The Effect Of MiR-125b On Malignant Melanoma Cells And The Research On The Relative Signal Pathway

Background:Melanoma is a highly invasive malignant tumor,it is for all types of cancer in the last thirty years one of the malignant tumors which have the fastest growing incidence.Although has experienced decades of research and drug development,but its therapeutic effect is not ideal,the molecular mechanism of tumor occurrence and development also has not been fully elucidated.Known miRNAs are small non-coding RNAs molecules that regulate the expression of genes in the transcription level,participate in biological control of the process of complex life,especially the occurrence and progress of tumor.Therefore,try to research melanoma miRNA and its target genes and related signaling pathways,provides a new basis for early diagnosis and treatment of melanoma.Objective:Preliminary experiments have confirmed that miR-125b is low expression in melanoma,and its target is MLK3.This experiment established stable malignant melanoma cell lines with the overexpression of miR-125b.We will observe the effection of overexpressed miR-125b on cell proliferation、apoptosis and cell cycle,to determine whether the miR-125b play a tumor suppressor role in melanoma,and to further explore the relative signaling pathways which miR-125b plays as a tumor suppressor on.To Clarify the molecular mechanism of how does miR-125b play as a tumor suppressor in melanoma,provides the basis for clinical treatment on melanoma.、Methods:Lentiviral vector carrying miR-125b gene has been successfully constructed and maintains high expression in primary melanoma cell A375 and invasive melanoma cell SK-MEL-5,the experimental groups transfected with miR-125bup virus are A375-125 bup and SK-125 bup,the negative groups transfected with no-load virus are A375-negative and SK-negative,the blank group not transfected.UsingRT-PCR method to detect the expression of miR-125b level in different group cells,MTT method todetect cell proliferation,Flow cytometry to detect cell apoptosis and cell cycle.Western-blotting technique to detect the expression level of target MLK3,and the key proteins(p-C-jun、C-jun、ERK1/2、MEK2、p-AKT、AKT1)in relative pathways.Results:1.RT-PCR results showed that the expression level of miR-125b in the experimental group compared to the negative group and the blank group is significantly increased(P<0.05),which confirms that the miR-125b gene was successfully over-expressed in in the experimental group;the expression level of miR-125b between the negative group and the blank group has no significantly difference(P>0.05).2.MTT results showed that the cell proliferation activity of A375-125 bup cells compared to the A375-negative cells and the blank cells is significantly decreased(P<0.05),while this activity between the A375-negative cells and the blank cells has no significantly difference(P>0.05).The trend of SK-MEL-5 cells is similar to the A375 cells.3.Flow cytometry results showed that the early stage of apoptosis rate of A375-125 bup cells compared to the A375-negative cells is significantly increased(P<0.05),the proportion of Cells in G0 / G1 phase increased obviously while the proportion in S phase decreased(P<0.05).The trend of SK-MEL-5 cells is similar to the A375 cells.4.The results of Western-blotting showed that the expression level of MLK3 in A375-125 bup cells is lower than in the A375-negative cells(P<0.05),as well as the key proteins like p-C-jun、C-jun、ERK1/2、MEK2、p-AKT and AKT1 are all lower expressed(P<0.05).The trend of SK-MEL-5 cells is similar to the A375 cells.Thus it can be seen that the overexpressed miR-125b inhibits the expression of target MLK3,maybe by blocking the C-jun、ERK and AKT pathways to play a role of tumor suppression.Conclusion:1.Lentivirus mediated-miR-125b overexpression system was constructedsuccessfully.The expression of miR-125b in Malignant melanoma cell transfected Successfully by lentivirus is up-regulated.2.The overexpression of miR-125b can inhibit cell proliferation,promote cell apoptosis,arrest the cell cycle in G0/G1 period and play a role in inhibiting tumor progression.3.The target of miR-125b is mlk3 protein,and have an effect on the C-jun、ERK and AKT pathway.4.Thus it can be seen that miR-125b is expected to become a therapeutic target for the treatment of malignant melanoma.