The Mechanism Of MiR-409-3p On Suppressing Invasion And Metastases Of Gastric Cancer And The Role Of MiR-202 In Chemotherapy Sensitivity To CML

PartⅠ The mechanism of miR-409-3p on suppressing invasion and metastases of gastric cancerBackground and Objective:Gastric cancer(GC)is one of the most common malignant tumors in China,which leading to the second death rate of all tumors.The morbidity and mortality in rural areas is higher than urban areas in China.Due to the early symptoms of gastric cancer are occult and lack of specific diagnostic techniques,most patients have been diagnosed in advanced stage,the survival rate of patients with advanced GC is still not satisfactory.Distant metastasis to lymphatics and peritoneum is still the main factors leading to treatment failure and poor prognosis of patients.Therefore,studying the mechanisms of the invasion and metastasis and exploring new therapeutic targets of GC are very important to improve the prognosis of GC patients.Micro RNA-409-3p(miR-409-3p)is a small,non-coding,multifunctional miRNA expressed by embryonic stem cells.It is one of the mature expressions of micro RNA-409,located in chromosome 14q32.31,which closely related to tumor progression and metastasis.It has been received extensive attention as to its important role in various processes such as tumor cell proliferation,apoptosis,angiogenesis,migration and invasion.In recent years,it has been found that miR-409-3p is down-regulated in variety of malignant tumors.It can regulate cell biological processes such as proliferation,apoptosis,differentiation,invasion,and metastasis of cancer cell by targeting different genes.MiR-409-3p specifically binds to a target sequence to achieve degradation of the target gene,and exerts its effection by suppressing the expression of the target gene at the translational level.GAB1 was a Grb2-associtated binder(Gab)family scaffolding adapter molecules,which played vital role in signals transduction such as growth factors,cytokines and antigenreceptors.It reported that GAB1 plays an important role in tumorigenesis,invasion and metastasis,especially in gastrointestinal tumors.whether miR-409-3p can regulate the invasion and metastasis of gastric cancer by targeting GAB1 has not been reported.To illuminate the molecular mechanisms of miR-409-3p suppressing the invasion and metastasis of gastric cancer,We explored following subjects:1.The expression of miR-409-3p in gastric cancer.2.Effect on invasion and metastasis after change miR-409-3p in gastric cancer cells.3.Screening-Identifying a functional target of miR-409-3p in gastric cancer.4.The effect of miR-409-3p on the invasion and metastasis of gastric cancer cells by interfering with the expression of target genes.Research methods:1.Real-time quantitative PCR(RT-q PCR)method was used to detect the expression of miR-409-3p in BGC823、MNK45、SGC7901、 MGC803 gastric cancer cells and normal gastric epithelial cells(GES1).The cell lines with relatively high and low expression of miR-409-3p were selected in gastric cancer cell for follow-up experiments.2.Real-time quantitative PCR(RT-q PCR)was used to detect the expression of miR-409-3p in 30 fresh gastric cancer tissues and their corresponding adjacent tissues.3.The synthesized miR-409-3p overexpressing mimics,miR-409-3p knockdown inhibitor and the corresponding negative control were transfected into BGC823 and MNK45 gastric cancer cell lines by Lipofectamine TM2000.To verify its effects by RT-q PCR method.4.CCK-8 assay detected the proliferation activity in gastric cancer cells after overexpression or knockdown of miR-409-3p expression.5.Transwell and Matrigel Transwell invasion assays were employed to detect migration and invasion in gastric cancer cells after over-expression or knockdown of miR-409-3p expression.6.According to reports in the literature,we first predicte and select the potential target genes for miR-409-3p from common and authoritative databases including Target Scan,miRanda,miRDB,and miRWalk.7.Real-time quantitative PCR(RT-q PCR)method was used to detect the expression of target genes related to proliferation,invasion and metastasis in gastric cancer cells after overexpression or knockdown of miR-409-3p expression.The potential target genes for miR-409-3p in gastric cancer were initially screened.8.The dual luciferase gene reporter system assay was employed to detect whether miR-409-3p has a true direct targeted binding effect on GAB1.9.The expression of GAB1 target gene in gastric cancer cells was detected by Western blot at the protein level after overexpression or knockdown of miR-409-3p expression.10.Transwell and Matrigel Transwell invasion assays were employed to detect migration and invasion in gastric cancer cells after silenced or overexpressed GAB1 gene expression.Research result:1.The expression of miR-409-3p in gastric cancer cell lines was significantly lower than that in normal gastric epithelial cells,and the difference was statistically significant(P<0.01).In each gastric cancer cell line,a relatively low expressing of miR-409-3p cell line BGC823 was selected,which was the highest expression of gastric cancer cell line MNK45.2.The expression of miR-409-3p was obviously decreased in gastric cancer tissues compared with the corresponding adjacent tissues(P<0.01).3.Overexpression or down-regulation of miR-409-3p expression has no significant effect of the proliferation in gastric cancer cells.4.Overexpression of miR-409-3p can inhibit the gastric cancer cells migration and invasion;in contrast,down-regulate the expression of miR-409-3p can promote gastric cancer cell migration and invasion.5.It is preliminarily predicted that miR-409-3p genes involved in proliferation,migration and invasion in gastric cancer are ZEB1、ELF2、CCND2、AKT1、and GAB1.6.The overexpression of miR-409-3p significantly inhibited the expression of GAB1 at the m RNA level in gastric cancer cells(P<0.01);Down-regulation of miR-409-3p expression significantly increased GAB1 m RNA expression in gastric cancer cells(P<0.01).7.The activity of luciferase in gastric cancer cells was significantly inhibited after overexpressing miR-409-3p,and miR-409-3p could bind to GAB1 gene 3’UTR with significant difference(P<0.05);Down-regulation of miR-409-3p expression enhanced the activity of luciferase in gastric cancer cells,and inhibited the binding of the endogenous miR-409-3p to GAB1 gene 3’UTR(P<0.01).8.The overexpression of miR-409-3p significantly inhibited the expression of GAB1 at the protein level in gastric cancer cells(P<0.001);Down-regulation of miR-409-3p expression significantly increased GAB1 protein expression in gastric cancer cells(P<0.001).9.Overexpression of miR-409-3p on the basis of high expression of GAB1 gene protein can promote the migration and invasion in gastric cancer cells;Down-regulation of miR-409-3p on the basis of interference with GAB1 gene expression can significantly inhibited the migration and invasion in gastric cancer cells.Conclusion:MiR-409-3p was lowly expressed in gastric cancer cells and gastric cancer tissues,which negatively correlated with the migration and invasion in gastric cancer cells;MiR-409-3p inhibit gastric cancer metastasis by down-regulating the migration and invasion in gastric cancer cells;miR-409-3p can suppresses gastric cancer cell invasion and metastasis partly by targeting GAB1.PartⅡ The role of miR-202 in chemotherapy sensitivity to CMLBackground and Objective:Chronic myelogenous leukemia(CML)is a malignant tumor formed by clonal proliferation of bone marrow hematopoietic stem cells.It accounts for 15-20% of adult leukemia,and usually affected is mainly seniors.Chinese CML patients are y oungerthan the western countries.Epidemiological surveys in several regions in Chi na show that the median age of onset on CML is 45 to 50 years,while in Wester n countries,the median age of onset on CML is 67 years.CML is a myeloprolifer ative disease which uniquely expresses a constitutively active tyrosine kinase,BCR/ABL.As a specific inhibitor of the BCR-ABL tyrosine kinase,imatinib becomes the firstchoice for the treatment of CML due to its high efficacy and low toxicity.H owever,there are still some patients developed a drug-resistance,However,there ar e still some patients developed a drug-resistance,or showed disease progression int o the CML accelerated phase,rapid change phase,the effect of tyrosine kinase inhi bitors and prognosis is poor.Therefore,the development of imatinib resistance limit s the long-term treatment benefits of it in CML patients.A growing evidence has revealed specific miRNAs in the pathogenesis of hem atological malignancies such as chronic lymphocytic leukemia(CLL),B-cell lympho mas,acute promyelocytic leukemias,acute lymphocytic leukemia(ALL),and CML.Previous studies reported that miR-202 was down-regulated in malignant tumor,ove rexpression of miR-202 inhibited tumor growth and might contribute to enhanceme nt of chemotherapy.In the present study,we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell linesResearch methods:1.Real-time quantitative PCR(RT-q PCR)method was used to detect the expression of miR-202 in K562、KU812、EM2、EM3、LAMA84、KCL-22、HL-60 CML cells and isolated leukocytes from a pool of healthy blood samples.2.Imatinib sensitive and resistant cells strains of the K562 CML cell line were constructed and survival cell clones were saved and collected for subsequent experiments.3.The synthesized miR-202 overexpressing mimics and the corresponding negative control were transfected into K562,KU812 CML cell lines by Lipofectamine TM2000.To verify its effects by RT-q PCR method.4.MTT,Brd U assay detected the proliferation activity in CML cells after overexpression of miR-202 expression.5.K562 or KU812 cells were treated with imatinib at 50,75 or 100 n M for 24 h,then the expressions of miR-202 were measured by RT-q PCR6.K562 imatinib sensitive and resistant cells were treated with imatinib at 0,0.2,0.4,0.8,1 or 2 μM for 24 h,followed by the measurements of cell viability by MTT assay.7.K562 imatinib sensitive and resistant cells were treated with imatinib at 0 or 2 μM for 24 h,followed by the staining of cell nuclei by DAPI.8.K562 imatinib sensitive and resistant cells were treated with imatinib at 0.4 μM for24 h,followed by the Western blot analysis.9.The expression of miR-202 was measured in K562 imatinib sensitive or resistant cells by q RT-PCR.Research result:1.The expression of miR-202 in seven CML cell lines was significantly lower than that isolated leukocytes from a pool of healthy blood samples,and the difference was statistically significant(P<0.01,P<0.05).K562 and KU812 cell lines were selected in the CML cell line for follow-up experiments.2.Overexpression of miR-202 significantly suppressed proliferation rates of CML cells.3.K562 and KU812 cells were treated with different concentrations of imatinib for24 hours,The expression of miR-202 was significantly decreased(P<0.05,P<0.01,P<0.001).4.The imatinib-resistant K562 cell line was successfully screened by MTT and DAPI methods,and K562 imatinib-sensitive and resistant strains were successfully constructed.5.The K562 parental cells showed a higher percentage of cleaved PARP than the resistant cells under imatinib treatments;Compared with K562 imatinib-sensitive strains,the expression of miR-202 in K562 imatinib-resistant strains was significantly downregulated(P<0.05)Conclusion:miR-202 was lowly expressed in CML cells;Overexpression of miR-202 significantly suppressed proliferation rates of CML cells;MiR-202 expression was significantly inhibited after imatinib treatment in CML cell lines;Imatinib-resistant CML cell lines exhibited lower levels of miR-202,suggesting that miR-202 may contribute to the sensitization of CML imatinib.