Effects Of MiR-204-5p On Proliferation,migration,invasion And Epithelial-mesenchymal Transition In Colorectal Cancer Cells

Objective: Colorectal cancer causes severe mental stress and economic burden on human beings.It can’t be ignored that the health and economic problems brought by colorectal cancer are very seriously.In United States,the incidence and mortality of colorectal cancer(CRC)rank third among all malignancies.Both the incidence and mortality of CRC have declined due to the increase of public health awareness,advances of diagnosis,treatment techniques and screening methods.However,the incidence and the mortality of CRC rank the top 5 among all malignant tumors in China.In addition,the incidence of CRC showed an upward trend year by year,and the younger adults showed an increasing trend.An integrated economic evaluation of common cancers,such as lung,breast,colorectal,esophageal,liver,and gastric,in urban areas was conducted by The China Urban Cancer Screening Project Team.The medical costs and related economic burdens of cancer patients in 37 three tertiary hospitals in 13 provinces and cities from 2012 to 2014 were investigated through multi-center cross-sectional surveys.The results showed that patients with CRC had the highest economic burden among all malignancies,with per capita expenditure of 10,978 dollars,and the patients with advanced CRC paid significantly more than the patients with early-stage CRC.Although there are many methods for diagnosing and treating CRC,the prognosis of patients with CRC is poor due to the early symptoms of CRC arenot obvious and the patients were not treated timely and properly.Therefore,the identifications and researches of the functional characteristics of CRC-related molecules can not only provide basis for early diagnosis of CRC,but also provide new targets and ideas for the treatment of CRC.Masuda et al.have reported that micro RNA(mi RNA)is expressing abnormally in colorectal cancer,and it is related to the degree of tumor differentiation,clinical stage,treatment effect and prognosis,suggesting that mi RNA is closely related to the occurrence and development of CRC and can be used for early diagnosis of CRC and prognosis prediction.Although many studies showed that mi R-204-5p is expressing lowly in multiple malignant tumors and inhibiting the proliferation,invasion,migration and epithelia-mesenchymal transition(EMT)of tumor cells,the studies of mi R-204-5p in CRC are rare.The previous experiment found that mi R-204-5p blocked the cell cycle of HT29 cells in G1 period,thus inbibitinh the proliferation of HT29 cells.In this study,we investigated the expression difference of mi R-204-5p between CRC tissue and normal intestinal mucosa.We also investigated the effects of mi R-204-5p on the proliferation,invasion,migration and EMT of CRC cells.Moreover,we explored the possible mechanisms of the effects of mi R-204-5p on CRC cells,aiming to provide a new basis and a target for early diagnosis and treatment of CRC.Methods:(1)Real-time quantitative PCR was used to detect mi R-204-5p expression in 23 pairs of fresh colorectal cancer tissues and normal tissues collected from The Affiliated Hospital of Southwest Medical University,and the correlations between mi R-204-5p expression and clinicopathologicalfeatures were analyzed.(2)The lentivirus over-expressing mi R-204-5p was used to transfect the colorectal cancer cell lines HT29 and Lo Vo.The transfection efficiency was judged by using fluorescence microscopy.Mi R-204-5p expression was detected by Real-time quantitative PCR.(3)The effects of mi R-204-5p over-expression on the proliferation,invasion and migration of HT29 and Lo Vo cells in vitro were detected by Plate clone formation assay,Scratch healing assay and Transwell invasion assay,respectively.(4)We analyzed biomarker expression of EMT-related marker,including E-cadherin and Vimentin by western blot in HT29 and Lo Vo cells after over-expression of mi R-204-5p.(5)The target genes of mi R-204-5p were predicted using Target Scan,mi RDB and mi RTar Base databases.The reliability and accuracy target genes were predicted by taking the intersection of the top50 genes in the three databases.The protein expression of target gene in cells after over-expression of mi R-204-5p was tested by western blot.Results:(1)The result of Real-time quantitative PCR showed that mi R-204-5p expression in colorectal cancer tissue was significantly lower than that in paired normal intestinal mucosal tissue(t=9.245,P < 0.001).Mi R-204-5p expression in colorectal cancer tissue was correlated with tumor size(2? =7.078,P=0.008),TNM stage(2? =9.603,P=0.002)and lymph node metastasis(2? =7.340,P=0.007),while not correlated with age(2? =2.718,P=0.099),sex(2? =2.718,P=0.099),degree of tumor differentiation(2? =1.343,P=0.511),location(2? =0.434,P=0.510)and serum CEA(2? =0.910,P=0.340).(2)The HT29 and Lo Vo cells in mi R-204-5p group and Lo Vo-204-5p group were transfected bylentivirus over-expressing mi R-204-5p,and the HT29 and Lo Vo cells in HT29-NC group and Lo Vo-NC group were transfected by lentivirus expressing green fluorescent protein(GFP).All the cells in HT29-204-5P,Lo Vo-204-5p,HT29-NC,and Lo Vo-NC groups were expressing green fluorescence.The result of Real-time quantitative PCR showed that mi R-204-5p expression in mi R-204-5p group was higher than that in HT29-NC group(t=-20.488,P <0.001).In addition,mi R-204-5p expression in Lo Vo-204-5p group was higher than that in Lo Vo-NC group(t=-59.078,P < 0.001).(3)The result of plate clone formation assay showed that the colony forming efficiency of mi R-204-5p group,Lo Vo-204-5p group,HT29-NC and Lo Vo-NC group were11±2.25%,14.67±3.33%,18.33±2.75% and 27.67±2.93%,respectively.The colony forming efficiency of mi R-204-5p group was significantly higher than that of HT29-NC group(t=20.172,P < 0.001),and the colony forming efficiency of Lo Vo-204-5p group was significantly higher than that of Lo Vo-NC group(t=5.077,P=0.001).(4)The result of scratch healing of assay showed that the scratch healing rates of mi R-204-5p group,Lo Vo-204-5p group,HT29-NC and Lo Vo-NC group were 16.03±2.35%,15.30±0.99%,25.41±4.36% and 37.79±4.70%,respectively.The scratch healing rate of mi R-204-5p group was significantly higher than that of HT29-NC group(t=3.280,P=0.031),and the healing rate of Lo Vo-204-5p group was significantly higher than that of Lo Vo-NC group(t=8.106,P=0.0012).(5)The result of scratch transwell invasion assay showed that the number of cells under the matrigel in mi R-204-5p group,Lo Vo-204-5p group,HT29-NC andLo Vo-NC group were 15.07±3.23,51.47±4.35,28.93±4.02 and 112.9±7.73,respectively.The number of cells under the matrigel in HT29 and Lo Vo group was much more than that in HT29-NC group(t=4.85,P=0.008),and the number of cells under the matrigel in Lo Vo-204-5p group was much more than that in Lo Vo-NC group(t=12.003,P<0.001).(6)The result of western blot showed that E-cadherin expression was upregulated in HT29(P < 0.001)and Lo Vo cells(P=0.016)after over-expression mi R-204-5p,while Vimentin expression was downregulated(P=0.002;P=0.013).(7)The Target Scan,mi RDB and mi RTar Base databases predicted that MAPRE2 was a potiential target of mi R-204-5p,and the western blot showed that MAPRE2 expression was down-regulated in HT29(P=0.001)and Lo Vo cells(P < 0.001)after overexpression of mi R-204-5p.Conclusions:(1)Mi R-204-5p expression in human CRC is down-regulated,and it could inhibit the proliferation,invasion,migration and EMT of CRC cells.(2)MAPRE2 may be a functional target gene of mi R-204-5p.