LncRNA SOX2-OT Targets MiR-654 To Regulate The Invasion And Metastasis Of Laryngeal Carcinoma

BackgroundLaryngeal cancer is a malignant tumor with a high incidence in the neck.Clinically,it can be divided into three types:glottic type(60.0%),supraglottic type and subglottic type.The incidence rate is slightly higher in men than in women.There are two types of laryngeal cancer:primary and secondary.Primary laryngeal cancer refers to the primary tumors that occur in the larynx,and squamous cell carcinoma is the most common clinically;while secondary laryngeal cancer refers to malignant tumors from other parts that metastasize to the larynx(rarely clinical).For diagnosed laryngeal cancer,surgery,chemotherapy or radiotherapy can be used for treatment.Different treatment methods have their own advantages and disadvantages,but the treatment effect is still not satisfactory,with low long-term survival rate and poor prognosis.The occurrence and development of laryngeal cancer are considered to be the result of the imbalance between oncogene and tumor suppressor genes.Therefore,actively seeking gene-level therapeutic targets has become a focus of current research.In recent years,with the continuous development of medical technology,more and more studies have found that less than 2%of genes in the human genome can be encoded and transcribed into proteins,most genes are difficult to encode proteins(called non-coding RNA)due to the lack of a complete open reading frame.Clinically,there are two types of short non-coding RNA and long non-coding RNA(LncRNA)according to the long-chain nucleotide length.Short non-coding RNA refers to non-coding RNA with a length of less than 200 nt;LncRNA belongs to a class of non-coding RNA with length of>200nt,which is mainly located in the nucleus and cytoplasm,and can directly participate in the occurrence and development of tumors through various regulatory methods such as apparent transcriptional regulation and post-transcriptional regulation.At present,there are many types of LncRNAs related to laryngeal cancer clinically,including:HOTAIR,MALAT1,H19,etc.,all of which have played an important role in the occurrence and development of laryngeal cancer.Enhancing the understanding of the role of LncRNA in laryngeal cancer can improve the early diagnosis and treatment of laryngeal cancer,and also improve the postoperative quality of life.The transcription factor SOX2 is a member of the SOX family,and it has been clinically found that the family has more than 20 members in humans and mice.SOX2 belongs to the group B member of the family,contains an exon,and the protein expression sequence is highly conserved.SOX2 can be expressed in a variety of normal tissues in adults,but it is abnormally expressed in many tumor tissues.At the same time,SOX2 is also an important factor in maintaining the self-renewal and differentiation potential of embryonic stem cells.Studies have shown that:SOX2-OT and SOX2 are abnormally expressed in a variety of tumors such as lung cancer,esophageal cancer,and breast cancer,and can directly participate in tumorigenesis,cell differentiation,and pluripotency.Overexpression of SOX2 is related to the malignant progression of laryngeal squamous cell carcinoma.It can be used as an important indicator of the prognosis of laryngeal squamous cell carcinoma.However,the clinical significance and biological function of LncRNA SOX2-OT in laryngeal cancer have not yet been elucidated.Compared with normal cells,tumor cells have the characteristics of infinite proliferation,transformation,and metastasis.Based on the above characteristics,clinical diagnosis and treatment are more difficult.At the same time,tumor metastasis is a process in which malignant tumor cells leave the primary tumor and reach secondary tissues or organs to continue to grow and proliferate,causing secondary tissues and organs to become cancerous.Therefore,tumor metastasis plays an important role in tumor mortality,disease occurrence,and development.The treatment of metastatic tumors is relatively difficult,on the one hand,the primary tumor needs to be considered in the treatment of metastatic tumor.;on the other hand,the clinical metastasis mechanism has not been clarified,especially in most patients,regional lymph node metastasis has occurred in the tumor cells before diagnosis,the patient even developed distant metastases.And SOX2-OT can participate in the invasion and metastasis of many tumor cells.At present,there are relatively few clinical studies on the regulatory mechanism of LncRNA SOX2-OT targeting miR-654 in the invasion and metastasis of laryngeal cancer.ObjectiveSOX2 belongs to the SRY-related SOX gene family and can maintain the pluripotency of self-renewal and undifferentiated embryonic stem cells.At the same time,the SOX2 gene is embedded in the LncRNA intron,which can cause SOX2 to overlap and transcribe to form SOX2-OT.However,the biological characteristics and mechanism of action in squamous cell carcinoma have not been studied.This study mainly explored the effects of SOX2-OT intervention on the proliferation,invasion,metastasis and apoptosis of squamous cell carcinoma in vitro,and initially explored its potential molecular mechanism to provide new targets for clinical treatment of laryngeal squamous cell carcinoma.Methods(1)Expression of LncRNA SOX2-OT in different cells.Laryngeal squamous cell carcinoma cells TU177,TU686,M4E,and AMC-HN-8 were purchased as objects,and they were divided into different test tubes.At the same time,a normal cell line NP69 was purchased as a control and measured by real-time fluorescence quantitative PCR(RT-qPCR).The expression level of LncRNA SOX2-OT in different cells was selected,and the cell line with the greatest difference was selected for subsequent experiments;Laryngeal squamous cell carcinoma cells in logarithmic growth phase were selected and transfected with Lipofectamine2000 to construct shRNA to interfere with the expression of LncRNA SOX2-OT and to form shRNA-NC,shRNA-SOX2-0T-1,and set control cells,the expression levels of miR-654 in different cells were determined by RT-qPCR.(2)LncRNA SOX2-OT targeted miR-654 to regulate the invasion and metastasis of laryngeal cancer.CCK8 technology was used to detect the cell activity of shRNA-NC,shRNA-SOX2-OT-1 and control cells at Oh,24h,48h,and 72h;the scratch test was used to detect the cell migration level of the three cells at 0h,6h,12h,and 24h;Transwell was used to detect the invasion ability of the three cells at Oh,6h,12h,and 24h;and the expression of three types of cells proliferation,migration and apoptosis-related proteins were determined by Western blot.Cell proliferation proteins include:CDK2,cyclinEl,PCNA and P21;cell migration proteins include:MMP7,MMP9;apoptosis-related proteins include:Bax,cleaved caspase-3,bcl-2.The effects of SOX2-OT on the proliferation,invasion,metastasis and apoptosis of laryngeal squamous cell carcinoma were observed.The dual luciferase reporter gene detection system of promega company in the United States was used to detect the targeting relationship between LncRNA SOX2-OT and miR-654,according to the experimental instructions;shRNA and miR-654 inhibitor were used to complete co-transfection,and the levels of cell proliferation and apoptosis after transfection were detected.CCK8 technique was used to detect the proliferation levels of shRNA-SOX2-OT-1,shRNA-SOX2-OT-1+miR-NC and shRNA-SOX2-OT-1+miR-654 inhibitor cells at Oh,24h,48h and 72h.Western blot was used to determine the expression of three apoptotic proteins,including Bax,bcl-2 and cleaved caspase3.The apoptosis rate was detected by flow cytometry.All data in this study were processed using SPSS20.0 software.ResultsPart 1.? After PCR amplification,gel electrophoresis enrichment,and PCR product recovery,the concentration of 1%gel electrophoresis was used to complete the detection of PCR and PCR recovered products.The results showed that the specific size band appeared at 969bP after PCR recovery.The identified target gene SOX2-OT was successfully amplified;white colonies were picked,PCR identification of the colonies was completed,and the corresponding fragments were further determined.The results showed that colonies 1,3,4,and 5 were all positive clones.Select positive clones to complete the corresponding sequence determination,and the final labeling is SOX2;the recombinant plasmid has specific bands in different target sizes;sequencing results show that the recombinant plasmid was successfully constructed and labeled as pEGFP-N1-SOX2;? shRNA-NC and shRNA-SOX2-0T-1 with green fluorescent markers were transfected into laryngeal cancer cells TU177 via Lipo2000,and perform continuous transfection for 24 hours.Observe the green fluorescence of different cells under an inverted microscope.The presence of green fluorescence in cell morphology indicated successful transfection,with the interference rate more than 60.0%;? The expression level of miR-654 in shRNA-SOX2-0T-1 cells was higher than that in control cells and shRNA-NC cells(P<0.05);shRNA-SOX2-OT-1 and shRNA-SOX-OT-1+miR-NC the expression level of miR-654 in cells was not statistically significant(P>0.05);both were higher than shRNA-SOX2-OT-1+miR-654-inhibitor cells(P<0.05);?LncRNA SOX2-OT mRNA expression levels of AMC-HN-8 and TU686 cells before cell transfection were not statistically significant(P>0.05);both were higher than M4E cells and NP69 cells(P<0.05);the level of LncRNA SOX2-OT mRNA before TU177 cell transfection were higher than the other four cells(P<0.05);the LncRNA SOX2-OT mRNA expression levels of shRNA-NC and control group cells were not statistically significant after TU177 transfection(P>0.05);both it were higher than shRNA-SOX2-OT-1 and shRNA-SOX2-OT-2 cells(P<0.05).Part 2:?The cell viability of TU177 control cells and shRNA-NC cells at 0h,24h,48h and 72h was not statistically significant(P>0.05);the cell activity of shRNA-SOX2-OT-1 cells at Oh,24h,48h and 72h was lower than control cells and shRNA-NC cells(P<0.05);? The scratch test results showed that the scratch distance of the TU177 control cells,shRNA-NC cells and shRNA-SOX2-OT-1 cells at Oh was not statistically significant(P>0.05)(the scratch distance of the control cells was slightly smaller);24h the scratch distances of control cells and shRNA-NC cells were not statistically significant(P>0.05);the scratch distances of shRNA-SOX2-OT-1 cells at 24h were greater than those of control cells and shRNA-NC cells(P<0.05);? Transwell test results showed that the migration and invasion rates of TU177 control cells and shRNA-NC cells were not statistically significant(P>0.05);the migration and invasion capabilities of shRNA-SOX2-OT-1 cells were lower than those of control cells and shRNA-NC cells(P<0.05);The results of crystal purple staining showed that there was no statistical difference in the proportion of cells passing through Transwell under crystal violet staining of control cells and shRNA-NC cells,but it was significantly larger than that of shRNA-SOX2-OT-1 cells(P<0.05),indicating that shRNA-SOX2-OT-1 cells have poor migration/invasiveness;? The results of Western blot analysis showed that the expression levels of CDK2,cyclinEl,and PCNA proteins in TU177 control cells and shRNA-NC cells were not statistically significant(P>0.05);the expression levels of CDK2,cyclinEl,and PCNA proteins in control cells and shRNA-NC cells were higher than those of shRNA-SOX2-OT-1 cells(P<0.05);the expression level of P21 protein was lower than that of shRNA-SOX2-OT-1 cells(P<0.05).Western blot results showed that TU177 control cells,shRNA-NC cells migration protein MMP7,MMP9 expression levels were not statistically significant(P>0.05);shRNA-SOX2-OT-1 cells migration proteins MMP7 and MMP9 expression levels were lower than control cells and shRNA-NC cells(P<0.05).The expression levels of apoptotic protein Bax and cleaved caspase-3 in TU177 control cells and shRNA-NC cells were not statistical significance(P>0.05),but it was significantly lower than those in shRNA-SOX2-OT-1 cells(P<0.05);the expression level of bcl-2 protein in control cells and shRNA-NC cells was not statistically significant(P>0.05),but it was higher than that in shRNA-SOX2-OT-1 cells(P<0.05).? Dual luciferase reporter gene test results showed that:3'UTR-MUT levels in SOX2-OT+ miR-654 NC cells and SOX2-OT+miR-654mimic cells were not statistically significant(P>0.05);The level of 3'UTR-WT of miR-654 NC cells was higher than that of SOX2-OT+miR-654mimic cells(P<0.05);miR-654 levels of control cells and shRNA-NC cells were not statistically significant(P>0.05),and was lower than that in shRNA-SOX2-OT-1 cells(P<0.05);? The co-transfection results of shRNA and miR-654 inhibitor showed that:the expression level of miR-654 and apoptosis level of shRNA-SOX2-OT-1 and shRNA-SOX2-OT-1+miR-NC cells were not statistically significant(P>0.05);all were higher than shRNA-SOX2-OT-1+miR-654 inhibitor cells(P<0.05).All three cells had no statistical significance for cell proliferation at the Oh time point(P>0.05);With the extension of time,the proliferation levels of the three cells were increased.The cell proliferation levels of shRNA-SOX2-OT-1+miR-654 inhibitor cells were higher than that of shRNA-SOX2-OT-1 and shRNA-SOX2-OT-1+miR-NC cells at 24h,48h and 72h.(P<0.05);the proliferation levels of shRNA-SOX2-OT-1+miR-NC and shRNA-SOX2-OT-1 cells at 24h,48h,and 72h were not statistically significant(P>0.05).Western blot results showed that the expression levels of apoptotic protein Bax and cleaved caspase3 protein in shRNA-SOX2-OT-1 and shRNA-SOX2-OT-1+miR-NC cells were not statistically significant(P>0.05),which were significantly higher than that of shRNA-SOX2-OT-1+ miR-654 inhibitor cells(P<0.05).The expression level of bcl-2 protein in shRNA-SOX2-OT-1 and shRNA-SOX2-OT-1+miR-NC cells was not statistically significant(P>0.05),which was significantly lower than that of shRNA-SOX2-OT-1+miR-654 inhibitor(P<0.05).Flow cytometry showed that the apoptosis rates of shRNA-SOX2-OT-1 and shRNA-SOX2-OT-1+miR-NC cells were not statistically significant(P>0.05).The apoptosis rate of shRNA-SOX2-OT-1+miR-654 inhibitor cells was significantly lower than that of shRNA-SOX2-OT-1 and shRNA-SOX2-OT-1+ miR-NC cells(P<0.05).Conclusion(1)LncRNA SOX2-OT is highly expressed in laryngeal squamous cell carcinoma cells and can directly participate in the occurrence and development of laryngeal cancer.It is expected to become a new therapeutic target for laryngeal cancer;(2)Lipofectamine2000 transfection was used to construct shRNA to interfere with the expression of LncRNA SOX2-OT,it can inhibit the activity of laryngeal carcinoma cells,weaken the invasion and metastasis of cells,and reduce cell proliferation and migration-related protein expression,and promote cell apoptosis;(3)LncRNA SOX2-OT can regulate the proliferation and apoptosis of laryngeal carcinoma cells by specific adsorption of miR-654,and can be used as a potential biological marker for laryngeal cancer.