The Enhancement Of Micro RNA-205-3p To Breast Cancer Cells Malignant Biological Behavior And Its Molecular Mechanism

Research background:Tumor is a life-threatening,serious disease,while the cancer burden in our country is increasing gradually.Each year about 1.6 million people were diagnosed with cancer,about 1.2 million people died of cancer.Breast cancer is one of the most common cancer in female currently.Since the 1970 s,the incidence of breast cancer in global has showed a trend of rising year by year,and the onset age tend to be younger in recent years.With the level of early diagnosis and comprehensive treatment level enhances unceasingly,5 years of survival rate of breast cancer in recent years had a great improvement,while the patients with metastasis prognosis is still poorly,5 years of survival rate is about 20%.Each year,about 30% of the patients with breast cancer metastasis,about 70% of them have bone metastasis.Invasive growth and distant metastasis is the main causes of death.Although,the current research help us have a better understanding of tumorigenesis and progression.There are still many unknown factors on the tumorigenesis and tumor progression of breast cancer that we need to find.Plenty of researches suggest that micro RNA plays an important role in diagnosis and treatment of cancer.Dozens of micro RNA can affect tumor progression and tumor metastasis in breast cancer had been found.mi RNA is a class of non-coding small RNA which about 20 ~ 24 nucleotides in length,which is short sequences of RNA of a set of non-coding proteins.Each mi RNA can have multiple target genes,also canregulate a same gene by micrornas interaction.Their m RNA3 ’UTR region combining with target genes to inhibit the protein synthesis or degradation of the target m RNA directly,to regulate the expression of genes.The expression of micro RNA associated with many kinds of cancer and they may have the effect of tumor-suppressor genes or cancer.At present,only a small number of the mi RNA biological function has been elucidated.It has been proved that mi R-205-3p is a kind of tumor suppressor genes which is negatively regulated the invasion and metastasis of tumor.Studies have reported that mi R-205-3p low expression in tumor tissue such as prostate cancer tumor,while restore the mi R-205-3p expression in prostate cancer can lead to interstitial cells transform to epithelial cells.Our lab found that mi R-205-3p is lower expression in breast cancer stem cells through gene analysis,therefore we decided to further study on its biological functions.The rapid growth of tumor cells is the prerequisite for malignant progress of solid tumor,it involves many abnormal expression and functional loss of target genes.Runx2,also called the core factor,which existing studies have shown that it plays the function of the cancer gene in a variety of malignant tumors.Whether the decrease of mi R-205-3p plays a role in the progress of breast cancer?Does mi R-205-3p has a connection with Runx2?In this article,we aim to explore the mechanism how mi R-205-3p regulate the breast cancer via cell biology methods.As well we will find the mechanism of mi R-205-3p in breast cancer.Research Objective:To explore the role of mi R-205-3p in breast cancer cells and find its target genes.Research Contents:The role of mi R-205-3p in breast cancer cell lines and mi R-205-3p and target genes Runx2 prediction and validation1.Measuring the level of mi R-205-3p expression in MCF-10 A,MB231 and MCF-7cell lines.2.Established the stable expression of mi R205 inhibitor in MCF-10 A cell using lentivirus vector,and established the stable expression of mi R-205-3p mimics and in MB231 and MCF-7 cell using lentivirus vector.3.Study the breast cancer cell biology function when established the stable expression of mi R-205-3p cells.Followed by:(1)MTT detected MCF-10 A,MB231 and MCF-7 cells proliferation.(2)Colony formation assay and soft agar semisolid colony forming assay is to observe the colony formation ability and the capacity of anchor-independent growth.(3)Transwell assay observed MB231 and MCF-7 cells invasion abilities.4.Analysis the EMT markers through Western blotting.5.Detect the Wnt/β-catenin signaling pathway by western blotting.6.Via Targetscan software to predict Runx2 target genes of mi R-205-3p and luciferase reporter gene experiments to verify.7.Detect the change of Runx2 expression after mi R-205-3p inhibition or overexpression by Western blot and PCR.Research results:1.mi R-205-3p has lower expression in breast cancer cells MB231 and MCF-7 cells compares to human normal breast epithelial MCF-10 A cells.2.Estabilished the stable expression of knockdown and overexpression mi R-205-3p cells.3.The effects of mi R-205-3p on the breast cells growth: MTT found that the proliferation capability of MCF-10 A cells was increased after inhibiting the expression of mi R-205-3p,but this capability of MB231 and MCF-7 cells was inhibited after the overexpression of mi R-205-3p.4.The impact of mi R-205-3p on breast cancer cell clone formation ability and anchorage-independent growth ability: The result of cloning shows that the size and rates of the clones decreased significantly after overexpression mi R-205-3p in MB231 and MCF-7,Size and number of soft agar colony formation also decreased significantly,which shows that mi R-205-3p inhibits breast cancer cell clonoy formation ability and anchorage-independent growth ability.5.The influence of mi R-205-3p on breast cancer cell invasion ability: Transwell results show that cell invasion ability is enhanced after knockdown the expression of mi R-205-3p in MCF-10 A,and cell invasion ability is decreased significantly after overexpression mi R-205-3p in MB231 and MCF-7.6.The relationship between mi R-205-3p and EMT: we observed cell morphology changed after overexpression mi R-205-3p in MB231.Detecting by WB shows the expression of vimentin were reduced and the expression of E-cadherin were increased.7.overexpression mi R-205-3p of breast cancer cells can reverse EMT via Wnt/β-catenin signaling pathway.8.Biological information prediction software analysis m RNA3’UTR region of Runx2 have a completely complementary base sequence with mi R-205-3p.Results showed that Runx2 is a target genes of mi R-205-3p.Conclusion:Overexpression mi R-205-3p in breast cancer cells can inhibit cell proliferation,invasion and colony formation ability,and could reverse EMT.mi R-205-3p plays the role of tumor suppressor genes in breast cancer.