| Background and Purpose Lung cancer is the leading cause of cancer death in men and in women worldwide.The main classification of lung cancer is non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Most patients with lung cancer are NSCLC,and most of them are advanced,which cannot be treated by surgery.Compared with SCLC,NSCLCs are more insensitive to radiation therapy and chemotherapy.In patients who are considered at high risk of lung cancer,only low-dose spiral CT scan is an early screening method that can alter mortality.Though new therapies have made progress,the prognosis of advanced NSCLC still unsatisfactory.Thus,it is indispensable to find new biomarker targets to improve the treatment of NSCLC patients.Micro RNAs(miRNA),with lengths of approximately 22 nucleotides,are a class of noncoding RNA molecules encoded by endogenous genes that degrade or inhibit the translation of the target m RNA 3’ untranslated regions(3’-UTRs)by complete or incomplete base pairing.Micro RNA-7(miR-7)was identified as a potential tumor suppressor targeting EGFR in glioblastoma in 2008.miR-7 plays a key role in both normal development and human diseases,such as diabetes,and in several types of solid tumors.miR-7 in lung cancer as a tumor-promoting factor or tumor suppressor factor is still controversial,which is worth further study.The production of miR-7 in nerve cells is regulated by Human Antigen R(HuR)and Musashi 2(MSI2).Both HuR and MSI2 are RNA-binding proteins,which are involved in tumorigenesis,development and metastasis by regulating the expression of multiple genes at the post-transcriptional level.However,whether HuR and MSI2 can modulate miR-7 in lung cancer cells is rarely reported.Metformin is a first-line medication for T2DM(type 2 diabetes mellitus)treatment.Metformin can lower blood glucose concentration by reducing hepatic gluconeogenesis and increasing glucose uptake in skeletal muscles;however,it does not cause hypoglycemia and rarely causes lactic acidosis.Research interest in metformin’s anticancer effects began in 2005 with Evans,who showed a lower risk of cancer in T2 DM patients after metformin therapy.In subsequent studies,metformin also reduced the risk for pancreatic cancer,lung cancer,endometrial cancer and so on.The downstream effect of metformin is the phosphorylation of AMP-dependent kinase(AMPK),an important sensor of cellular energy charge;its activation inhibits AKT and m TOR signaling.In this study,we investigated the effect of Metformin and miR-7 on the development of NSCLC in vitro and in vivo,and explored the mechanism of Metformin regulated miR-7 expression in NSCLC A549 and H1299 cells.The purpose of this paper was to provide a potential treatment approach for NSCLC by metformin,to provide an experimental basis for miR-7 as markers,for miR-7,HuR and MSI2 as therapeutic targets for lung cancer.Method1.In Vitro,metformin inhibits the proliferation,migration and invasion of NSCLC cells by up-regulating miR-7 expression(1)CCK8 test,Transwell migration test,scratch test and Transwell invasion test were used to examine the effects of metformin on the proliferation,migration and invasion of A549 and H1299 cells.(2)miR-7 mimic and inhibitor were transfected into A549 and H1299 cells,and then the effects of miR-7 on the proliferation,migration and invasion of A549 and H1299 cells were examined by CCK8,Transwell migration and Transwell invasion assay.(3)q PCR assay was used to detect the effect of metformin on the expression of miR-7 in A549 and H1299 cells.(4)The miR-7 target genes were predicted by bioinformatics.Western blot was used to detect the effect of metformin and miR-7 on the expression of NF-κB/p65 、MAPK/Erk and AKT/m TOR signaling pathways in A549 and H1299 cells.2.The mechanism of metformin mediated HuR and MSI2 regulation of micro RNA-7expression in vitro(1)Constructed and identified the overexpression vectors of HuR and MSI2,then transfected vectors into A549 and H1299 cells and detect the expression of HuR and MSI2.(2)Transfected the si HuR and si MSI2 to A549 and H1299 cells and detected transfection efficiency of siRNAs.(3)q PCR assay was used to detect the effect of overexpression and knock down of HuR and MSI2 on the expression of pri-miR-7,pre-miR-7 and miR-7.(4)q PCR assay was used to detect HuR,MSI2,pri-miR-7 and pre-miR-7 in A549 and H1299 cells treated with metformin.(5)Immunofluorescence assay was used to detect the localization of HuR and MSI2 cells after metformin treatment.3.The effects of metformin on the development of subcutaneous lung cancer and the regulation of the expression of miR-7,HuR and MSI2 in vivo.(1)Establishment of C57BL/6 mice lung cancer subcutaneous tumor model.The contents of miR-7,HuR and MSI2 were detected by q PCR and Western blot.The changes of cell morphology,the expression of HuR and MSI2 and cell localization were detected by HE staining and immunohistochemistry.(2)Establishment of subcutaneous tumor model of lung cancer in nude mice.Detection of miR-7,HuR and MSI2 expression in plasma by q PCR.4.Detect the expression of miR-7,pri-miR-7,pre-miR-7,HuR and MSI2 in lung cancer tissues and analyze the difference of HuR and MSI2 expression in different types of lung cancer by bioinformatics(1)The expression of miR-7,pri-miR-7,pre-miR-7,HuR and MSI2 were detected by q PCR.(2)The protein expression of HuR and MSI2 were detected by Western blot.(3)Analyze the expression of HuR and MSI2 in lung adenocarcinoma and squamous cell carcinoma by bioinformatics.(4)Bioinformatics analyze the correlation between HuR and MSI2 and the correlation between HuR and MSI2 in patients with lung adenocarcinoma and squamous cell carcinoma.Result1.In Vitro,metformin inhibits the proliferation,migration and invasion of NSCLC cells by up-regulating miR-7 expression(1)Metformin inhibited the proliferation migration and invasion of A549 and H1299 cells in a concentration-and time-dependent manner.(2)miR-7 mimic significantly inhibited the proliferation,migration and invasion of A549 and H1299 cells,and miR-7 inhibitor had the opposite effect.(3)Metformin can increase the expression of miR-7 in A549 and H1299 cells.(4)Bioinformatics predicted that m TOR,Erk and NF-κB/p65 were the target genes of miR-7,and both metformin and miR-7 significantly inhibited the expression of NF-κB/p65,MAPK/Erk and AKT/m TOR related proteins in A549 and H1299 cells.2.The mechanism of metformin mediated HuR and MSI2 regulation of micro RNA-7expression in vitro(1)HuR and MSI2 overexpressed plasmid vectors were successfully constructed.After transfected into A549 and H1299 cells,the q PCR and Western blot results showed that both HuR and MSI2 m RNA and protein were significantly overexpressed in A549 and H1299 cells.(2)Transfection of si HuR and si MSI2 to A549 and H1299 cells,q PCR and Western blot assay results showed that the m RNA and protein contents of HuR and MSI2 were significantly inhibited in A549 and H1299 cells.(3)q PCR results showed that the expression of pri-miR-7,pre-miR-7 and miR-7 in the cells transfected alone or cotransfected with p HuR and p MSI2 decreased with the control group,co-transfection showed a more significant reduction,but the inhibition was reversible when co-transfected with p HuR + p MSI2 and metformin.The pri-miR-7,pre-miR-7 and miR-7 contents in A549 and H1299 cells were significantly increased after transfection alone and co-transfection with si HuR and si MSI2.(4)Metformin significantly inhibited the m RNA and protein contents of HuR and MSI2 in A549 and H1299 cells,and promoted the expression of pri-miR-7 and pre-miR-7.(5)Immunofluorescence assay results showed that HuR was mainly expressed in the nucleus,MSI2 was expressed in both cytoplasm and nucleus,and the expression of MSI2 in nucleus was higher than that in the cytoplasm.After merging,the yellow fluorescence can be seen in the nucleus.3.The effects of metformin on the development of subcutaneous lung cancer and the regulation of the expression of miR-7,HuR and MSI2 in vivo(1)The results of the C57BL/6 mice lung cancer subcutaneous tumor experiment showed that metformin had an inhibitory effect on the volume of subcutaneous tumor,and the inhibitory effect was more significant at high concentration.The q PCR assay was used to detect miR-7 expression in tumor tissues.The results showed that metformin could promote miR-7 expression in subcutaneous tumors.Western blot results showed that metformin inhibited the expression of HuR and MSI2 in subcutaneous tumors.HE results showed that metformin had no significant effect on the morphology of subcutaneous tumor cells,but could reduce angiogenesis in subcutaneous tumor tissues.Immuno-histochemical experiments showed that metformin significantly attenuated the expression of HuR and MSI2 in subcutaneous tumor tissues.(2)HuR or HuR+MSI2 could promote the growth of subcutaneous tumor in nude mice.q PCR results showed that the level of miR-7 in the plasma of the nude mice was significantly increased,while the expression of HuR and MSI2 was significantly decreased after feeding with metformin.4.Detect the expression of miR-7,pri-miR-7,pre-miR-7,HuR and MSI2 in lung cancer tissues and analyze the difference of HuR and MSI2 expression in different types of lung cancer by bioinformatics(1)The levels of miR-7,pri-miR-7 and pre-miR-7 in lung cancer tissues were significantly lower than those in adjacent tissue,and the m RNA levels of HuR and MSI2 were significantly higher than those in adjacent tissue.(2)The protein expression of HuR and MSI2 were significantly higher than those in adjacent tissue.(3)The expression of HuR and MSI2 by Encori analysis was higher than that in normal lung tissues.(4)GEPIA analyzed the correlation between HuR and MSI2,the results showed that HuR and MSI2 had weak correlation directly.The prognostic analysis of HuR and MSI2 in lung adenocarcinoma and squamous cell carcinoma showed that the survival rate of lung cancer patients with high expression of HuR protein was decreased.Conclusion Our study found that metformin and miR-7 can effectively inhibit the proliferation,migration and invasion of NSCLC A549 and H1299 cells,and metformin can up-regulate the expression of miR-7 by inhibiting the content of HuR and MSI2.These results provide a potential treatment approach for NSCLC by metformin,to provide an experimental basis for miR-7,HuR and MSI2 as therapeutic targets for lung cancer. |
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