| Colorectal cancer is the third most common cancer,which is the world’s fourth largest lethal tumor, and has the second largest 5-year survival rate. There are about 1,361,000 people are diagnosed with colorectal cancer and 694,000 patients died from Colorectal cancer every year. As one of the most popular carcinoma over world, Colorectal cancer can metastasis to visceral organs, such as liver,lung and Peritoneum. Metastasis is the most important factor that can lead death of patients. Recent years, chemotherapy and molecular targeted therapies of colorectal cancer have made considerable progress, but the transfer to their clinical treatment has no substantive breakthrough. The prognosis of colorectal cancer is still not optimistic. Therefore, to further clarify the invasion and metastasis of colorectal cancer occurrence, development and molecular mechanisms are still the important direction for their research.Micrornas (miRNA) are about 18 to 25 base non-coding RNAs, which widely exist in cells. They regulate the expression of target genes by cutting and inhibiting mRNA (2-5). MiRNAs play an important role in a variety of important cell activities, such as cell proliferation, apoptosis and differentiation. In the past 20 years, more and more MiRNAs were reported that they are involved in the development of tumor. And a variety of MiRNAs were detected their abnormal expression in tumors, such as breast, lung, colorectal, pancreatic cancer and ovarian cancer. MiRNAs regulate the expression of oncogenes or tumor suppressor genes in cancer. Therefore, studies of MiRNAs in tumor function will help us to understand the mechanisms of cancer, the diagnosis and treatment.MiR-34a is a member of the family miR-34. MiR-34 family consists of three members:MiR-34a, MiR-34b and MiR-34c. Previous study showed that MiR-34 family is in a wide variety of tumors by targeting a variety of genes in recent years, and play the role of tumor suppressors. MiR- 34 family regulate the proliferation, apoptosis, invasion and metastasis of tumor cells. It was reported in a wide variety of tumors which has the CPG promoter with miR-34a silence, and colorectal cancer patients serum MiR-34a expression is also lowered. So far, however, only two target genes of mi -34a were found in colorectal cancer and, the mechanism of miR-34a in the colorectal cancer still has no detailed research.To clarify the role of miR-34a in the proliferation, invasion and metastasis of colorectal cancer, the main contents are as follows:(1) To investigate the impact of miR-34a on the biological behavior of colorectal cancer cells;(2) To predict and filter target genes of miR-34a in colorectal cancer, clarifing the role of them in proliferation, invasion and metastasis of colorectal cancer;(3) To detect the expression of miR-34a in colorectal cancer clinical specimens, analyze its relationship with clinical indexes and analyze the correlation of miR-34a and target genes in colorectal cancer.Methods1.Effect of miR-34a on the biological behavior of colorectal cancer cells.(1) Endogenous expression of miR-34a in 6 CRC Cell lines by real-time qpcr. Screening out miR-34a high expression and low expression cell lines as a follow-up to the interference and over-expression experiments.(2) Screening effective inhibitor and mimics of miR-34a in CRC cell lines by real-time PCR.(3) Detecting the influence of inhibiting and over-expressing miR-34a in CRC cell lines by CCK8 assay, Boyden cell invasion detection chambers, cell cycle detection assays and cell apoptosis detection assay.(4) Using commercial miR-34a express lentivirus package to establish miR-34a stable expressing CRC cells, and verify its expression efficiency by real-time PCR.(5) Observing the influence of miR-34a over-expression cells on subcutaneous tumor sizes and in vain tumor migration ability in SPF class BALB/c nude mice models.2.Prediction, screening and validation of the target genes of miR-34a in colorectal cancer.(1) Four commonly databases, including MiRanda, TargetScan, DIANA-microT and Pictar, were used to forecast the targets with the search term of miR-34a. Targets with high predictive values were selected as candidate targets. The genes were identified as targets of miR-34a by the Luciferase reporter system and western blot.(2) After expression of miR-34a in colorectal cancer cell lines, we restored the expression of target genes. CCK8, Boyden chamber invasion experiment, cell cycle and cell apoptosis were used to detect whether target genes can effectively reverse the inhibition of miR-34a in colorectal cancer.3.Expression of miR-34a and the targets in colorectal cancer and the correlation between them.The expression of miR-34a and FMNL2/E2F5 were measured by real-time qpcr or western-blot in 30 pairs of clinical samples, and analysis the correlation between miR-34a and it’s targets was analysed at the same time.Results1. miR-34a inhibits cell proliferation, invasion and metastasis of colorectal cancer.(1) Endogenous expression of miR-34a in 6 CRC Cell lines by real-time PCR,through the single factor analysis of variance Levene test F (F=767.9,P<0.0001), the results showed miR-34a expression level in 6 strains was significant differences in colorectal cancer. MiR-34a expression quantity highest in HCT116 cell line, which was significantly higher than the other five CRC cell lines, and its expression in LOVO cell line was the lowest.(2)Commercialization inhibitor and mimics of miR-34a were transfected into CRC cells, and the result of real-time per showed that the inhibitor efficiency were 0.324 and 0.324 (p< 0.001, p< 0.001),and the mimic efficiency were 3.824 and 3.658 (p=0.0069,p<0.001). Both of them are quite effiency。(3) CCK8(cell counting kit 8) method was used to detect the change of proliferation ability in vitro in SW480/NC and SW480/miR-34a mimic, LOVO/NC and LOVO/miR-34a mimic, SW480/NC and SW480/miR-34a inhibitor,HCT116/NC and HCT116/miR-34a inhibitor tumor cells, respectively, and the growth curves were drew. Factorial analysis results showed that there was significant difference on the level of growth time between miR-34a over-expression and negative control cell lines (P<0.001, P<0.001), while the ability of cell proliferation had significantly differences between cell groups (P<0.001, P<0.001). Time and group interaction effect had significant difference (P<0.001, P<0.001). There was significant difference on the level of growth time in miR-34a knocked down cell lines (P<0.001, P<0.001), the ability of cell proliferation was significantly difference between cell groups (P<0.001, P<0.001). The result showed that compared with the NC group, miR-34a over-expression group proliferation increased slowly, and in contrast, miR-34a inhibited cell groups promoted cell growth significantly when compared to the NC control group.(4)In the miR-34a high expression colorectal cancer cell lines HCT116 and SW480, we transient transfected miR-34a inhibitor, respectively; while miR-34a low expression colorectal cancer cell lines LOVO and SW480 were transfected with miR-34a mimics. Boyden Transwel chamber test was ued in vitro to see the invasion ability changes. Results showed that after the interference miR-34a, compared with NC group, miR-34a SW480 and HCT1 16 cell number in membrane inhibitor group were decreased significantly (t= 7.165, P= 0.002; t= 4.562, P= 0.0103). Compared with NC group, after expressing miR-34a, LOVO and SW620 cell number of wear membrane increased significantly (t= 3.539, P< 0.00224, t= 0.00224, P= 0.031). Combined with interference and over-expression group, we can draw the following conclusion that miR-34a can inhibit colorectal cancer cell in vitro invasion ability.(5) In two strains of colorectal cancer cells SW480 and LOVO, compared with NC group, G1 phase cell proportion of miR-34a group increased significantly (t= 5.477, P= 5.477, t= 8.951, P= 0.001). S phase of cell ratio decreased significantly (t= 3.687, P= 0.0211; t= 9.39, P< 0.001); G2/M phase cells proportion increased significantly (t= 2.665, P= 0.021, t= 8.48, P= 0.001). And compared with NC group in SW480 and HCT1 16 cell lines, G1 phase of the cell ratio of miR-34a inhibtor group decreased significantly (t= 9.825, P= 0.0102; t= 6.075, P= 0.026). S phase of cell proportion increased significantly (t= 4.838, P= 0.0402, t= 16.27, P= 0.0037). G2/M phase cells ratio decreased significantly (t= 17.63,< 0.001, t= 0.001, p= 0.005). Prompting miR-34a can inhibit cancer cell evolution from G1 to S phase, thus inhibiting cell proliferation ability.(6)Giving the interference fragment miR-34a inhibitor to SW480 and HCT116, and to give miR-34a mimics in LOVO and SW480, after 48 hours, the flow cytometry instrument was used to detect the apoptosis of colorectal cancer cells. The results showed that compared with LOVO/NC and SW480/NC group, early apoptosis rate of LOVO/miR-34a and SW480/miR-34a group increased significantly (t= 113.1, P < 0.001, t= 300, P< 0.001; figure 2-6-11) in table 1. And compared with SW480/ NC, HCT116/NC group, early apoptosis rate of SW480/miR-34a inhibitor, HCT116/miR-34a inhibitor group was significantly lower (t= 25.73, P< 0.01, t= 0.01, P< 0.001), the result showed that miR-34a can promote the occurrence of colorectal cancer cell apoptosis.(7)Successfully build SW480/miR-34a stability Expression cells, and quantitative PCR had verify its expression efficiency of 17.41 (t= 22.04, p< 0.001).(8)Compared with the control group, over-expression of miR-34a significantly inhibited tumor growth in vivo (t=3.244, P= 0.018). Ki67 detection line, SW480/ Ki67 positive cell percentage of subcutaneous tumor miR-34a group was obviously lower than the control group SW480/NC (t= 30.07, p< 0.001), the above results showed that the miR-34a inhibited tumor cell proliferation in the body.(9) MiR-34a expressing SW480 cells and control cells were injected through tail vein,results show that the two groups in the nude mice liver, kidney, intestine, brain and other organs has not been found on the microscopic metastases, whereas in the lung surface shows obvious metastases. Lung metastasis in nude mice SW480/NC group rate was 100%(6/6); SW480/miR-34a group of lung metastasis rate was 66.6%(4/6); SW480/occurred pulmonary metastases, the number of miR-34a group was obviously less than control group (t= 2.789, P= 2.789), and miR-34a transfer ability can inhibit colorectal cancer cell in the body.2.MicroRNA-34a targets FMNL2 and E2F5 and suppresses the progression of colorectal cancer.(1)Using miRanda, TargetScan and DIANA-microT, Pictar4 to predict the targets of miR-34a, the prediction of higher reliability and accuracy of relevant miRNA target genes were picked out.(2)Luciferase report system test results showed that:FMNL2 miR-34a-3’UTR and E2F5-3’UTR luciferase activity were significantly reduced(F= 21.75, P= 0.0018, F = 6.851, P= 0.028; F= 17.23, P= 0.003, F= 14.23, P= 0.029), which verifies that miR-34a can combined with the 3’UTR of FMNL2 and E2F5.(3)Western blot detected the expression of target genes after inhibiting or over-expressing miR-34a, the results showed that when miR-34a was expressed in colorectal cancer cell lines, FMNL2 and E2F5 protein expression were down-regulated. And after the interference of miR-34a, target genes FMNL2 and E2F5 expression were up-regulated,.which indicated that FMNL2 and E2F5 are direct targets of miR-34a in colorectal cancer.(4)CCK 8 was used to test SW480 cell line in the NC group, the miR-34a group, and miR-34a/FMNL2/E2F5 growth curve was drawn as in vitro proliferative ability at the same time. Factorial ANOVA statistical results showed that compared with NC group, the proliferation rate of miR-34a group was obviously lower (P< 0.001), while miR-34a/FMNL2 and miR-34a/E2F5 group could effectively reverse the inhibition of miR-34a on colorectal cancer cell proliferation (P< 0.001; P< 0.001), the results showed that the miR34 through FMNL2 and E2F5 inhibited colorectal cancer cell proliferation, and FMNL2 and E2F5 could effectively reverse its inhibitory effect on colorectal cancer cell proliferation.(5)Boyden Transwell chamber was used to detect the in vitro invasion ability of NC, miR-34a mimic;miR-34a/FMNL2 and miR-34a/E2F5 cells. According to the results, miR-34a/E2F5 and miR-34a/FMNL2 group were effectively reverse the inhibition of miR-34a on colorectal cancer cell invasion ability(F= 202, P< 0.001; F= 218, P< 0.001).(6)Flow cytometry instrument was used to detec the effects of NC, miR-34a mimic; miR-34a/FMNL2 and miR-34a/E2F5 on cell cycle. Independent sample t-test analysis showed that:in SW480, after expressing miR-34a miR-34a group compared with NC group, G1 phase cell proportion increased significantly; Significantly reduce the proportion of the S phase cells, G2/M phase cells proportion increased significantly. After expressing miR-34a and restore FMNL2 and E2F5 expression, G1 phase of the cell ratio decreased significantly (p< 0.001, p< 0.001); S phase of cell proportion increased significantly (p=0.001, p< 0.001); G2/M phase cells ratio decreased significantly (p= 0.005, p< 0.001 figure 3-5); miR-34a can inhibit cancer cell evolution from G1 to S phase, thus inhibiting cell proliferation, and FMNL2 and E2F5 can effectively reverse miR-34a for colorectal cancer cell cycle arrest, to reverse its proliferation.(7)Flow cytometry instrument was used to detect the effects of NC, miR-34a mimic; miR-34a/FMNL2 and miR-34a/E2F5 on colorectal cancer cell apoptosis. Results showed that, compared with SW480/NC,early apoptosis of SW480/miR-34a group increased significantly, which indicated that miR-34a can promote the occurrence of colorectal cancer, apoptosis, and overexpression of miR-34a and FMNL2/E2F5 expression at the same time could effectively reverse miR-34a on the apoptosis of colorectal cancer cells (figure 3-6, table 3-4, F= 1867, p< 0.01, F= 384.1, p< 0.001).3.MiR-34a is down-regulated in CRC tissues and negatively correlates with FMNL2 and E2F5 expression.The expression of miR-34a was quantified in 30 pairs of CRC tissues and the corresponding noncancerous tissues using qRT-PCR. The results revealed that miR-34a expression level was markedly down-regulated in CRC tissues compared with matched normal tissues. Clinicopathological analyses showed that miR-34a expression correlated closely with lymphatic metastasis. We also examined the expression correlations between miR-34a and FMNL2 or E2F5. Compared with the matched normal tissues, both E2F5 andFMNL2 were up-regulated in CRC tissues There were inverse correlations between expressions of miR-34a and two target genes, E2F5 and FMNL2 in CRC tissues (,spearman correlation test,r=-0.971, P<0.0001; r =-0.9216, P< 0.001). Taken together, these results demonstrated that reduced miR-34a expression along with increased E2F5 and FMNL2 protein expression were frequent events in human CRC tissues.Conclusion1. miR-34a supresses cell proliferation, invasion and metastasis of colorectal cancer.2.MiR-34a directly targets FMNL2 and E2F5, and reintroduction of FMNL2 or E2F5 impairs miR-34a-induced suppressive effects on CRC cell behaviors3.MiR-34a is down-regulated in CRC tissues and negatively correlates with FMNL2 and E2F5 expressions. |
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