| [Objective and significance]Breast cancer is the most commonly found carcinoma of female breasts, and also one of the most common malignant tumor in women, whose incidence and mortality has been increasing in recent years, ranking the top of the female carcinoma. Currently, the common treatments for breast cancer are the traditional surgical treatment, supplemented by local or systemic postoperative radiotherapy, chemotherapy and endocrine therapy. Though these therapies can achieve a higher survival rate and even in some patients, a clinical cure, its recurrence and distant metastasis remained formidable, presenting challenges and difficulties to scholars. Gene therapy is a newly developed treatment for breast cancer following surgery, radiotherapy, chemotherapy and endocrine therapy, which have been applied in clinical practice with favorable therapeutic efficacy. For instance, Herceptin (Trastuzumab), a product by Roche, has been applied to treat metastatic breast cancer with overexpression of Her-2/neu, and shed light on the treatment of breast cancer. Although preliminary success of gene therapy has been achieved in treatment of breast cancer, there are still many issues to be resolved, with one of the most important issues being the singular target gene. Therefore, searching for some new target genes has been demonstrated to be greatly important. Phosphatase of regenerating liver-3 (PRL-3), a small molecular protein weighing only 20KD, belongs to the PRL family which includes members of PRL-1, PRL-2, and PRL-3, with homology of about 76-87%. PRL-3 is also known as PTP4A3, with its coding genes being located on chromosome 8q24.3 and its first-level structure composed of 173 amino acids and secondary structure composed of 4 a-helices,5β-sheet and a P loop. In recent years, the potential relationship between the biological features of cancer and the members of the PRL protein family, has gained focused attention from both basic biological and clinical researches. It has been appreciated that protein tyrosine phosphatase regulates protein activities via mediating the phosphorylation and dephosphorylation of the protein tyrosine residues, and participates in various cellular activities. In normal human bodies, PRL-3 only has different expression in the cardiac and skeletal muscle cells. Recent studies have demonstrated that PRL-3 has high expression in cancers of liver, ovaries, colorectum, stomach and lungs, while PRL-3 expression was also found in invasive colorectal and breast cancers as well as in breast carcinoma vessels. The findings indicate highly expressed PRL-3 in tumor tissue is closely related to tumor metastasis and PRL-3 protein is a reliable factor predicting prognosis of breast cancer. Qian Li-ping, et al, used RNA interference (RNA interference, RNAi) to synthesize PRL-3 gene for small interfering RNA (small interfering RNA, siRNA), which was then transfected to treat colorectal cancer cells HCT116. Its effects on cancer cells invasion were observed both in vivo and in vitro. The results showed that PRL-3 gene played an important role in invasion of colorectal cancer cells, and the down regulation by siRNA transfection inhibited the invasive ability of colon cancer cells both in vivo and in vitro. Similar conclusions have also been achieved from studies of other tumors. That is, silencing the expression of PRL-3 genes in cancer cells by RNAi can inhibit the proliferation and metastasis of cancer cells.Based on the previous studies, with PRL-3 gene as the target gene we used RNAi to synthesize PRL-3-shRNA (short hairpin RNA) template chain in vitro, to which the carrier pGenesil-1.1 and PRL-3-shRNA were connected to establish recombinant plasmid. After its transfection to MCF-7 cells mediated by liposome Lipofectamine 2000 and silencing PRL-3 expression in MCF-7 cells by PRL-3-shRNA, the proliferation and apoptosis of MCF-7 cells were observed in vitro. Our study is to provide new insights to the gene therapy for breast cancer, with certain scientific significance and clinical value.[Statistical methods]Statistical analysis was performed using SPSS 13.0 software package. All data are presented as mean±standard deviation (x±s). One-way ANOVA was used to determine the statistical significance. The homogeneity of variance was firstly tested. If the equal variances assumed, the groups were compared by LSD; if not assumed, the heterogeneity of variance was corrected by Welch approximation method and the Brown-Forsythe method; if the equal variances was still not assumed, the groups were compared by Dunnett's T3. Repeated measures ANOVA was used to test the interaction between the groups and time points in MTT test. P<0.05 was considered statistically significant.[Methods and Results]Chapter 1 Design and construction of the shRNA expression vector targeting PRL-3[Objective] To synthesize three recombinant plasmids as the PRL-3-shRNA expression vector for MCF-7 cells transfection to silence PRL-3 genes expression.[Methods] Three shRNA sequences targeting PRL-3 were designed and synthesized respectively to obtain the RNA interference sequence segment targeting PRL-3. They were then inserted into the plasmid pGenesil-1.1 containing green fluorescent protein (EGFP) and the stability selection marker to construct recombinant plasmids pGenesil-1.1-PRL-3-shRNA-1, pGenesil-1.1-PRL-3-shRNA-2, and pGenesil-1.1-PRL-3-shRNA-3. After their enzyme digestion and sequencing approval, they were amplified in great amount.[Results] We used plasmid extraction kit to extract the recombinant plasmid, followed by SacⅠenzyme digestion and 1% agarose electrophoresis identification. SacⅠrestriction site was designed in the three target genes respectively, and plasmid pGenesil-1.1 contains a Sac I restriction site. Therefore, the recombinant plasmid can be digested into a small band containing 916 bp DNA by Sac I due to the correct connection. Enzyme digestion approved that the three recombinant plasmids were in line with the design requirements. Based on the sequencing results from Shanghai Invitrogen Biotechnology Co. Ltd., it was approved that the sequence was completely correct.Chapter 2 Effects of RNA interference targeting PRL-3 on proliferation and apoptosis of MCF-7 human breast cancer cells2.1 MCF-7 cell transfection[Objective] To detect the transfection of MCF-7 cells by liposome (lip2000) mediated recombinant plasmid.[Methods] 48 h after transfection, the transfected MCF-7 cells by liposome-mediated plasmid were detected using fluorescence microscopy[Results] Liposome (lip2000) mediated plasmid was successfully transfected into MCF-7 cells.2.2 Detection of PRL-3 gene expression after transfection using Real-time PCR[Objective] 72 h after transfection, mRNA expression of PRL-3 genes in MCF-7 cells was detected, and one of the most efficient recombinant plasmid in silencing mRNA expression of PRL-3 genes was selected.[Methods] The experiment was conducted in 5 groups, namely blank control group, negative control group, PRL-3-shRNA-1 group, PRL-3-shRNA-2 group and PRL-3-shRNA-3 group. The latter four groups were transfected negative control plasmid, the recombinant plasmids pGenesil-1.1-PRL-3-shRNA-1, pGenesil-1.1-PRL-3-shRNA-2, and pGenesil-1.1-PRL-3-shRNA-3 respectively. By using real-time PCR with GAPDH as a reference gene, expression of PRL-3 gene in each group was detected 72 h after the respective transfection.[Results] Real-time PCR showed that liposome mediated recombinant plasmid was successfully transfected into MCF-7 cells. Represented by the data of samples 2'ΔΔCt, the results indicated that the primer could be amplified for the specific bands under such conditions, and the best interference vector was PRL-3-shRNA-2. Therefore, the recombinant plasmid pGenesil-1.1-PRL-3-shRNA-2 was used to perform the subsequent experiments.2.3 Expression of PRL-3 protein after transfection detected by Western blot[Objective] To detect the expression of PRL-3 protein in MCF-7 cells at 72 h after transfection.[Methods] The subjects were divided into 3 groups, namely the experimental group, the negative control group and the blank control group. The first two groups were transfected with recombinant plasmid pGenesil-1.1-PRL-3-shRNA-2 and the negative control plasmid respectively. In this study, the expression of the PRL-3 protein in each groups were detected by Western blot 72 h after the transfection.[Results] Western bolt indicated that all groups had consistent developing ofβ-actin expression, which was an internal reference. However, but the PRL-3 protein in the PRL-3-shRNA-2 cells was significantly weaker than the negative control group and blank control group. Statistical analysis indicated the relative gray value had significant differences (F-608.256, P<0.01) between the PRL-3-shRNA-2 group (0.500±0.0158), the negative control group (0.910±0.026) and the control group (0.894±0.021).2.4 Cellular proliferation of MCF-7 cells after transfection by MTT method[Objective] To detect the cellular proliferation of MCF-7 cells after transfection.[Methods] In this study, the experimental groups were divided the same as in the section of 2.3 experiment. Cells were planked (six-well plate) till their being cultured with at least 90% fusion rate, with 25 million cells per well. And the transfection was conducted in the same procedures as in the section of 2.3 experiment. The detection was performed every 24 h in the following procedures:Each well was added into freshly prepared MTT 10 ul at the concentration of 10 mg/ml for continued culture of 4 h. After the supernatant fluid was discarded, each well was additionally added into 0.02 ml freshly prepared DMSO. Automated enzyme-linked immunoassay was used to determine the absorbance of each well, the culture medium not containing cells as the blank control. The mean value of 4 parellel wells was calculated. And the experiment was repeated for 3 times.[Results] The proliferation of MCF-7 cells analyzed by MTT method indicated that the difference between the three groups have statistical significance (F=39.112, P<0.01), with the growth curve of the negative group and the control group overlapping, but that of the experimental group significantly rightwards indicating significantly slowing down growth rate. The value of optical-density of the experimental control cells gradually increased along with prolonged transfection time. At ld,2d,3d and 4d after transfection, the value of optical-density were (0.194±0.019),(0.307±0.015),(0.453±0.020),(0.699±0.045) respectively, with statistical significance (P=0.494, P=0.940). The four repeated factors between different experimental days had statistical difference (F=776.185, P<0.01). Different experimental days and different groups showed interactive effects (F=9.591, P<0.01).2.5 Apoptosis rate of MCF-7 cells after transfection by Flow cytometry[Objective] To detect the apoptosis rate of MCF-7 cells at 48 h after transfection.[Methods] In this study, the experimental groups were divided the same as in the section of 2.3 experiment. Cells were planked (six-well plate) till their being cultured with at least 90% fusion rate, with 25 million cells per well. The cells were cultured for 16 h in 6-well plates and then transfected with the plasmid, with negative fragment of plasmid as control.48 h after transfection, the cells were firstly digested with trypsin for PBS cleansing once, and then fixed 4 times with 75% ethanol, followed by overnight fixing, no less than 16 hours. The PI dye (0.5mg/ml) were used for staining, which were added to the cells of severe suspension and PBS at the ratio of 1:10 for homogeneity and reaction for 5 min in darkness. Finally it was centrifuged 5 min in 1000rpm. Subsequently, the supernatant was suctioned off and the residues were washed once again with PBS, centrifuged for 5 min in 1000rpm, resuspended with PBS 500μl and analyzed by flow cytometry. The experiment was repeated 3 times.[Results] 48 h after transfection, the apoptosis rate increased in the experimental group compared to the negative control group and the blank control group (F= 29.064, P=0.001). And the apoptosis rate of the latter two groups had no significant difference (P=0.26). In this study, the apoptosis rate of the experimental group, compared with the negative group and the control group, the difference in G1 and S phase indicated statistical significance (G1:F=15.886, P=0.004; S:F=7.253, P= 0.025), but G2 phase had no statistical difference (F=0.160, P=0.856). Pairwise comparisons indicated that between the negative control group and the blank control group, there were no significant difference in G1 and S phases (P> 0.05). But comparison between the experimental group and negative control group or blank control group indicated that G1 phase of the experimental group increased significantly, while the S phase decreased significantly, suggesting a slower shift from G1 phase to S phase in the experimental group.[Conclusion]In this study, the breast cancer MCF-7cells was adopted as the study model. We constructed PRL-3-shRNA carrying specific interfering segment for PRL-3 for RNA interference to silence the expression of PRL-3 genes. According to the results of this study, we clarified the following issues:1) The expression of PRL-3 in MCF-7 cells can be inhibited via silencing PRL-3 gene by PRL-3-shRNA.2) Silencing PRL-3 gene by PRL-3-shRNA can inhibit the proliferation of MCF-7 cells.3) Silencing PRL-3 gene by PRL-3-shRNA can promote the apoptosis of MCF-7 cells. In view of these results, we came to the following conclusions:PRL-3-shRNA, which carries PRL-3-specific interfering sequence can effectively silence the PRL-3 gene, inhibit the proliferation and invasive ability of MCF-7 cells, and promote their apoptosis. These results indicate that the PRL-3 protein play a role in cell proliferation, differentiation, cell migration, and other life activities. It is closely related to pathological processes of the breast cancer MCF-7 cells, such as their proliferation and metastasis. |
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