Effects Of Inhibiting MYO6_LISplicing Variant With SiRNA On The Maligant Phenotype Of Human Lung Cancer Cell Line A549 And Relative Mechanisms

Lung cancer is one of the most common and lethal malignancies in the world.Because of the complex biological characteristics,high degree of malignancyand the limited validity of treatment,lung cancer has become one of the most principle diseasesthreatening people's life and health.It has been found that there are many splicing variants involved in lung cancer,such as Bcl-xL,CD44v6,Cyclin Dlb and VEGF189.The splicing variants of these tumor-related genes play important roles on maintaining the malignant characteristics of lung cancer cells.In view of this,abnormal alternative splicing genes of lung cancer associated with lung cancer development is of great significance in the diagnosis and treatment of lung cancerMY06,also called myosin 6,was expressed in the human embryo,heart,kidney,brain,intestines,cochlea and other tissue cells.Our previous study showed that the differential expression of MY06 gene splicing variants occurred in many tumor tissues,compared to normal tissue throughthe Second Generation Sequencing analysis.However,the effects of MY06 gene specific splicing on tumor biology and related mechanisms have not been reported.In particular,the effects of MY06LI on the malignant phenotype of lung cancer cell line A549 is not clear.Therefore,we used RNA interference to inhibit the expression of MY06LI splicing variant,to detect its influence on malignant phenotype of A549 cells and investigate the related regulatory mechanisms,which can lay a solid theoretical foundation for tumor diagnosis and treatment with MY06LIsplicing variant as the target.The main results are as follows:1.The expression of MY06 genesplicing variant in various cell lines.RT-PCR combined with DNA sequencing was used to detect the expression of MYO6LI and MY06NI in lung cancer cell line A549.Compared with normal pulmonary epithelial cell16HBE,MY06LIsplicing variant was highly expressed in A549 cells.In addition,MY06 also has a variety of variable splicing in hepatocellular carcinoma,uterine cancer and ovarian cancer cell lines.Since high expression of MY06LI is more prominent in lung cancer cells,we selected lung cancer to carry out related research.2.Identification of siRNAs knockout effects of MY06LI splicing variant.Two siRNA sequences targeting MY06LIsplicing variant,LI-siMY06-1 and LI-siMY06-2 were designed and commercial siRNA to interference MY06 all splicing variants,All-siMY06 was also applied.RT-PCR and western blot were used to detect the effects of siRNAs on the mRNA and protein levels of MY06.Theresults showed that the siRNAs used in this study all have the expected interference effects and can be used in the follow-up study.3.Effects of silencing MY06LIsplicing variant with siRNAs ontheproliferation of lung cancer cell line A549 and relative mechanisms.MTT assay was used to detect the effects of siRNAs on the viability of A549 cells.siRNAstargeting MY06LI significantly inhibited the activity of A549 cells as well as the All-siMY06.Then,Ki67 immunofluorescence staining,BrdU incorporation and cell plate cloning experimentsfurther demonstratedthat siRNAs inhibited the expression of MY06LI significantly inhibited the proliferation ofA549 cells.Western blot analysis showed that inhibitory effects of siRNAs on A549 cells proliferation could be related to the suppression of ERK1/2 MAPK,p38/MAPK and P13K-AKT and other proliferationrelated pathways.4.Effects of silencing MY06LIsplicing variant with siRNAs on the apoptosis of lung cancer cell line A549 and relative mechanisms.First,DAPI immunofluorescence staining and flow cytometry analysis with Annexin V-FITC/PI double stainingshowed thatsiRNAs inhibited the expression of MY06LIcouldinduceapoptosis ofA549 cells.Then,we further demonstrated that MY06LI knockdown induced apoptosis of A549 cells through triggering all three apoptotic pathways including death receptor pathway,mitochondrial/cytochrome C pathway and endoplasmic reticulum stress.5.Effects of silencing MY06LIsplicing variant with siRNAsonthemigration and invasion of lung cancer cell line A549 and relative mechanisms.First,scratch experiments and Transwell cell migration experiments showed that siRNAs interference of the expression of MY06LI coulddecrease the migration ability of A549 cells.Subsequently,Transwell cell invasion experiments demonstrated that siRNAs targeting MY06LIcould also inhibit the invasion of A549 cells.Finally,western blot analysis showed that siRNAs interfering MY06LIregulatedEMT-related protein expression and reversed the EMT process of A549 cells which might be one of its possible mechanisms to inhibit the migrationandinvasion of A549 cells.In summary,siRNAs inhibiting the expression of MY06LI showed similar effectsto the siRNA that knockdown the expression of total MY06 in lung cancer cell A549.All these indicate that MY06LI splicing variant maybe play a leading role in regulating cell proliferation,resistance to apoptosis and migration and invasion in A549 cells.