Mechanism Of Heme Oxygenase 1 On Proliferation And Apoptosis Of Melanoma Cells

Background: Melanoma originated in menanocytes,incidence of a disease of malignant tumor in humans is about 2.0 % or even higher across the world,causeing significant deaths annually,In recent decades,research showed that the incidence of malignant melanoma have an ascending trend.Current surgical resection is the most common clinical treatments.In addition,there are many melanoma patients given cellular immune therapy,its treatment effect is relatively more significant and lasts for longer.However,most melanoma patients are much resistant to conventional therapy,which appealing to define a more effective way of melanoma treatment.Following the available and recent literature on tumor research,we found that there are a variety of genes closely related to the growth of melanoma in our body e.g.Heme oxygenase-1(HO-1),which is one of the well studied gene.HO-1 is widely targeted as a check point in chemotherapies.HO-1 mainly degrade hemoglobin to release biliverdin,CO and Fe2+ is involved in maintaining cellular oxidative stress,reduce the body's oxidative damage,along with inflammatory responses,regulation of apoptosis,cell proliferation and so on.It has been confirmed that HO-1 is highly expressed in various human malignancies such as liver cancer,lung cancer,breast cancer and skin cancecr etc.Abnormal expression of HO-1 in tumor can be affected by signaling pathways network,and then regulate cell proliferation and apoptosis.The exact function of HO-1 in melanoma is rarely reported,I herein have attempted to further explore the mechanism of HO-1 expression in melanoma by CRISPR / Cas9 technique,which is a gene editing technology and is characterized by the ability to target transformation and modification of genes for improved and enhanced gene therapy.I constructed HO-1 interference,for both overexpression and knockout vectors,and transfected into human malignant melanoma A375 cells to explore its biological effects in regulation of PI3K/AKT signaling pathway.Objects: The effect of HO-1 on cell proliferation,apoptosis and metastasis of cancer cells.Furthermore,we will study the involvement of PI3K/AKT signal pathway and its biological effects.Methods: Uses:Study of A375 cells by high-throughput sequencing means to detect whether the knock out HO-1 is successful.The expression of HO-1 and related signal pathways was detected by Western blotting,Q-PCR,and immunofluorescence assay.CCK8 and clone formation experiments tested three different stable cell lines for their proliferation rates.Scratch test also used for three different stable cell lines to cellular migration distance.Flow cytometry technique detected cell cycle arrest and apoptosis.Nude mice were put into tumor experiment testing HO-1 effects on tumor growth.Results: 1.Identification of stable cell line Western blotting showed that HO-1 protein was seen overexpressed in stable cell lines such as A375 cells was significantly higher than that in the control group,and similar results also shown by immunofluorescence assay.The protein levels of HO-1 distribution in stable cell lines have a certain degree of reduction.High-throughput sequencing results showed that HO-1 had a frameshift mutation and knockout were successful.2.HO-1 overexpression has the effect of promoting the proliferation of A375 cells,HO-1 interference had no significant difference in A375 cell's proliferation.HO-1 knockdown significantly reduced the proliferation of A375 cells.The results of CCK8 showed that there was no significant difference in cell proliferation between HO-1 overexpression and interference in the 96-hour period compared with the control group.HO-1 knock-outs of the stable cell lines showed decline in cell proliferation compared with the control cells.Crystal violet staining showed that the number of HO-1 overexpressing cells was more than that of the control group and there was no significant difference in the number of cell proliferation between HO-1 interfering cell lines and the control group overexpression and compared.But the number of HO-1 knockout cells was much less than that of the control group.The results of tumor formation in nude mice showed that HO-1 overexpression has the effect of promoting tumor growth and HO-1 interference had little effect on the proliferation of A375 cells,but HO-1 knockout significantly inhibited the proliferation of A375 cells.3.HO-1 had no effect on A375 cell migration.The results of cell scratches showed that there were no significant differences in cell migration distance among three different stable cell lines at 12,24,36 and 48 hours,and did not effectively inhibit the migration of A375 cells,This indicating that HO-1 had little effect on the migration ability of A375 cells.4.HO-1 knockout inhibited the proliferation of A375 cells and in nude mice apoptosis The results of flow cytometry showed that the HO-1 overexpression has the effect of promoting cell proliferation and promoting cell apoptosis.The interferenc of HO-1 had little effect on the apoptosis of A375 cells.But HO-1 knockout could significantly promote the apoptosis of A375 cells and the cell cycle arrest in S phase,it indicated that knockout HO-1 inhibited cell proliferation.5.HO-1 knockout activates PI3 K / AKT signaling pathway The results showed that PI3 K,AKT,Bax,P21 and other signaling molecules were detected by Western blotting and Q-PCR.The results showed that the expression of PI3 K and AKT was significantly decreased in HO-1 knockout cell line,and the expression of Bax and cyclin D1 was not increased Therefore,I hypothesized that HO-1 mainly induced the cell cycle arrest and induced apoptosis of A375 cells through PI3 K / AKT signaling pathway.Conclusion: The expression of heme oxygenase-1(HO-1)in human malignant melanoma A375 cells was altered by different gene editing methods.The results showed that overexpression and interference of HO-1 could not inhibit the proliferation rather it promoted apoptosis of A375 cells.On the other hand,knockout HO-1 could significantly inhibited the proliferation of melanoma cells and promote apoptosis.The PI3K/AKT signaling pathway to control.In addition,HO-1 had no effect on the migration ability of A375 cells.