The Function And Mechanism Of LncRNA H19 In MSCs Contributing To Breast Cancer Cell Metastasis Under Hypoxia Environment By Microfluidic Chip

Objective: Breast cancer is on the top during female malignant tumor in our country even in the world.It has seriously threatened female health.Study shows that the morbidity,fatality rate and prevalence rate of female breast cancer during the past five years are gradually increasing.Therefore,the breast cancer'control and therapy is no time to delay.To find the target spot for treating breast cancer is of great importance.Long noncoding RNA whose length is between 200 and 100000 nt isn't coding protein.But it takes part in much regulation in cell.H19 gene whose overall length is2.5kb has five exons and four introns.The mature H19's overall length is 2.3kb.Because lacking obvious open reading frame,H19 is called noncoding RNA.Although H19 RNA is detected in cytoplasm and nucleus,H19 RNA is mainly in cytoplasm.H19 RNA functions as regulatory RNA or ribose regulon.Metastasis is the most important biology characteristic of malignant tumor that imperils lives.It means the key transition of tumor development.Distant metastasis usually means that tumor enters into late stage and now it is hard to cure tumor only by local treatment.In the process of clinical treatment,distant metastasis has appeared when approximately 1/3 patients are explicitly diagnosed as malignant tumor at first.For fairly proportion of entity malignant tumor patients,once metastasis occurs,it usually means a poor clinical prognosis.Hence having a good knowledge of the whole process of invasion and metastasis is good to design more ideal tumor markers.It has important significance for the early diagnosis and early treatment of malignant tumor.Because malignant entity tumor grows quickly,hypoxia and acidity increase in the tumor microenvironment.Cancer cells may cope with hypoxia and acidity environment quickly,regulate the pH value inside and outside of cell,secrete angiogenic factors,promote the proliferation and migration of vascular endothelial cell and thus induceneovascularization making tumor can gain enough blood and oxygen in order to create conditions for tumor's invasion.In the many complicated external factors,hypoxia that promotes growth to outside and resistance to apoptosis is the change of microenvironment.The responses of tumor cell answering to hypoxia include increasing the stability of transcription complex HIF-1? and then promote angiogenesis,anaerobic metabolism,cell survival and invasion.MSC is a stromal cell which has strong refresh function and immune modulating function.Meanwhile,MSC is the important sustentacular cells for hematopoietic stem cell and progenitor cell in marrow.MSC supports hematopoiesis by secreting many cytokines.MSC is the important ingredient in tumor stroma and it also is regarded as the precursor for tumor-associated fibroblasts.Study shows that MSC has interaction with many immune cells such as T cell,B cell,DC,mononuclear macrophage and NK cell.Experiment indicates that MSC can attract more monocyte,macrophage and neutrophile granulocyte by secreting vast cytokines in order to affect the growth of tumor.Microfluidic chip is made up of microchannels with controlled fluid running through the whole system and it can replace the general biochemistry laboratory.It can miniature sample preparation,reaction and separation on a chip.As it has the merit of high throughput,integration,regulable system and manageable system,it is a beneficial platform for simulating microenvironment to study tumor metastasis.The macromolecule organosilicon compound PDMS that microfluidic chip use has good biocompatibility and permeability for oxygen and carbon dioxide.Cell culture,stimulation,sorting and dissociation can be implemented on the chip platform.Microfluidic chip has become extremely important research platform for cytobiology.Puting different cells in microfluidic chip to co-culture,thus it is better to study the interaction amongst cells.This method has been reported in the lung cancer cells and macrophages.In this study,we use the microfluidic chip technology to explore the role and mechanism of LncRNA H19 in breast cancer metastasis that MSCs mediated underanaerobic environment.Methods: 1.Draw the microfluidic tumor metastasis model and use camera take pictures of microfluidic chip.Explain every part on the model detailedly.Explain the constituent in each passageway on the microfluidic chip.2.Isolate MSCs from the infant's umbilical cord and then do the primary cell culture of MSCs.Then we authenticate the cells we culture whether is MSCs by fow cytometry.3.We use microfluidic chip to construct tumor metastasis model in hypoxic microenvironment.Put the MSCs into the passageway of microfluidic chip and respectively put them into the normal and 1%O2 hypoxia cell incubators to cultivate.In the end,we use IF to detect the hypoxia environment in the microfluidic chip.4.We use plasmid to pack different virus which can respectively express RFP and GFP.Then we use the virus that express RFP infect the breast cancer cell line MDA-MB-231 and the virus that express GFP infect the MSCs.In the end we gain the breast cancer cell strain that is marked with RFP and the MSCs cell strain.5.Put the MDA-MB-231 that is marked with RFP and the MSCs that is marked with GFP respectively into the microfluidic chip.Then put them respectively into normal and hypoxia incubators to cultivate in order to observe the cells' migration under different conditions.6.Mix the MDA-MB-231 that is marked with RFP and the MSCs that is marked with GFP at the ratio of 1:1 and then put the mixture into the microfluidic chip.Then put them respectively into normal and hypoxia incubators to cultivate in order to observe the role of MSCs in breast cancer cell's metastasis under different conditions.7.Use FISH detect the location of LncRNA H19 in the MSCs.Extract respectively the total RNA in the cytoplasm and nucleus by nucleocytoplasmic separation assay and then use RT-PCR detect the relative transcript level of LncRNA H19 respectively in the cytoplasm and nucleus.Collect the MSCs that has been put in 1%O2 hypoxia environment 0h,12 h,24h,36 h and 48 h and then detect the relative transcript level of H19 and MMP1 respectively by RT-PCR.After knocking down the H19,detect the relative transcript level of H19 and MMP1 respectively by RT-PCR in MSCs.Uselet-7mimic and let-7inhibitor transfect MSCs respectively and then detect the relative transcript level of MMP1 by RT-PCR.Use luciferase report gene assay detect the role of MMP1 to let-7 binding site.8.Use the virus that is marked with GFP and can knock down H19 infect MSCs and then put it into the microfluidic chip.And then put it into 1%O2 hypoxia incubator to cultivate in order to observe the cell's metastasis.Mix the MDA-MB-231 that is marked with RFP and the shH19's MSC that is marked with GFP at the ratio of 1:1 and then put the mixture into the microfluidic chip.Then put them into hypoxia incubators to cultivate in order to observe the two different cells' metastasis.Results: 1.Put DMEM which contains 1%FBS into the left red passageway,put DMEM which contains 10%FBS into the right red passageway and put matrigel which contains 1%FBS into the middle blue passageway.Bond the PDMS chip with glass slide firmly.2.Isolate MSCs from the infant's umbilical cord and do the primary cell culture of MSCs.Use flow cytometry detect the cell's surface molecular markers.Results are as follows: mesenchymal cell's molecular markers: CD90(+),CD13(+),CD73(+),CD105(+);cell adhesion molecular markers: CD29(+),HLA-ABC(+),CD44(+);hematopoietic cell molecular markers: CD45(-),CD34(-);endothelial cell's molecular markers: CD133(-),CD31(-).These results meet MSCs' requirements of surface molecular markers.3.Put the bonded microfluidic chip into the 10 cm dish.The result of IF shows that the expression level of HIF-1? in the hypoxia condition increases.4.Construct the MDA-MB-231 that is marked with RFP,the MSCs that is marked with GFP and shH19's MSC that is marked with GFP.The results show that before and after constructing the cell line,the cell's morphology doesn't change.5.After observing the cells for 4 days,the results of microfluidic show that in the hypoxia condition MDA-MB-231 and MSCs' s migration distances are larger than the control group.6.After observing the cells for 4 days,the results of microfluidic show that in thenormal and hypoxia condition MSC s is faster than MDA-MB-231.In the hypoxia the speed of MSCs and MDA-MB-231 is faster than the control group.7.The result of FISH shows that H19 is mainly in the cytoplasm.In the nucleocytoplasmic separation assay,the result of RT-PCR shows that the relative expression level of H19 in the cytoplasm is higher than nucleus.The results of RT-PCR indicate that the relative expression level of H19 and MMP1 are gradually increasing along with extension of hypoxia time.The increase range of H19 is larger than MMP1.The result shows that let-7 can reduce the expression quantity of MMP1.The result of luciferase report gene assay indicates that MMP1 can combine with let-7.8.After observing the cells for 3 days,the results of microfluidic show that in the hypoxia condition,the migration distance of MSCs decreases after knocking down H19.In the group of the mixture of MSCs and MDA-MB-231,the migration distance of MSCs decreases after knocking down H19 in the hypoxia condition,meanwhile the migration distance of MDA-MB-231 also decreases.Conclusions: The study shows that MSCs can promte the migration of breast cancer cell and in the hypoxia condition the effect is more obvious.The abnormal activation and over expression of LncRNA H19 are of important factors leading to the migration of breast cancer cell that MSCs induce.