| As the Cervical cancer with the characteristics of high incidence, high invasive and metastatic and it trends to be gradually much younger, it becomes the second most common female malignancy cancer among women worldwide. The prognosis of cervical cancer patients is related to the stage of cancer. The prognosis of invasion metastasis patients is poorer, while the tumor confined patients’ prognosis is better. An increasing number of evidence has indicated that high-risk human papillomavirus(HR-HPV) type serve as an important role in the development of the precursors of cervical cancer. However, not all of those who are infected with HPV develop into cervical carcinoma. It indicates that factors other than HPV viral proteins also contribute to the progression of cervical cancer. The cervical cancer which has a poor prognosis is usually associated with pelvic lymph node involvement and the tumor cells may have metastasis. In the process of invasion and metastasis,the cancer cells acquire the ability to break through the basement membrane, single cell or groups of cancer cells begin to invade the adjacent stroma.In the initial stage of this process, the inter- polarized epithelial cells change to mesenchymal cell morphology, which is called epithelial- mesenchymal transition. EMT is associated with tumor transmission and diffusion. Presently, little is known about how the cervical cancer cells acquire the ability to invade the surrounding tissue, and the role of EMT in cervical cancer cells and tissues is little known.Macrophage migration inhibitory factor(MIF), which is defined as a proinflammatory cytokine, it involves in many pathological damage processes. It has been found that it is significantly higher in various cancers such as breast cancer, prostate cancer, lung cancer, ovarian cancer and cervical cancer and so on. Our previous studies have found that MIF is lower expressed in cervical cancer Si Ha cells and higher expressed in cervical Caski cells. We have also confirmed that MIF can promote the proliferation and metastasis of cervical cancer cells in vitro and inhibit apoptosis, therefore promote the invasion and metastasis of cervical cancer. Some scholars have confirmed that MIF’s overexpression can promote the occurrence of EMT in pancreatic cancer cells and promote their invasion and metastasis.And after RNA interfere MIF, it can inhibit the occurrence of EMT of pancreatic cancer cells and reduce the invasion and migration of cancer.However, little information of the correlation between MIF and EMT in cervical cancer is available. Objective:1. To detect the expression of MIFm RNA and protein after overexpression MIF in human cervical carcinoma Si Ha Cells and EMT-related marker E-cadherin, Snai, Twist and Vimentin;2. To detect the expression of MIFm RNA and protein and EMT-related marker E-cadherin, Snai, Twist and Vimentin after knocking down of MIF gene and RNA interference technology in human cervical carcinoma Caski Cells. Methods:1. Recombinant plasmid of p EGFP-N1-MIF, p Genesil-MIF si RNA, p EGFP-N and p Genesil-1 were constructed and identified, then plasmids were extracted for transfection respectly;2. Low expression of MIF cervical cancer Si Ha and high expression of MIF cervical cancer Caski cells and cells and were cultured;3. Recombinant eukaryotic expression plasmid p EGFP-N1-MIF and p EGFP-N1 were transfected into human cervical cancer cells Si Ha cells, p Genesil-MIF si RNA and p Genesil-1 were transfected into human cervical cancer Caski cells using the lipofectamine, while. After transfection, we observed the transfection efficiency under an inverted fluorescence microscope;4. Fluorescent quantitation polymerase chain reaction and western blot were used to detect the the expression of MIF m RNA and protein in each group, cervical cancer Si Ha cells and Caski cells were used in blank groups;5. Fluorescent quantitation polymerase chain reaction and western blot methods were used to detect the expression of EMT related markers such as E-cadherin, Vimentin, Snail and Twist before and after transfection in each group. Results:1. The growth of Si Ha and Caski cell lines are vigorous;2. Recombinant plasmid of p Genesil-MIF si RNA, p EGFP-N1-MIF, p Genesil-1 and p EGFP-N were constructed successfully;3. The eukaryotic expression vector p EGFP-N1-MIF could be observed green fluorescence under inverted fluorescence microscope after transfecting Siha six hours, the fluorescence intensity was the strongest after transfecting Siha 48 hours,and then gradually decreased.The eukaryotic expression vector p Genesil-MIF si RNA was the same;4. For Si Ha cells, western blot method was confirmed that compared with negative plasmid transfection group( transfected with plasmid p EGFP-N1) and control group(Siha), for the experimental group which was transfected with plasmid p EGFP-N1-MIF, the MIF protein expression was higher than that of the control group(P <0.05), while compared to the control group,the expression of MIF protein in negative plasmid group did not change significantly(P> 0.05), which proved that the negative control group plasmid had no effect on MIFm RNA and protein of Si Ha cells;5. For Caski cells, PCR and western blot methods were confirmed that compared with negative plasmid transfection group( transfected with plasmid p Genesil-1) and control group(Caski), for the experimental group which was transfected with plasmid p Genesil-MIF si RNA, the MIFm RNA and protein expression were lower than that of the control group(P <0.05), while compared to the control group,the expression of MIFm RNA and protein in negative plasmid group did not change significantly(P> 0.05), which proved that for the negative control group plasmid had no effect to MIFm RNA and protein;6. Detection of the EMT-related indicators: For Si Ha cells, compared with the control group, after the transfection with p EGFP-N1-MIF, the epithelial marker E-cadherin m RNA and protein expression levels reduced significantly(P <0.05), mesenchymal markers Vimentin, snail and Twist m RNA and protein levels increased significantly(P <0.05), while compared with the control group, the expression of m RNA and protein of each index in the negative group did not change significantly(P> 0.05);While for Caski cells, compared with the control group, after the transfection with p Genesil-1-MIFsi RNA, the epithelial marker E-cadherin m RNA and protein levels increase significantly(P <0.05), mesenchymal markers Vimentin, snail and Twist m RNA and protein expression levels were significantly reduced(P <0.05), while compared with the control group, the expression of m RNA and protein of each index in the negative group did not change significantly. Conclusions:1. When cervical cancer Siha cells overexpressed MIF, the expression level of epithelial maker E-cadhein decreased, but the mesenchymal maker Vimentin, snail and Twist increased;2. When silence the MIF gene in cervical cancer Caski cells, the expression level of epithelial maker E-cadhein increased, but the mesenchymal maker Vimentin, snail and Twist decreased;3. Overexpression of MIF can promote the occurrence of EMT in cervical cancer Si Ha cells; and after the silence of MIF in cervical cancer Caski cells can inhibit the occurrence of EMT. |
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