignant tumor diseases and prognostic assessment.As the focus of attention for modern medicine,Mi RNA-200 family includes the following five key members: 1)mi RNA-200 a,2)mi RNA-200 b,3)mi RNA-200 c,4)mi RNA-141,5)mi RNA-429,which is featuring with high conservatism,strong timing,gene clustering and good tissue specificity.For such malignant tumors as liver cancer and renal cell carcinoma,the aforesaid family shows a decreased expression level.However,it has an increased expression in such malignant tumors as bladder cancer,cervical cancer etc.It has been proved that all the five members of the mi RNA-200 family can be correlated jointly and individually.In the case of joint action,expressions of ZEB1 and ZEB2 are regulated.In individual action,these five members can restraint the occurrence of EMT.However,clear research findings concerning the role of the family in common human malignant tumors have not yet concluded.This research conducts a test on the expression level of hot gene mi RNA-141 from mi RNA-200 family in colorectal cancer and two adjacent normal tissues by applying real-time quantitative PCR technology.A study on the correlation between such expression level and pathological parameters of patients with colorectal cancer is implemented at the same time.Real-time quantitative PCR technology is applied to test the expression content of mi RNA-141 PCR in colorectal carcinoma HT29 cells.Transient transfection is applied to transfer mi RNA-141 mimics or negative control into colorectal carcinoma HT29 cells,in order to build up mi RNA-141 model with high expression.Furthermore,MTT method is used to test the proliferative status of the cancer cells and analysis is carried out on the periodic changes of colorectal carcinoma HT29 cells by using the flow cytometer.Finally,observation on the transfer ability of colorectal carcinoma HT29 cells is conducted by means of cell wound healing assay.Influence of mi RNA-141 on the cell biological behavior by colorectal cancer is analyzed.Thus,theoretical basis is concluded for further study on the role of mi RNA-141 in the occurrence and development of colorectal cancer.Bioinformatics software is applied to conduct a preliminary prediction on potential target genes in mi RNA-141 and screening of potential target genes is implemented.At last,target genes in colorectal carcinoma HT29 cells is verified through dual-luciferase reporter gene system,in order to carry on feasibility research of these genes as target tumor markers and tumor treatment,in hope of providing certain experimental basis for some clinical challenges and laying a foundation for further study on the molecular mechanism of mi RNA-141.Part One Expression of micro RNA-141 in colorectal cancer and its correlation with clinicopathological featuresObjective: To explore the different expression levels of mi RNA-141 in colorectal cancer and its adjacent normal tissues.Then,to explore the relationship between the expression level of mi RNA-141 and the pathological parameters of patients with colorectal cancer.Methods: Selection of patients with colorectal cancer in 58 patients after resection by quantitative Real-time PCR method for colorectal cancer and adjacent normal expression levels of mi RNA-141 in two tissues were detected by T test or Wilcoxon rank sum test to sample differences between the results,at the same time using Wilcoxon rank sum test,explore the correlation factor expression and colorectal cancer the pathological parameters exist.Results: 1 We detected the expression level difference of the mi RNA-141 in 58 cases colorectal cancer tissues,also their matched adjacent tissues were determined by quantitative Real-time PCR method.The expression of mi RNA-141 in 58 cases of colorectal cancer in cancer tissues compared with adjacent normal tissues were significantly down-regulated（Z-6.314,P<0.001）,mi RNA-141 84.48%（49/58）specific expression in cancer tissues compared with normal tissues were significantly lower.2 By Wilcoxon rank sum test,we analyzed of the relationship between the level of expression of mi RNA-141 and pathological parameters in patients with colorectal cancer.The results showed that there was a significant correlation between the detection level of mi RNA-141 and lymph node metastasis and tumor depth（P<0.05）in 58 patients with colorectal cancer.However,the gender,age,TNM stage,histological grade,distant metastasis and intravascular cancer embolus,it is no significant correlation（P> 0.05）.Part Two Effects of mi RNA-141 on the biological behavior of human colorectal cancer cell lines and the regulation mechanismObjective: To study the effect of mi RNA-141 on the reproduction,cycle and migration ability of HT29 cells in colorectal cancer.Methods: Quantitative Real-time PCR method was used to detect the expression of 3 colorectal cancer cells.The mi RNA-141 mimetic or negative control was transferred to colorectal cancer cell line by transient transfection method.From MTT method,flow cytometry instrument and wound healing assay,we can detected the cell proliferation,the cell cycle change and the tumor cell migration ability.Results: 1 Different expression of mi RNA-141 in 3 colorectal cancer cell lines.By quantitative Real-time PCR method to identify 3 strains of colorectal cancer cell（HCT8,HT29 and HCT116）levels were detected within mi RNA-141,and the detection value of adjacent normal tissues as control,which indicated that the expression level of mi RNA-141 in colorectal cancer cell lines were significantly downregulated in 3 cell lines,reduce the times were 9.92 times,7.28 times and 2.95 times（P<0.05）.2 The HT29 cell was found to be a suitable cell in vitro cell model.We detected the expression level of mi RNA-141 in HT29 cell transferred the mi RNA-141 mimics or negative control after 48 hours.The results displayed that the expression level of mi RNA-141 transferred the mi RNA-141 mimics was significantly higher than those in the negative control and blank control group,the median change about 10.5 folds（P<0.05）.These suggested that the mi RNA-141 transient transfection was successful and could do the next studies.3 The effect of high level of mi RNA-141 on the proliferation of HT29 cells.Transfection of mi RNA-141 and negative control 24,48 and three hours after 72 h,by the method of MTT on HT29 cells proliferation were made century detection results: 24,48 and 72 h after transfection,mi RNA-141 mimics experimental group in each period was followed by 0.307 ± 0.010,0.361 ± 0.015 and 0.461±0.028,while the OD values of negative control group were 0.338±0.006,0.503±0.008 and 0.774±0.011.Three points after transfection,the rate of inhibition in mi RNA-141 mimics group were 10.70±1.87%%,30.34±1.25% and 40.89±3.16%,while the rate of inhibition in negative control group were 1.59±1.18%,2.92±1.55% and 0.86±0.37%.They had significant difference in each time point（P<0.05）.4 Effect of high level expression of mi RNA-141 on cell cycle of HT29 cells.Flow cytometry was used to detect the HT29 cell cycle after transfection of mi RNA-141 into 48 h.The ratio in G1 phase,S phase and G2 phase of mi RNA-141 mimics group were 73.77±0.84%,8.97±0.44% and 18.46±1.39%,while the ratio of negative control group were 42.11±0.81%,43.56±1.26% and 14.73±2.05%,respectively.There was significant difference in G1 phase and S phase（P<0.05）.The G1 phase arrest suggested that over-expression of mi RNA-141 in HT29 cell could inhibit the cell cycle from G1 phase to S phase.5 Overexpression of mi RNA-141 in colorectal cancer cells HT29 can weaken the ability of cells to migrate in vitro.the tumor cell migration ability of mi RNA-141 mimics group was significantly decreased compared to the negative control group（P<0.05）.The width in mi RNA-141 mimics group was 345.40±17.34μm,while the width in negative control group was 270.61±18.31μm.There was significant difference between mi RNA-141 mimics group and negative control group（P<0.05）.Part Three The predict and verify of mi RNA-141 target genesObjective: To investigate whether MAP4K4 can be used as an important target gene of mi RNA-141,For further research on molecule mechanism of mi RNA-141 make a foundation.Methods: The target genes of mi RNA-141 were predicted and analyzed by bioinformatics software,and the reliability of target gene was confirmed by double luciferase reporter assay.Results: 1 Bioinformatics prediction of mi RNA-141 target genes.Targeting mi RNA gene,we will choose the most bioinformatics software in the screening,to forecast the biological information of Target Scan,Pic Tar,mi Randa and mir Base and many other types of software,the specific results: mi RNA-141 equivalent to human chromosome 12 and is located to 12: 6964097-6964191 [+].There were 1016,465 and 7721 target genes of mi RNA-141 by Target Scan,Pic Tar and mi Randa bioinformatics software.Comprehensive analysis we chose MAP4K4（NM004834）as the research subject.We found that MAP4K4 3’UTR has two binding sites of mi RNA-141,so we will build the report carrier of MAP4K4 3’UTR and verify the target gene by dual luciferase report gene assay.2 We verified that the MAP4K4 is a target gene of mi RNA-141 by the classical method.Firstly,the MAP4K4 3’UTR vector and MAP4K4 mut 3’UTR vector were successfully constructed.We divided into four groups through transferring the MAP4K4 3’UTR/ MAP4K4 mut 3’UTR and mi RNA-141 mimics/ mi RNA-141 NC in HT29 cell.They were MAP4K4 3’UTR+mi RNA-141 mimics group,MAP4K4 3’UTR + mi RNA-141 NC group,MAP4K4 mut 3’UTR + mi RNA-141 c mimics group and MAP4K4 mut 3’UTR + mi RNA-141 NC group.The dual luciferase report gene assays showed that 48 h after transferred,the luciferin protein expression level of MAP4K4 3’UTR + mi RNA-141 mimics group was reduced compared to other groups（P<0.05）.The results showed that mi RNA-141 could targeted inhibit the activity of MAP4K4 3’UTR,suggesting that MAP4K4 was the target gene of mi RNA-141.Conclusion: 1 The expression level of the mi RNA-141 was down-regulated in colorectal cancer tissues compared with adjacent normal tissues,suggesting that the down-regulated of the mi RNA-141 might become a therapeutic and preventive tool in patients with colorectal cancer,but might not be a promising biomarker in the early diagnosis of colorectal cancer.2 Lower levels of mi RNA-141 were associated with invasive depth and lymph node metastasis,suggesting that mi RNA-141 might play an important role in the occurrence and development of colorectal cancer.3 mi RNA-141 in the colorectal cancer cell lines HCT8,HT29 and HCT116 were significantly decreased,and the detection of colorectal cancer in colorectal cancer tissues were consistent with the expression of the 3 cell lines.4 The overexpression of mi RNA-141 in colorectal cancer cell line HT29 can inhibit cell proliferation.The results indicate that mi RNA-141 may be an important regulatory molecule suitable for cell proliferation,as an effective target for clinical treatment of colon cancer.5 mi RNA-141 can change delay cells from the previous G1 period of S in colorectal cancer cells overexpressing HT29,so that mi RNA-141 can be adjusted in or on the cell cycle,and thus play the corresponding inhibitory effect on cell proliferation.6 The over-expression mi RNA-141 in HT29 cell could decrease the tumor cell migration ability,suggesting that the down-regulation of mi RNA-141 might affect the cell migration ability and could provide the proof for searching the molecular targets of treatment in colorectal cancer.7 The mi RNA-141 could targeted inhibit the activity of MAP4K4 3’UTR,verifying that MAP4K4 was the target gene of mi RNA-141.This suggested that mi RNA-141 has the inhibitory function to MAP4K4,and may exert its biological behaviors by MAP4K4.Thus they may affect the occurrence,development,invasion and metastasis of colorectal cancer.For further clarify the molecule mechanism of mi RNA-141 provide a foundation in colorectal cancer. |
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