| Tumor is a single cell to lose the normal regulation of its growth in genetic or epigenetic level affected by various oncogenic factors such as biological, physical and chemical factors, abnormally clone and produce neoplasm. Tumor is often divided into two broad categories benign and malignant. Typical "cancer" is the uniform title of all malignancies in organisms. In recent years, cancer incidence and mortality rates are rising. According to World Health Organization statistics, the number of newly diagnosed cancer patients is more than 12 million around the world, and 1 out of every 8 deaths people die of cancer than AIDS, which is more than the total number of deaths cause by tuberculosis and HIV in 2007. The number of new cancer patients reach nearly 2 million in the mainland of China, about 1.5 million people died caused by cancer, and incidence increased at an annual rate of more than 2%. By 2020, The number of new cases will reach 3.5 million and the number of deaths caused by malignant neoplasms will reach 2.63 million in China. National Health and Family Planning Commission of China reported that cancer has exceeded the cardio-cerebral vascular diseases, become the first cause of death from urban to countryside of China, its rate has risen from 12.6% in 70s to 26.7% in 2007 accounting for the total death (panshumei,2010).With unremitting efforts of global oncological scientists for many years, scholars have found that cancer is not a single disease, but rather the sum of hundreds of diseases. In essence, cancer is a genetic disease. Tannishtha et al (2001) proposes two theories for the origin of tumor models:clonal evolution theory model and the cancer stem cell theory model. Clone evolution theory think, various intrinsic genetic and exogenous environment of carcinogenic factors collaboratively or continuously work to cause DNA injury, to activate proto-oncogenes and inhibit tumor suppressor genes, plus abnormal changes of apoptosis regulation genes and (or) DNA repair genes, then caused chromosome variation and genomic unstability causing cell transformation. Transformed cell clonally proliferate in signal cell, after long period of evolutionary process, one of which relatively unrestricted grow with additional mutations, selectively formed sub-clones with different characteristics, acquiring the transfering ability and ultimately forming malignant tumors. Cancer stem cell theory sought that stem cell undergo genetic mutations under the influence of a variety of internal and external factors, avoid the restriction of the regulation mechanisms of body for cell proliferation and differentiation, shows relatively autonomic and sustainable growth forming cancer stem cells. Only very small amounts of cancer stem cells possess infinite proliferation, self-renewal capacity and the ability to differentiate into other types of cells forming malignant tumors in the tumor. Currently the two theories have been supported by a lot of evidence, suggesting it may also play a role in the process of tumorigenesis and development.Although the origin of tumor cells is controversial, the oncological scientists have reach an aggrement about the basic characteristics of malignant tumor cells. Hanahan and Weinberg’(2011)(Hallmarks of cancer:the next generation)articles summarized 10 basic features for cancers:1. Sustaining Proliferative Signaling; 2. Evading Growth Suppressors; 3. Resisting Cell Death; 4. Enabling Replicative Immortality; 5. Inducing Angiogenesis; 6. Activating Invasion and Metastasis; 7. Avoiding Immune Destruction; 8. Tumor Promotion Inflammation; 9. Deregulating Cellular Energetics; 10. Genome Instability and Mutation. The former six characteristics were obtained in the multi-step process of carcinogenesis, these characteristics make cancer cells to survive, proliferation and the transfer. The later four characteristic could be considered accessary features of the former six characteristics, their existance create an appropriate conditions and environments for tumor cells production and survival.Two theoretical models for the origin of tumor are based on the assumption that cancer is come from the somatic cells or stem cells mutations, these changes include gene amplification, deletion, mutation, rearrangement, epigenetic change (b Vogelstein and Kinzler KW,2004). The genetic changes mainly included the following aspects:1. the protooncogene activation (RAS,C-MYC),2. Loss of tumor suppressor gene functionality (such as RB, TP53),3. The non-coding RNA abnormal expression (such as miRNA,lncRNA),4. Loss of genes maintaining genome stability (such as ATM, BRCA1, etc). The epigenetic changes mainly includes the following aspects:1. DNA methylation and demethylation,2. Histone modification (methylation, ubiquitylation and acetylation),3. Chromatin modifications. These genetic and epigenetic changes constitute the molecular biological basis of tumorigenesis, also become the most-wathed core issues in cancer research. In recent years, along with the rapid development of computing technology and bioinformatics, sequencing technology and equipment is making breakthroughs and updates unceasingly, sequencing costs have fallen sharply. High throughput and massive tumor genome sequencing, microarray, protein chips and other advanced technologies are increasingly being applied by cancer researchers. Thomas RK et al (2007) found in 242 cases with non-small cell lung cancer (NSLNC) samples,40% of East Asia patients and 10% of Caucasian patients have the EGFR mutation. These findings together with other over 50 types of cancer gene maps were included in Onteraational Cancer Genome Sequencing project ICGC (The International Cancer Genome Consortium) (Hudson TJ et al,2004). With the completion of "Cancer Genome Project", each patient’s cancer genome could be identified through blood test in the future, which can be customized for each patient individualized treatment plan, the successfully implement precision medical. For example, once one of the mutations in the EGFR gene is detected in lung cancer patients, using "gefitinib" and "Erlotinib", two specific inhibition drugs of EGFR for the patient, will achieve a good effect (Dancey JE et al,2012). Nearly 50 years, with great progress has been achieved in cancer research, researchers better understand the inner mechanism and external features of carcinogenesis. With the appearence and development of the Third-generation sequencing technology and other technologies, personalized treatment get more and more attention. The treatment effect for tumor has been obviously improved, accompanied by the successful development of trastuzumab and HPV virus vaccine. Even though, cancer is still one of the main diseases threating human life and health, the recurrence and metastasis of cancer cells caused by the drug-resistance are still two stubborn illnesses plaguing us. American Association for Cancer Research’s survey predicted that close to one-second men and one-third women will suffer from cancer in his lifetime. There may be 17 million people worldwide die of cancer annually by 2030, cause huge economic losses of nearly $ 900 billion in world, and thus achieving thorough treatment of cancer is still a long way to go (AACR Cancer Progress Report,2011).According to the International Agency for research on cancer (IARC) statistics, there are more than 1.7 million cases of breast cancer in the world and more than 520,000 dead cases in 2012. In China, new patients with breast cancer accounted for 12.2% of the global rate of new breast cancer patients, breast cancer deaths accounted for 9.6% of breast cancer deaths globally. Because of increased socio-economic status of the people and the implementation of the family planning policy, China breast cancer patients in the region is rising compared to the global rate of breast cancer patients. As one of the highest rates of morbidity and mortality in female malignant tumor, breast cancer seriously threaten human life and health, therefore breast cancer has become one focus of cancer screening and prevention. About 30% patients even accept early diagnosis and treatment, who will eventually undergo recurrence and metastasis. If tumor metastasis has occurred at diagnosis, patients initially can be effectively controlled through conventional therapy, however, most of whom over time will ultimately lead to condition deterioration. In order to improve the diagnosis of breast cancer and reduce mortality, and develop new diagnostic and therapeutic measures, we need to fully grasp molecular biological characteristics on occurrence and progression of breast cancer.Micro-RNAs (microRNAs), a kind of 19～25 nucleotides in length, is endogenous small non-coding RNA molecules, which play an important regulatory roles in many biological processes, such as embryonic development, tissue formation, virus defense, cell proliferation, tumor occurrence and development. miRNA can target mRNA 3’-UTR in specific sites, curb its translation process or induced its degradation, thereby participate in the regulation of gene expression. miRNA can participate in multiple signaling pathways of tumor, is closely related to tumor development, proliferation, invasion, metastasis. At present, dysregulation of miRNAs expression in breast cancer has been reported extensively, such as miR-200, let-7 and miR-10b. Therefore, further research on the expression and function of miRNA expression in breast cancer will help us to better understand the molecular mechanisms of the development of breast cancer and to find new strategies for effective control of tumor development.MiR-204-5p length is 22bp, encoded by the 6th introns of TRPM3 genes located in the long arm of 9th chromosome, its biological function research involve in cell growth, proliferation, migration, apoptosis of a variety of tumor and regulation of lens and retina development. Numerous studies have reported that miR-204-5p could inhibit tumor cell on various aspects of biological functionality through a variety of key proteins. Chung TK et al confirmed that miR-204-5p can inhibit the FOXC1 gene to inhibit endometrial cancer cell migration and invasion. Mikhaylova O’research showed that miR-204-5p is a type of VHL-regulated tumor suppressor in renal clear cell carcinoma inhibiting macroautophagy of renal clear cell carcinoma by regulating MAP1LC3B. What’s more, Hall DP et al reported that TRPM3 and miR-204-5p form a loop to regulate renal cell carcinoma autophagy. Also, several studies reported that miR-204-5p are involved in the development and progression of breast cancer.Sixl was firstly discovered in Drosophila, as one members of the Six family, played a extensive and powerful role similar to the family members. So far, Six family genes are already found from invertebrates to higher mammal, of which Sixl received the most attention and the most widely research, mainly involved in the development of various tissues and organs, such as muscle, kidney, auditory system, sense organs, and facial structure. Sixl mainly through interactions with proteins such as Eyal/Eya2 involved in the formation of the Visual system. In addition, the role of Sixl in tumor formation also has attracted much attention. Lots of research reports confirmed that Sixl is involved in a variety of human malignancies, including breast cancer, ovarian cancer, endometrial cancer, hepatocellular cancer, Rhabdomyosarcoma, renal cell carcinoma and colorectal cancer. Six1 involved in the occurrence and development of breast cancer through TGF-P signaling pathway, Cyclin Al/Cyclin D1, VEGF-C and other key proteins. Sixl involved in ovarian Carcinoma and cervix carcinoma through the TRAIL signaling pathway. Six1 involved in the development and progression of colorectal cancer through key protein ZEB1. Six1, through known and unknown signaling pathways, involved in the development of a variety of tumors and have extensive affect on the biological function of a variety of tumors. Therefore, Sixl research is of great significance for understanding the mechanism of Tumorigenesis, as well as provide potential targets for cancer diagnosis and treatment.miR-204-5p own powerful function, but few works reported about breast cancer, so it is necessary to conduct further study. Our work use bioinformatics technologies by online bioinformatics software TargetScan, mirDIP and miRDB collaborative screen potential downstream target genes of miR-204-5p, and selected 100 potential targets for high scores, removing existing research reports confirmed targets. Further, we validate the selected target genes using dual-luciferase and immunoblotting techniques. By using clinical specimens, we perform in situ hybridization technique to detect the expression levels of miR-204-5p in breast cancer tissues, and perform immunohistochemical technique to detect the corresponding target genes expression levels in the adjacent section of breast cancer. In vitro cell function experiments to investigate the impact of miR-204-5p on breast cancer cell proliferation, migration, invasion and other biological behaviors. Because of transcriptional regulation function Sixl possess, this experiment further detect possible effect of Sixl on miR-204-5p and relative gene expression using technique real-time fluorescent quantitative PCR, so as to clarify the molecular mechanisms of Sixl’s role in breast cancer. Results of this research will deepen our knowledge about the mechanisms for the occurrence and development of breast cancer, provide new strategies for early diagnosis and treatment of breast cancer, or provide a key molecular targets for drug discovery of such disease.Methods1. miR-204-5p downstream target genes prediction, validation and the research on the regulation of target genes Sixl(1) Using online softwares includes a TargetScan, mirDIP, and miRBase to predict possible miR-204-5p targets.(2)MCF-7 cell total RNA was used as template, on which amplify Sixl mRNA sequence of 3’-UTR and conbined with psiCHECKTM-2 vector to build psiCHECKTM-2-Six1-3’-UTR wild-type recombinant plasmids.(3) Subcloning was based on psiCHECKTM-2-Six1-3’-UTR recombinant plasmid template. By using the method of SOE, Sixl-3’-UTR mutants were constructed for the miR-204-5p binding sequences to build recombinant mutant plasmids including psiCHECKTM-2-Sixl-3’-UTR-Mut-l+Mut-2, psiCHECKTM-2-Six 1-3’-UTR-Mut-1 and psiCHECKTM-2-Sixl-3’-UTR-Mut-2 psiCHECKTM-2-Sixl-3’-UTR-TM,.(4) Dual-luciferase reporter detection system was used to identify the target genes of miR-204-5p. After the miR-204-5p mimic and mimic NC transfected 293T cells, MCF-7, MDA-MB-231 respectively, fluorescent signals were collected in each group 48 hours later using one-way ANOVA statistical method for analysis.2. The expression and significance of miR-204-5p and of Sixl in breast cancerFirstly,30 fresh breast cancer clinical specimens were collected, then in situ hybridization technique (in situ hybridization, ISH) was used to detect miR-204-5p expression and immunohistochemical (immunohistochemistry, IHC) was used to detect Sixl in the adjacent section of same breast cancer tissue, so as to analyze the relationship and clinical significance of the both.3. The effect of miR-204-5p on biological function of breast cancer cells(1) The experiment diveded into two groups:MCF-7-NC (negative control) group, MCF-7-miR-204 cell group (the experimental group).(2) MTT assays was performed to draw the 0h,12h,24h and 48h cell growth curve in each group to assess the effect of miR-204-5p for MCF-7, MDA-MB-231 proliferation.(3) Scratch assay test MCF-7 cell migration in each group to assess the effect of miR-204-5p overexpression on MCF-7 cell migration.(4) Matrigel-Transwell used to detect and get breast cancer cell invasion result in each group, so as to evaluate the impact of miR-204-5p on MCF-7, MDA-MB-231 cell invasiveness.4. The study on the molecular mechanism of Sixl/miR-204-5p feedback loopObjective:to study the miR-204-5p target regulate Sixl related molecular pathways, and we firstly hypothesize and investigate that upstream miR-204-5p reversely regulated by its target Sixl gene.(1) MCF-7 cell total RNA was used as template to amplify Sixl mRNA sequence of CDs and construct the recombinant plasmid pcDNA-3.1-Six1.(2) After NC, Sixl-siRNAl, Sixl-siRNA2, pcDNA3.0-Sixl transfected MCF-7 cells, Real-time fluorescent quantitative PCR assay was used to detect miR-204-5p expression and analyse its significance.3. After MCF-7 cells transfected by mimics NC, miR-204-5p mimics, siRNA1, siRNA2, Sixl+siRNA1, and Sixl+siRNA2, Western blotting Assay was used to detect Sixl protein and invasiveness-associated protein CDH1 (E-cadherin) expression levels.Results1. Sixl is a downstream target genes of miR-204-5p and its research on the targeting mechanism(1) Bioinformatics analysis results:three online database TargetScan, miRDIP and miRBase forecasted and screened out the miR-204-5p target genes Sixl, results showed that there are two 7 bp sites in Sixl 3’-UTR matched by miR-204-5p.(2) Dual luciferase reporter gene proved that miR-204-5p could target Sixl 3’-UTR.(3) Western Blot Assay showed that miR-204-5p downregulated Sixl protein expression.(4)Western blotting found exogenous Sixl without the 3’-UTR sequence is not affected by miR-204-5p, meanwhile miR-204-5p inhibition for Sixl can be reversed by introducing exogenous Six1 CDs.2. The miR-204-5p is negatively associated with Sixl in breast cancerSixl show low expression in high miR-204-5p expression, meanwhile Sixl show high expression in low miR-204-5p expression in the same breast cancer tissue samples. The miR-204-5p expression is negatively related with Sixl expression in breast cancer.3. miR-204-5p promate the migration and invision of breast cancer cells(1) After miR-204-5p mimic successfully transfected MCF-7 cells, quantitative real-time PCR (Real-time PCR) detected significant upregulation of miR-204-5p in MCF-7 cells. After miR NC successfully transfected MCF-7 cells, miR-204-5p expression decreased significantly in NC group compared with that experimental group.(2) MTT assay results showed, experimental group compared with the NC group by using independent samples t-test analysis for the effects of miR-204-5p mimic introduction on cell proliferation:24h T=0.636, P=0.543; 48h T=0.842, P=0.424; 72h T=1.716, P=0.125; all p values greater than 0.05. Experimental results showed that the miR-204-5p expression level changes had no effect on MCF-7 cell proliferation, miR-205-5p is not a breast cancer cell proliferation-associated genes.(3) Scratch assay results showed, after 24h culture in serum-free, scratch distance significantly reduced in the negative control group compared with that in experimental group, the experimental group showed obvious scratches. Experimental results showed that the miR-204-5p could promotes MCF-7 cell migration.(4) Matrigel-Transwell results showed that transfer ability of MCF-7 cells in experimental group was significantly weaker than that NC group. The same results also appeared in MDA-MB-231 cells. Experimental results were analysed by the independent samples t-test, T=15.469, P=0.000, which has significant differences.In vitro experiments showed that exogenous miR-204-5p does not affect the migration ability of breast cancer cell, but the exogenous miR-204-5p overexpression significantly inhibited breast cancer cell migration and invasion, miR-204-5p act as a tumor suppressor gene.4. Six1/miR-204-5p form negative feedback-loop(1) The miR-204-5p expression in Six1-siRNA transfected group was significantly higher than that in the NC group and Six1-overexpression group, which showed that Six1 could inhibit the expression of miR-204-5p genes.(2) The Six1 protein expression reduced significantly in miR-204-5p mimic group compared to that in mimic NC Group, which indicated that intrinsic miR-204-5p expression is negatively correlated with intrinsic Six1 expression in breast cancer.Six1 can inhibit the miR-204-5p expression in breast cancer. Meanwhile, miR-204-5p upregulate CDH1 protein expression, through inhibition of the Six1 expression, to inhibit breast cancer cell migration and invasion. Therefore, We firstly found that miR-204-5p and Six1 form a double-negative feedback-loop.. |
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