The Role Of M3 Machr And Its Mechanisms On The Proliferation And Migration Of NSCLC H1299 Cell

The incidence and mortality of lung cancer are the highest in human malignancies,and it is increasing year by year.The prognosis of lung cancer is poor and the five-year survival rate is low.According to histological classification,lung cancer can be divided into small cell lung carcinoma?SCLC?and non-small cell lung carcinoma?NSCLC?,and 85%lung cancer is NSCLC.In the early stages of NSCLC,surgical resection is mainly used.In advanced stage and metastatic patients,traditional systemic chemotherapy is mainly used,such as platinum drugs,but it can produce serious side effects,and the treatment effect is poor,and the five survival rate is just 5%.Therefore,the development of new treatment strategies is imminent.In recent years,targeted therapies have produced promising clinical results in advanced and metastatic lung cancer.This has led to an in-depth study of the multi-target therapies in lung cancer.Tobacco is an important factor that causes lung cancer.Nicotine that is important chemical contained in tabacco,and it can act on nicotinic acetylcholine receptor?nAChR?on lung cancer cells,activate the downstream signal pathway and promote the lung cancer.At the same time research found that acetylcholine?ACh?can also be synthesized by non-neuronal cells and released into the extracellular fluid in an autocrine and paracrine manner.It can bind on acetylcholine receptors,and involves in the regulation of cell proliferation,differentiation and other basic biological activities.We named it non-neuronal cholinergic systems.In addition,the researchers observed that many tumor cells,including lung cancer,can synthesize and secrete acetylcholine,and expresses nAChR and mAChR?Muscarinic acetylcholinee receptor?.Exogenous acetylcholine receptor agonists and endogenous ACh can activate nAChR and mAChR and promoted the proliferation of NSCLC cells.We think the neurocholinergic system is a very important signaling pathway system for the proliferation of tumor cells.Further studies have shown M3R is significantly upregulated and highly correlated with poor prognosis of tumors and in many tumors.So M3R has gradually attracted researcher`s attention and become a hotspot for tumor cell target research.Our previous data has confirmed M3R was highly expression in a variety of NSCLC by reverse transcription-polymerase chain reaction?RT-PCR?.It indicates that NSCLC can autocrine or paracrine ACh and activate M3R,and involves in cancer's proliferation.However,whether M3R can become a new therapeutic target for NSCLC need futher study.In this study,M3 receptors were antagonized and knocked out to study the improtant role of M3R in H1299 cells.SRB?Sulforhodamine B,sulforhodamine B?,real-time cellular analysis?RTCA?,flow cytometry?FCM?,Scratch assays,antibody microarrays and Western Blot assays were used to demonstrate the importance role of M3R in the proliferation and metastasis of H1299 cells.The results are as follows:?1?SRB method was used to observe the effect on growth and proliferation of H1299 cells of M3R antagonist R2-8018[R,R,-Peuexredrazine]and darifenacin in the concentration range of 1.25-80?M.The results show that R2-8018 and darifenacin can inhibit the proliferation of H1299 cells in a concentration-dependent manner.We then observed the same results using the RTCA method and obtained a real-time inhibition curve.?2?Exogenous administration of acetylcholine and carbachol failed to promote the proliferation of H1299 cells by RTCA.?3?The effect of R2-8018 on cell cycle phase of H1299 cell was measured by FCM,and it was observed that R2-8018 could induce H1299 G₀/G₁ phase block in a time and concentration dependent manner.?4?Scratch test results showed M3R antagonist significantly inhibited H1299 cells migration,suggesting that M3R not only participates in the proliferation,but also participates in the migration of H1299 cells.?5?Knocking out M3R gene by CRISPR/Cas9 technology,T7 mismatch enzyme detection showed that M3R knockout was successful.?6?RTCA method showed that the growth activity of H1299 cells was significantly reduced after M3R knockout compared with wild type H1299cells.?7?RTCA method showed that exogenous acetylcholine and M3R antagonist R2-8018 and darifenacin had no significant effect on the growth and proliferation of M3R KO H1299 cells.?8?The results of cell cycle detection by FCM were consistent with the results of antagonizing M3R.Knockout of M3R gene can induce H1299 cells to produce G₀/G₁ phase arrest.?9?Scratch test results show that knockout of M3R can significantly reduce the migration ability of H1299 cells,and the result was consistent with the result of antagonizing M3R.?10?Antibody microarray results showed that,compared with wild-type H1299 cells,phosphorylated EGFR was significantly decreased after M3R knockout,and several key proteins in MAPK and PI3K/AKT signaling pathways were significantly changed.It is suggested that antagonizing and knock out M3R can influence by decreasing EGFR phosphorylation and inhibiting the activation of MAPK and PI3K/AKT,and then inhibiting the growth and proliferation of H1299 cells.?11?Further studies by Western Blot showed that antagonism and knockout of M3R can effectively inhibit the expression of PKC and EGFR proteins and the activities of MEK/ERK1/2 and PI3K/AKT pathways,but have little effect on P38MAPKs.We believe that antagonism and knockout M3R play a role mainly through EGFR/MEK/ERK1/2 and EGFR/PI3K/AKT signaling pathway.?12?Western Blot was used to observe the expression of related proteins that regulate the cell cycle.The results showed that R2-8018 antagonized and knocked out M3R can signifcantly affect the expression of cell cycle protein?Cyclin?and cell cycle dependent kinases?CDK?,and increased the expression of cell cycle dependent kinase inhibitor?CDKI?.This result well explained the effect of R2-8018 and knock-out of M3R-induced cyclic arrest of H1299 cells.?13?The expression of matrix metalloproteinase 2?MMP2?in H1299 cells was significantly decreased after R2-8018 antagonism and knockout of M3R by Western Blot method,indicating that antagonism and knockout of M3R can inhibit the migration of H1299 cell by decreasing the expression of MMP2.Conclusion:Antagonism and knock-out of M3R can significantly inhibit the proliferation and migration of H1299,and result in G₀/G₁ arrest of H1299cells.Antagonism and knockout of M3R can inhibit the phosphorylation of EGFR and inhibit the activation of MEK/ERK1/2 and PI3K/AKT pathways.Finally,it inhibits the progression of the cell cycle and exerts tumor inhibitory effects.It also inhibits the expression of MMP2 and the migration of H1299cells.This study can provide some research data for confirming M3 receptor as an anti-tumor target.However,because EGFR is a very well-defined lung cancer target,We guess that combination therapy with M3R antagonists and EGFR antagonists may achieve well treatment effect targeting non-small cell lung cancer.