MicroRNA-21 Promote Cell Metastasis Of Human Cervical Cancer By Modulating Epithelial-mesenchymal Transition

BackgroundCervical cancer is the second common malignant tumor on female reproductive system.There are more than 500,000 new cases all around the world every year. About 80% new cases happen in developing countries.In our country,there are more than 130,000 new cases each year, more than 60,000 patients died due to cervical cancer each year.With the application of the HPV vaccine and the early stage cervical cancer screening methods, the incidence of cervical cancer decreases However, the HPV vaccine isn’t applicated worldwide, the screening plan is not carried out on economic backward areas.The mortality of cervical cancer doesn’t decrease. Invasion and metastasis is still the leading cause of its poor prognosis. Therefore, systematic studing the moleular mechanisms of the invasion and metastasis of cervical cancer is important to carry out individualized treatment and targeted intervention.MicroRNAs and their relationship with tumors are always hotspot in the tumor researching area in recent years. MicroRNAs are 18 to 25 nt length endogenous non-coding single-stranded RNA molecules. They target mRNA 3’noncoding region (3’ UTR). Complementary pairing led to the degradation of mRNA and imcomplementary pairing led to translation inhibition of mRNA. MicroRNAs participate in a series of important life processes, including cell proliferation, differentiation, apoptosis, and so on. MicroRNAs regulate one third of human gene expression. They are closely related to the occurrence, development, and metastasis of tumor. MicroRNA-21 (miR-21) is one of important microRNAs, which has been found high expression in a variety of solid malignant tumors.MiR-21 modulates a variety of tumor suppressor,such as PDCD4、PTEN、maspin、 TPM1、BMPRⅡ、sprouty 2,etc. It influences tumor cell proliferation, infiltration and antiapoptotic. MiR-21 plays an important role on the developing process of tumor and has been developed as a potential cancer therapeutic target.A large number of studies have shown that the expression level of miR-21 increased in cervical cancer. As known to us, high-risk human papilloma virus (HPV) infection is a major cause of cervical cancer.HPV integrated into the genome of host cell and lead to cell genetics and epigenetics change and malignant transformation. MiR-21 gene locates on chromosome 17q23.2 FRA17B fragile site area. This area is also the integration site of HPV16 gene. So the high levels of miR-21 is due to its location in cervical cancer Another study found that in cervical cancer cell line Hela, inhibiting the expression of miR-21 can inhibit the proliferation of Hela cell significantly.Yao et al studied cervical squamous cancer cells and found that miR-21 regulated target gene CCL20 to influence cervical cancer cell proliferation, apoptosis and migration.In addition, the expression quantity of miR-21 was also associated with the disease progression and lymph node metastasis of cervical cancer patients. These studies illustrated that miR-21 was closely associated with cervical cancer occurring.However the mechanisms of miR-21 participate in the invasion and metastasis of cervical cancer is not fully clear.Big progress has been made on the studies about the mechanism on invasion and metastasis of tumor in recent years. Tumor metastasis is a multi-step, muti-factors process. The process includes local tumor cell infiltrating, wearing into the blood vessel walls, tranferring, moving out the vessel wall and planting. Epithelial mesenchymal transition (EMT) may be the initiate process of tumor invasion and metastasis. EMT means the characteristics of epithelial cells transform to stroma cells accompany with loss of cell polarity and gain of stroma cell features. We have found lots of EMT process in the human body, including embryogenesis, organ differentiation and tissue inflammation, wound healing. EMT plays a key role on the invasion and metastasis process of tumor cells.MicroRNAs regulate nearly one third of the human gene expression, so it may be related to the gene expression regulation on EMT process.Present studies have confirmed that several microRNAs regulated the EMT process on tumors.A study on pancreatic cancer, miR-200c positively regulated E-cadherin, which inhibited the invasion and metastasis of tumor.Schickel et al also confirmed that miR-200 could modulate Fas related phosphorus esterase-1 (FAP-1), to inhibite tumor EMT process. MiR-34a was considered to be a potential tumor inhibitor due to it low expression level in tumor tissues. The downstream target genes of miR-34a, such as SIRT1, Notchl and Snaill also involved in the process of EMT. Zhen et al had found that miR-21 regulated biological behavior by inducing EMT in human cholangiocarcinoma.Research has proved that miR-21 participated in cervical cancer metastasis, but its specific mechanism is unclear.The process of EMT play a key role on tumor metastasis.We hypothesis that miR-21 may promote human cervical cancer metastasis by modulating cell epithelial-mesenchymal transition. This study has not seen in recent research reports.ObjectivesThe objectives of this study is to find out the relationship between miR-21 and cervical cancer invasion and metastasis, the relationship between the EMT process and cervical cancer invasion and metastasis, the relationship between miR-21 and the EMT process in cervical cancer. The another objective is to test the theory that miR-21 promote human cervical cancer metastasis by modulating cell epithelial-mesenchymal transition and to provide experimental data and theoretical basis to carry out the individualized treatment and gene targeting interventions in cervical cancer.Materials and methods1.Tissue samples experiments1.1 The fresh tissue samples were obtained from 45 cervical cancer patients who underwent operations. Each specimen included carcinoma tissue,corresponding normal cervical tissue and suspicious metastasis lymph node tissue.If the lymph node tissue was confirmed positively by pathological test, it was used in the experiment.15 cases with lymph node metastasis carcinoma were used in our study. Real-time quantitative PCR was used to detect the relative expression level of miR-21 in the cervical cancer tissues and the corresponding normal cervical tissues and 15 cases lymphnode metastasis cancer tissues. The relationship between the expression level of miR-21 and clinicopathological parameters was analysed.1.2Real-time quantitative PCR was used to detect the relative expression quantity of ZEB1mRNA in cervical cancer tissues and normal cervical tissues and lymph nodes metastatic carcinoma tissues.The relationship between the expression level of ZEB1 mRNA and clinicopathological parameters was analysed. At the same time, the relationship between the expression level of miR-21 and ZEB1 mRNA was analysed,too.1.3Immunohistochemical technique was used to detect the EMT related proteins ZEB1, E-cadherin and Vimentin in cervical cancer tissues.The relationship between EMT related proteins and clinicopathological parameters of cervical cancer was analysed to explore whether the EMT process happened in the process of cervical cancer metastasis.2. In vitro cells experiments2.1 The cervical cancer cell lines (Hela and Siha) were used in this experiment, Mimic control, miR-21 mimic, inhibitor control or miR-21 inhibitor were transfected into the cells using Lipofectamine 2000 Reagent. Real-time fluorescent quantitative PCR method was used to detect miR-21 expression level of the cells after transfection.2.2 Wound healing assay and Transwell assay were used to test the invasion and migration ability of the cells after transfection.2.3 Real-time fluorescent quantitative PCR technique was used to detect the expression level of the EMT related transcription factors ZEB1 mRNA and Snail mRNA of the cells after transfection.Western Blot technique was used to detect the expression level of EMT related protein E-cadherin, Vimentin, N-cadherin after transfection.Results1.Tissue samples experiments results1.1 Real-time fluorescent quantitative PCR showed that the relative expression level of miR-21 was higher in cervical cancer tissues than in normal cervical tissues (P< 0.05). The relative expression level of miR-21 in cervical cancer tissues was lower than those in lymphatic metastatic carcinoma tissues (P<0.05). And the expression level of miR -21 was significantly correlated with muscular infiltration depth, parametrial invasion,lymphnode metastasis (P<0.05), was not correlated with age, tumor size, pathologic types (P>0.05). The relative expression level of miR-21 was higher in HPV16 positive cervical cancer tissues than non-HPV16 positive tissues (P <0.05).1.2Real-time fluorescent quantitative PCR showed that the relative expression level ZEB1 mRNA was higher in cervical cancer tissues than in normal cervical:tissues (P <0.05). The relative expression level of ZEB1 mRNA in cervical cancer tissues was lower than those in lymphatic metastatic carcinoma tissue (P<0.05). And the expression level of ZEB1 mRNA was significantly correlated with muscular infiltration depth, lymph node metastasis (P<0.05), was not correlated with age, tumor size, parametrial invasion and pathologic types (P>0.05). Further correlation analysis found that in cervical cancer miR-21 expression level was significantly correlated with ZEB1 mRNA (rs=0.841,P<0.05).1.3Immunohistochemical show that the expression level of ZEB1 protein in cervical cancer tissue was higher than in normal cervical tissue and was also significantly correlated with muscular infiltration depth, lymph node metastasis (P<0.05), was not correlated with age,tumor size, parametrial invasion and pathologic types (P>0.05). Compared with the normal cervical tissue,the expression level of E-cadherin in cervical cancer tissues was lower and was significantly correlated with lymph node metastasis (P<0.05). On the contrary, the expression level of Vimentin in cervical cancer tissues was higher (P<0.05)and was significantly correlated with lymphnode metastasis too(P<0.05). Further statistical analysis found that in cervical cancer tissues the expression level of ZEB1 protein was correlated with E-cadherin, was not correlated with Vimentin.2.1n vitro cells experiments results2.1 The results of qRT-PCR showed that the level of miR-21 in Hela and Siha cells increased after being transfected with miR-21 mimic and the level of miR-21 in Hela and Siha cells decreased after being transfected with miR-21 inhibitor.2.2Wound healing assay showed that the migration ability of Hela and Siha cells increased after being transfected with miR-21 mimic and the migration ability of Hela and Siha cells decreased after being transfected with miR-21 inhibitor.2.3Transwell experiment showed that the invasive ability of Hela and Siha cells increased after being transfected with miR-21 mimic and the invasive ability of Hela and Siha cells decreased after being transfected with miR-21 inhibitor.2.4The results of qRT-PCR showed that the level of ZEB1 and Snail mRNA in Hela and Siha cells increased after being transfected with miR-21 mimic and the level of ZEB1 and Snail mRNA in Hela and Siha cells decreased after being transfected with miR-21 inhibitor (P<0.05).2.5Western Blot showed that the level of E-cadherin in Hela and Siha cells decreased after being transfected with miR-21 mimic and the level of E-cadherin in Hela and Siha cells increased after being transfected with miR-21 inhibitor However,the level of Vimentin, N-cadherin in Hela and Siha cells increased after being transfected with miR-21 mimic and the level of miR-21 in Hela and Siha cells decreased after being transfected with miR-21 inhibitor.Conclusions1. The relative expression level of miR-21 was higher in cervical cancer tissues than in normal cervical tissues. The relative expression level of miR-21 in cervical cancer tissues was lower than those in lymphatic metastatic carcinoma tissue. The expression level of miR-21 was significantly correlated with muscular infiltration depth, parametrial invasion,lymph node metastasis and HPV16 infected or not.These results showed that miR-21 played a key role on the development and metastasis in cervical cancer.MiR-21 promotes cervical cancer may be associated with HPV16 infection.2. The relative expression level of ZEB1 mRNA was higher in cervical cancer tissues than in normal cervical tissues and higher in lymphatic metastatic carcinoma tissues than in cervical cancer tissues The expression level of ZEB1 mRNA was significantly correlated with muscular infiltration depth, lymph node metastasis The expression level of ZEB1 protein was also higher in cervical cancer tissues. The expression level EMT inhibitor E-cadherin protein was lower in cervical cancer tissues. The expression level of EMT promoter Vimentin was higher in cervical cancer tissues.These results showed that EMT process may occur on the development and metastasis process of cervical cancer.3.In cervical cancer tissues the expression level of ZEB1 protein was correlated with E-cadherin and miR-21 expression level was correlated with ZEB1 mRNA..In vitro experiments further confirmed that increasing miR-21 level in cell lines promoted the ability of invasion and metastasis of cervical cancer cells and reducing the amount of miR-21 in cell lines inhibited the ability of invasion and metastasis of cervical cancer cells.Further analysis found that miR-21 positively regulated the EMT related transcription factors ZEB1 and Snail,then increased the level of EMT promoting protein Vimentin, N-cadherin and decreased the level of EMT inhibiting protein E-cadherin.In conclusion, miR-21 promote cell metastasis of human cervical cancer by modulating epithelial-mesenchymal transition.