| BACKGROUND:Colorectal carcinoma (CRC) is the third most common cancer in western countries and one of the most common cancers in China. Though its mortality rates has decreased slightly over the past three decades because of increased early diagnosis and advances in treatments, with the living standard improved and living style changed, the CRC incidence is steadily rising. Invasion and metastasis are the main causes that affect the therapeutic efficacy and lead to death of patients. Therefore, it is very essential to explore the factors that closely correlated with genesis, development, diagnosis and therapy of CRC.The carcinogenesis and progression of malignant tumors is a complex biological process involving multiple factors and steps. Study on genesis, development, invasion, metastasis and novel therapy of cancer has being a research focus in the medical field. MicroRNAs (miRNAs) have been a hot topic in cancer research for the few years. miRNAs are a large family of short18to24-nucleotide (nt) long single-stranded noncoding RNAs identified in many eukaryotes. It is transcribed from DNA, and instead of being translated into protein, it regulates the functions of other genes in protein synthesis. miRNA pairs with complementary or near-complementary mRNA and induces cleavage or inhibits the translation of the target mRNA. miRNAs that bind to their mRNA target with perfect complementarities induce target mRNA cleavage, but miRNAs that bind with imperfect complementarities modulate gene expression by inhibiting protein translation. The interaction between miRNAs and genes is complex. A miRNA can regulate many genes, and many miRNAs can regulate a gene. In addation, genes can regulate miRNAs.miR-200family has been a hot topic in cancer research for the past few years. miR-200a is a member of the miR-200family (miR-200a, miR-200b, miR-200c, miR-114, miR-429). The expression of miR-200a is down-regulated in many types of cancer and has been shown have a key role in invasion and metastasis of cancer by regulate epithelial-mesenchymal transition (EMT). EMT is regulated by various factors. Cancer cells underwent EMT through a variety of factors. It mainly includes the up-regulated expression of transcription factors and the involvement of signaling pathway. Many studies demonstrate that AKT and ERK signaling pathway play improtant roles in the development of EMT in CRC. miR-200a specifically modulates AKT signaling in hepatic carcinoma and pancreatic cancer cell lines by target gene. Howeve, the relationship among miR-200a, EMT and AKT/ERK signaling pathwany have no report in CRC. miR-200a role as a tumor suppressor gene in many cancer. Interestingly, some reports showed miR-200a is down-regulated in CRC, while it is up-regulated in other reports. There are few researches about effects and mechanisms of miR-200a in CRC. On the basis of our experiments, inhibitor/miRNA-200a or mimics/miR-200a is transiently transfected into HCT116cell and SW480cell or LoVo cell and SW480cell to gain-of-function or loss-of-function of miR-200a, respectively, then we detected the changes of biological behavior in CRC cells and investigated the molecular mechanism of miR-200a in CRC.EMT was originally described during embryogenesis as a developmental process such as gastrulation, renal organogenesis, and the formation of neural crest. Then EMT has been considered essential for cancer invasion and metastasis and the EMT process is necessary to the conversion of benign tumor to aggressive and highly invasive cancer. However, it is still controversial whether transformation of a noninvasive tumor into a metastatic tumor truly represents an EMT. The main argument for the lack of a role of EMT in cancer is that metastases seem histopathologically similar to the primary tumors from which they are derived, one research showed that EMT and non-EMT cells cooperate to complete the spontaneous metastasis process. EMT cells are responsible for degrading the surrounding matrix to lead the way of invasion and intravasation. Non-EMT cells then enter the blood stream and reestablish colonies in the secondary sites. Even have research showed that EMT is a fallacy. To resolve this apparent contradiction, a mesenchymal-epithelial transition (MET) process in the metastatic sites has been postulated as a part of the process of metastatic tumor formation. According to this hypothesis, EMT cells change their adhesive properties, activate proteolysis, and become motile, allowing them to leave the primary tumor and establish secondary tumor in distant sites. During the colonization of distant sites, a reverse process MET takes place and metastatic cancer cells again acquire the epithelial phenotype. MET is an attractive hypothesis that can explain the histopathological similarity between primary and metastatic tumors. However, direct experimental data supporting MET in cancer metastasis are still lacking. So, We performed this study to investigate the difference of epithelial-mesenchymal transition in human colorectal carcinoma and its lymphatic metastasis and colon cancer cells SW620and SW480, moreover, we compared histomorphology and protein expression of E-cadherin and Vimentin among primary, metastatic carcinomas and their emboli to explore whether cancer cells abide by the epithelial-interstitial transformation mechanism in the process of metastasis.In order to explore the effects of miR-200a and EMT in colorectal cancer and search therapeutic method, we mainly undertake these researches as follows.OBJECTIVE:1. To explore the changes of biological behavior in CRC cells of miR-200a in CRC by gain-of-function or loss-of-function of miR-200a.2. To investigated the molecular mechanism and the relationship among miR-200a, EMT and AKT/ERK signaling pathwany in CRC by gain-of-function or loss-of-function of miR-200a.3. To explore whether cancer cells abide by the epithelial-mesenchymal transition in the process of metastasis by comparing histomorphology and protein expression of E-cadherin and Vimentin among primary, metastatic carcinomas and their emboli.4. To explore the difference between epithelial-mesenchymal transitions in human CRC as well as its lymphatic metastasis and CRC cell lines SW480and SW620, and understand the role of EMT in metastasis.METHODS:1. Inhibitor/miR-200a and mimics/miR-200a were transiently transfected into HCT116, SW480and LoVo, SW480, respectively. Then we detected the inhibition or up-regulation efficiency by quantitive real time PCR. CCK-8assay were used to detecte the cell proliferation. Assessment of apoptosis using a TUNEL assay. The mobile ability of cells were detected by transwell migration assay. homogenous adhesion experiment were used to detecte the ability of cell-cell adhesion. E-cadherin and Vimentin were determined by quantitive real time PCR and immuocytochemistry. Correlated proteins (PTEN, p-AKT, p-ERK1/2, AKT, ERK2, CD147, MMP-9) were detected by western Blot.2. The binding sites of has-miR-200a and PTEN predicted by bioinformatics approach.3. A total of68tissue specimens in59cases of primary adenocarcinoma or squamous cell carcinoma and their lymphatic metastasis were collected, of which there were13well differentiated,11moderately differentiated,30poorly differentiated tumors and14lymphatic metastases. The morphology and the expression of E-cadherin and Vimentin were assessed by HE stain and immuocytochemistry.4. The expression of E-cadherin and Vimentin mRNAs in human CRC cell lines SW480and SW620were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). The expression of E-cadherin and Vimentin proteins in30pairs of human primary CRC and its lymphatic metastasis were detected by immunohistochemistry. The morphology of primary carcinoma and its lymphatic metastasis, SW480and SW620cells were observed by HE staining.RESULTS:1. The effect to HCT116and SW480by repressing the expression of miR-200aThe expression of miR-200a in HCT116and SW480were declined50.9%and54.4%respectively by transient transfection. CCK-8assay showed that after decreasing the expression of miR-200a, the proliferation of HCT116and SW480apparently increased as compared blank group or negative control group (F=4.009, P=0.031; F=6.122,P=0.004; respectively). Transwell migration assay indicated that the HCT116and SW480crossing the membrane were apparently increased after inhibiting the expression of miR-200a as compared blank group or negative control group (F=102.452, P=0.000; F=41.632, P=0.000; respectively). TUNEL assay indicated that the percent of apoptotic cells in HCT116and SW480apparently decreased as compared blank group or negative control group (F=19.932, P=0.000; F=34.720, P=0.000; respectively). Homogenous adhesion experiment showed the adhesive ability of HCT116and SW480apparently decreased as compared negative control group (t=8.060,P=0.000; t=3.668, P=0.004; respectively). qRT-PCR indicated that the expression of E-cadherin in HCT116and SW480were significantly decreased after inhibiting the expression of miR-200a as compared negative control group (t=-27.163, P=0.001; t=-5.079, P=0.037; respectively), while Vimentin were significantly increased (t=75.054, P=0.000; t=14.068, P=0.005; respectively). Immuocytochemistry results showed that the expressions of E-cadherin were significantly decreased, while Vimentin were significantly enhanced. Western Blot showed that inhibition of miR-200a up-regulated p-AKT, p-ERK1/2, CD147and MMP-9expression, down-regulated PTEN expression, but has no effect on total AKT and ERK2expression.2. The effect to LoVo and SW480by increasing the expression of miR-200aThe expression of miR-200a in LoVo and SW480were increased2053times and102times respectively by transient transfection. CCK-8assay showed that after increasing the expression of miR-200a, the proliferation of LoVo and SW480apparently decreased as compared blank group or negative control group (F=101.928, P=0.000; F=27.583, P=0.000; respectively). Transwell migration assay indicated that the LoVo and SW480crossing the membrane were apparently decreased after up-regulating the expression of miR-200a as compared blank group or negative control group (F=119.464, P=0.000; F=58.670,P=0.000; respectively). TUNEL assay indicated that the percent of apoptotic cells in LoVo and SW480apparently increased as compared blank group or negative control group (F=25.620, P=0.000; F=25.159, P=0.000; respectively). Homogenous adhesion experiment showed the adhesive ability of LoVo and SW480apparently increased as compared negative control group (t=-4.250, P=0.005;t=-4.190, P=0.006). qRT-PCR indicated that the expression of E-cadherin in LoVo and SW480were significantly increased after up-regulating the expression of miR-200a as compared negative control group (t=15.006,P=0.004;t=23.844, P=0.002), while Vimentin were significantly decreased (t=-6.010, P=0.027; t=-9.081, P=0.012). IHC results showed that the expressions of E-cadherin were significantly increased, while Vimentin were significantly decreased. Western Blot showed that over-expression of miR-200a down-regulated p-AKT, p-ERK1/2, CD147and MMP-9expression, up-regulated PTEN expression, but has no effect on total AKT and ERK2expression.3. Targetscan software identified PTEN as a potential target of miR-200a. 4. The overall morphology of the primary cancers and their tumor emboli was similar. Among54primary cancers,50cases were positive for E-cadherin and22cases were positive for Vimentin. Fifty-one cases were positive for E-cadherin and22cases were positive for Vimentin in the tumor emboli, with no statistical difference (Z=-0.248, P=0.804; Z=-0.199, P=0.842, respectively). Among14cases of lymphatic metastasis,12cases were positive for E-cadherin and6cases were positive for Vimentin, and the tumor emboli in12cases were positive for E-cadherin and7cases were positive for Vimentin, with statistical difference (Z=-1.727, P=0.084; Z=-0.154, P=0.878, respectively). There were no significant difference of E-cadherin and Vimentin protein expression between the cancer tissue and its emboli (Z=-0.824,P=0.410; Z=-0.222, P=0.824, respectively). A subset of tumor cells in cancer emboli expressed E-cadherin at a high level without Vmentin expression, whereas other cells in tumor emboli showed an opposite expression pattern.5. The expression levels of E-cadherin and Vimentin mRNAs in SW620cells were significantly higher than that in SW480cells (t=10.837, P=0.008; t=4.796,.P=0.041, respectively). There were no significant difference in E-cadherin and Vimentin protein expression between primary colorectal carcinoma and its lymphatic metastasis (Z=-0.235, P=0.814; Z=-0.479, P=0.632). However, E-cadherin protein expression in metastatic lymph nodes were higher than in primary carcinoma (4/30), whereas Vimentin protein expression in metastatic lymph nodes was lower than in primary lesions (6/30), but there were no statistically significant difference. The histopathology for colorectal carcinoma with lymphatic metastasis was similar to primary colorectal carcinoma, there was also no difference between both cell lines in terms of morphology.CONCLUSIONS:1. miR-200a role as a tumor suppressor gene in CRC. Down-regulation of miR-200a in CRC can promoted cells proliferation, migration and invasion but depressed cells apoptosis and cell-cell adhesion. All of these eventually influence the progression of CRC.2. miR-200a have a role in the regulation of EMT, AKT and ERK pathway activities and Correlated proteins includes PTEN, CD147, MMP-9. All of these eventually regulate the development of EMT and then changed CRC cell biology: proliferation, migration, adhesion, apoptosis, invasion and metastasis.3. There is no significant difference of EMT characteristics among primary cancer, lymphatic metastases and their cancer emboli. Cancer thrombus contains both EMT cells and non-EMT cells. We presume that EMT cells change their adhesive properties, activate proteolysis and became motile, allowing them to leave the primary tumor and enter the vessels. Part of the EMT cells takes place MET in vessels, so as to cancer thrombus contains both EMT cells and non-EMT cells. The EMT cells that not takes place MET is the reason of metastasis. After establish secondary tumor in distant sites, a reverse process MET takes place and metastatic cancer cells again acquire the epithelial phenotype, however, part of the cancer cells still keeps EMT feature. So, there is no significant difference of EMT characteristics between primary cancer and lymphatic metastases, and the EMT cells that not takes place MET in secondary tumor is the reason of metastasis in further distant sites.4. There was no significant difference of EMT characteristics in either primary colorectal carcinoma versus its lymphatic metastasis or in SW480versus SW620. We presume the mechanism is same as conclusion3and further studies are required to elucidate the role of EMT. |
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