| Background:Malignant neoplasms are currently the leading cause of human death worldwide.Lung cancer is the most common malignancy in both new cases and deaths.The pathogenesis of lung cancer is extremely complex,and its high invasive and metastatic nature makes its prognosis poor.The invasion and metastasis of lung cancer are multifactorial and multistep,and are affected and regulated by multiple extracellular and extracellular factors.The complex mechanism needs to be further clarified.With the development of non-coding RNA,the role of long non-coding RNA has attracted great attention.Long non-coding RNA is a kind of non-coding RNA whose transcript length is generally greater than 200nt and has no protein-coding potential.There are a large number of long non-coding RNA,but as a new research field,most of the functions of long non-coding RNA are still unclear,and only a few have been found to be related to tumorigenesis and development.These long non-coding RNAs regulate the biological behavior of tumor cells in terms of genomic stability,substance metabolism,and post-translational modifications of proteins.With the rapid development of transcriptome analysis technology in recent years,people have gained a new understanding of non-coding RNA,a large number of non-coding RNA with multiple functions has been found.It can regulate gene expression by chromatin modification and remodeling histone modification and nucleosome localization.Objective:TM4SF1-AS1 was used to screen and identify differentially expressed lncRNA that were significantly more expressed in high-metastatic cell lines than low-metastatic cell lines(95C and 95D)with gene chip.This study aims to further clarify the biological function,molecular mechanism and clinical significance of TM4SF1-AS1 in lung cancer,with the expectation to provide a fundamental basis for the diagnosis and treatment of lung cancer.Methods:(1)Screening of metastasis-related lncRNA:a.Screening of differentially expressed lncRNAs by gene chip;b.Enrichment analysis of differentially expressed mRNA.(2)Objective lncRNA identification:q-PCR assay was used to test the chip results.(3)TM4SF1-AS1 expression in lung cancer tissues and adjacent tissues:a.We selected 43 cases of lung cancer and their paired adjacent tissues to detect the expression of TM4SF1-AS 1 by q-PCR;b.The correlation between the expression of TM4SF1-ASland clinicopathological factors such as age,gender,tumor stage,lymph node metastasis,tumor size and tumor differentiation were analyzed based on clinical information.(4)Cell invasion and migration detection:a.To establish stable cell lines for TM4SF1-AS1 overexpression or knockdown;b.The effect of TM4SF1-AS1 on the invasion and metastasis ability of lung cancer cells was analyzed by using scratch test and Transwell test.(5)Detection of EMT-related markers:The expression of EMT-related markers was detected by Western blot.(6)Study on the potential molecular mechanism of TM4SF1-AS1 involved in the invasion and metastasis of lung cancer cells:a.RNA-seq technique was used to screen TM4SF1-AS1 related genes;b.Enrichment analysis of differentially expressed genes C.Expression of proteins related to PI3K/AKT signaling pathway was detected by Western blot.Results:(1)screening of metastasis-related lncRNACompared with 95C,582 genes were up-regulated and 164 genes were down-regulated in 95D.Through enrichment analysis,we found that the mRNA of these differentially expressed genes were related to cell connection,cell adhesion and basement membrane formation(2)Objective lncRNA identificationLncRNA screening should be conducted according to the following principles:(a)The length should be within 400-2000;(b)The corresponding variation multiple was at least 2 times,and there was no coincidence with the mRNA position;(c)Expression abundance.The selected 4 lncRNA sequences were verified by q-PCR.The test results showed that TM4SF1-AS1 had the most obvious up-regulation effect in 95D and its expression was relatively stable in repeated tests,which was consistent with the results of the chip.Therefore,TM4SF1-AS1 was selected for subsequent experiments(3)The expression of TM4SF1-AS1 in lung cancer tissues and its relationship with clinicopathological factorsThere was no significant difference in TM4SF1-AS1 expression in lung cancer tissues and adjacent tissues.However,our study also found that in lung cancer patients,TM4SF1-AS1 expression in lung cancer tissues with positive lymph node metastasis was higher than that in lung cancer tissues with negative lymph node metastasis,the results were statistically significant(p<0.01).TM4SF1-AS1 expression in TNM stage II and III was higher than that in TNM stage I,and the results were statistically significant(p<0.01,p<0.001).TM4SF1-AS1 expression was higher in middle and poorly differentiated lung cancer tissues than in highly differentiated lung cancer tissues,and the results were statistically significant(p<0.01).Then we analyzed the relationship between the expression of TM4SF1-AS1 and clinicopathological factors,and found that the expression of TM4SF1-AS1 was closely related to tumor differentiation,TNM stage and lymph node metastasis,but had nothing to do with gender,age,histological type and tumor size.(4)Effect of TM4SF1-AS1 on invasion and metastasis of lung cancer cellsA.Construction of stable cell linesThe NCI-H1299 cells were used to construct the TM4SF1-AS1 overexpressed cell model,and the NCI-H1650 cells were used to construct the TM4SF1-AS1 interfering cell model.B.Detection of cell invasion and metastasis abilityThe invasion and metastasis ability of cells were detected by the scratch test and Transwell test.Compared with the negative control group,the number of cell migration was significantly increased in the TM4SF1-AS1 overexpression group 24h after the scratch(p<0.001),and the number of cell metastasis was also significantly increased in the TM4SF1-AS1 overexpression group(p<0.001).Next,we used the cells of the interference-negative control group and the cells of the TM4SF1-AS1 interference group for the scratch test and Transwell migration and invasion test.In the scratch test,compared with the interference negative control group,the cell migration number of TM4SF1-AS1 interference group was significantly reduced 24h after the scratch(p<0.001).The results of Transwell showed a significant decrease in the number of cell metastases(p<0.001).(5)TM4SF1-AS1 is involved in the study of the potential molecular mechanism of invasion and metastasis of lung cancer cellsA.TM4SF1-AS1 promotes the EMT process in lung cancer cellsWe detected the expression changes of epithelial mesenchymal markers in TM4SF1-AS1 overexpression and interference cell lines by Western blot.The results showed that compared with the negative control group,the expression of the intermediate marker Vimentin and the transcription factors Snail and Twist were also significantly up-regulated in the TM4SF1-AS1 overexpressed group.However,E-cadherin,one of the important epithelial markers,was significantly down-regulated in cells of the TM4SF1-AS1 overexpression group.In contrast,the expression of Vimentin,Snail and Twist was down-regulated in the interfered cell lines,while the expression of E-cadherin was up-regulatedB.RNA-seq differential gene screeningIn order to reveal the mechanism of TM4SF1-AS1 promoting invasion and metastasis of lung cancer cells,we used RNA-seq technology to screen genes related to TM4SF1-AS1 from highly expressed TM4SF1-AS1 cell lines and negative control cell lines.A total of 4244 different genes were identified,including 2355 up-regulated genes and 1909 down-regulated genes.C.Regulation of PI3K/AKT signal transduction pathway by TM4F1-AS 1Since the differentially expressed genes contain TM4SF1 and many important factors of the PI3K/AKT signaling pathway,we further studied the effect of TM4SF1-AS1 on the PI3K/AKT signaling pathway.Western blot was used to detect the expression of PDK1,AKT,mTOR,PI3K,p-PI3K,p-mTOR and p-Akt in the overexpressed TM4SF1-AS1 cells and the negative control cells,respectively.The expression levels of PDK1,mTOR,p-mTOR,p-PI3K and p-Akt in the cells overexpressing TM4SF1-AS1 were higher than those in the negative control group.In addition,Western blot showed that TM4SF1 expression was also increased in cells overexpressing TM4SF1-AS1Conclusion:1.Gene chip technology can be used to identify the genes associated with lung cancer metastasis.2.Enrichment analysis of differentially expressed genes showed that the differentially expressed genes were closely related to lung cancer metastasis3.TM4SF1-AS1 expression in lung cancer tissues is positively correlated with lymph node metastasis,clinical stage and tumor differentiation.4.TM4SF1-AS1 can promote the invasion and metastasis of lung cancer cells in vitro.5.The ability of TM4SF1-AS1 to promote the invasion and metastasis of lung cancer cells in vitro may be realized by promoting the EMT process of lung cancer cells.6.TM4SF1-AS1 may promote metastasis of lung cancer cells through PI3K/AKT signaling pathway. |
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