The Experimental Research On Decreaseing Invasion And Metastasis Ability Of Colonic Cancer By Inhibiting Of MicroRNA-21Expression

In this study, the human colonic cancer cell line were modified by plasmid vector, miRZip21, to inhibit the expression of endogenous microRNA-21. Then we explored the mechanism of invasion and metastasis of Colon Carcinoma by detecting the colonic cancer cells’ expressive levels of mRNAs and proteins of TIMP-3, RECK, BMPRⅡ and PCDH17(microRNA-21’s targets) and observing the colonic cancer cells’biological behavior with low expression of microRNA-21in vivo and in vitro.Part I The Research on The Expression levels of microRNA-21in different human colonic cancer cell linesObjective:To explore the expression levels of endogenous microRNA-21in different human colonic cancer cell lines and to determine the adaptive type colonic cancer cell lines to carry out the next stage experiment. Methods:4differernt human colonic cancer cell lines, SW480, SW620, HCT-116and LoVo were cultured and the expression levels of endogenous microRNA-21in4cell lines were detected by TaqMan" microRNA Assays. Results:The growth characteristics of4cell lines were different as well as the expression levels of microRNA-21. The high level expressions of microRNA-21were in SW480and HCT-116and the low amount of microRNA-21were in LoVo and SW620. Conclusion:(1) The colonic cancer cell lines established from the primary tumors express the high level of endogenous microRNA-21and the cell lines initiated from the metastatic tumor have small amount of endogenous microRNA-21.(2) The colonic cancer cell line of HCT-116was the adaptive target of this research on the invasion and metastasis of colonic cancer since SW480is not exprssing Matrilysin, a metalloproteinase associated with tumor invasiveness.Part II The Effect on Invasion and Metastasis Ability of Colon Carcinoma by Inhibiting the Expression of microRNA-21in Vitro Objective:to explore the effect on invasion and metastasis of colonic cancer and to detect the expression of mRNAs and proteins of the microRNA-21target genes, which are relative to the invasion and metastasis of cancer, by downregulating the microRNA-21expression level of HCT-116cells. Methods:(1) Plasmid miRZip21, miRZipTM anti-microRNA-21expression lentiector, were used to transfect HCT-116cells by Gene porter3000. The expression of microRNA-21was assessed to identify the function of miRZip21.(2) The microRNA-21target genes associated with invasion and metastasis were predicted by softwares of Target Scan and PicTar.(3) The expression levels of microRNA-21in HCT-116cells transfected with Plasmid miRZip21was detected by TaqMan(?) microRNA Assays. The proteins of TIMP-3> RECK. BMPR-Ⅱ and PCDH17in HCT-116cells transfected with Plasmid miRZip21were analyzed by Western blotting.(4) The ability of migration and invasion of the HCT-116cells, transfected with Plasmid miRZip21were detected by the Scratch Migration Assay and the Cell Invasion Assay. Results:(1) The microRNA-21level of HCT-116cells descented to about60%after the cells transfected with the plasmid miRZip21by Gene porter3000.(2)4genes:TIMP-3、RECK、BMPRⅡ and PCDH17were the gene targets of microRNA-21predicted by softwares of Target Scan and PicTar.(3) The mRNA levels of TIMP-3、RECK、BMPRⅡ and PCDH17upregulated to2.3fold,2.7fold,1.3fold and1.2fold respectively after inhibitig the microRNA-21expression of HCT-116cells. The changes of TIMP-3and RECK are significantly different (P<0.05). The anlysis of Western Blotting showed that the priteins of TIMP-3and RECK upregulated to about3.5fold and2fold respectively after inhibiting the microRNA-21expression of HCT-116cells(P<0.05).(4)Significant downregulation of invasion and migration were observed in anti-microRNA-21-transfected HCT-116cells(P<0.05). Conclusion:(1) Plasmid miRZip21, miRZipTM anti-microRNA-21expression lentiector, can downregulate the level of microRNA-21when transfecting HCT-116cells.(2) MicroRNA-21has more than one target genes. TIMP-3and RECK, which are relative to the invasion and metastasis of cancer, were upregulated to a certain extent respectively after inhibiting the expression of microRNA-21. The regulation of microRNA-21to its targets happens on two domains, the mRNAs and the proteins.(3) The degrees of microRNA-21to inhibit its gene targets are different, likely corresponding to its ability of combining with its targets.(4) Inhibiting the microRNA-21expression can downregulate the metastatic and invasive ability of HCT-116cells in vitro.Part Ⅲ The Research on Decreasing Invasion and Metastasis Ability of Colon Carcinoma by Inhibiting the Expression of microRNA-21in VivoObjective:To establish mouse model of heterotopic colonic cancer with HCT-116cells and to explore the biological behaviors of the heterotopic colonic cancer by inhibiting the expression of microRNA-21in vivo. Methods:(1)4-6week-old female BalB/c-nu mice were utilized to establish heterotopic colonic cancer model by the mice’s subcutaneous injection of the left inside lower limb with the colonic cancer cells. The mice were divided into2groups:Group A implanted with the HCT-116cells transfected with the plasmid miRZip21in vitro and Gropg B implanted with HCT-116cells.(2)The mice of group B invidided into3subgroups randomly:group B1(miRzip21), group B2(blank-plasmid control) and group B3(control). The mice of Group B1and Group B2received transfection in vivo with the plasmid miRZip21and the plasmid Leti3according to the experiments design when the volumes of the tumor increased to60mm3. The mice of Group A and Group B3recived no further treatments. The tumors’growth and metastasis were observed after the tumors implantation for about6weeks.(3) The tumors were collected after6weeks to detect the levels of microRNA-21and the expression of the protein, TIMP-3and RECK by TaqMan(?) microRN A Assays and imrnunohistochernistry(IHC) and the metastasis of the heterotopic colonic cancer in mice were observed. Results:(1)100%of mice formed heterotopic colonic cancer by receiving subcutaneous injections with HCT-116cells.(2) The volumes of mice colon tumors between the group A and the group B1were likely at the3th week after group B1receiving the plasmid miRZip21transfection(P>0.05). But, at the other time the tumor volumes of groups A were smaller than that of group B1(P<0.05).(3) The volumes of both groups A and group B1were smaller than that of group B2and group B3(P<0.05, P<0.05).(4) The microRNA-21expression levels of2groups received miRZip21plasmid transfection in vivo and in vitro were close(P>0.05), while were lower than that of Group B2and Group B3(P<0.05, P<0.05).(5) The numbers of inguinal region metastasis lymph nodes in groups B2(5/8) and B3(6/8) were more than that in the Groups A (3/8)and Group B1(3/8). All groups had no metastasis in lungs, livers and spleens, but some abdominal swelling lymph nodes can observed in several mice of GroupB2(2/8) and Group B3(3/8).(6) The results of IHC showed that the protein expressions of TIMP-3and RECK were upregulated significantly in groups inhibiting the level of microRNA-21comparing to control groups (P<0.05). Conclusion:(1) Subcutaneous injection with HCT-116cells successfully forms heterotopic colonic cancer in BalB/c-nu mouse, and is a convenient method to form animal model of heterotopic colonic cancer in nude mouse.(2) Decreasing the timors’growth via inhibiting the microRNA-21expression of the colonic cancer indicates that microRNA-21’s target genes including some others genes, which involved in the proliferation of the colonic cancer.(3) Transfection with miRZip21plasmid at the colonic tumor in vivo can receive the likely results of transfection colonic cancer cells in vitro before establishing mouse model, which indicated that miRZip21plamid can be uesd as a vector for gene therapy about basic and clinic research.(4) Treatment by inhibiting the microRNA-21expression can decrease the growth of the colon tumor and increase the expression levels of the tumor suppressor gene:TIMP-3and RECK.(5) The results of experiments on decreasing invision and metastasis ability of colonic cancer by inhibiting the microRNA-21expression in vivo and are consistent with that in vitro.(6) microRNA-21might be one of the target sites in the gene treatment for colonic cancer.