Effects Of Lentiviral Vector-mediated CXCR7shRNA On The Biological Behaviors Of Colon Cancer

The occurrence and deterioration of malignant tumor involve abnormal expression of a number of chemokine receptors.CXCL12 and its receptor CXCR4 are the key factors in tumor growth and metastasis. Recent study of prostate cancer showed that the expression of CXCR4 inhibited could only partially block the malignant growth and metastasis. Further study found that CXCR7(orphan receptor RDC1),with high affinity to CXCL12,is expressed in many tumor cell lines and directly involved in regulating the occurrence and deterioration of tumor.The signal pathway of CXCR7 is unclear and presumed to activate cells by a structural activate mechanism of reversible binding with its ligand.CXCR7 can enhance growth,adhesion and invasion of tumor cell in vitro.Tumorigenicity was significantly increased by transfecting CXCR7 sequence into breast cancer cells MDA-MB435s,but suppressed by transfecting CXCR7siRNA into other breast cancer cells 4T1 in vivo.The small molecule antagonist of CXCR7 could also significantly suppress the tumor growth.Present investigation of the effects of CXCR7 on biological behavior of tumor only involve in breast cancer,lung cancer,prostate cancer, bladder cancer and so on.This article intended to study "CXCR7 and colon cancer".First of all,CXCR7 was envisaged as a promotive factor of colon cancer,high-expression in colon cancer tissue and to associate with proliferation and migration of colon cancer cell.Furthermore,it was also assumed that gene silence of CXCR7-targeting could effectively inhibit malignant progression of colon cancer.Accordingly,the design of this experiment was as follows:1.To investigate the expression levels of CXCR7 in human colon cancer,normal colon tissue and colon cancer cell lines and to explore the relationships between CXCR7 expression level and colon cancer,lymph node metastasis and tumor differentiation.2.Designing and screening the siRNA sequence which can effectively inhibit the expression of CXCR7 of human colon cancer cell for constructing the recombinant lentiviral vector of CXCR7shRNA (lentivirus-CXCR7shRNA,LV-CXCR7shRNA).3.Research on the regulatory roles of LV-CXCR7shRNA on proliferative and migratory activities of human colon cancer cell for exploring the effect of CXCR7 on biological behaviors of colon cancer from the cellular level.4.Research on the therapeutic effect of LV-CXCR7shRNA on bearing-tumor nude mice of human colon cancer,which further confirm the role of CXCR7 on biological behaviors of colon cancer from in vivo of animals.Part one To investigate the expressive significance of CXCR7 in human colon cancer tissues and cell linesObjective Study the expression level and significance of CXCR7 in human colon cancer tissues,normal colon tissues and cell lines.Methods1.Expressive level of CXCR7 in human colon cancer tissues:the expression levels of CXCR7mRNA and protein were determined by Real-time fluorescent quantitative PCR(FQ-PCR) and Western blotting (WB) in human colon cancer tissues and normal colon tissue, respectively.Data were analyzed together with lymph node metastasis and tumor differentiation.2.Expression level of CXCR7 in human colon cancer cell lines:the expression levels of CXCR7mRNA were determined by FQ-PCR in HT-29 and SW480 of human colon cancer cell lines and the higher was selected for further experiment.Results1.The medians of CXCR7mRNA relative expression were 1.14 and 0.22,with statistically significant(P＜0.05),in human colon cancer and normal colon tissue,respectively.The relative expression levels of CXCR7 protein were 0.245±0.0591 and 0.17±0.064 in colon cancer and normal colon tissue,the difference was statistically significant(P＜0.05).2.The medians of CXCR7mRNA relative expression were 1.455 and 0.74 in colon cancer tissues with and without lymph node metastasis, respectively.The difference was statistically significant(P＜0.05).The relative expression levels of CXCR7 protein were 0.276±0.055 and 0.2±0.026,with significance difference(P＜0.05),in above two tissues, respectively. 3.The expression levels of CXCR7mRNA and protein were no significant difference(P＞0.05) in tumor tissues with different differentiations.4.The expression level of CXCR7mRNA of human colon cancer cell line HT-29 was higher than that of SW-480.Conclusions1.The expression of CXCR7 is significantly up-regulated in the colon cancer tissue with higher expressions as lymph node metastasis.2.The expressions of CXCR7 are not difference in differentiations.3.HT-29 cell was selected for higher expression of CXCR7mRNA for further experiment.Part two To construction the recombinant lentiviral expression vector of CXCR7shRNAObjectiveScreening effective sequence of CXCR7siRNA and constructing the LV-CXCR7shRNA.Methods1.Screening effective sequence of CXCR7siRNA in human colon cancer cell:3 siRNA interference sequences of CXCR7-targeting gene were designed for constructing recombinant plasmid-shRNA,respectively. A small amount of shRNA lentiviral vectors of 3 kinds of plasmid-shRNA was constructed and then transfected into human colon cancer cells HT-29,respectively.CXCR7 expressions of mRNA and protein were detected by FQ-PCR and WB,respectively.Then the effects of gene silence were determined for screening the effective target.2.Constructing LV-CXCR7shRNA:to construct the recombinant plasmid using the selected target sequence of siRNA for constructing a large number of LV-CXCR7shRNA,and then detecting the titer of virus.Results1.FQ-PCR detected the effects of 3 siRNA sequences on CXCR7 gene silencing.The decline of CXCR7mRNA was most marked(72%) by LV-CXCR7shRNA-2.This result was consistent with that of WB.2.To construct the lentvirus-shRNA vector using the siRNA sequence of 72%silence effect.The titer of virus detected was 4.0×10~8 TU/mL.ConclusionsLV-CXCR7shRNA vector was constructed successfully which effectively reduced the expressions of CXCR7mRNA and protein in human colon cancer cell HT-29. Part three Effects of gene silence of CXCR7-targeting on biological characteristics of human colon cancer cellObjectiveTo study the proliferative and migratory activities of human colon cancer cell after CXCR7-targeting inhibition.Methods1.Human colon cancer cells HT-29 were divided into 3 groups: experimental group,negative control group and blank control group.The experimental group was administrated with LV-CXCR7shRNA,negative control group with LV-shRNA negative control and blank control group without the any.2.CXCR7mRNA and protein of every group were detected using FQ-PCR and WB methods,respectively.3.The proliferative and migratory activities of every group were detected using cell active assay(MTT) and Transwell cell migratory assay.Results1.Expression levels of CXCR7mRNA and protein:CXCR7mRNA was lower expression in experimental group than the negative control group and blank control group(P＜0.05),but no significant difference (P＞0.05) between the two control groups.These results were consistent with protein expression.It is prompted that LV-CXCR7shRNA can effectively silence the gene expression of CXCR7 in HT-29 cell.2.Cell activity assay(MTT):the cell growth curve of experimental group was flatter and the level of cell growth was significantly lower(P ＜0.05)than the negative control group and blank control group.The cell growth curve of negative control group was lower than blank control group.These results suggest that the gene silencing of CXCR7-targetting significantly inhibit the proliferative activity of colon cancer cell.In addition,lentiviral vector has potential cytotoxicity.3.Transwell cell migratory assay:the cell populations of penetrating the filtrative membrane in experimental group were significantly lower(P＜0.05)than negative control group and blank control group,with no significant difference(P＞0.05) between the two control groups, suggesting that inhibition of CXCR7 expression suppress migratory activity of colon cancer cell.ConclusionsGene silence of CXCR7-targeting suppresses the proliferative and migratory activities of colon cancer cell.Part four The therapeutic effect of recombinant lentiviral-CXCR7shRNA vector on planted subcutaneous tumors of human colon tumor-bearing nude miceObjectiveTo explore the growth inhibition of planted subcutaneous tumors in human colon tumor-bearing nude mice using lentivirus-CXCR7shRNA, for enlightening further study of CXCR7-targeting gene therapy toward human.Methods1.To establish the animal model of human colon tumor-bearing mice and calculate the tumor-formation rate.2.Tumor-bearing nude mice were randomized into 3 groups and each group contained 8.Treatment groups:injecting 50μL of LV-CXCR7shRNA into tumor;Negative control group:injecting 50μL of LV-shRNA negative control into tumor;Blank control group:injecting 50μL PBS into tumor.3.Tumor volumes were measured per 3 days after administration for mapping tumor growth curves.4.To sacrifice animals after 24 days,remove tumors,measure tumor volumes and weights and calculate inhibition rates,respectively.5.CXCR7mRNA and protein of tumors were measured by FQ-PCR and WB.Results1.The model animals of human colon tumor-bearing mice were established successfully by 100%.2.As compared with negative control and blank control group,the tumor growth of treatment group was slow and the tumor volume and weight of treatment group decreased significantly(P＜0.05) with 38% and 34.04%tumor-growth-inhibition rates,respectively.3.As compared with negative control and blank control group,the expression of CXCR7mRNA and protein in tumors of treatment group were down-regulated significantly(P＜0.05),with no significant difference(P＞0.05) between the negative control group and blank control group.It was prompted that lentiviral-CXCR7shRNA effectively inhibited the expression of CXCR7 in tumors of human colon cancer-bearing nude mice.ConclusionsThe LV-CXCR7shRNA can effectively inhibit the growth of tumor of human colon tumor-bearing nude mouse.