| Background:Lung cancer is the leading cause of cancer death in men in both developed and developing countries,and it has surpassed breast cancer and has become one of the leading causes of cancer deaths among women in developed countries.Lung cancer is divided into two major histological types: small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Of these,85% were NSCLC.Despite the rapid development of lung cancer diagnosis and treatment in recent years,the 5-year survival rate has not been significantly improved.The key reason for high mortality is the metastatic potential of the tumor.Therefore,better understanding of the molecular mechanisms of invasion and metastasis of tumor cells is a necessary condition for more effective NSCLC management.However,so far no tumor molecular markers can accurately predict the lung cancer metastasis.Therefore,screening and identification that can accurately predict the relevant biomolecular markers of lung cancer metastasis is an important part of the current lung cancer research process.Over the past few decades,many molecular epidemiological studies have shown that mutations in certain protein-coding genes(EGFR,KRAS)play an important role in the pathogenesis of NSCLC.However,recent progress showed that another important type of gene,a non-coding gene,may also become a new player in cancer progression.Non-coding RNAs are a class of RNA molecules that do not have protein encoding capabilities.Long non-coding RNA(lncRNA)is a type of RNA that is defined as having a length of more than 200 nucleotides.Although lncRNA does not have protein encoding ability,it is involved in the regulation of gene expression through transcription,apparent modification and post-transcriptional regulation and other pathways.In the study of malignant tumors such as lung cancer,colorectal cancer,and gastric cancer,abnormal expression of lncRNA is often found and may be associated with the development of malignant tumors.In this study,we first used the human lncRNA chip technology to screen differentially expressed lncRNAs in 5 pairs of lung cancer tissue samples that were negative and positive respectively.Based on the final analysis results of the lncRNA chip,we then selected linc00675,which was more prominently expressed in 5 pairs of lung cancer tissue samples,as our focus of this experiment.Through statistical analysis of clinical information,cell function experiments,vivo animal experiments and molecular biology experiments,we explore the relationship between linc00675 and clinical prognosis,the functional impact of lung cancer cell lines,the potential molecular mechanism of linc00675.Methods:Firstly,we used lncRNA microarray technology to screen differentially expressed lncRNAs in 5 pairs of lung cancer tissue samples that were negative and positive respectively.Among them,the expression of linc00675 with more obvious differences was the focus of our research in this experiment.The qRT-PCR experiment further validated the analysis of 30 pairs of linc00675 in lung cancer tissues that were negative and positive respectively.Next,we further increased the sample size of the tissue and collected and improved the sample data in the sample.We detected the expression level of linc00675 in 110 lung cancer tissue samples by qRT-PCR assay and analysed the correlation with clinical pathological factors and prognosis of clinical patients.Firstly,the sequence of up-regulation and down-regulation of linc00675 was designed.Lentivirus-infected lung cancer cell lines A549 and Calu3 were used to construct a cell line that stably up-regulated and down-regulated linc00675.MTT assay was used to detect the proliferative activity of A549 and Calu3;scratch test and transwell assay were used to detect the migration and invasion ability of A549 and Calu3;nude mice xenograft lung metastasis model was used to observe the effect of linc0067 on lung cancer cell metastasis in vivo.Western blot was used to study the molecular mechanism of linc00675 to promote the phenotype of lung cancer cells.Results:The qRT-PCR analysis of 30 pairs of validation samples showed that the expression of linc00675 was lower in lung cancer tissues with positive lymph node metastasis compared with lung cancer tissues with negative lymph node metastasis(P<0.05).By analyzing the clinicopathological factors and the expression level of linc00675 in 110 lung cancer patients,we found that the expression of linc00675 was correlated with TNM stage and lymph node metastasis in lung cancer patients(P<0.05).The Kaplan-Meier survival curve showed that the overall survival(OS)and disease-free survival(DFS)of patients with low expression of linc00675 were significantly lower than those of patients with high expression of linc00675(P<0.05,P<0.05).Multivariate Cox regression models showed that linc00675 expression was an independent prognostic factor for lung cancer patients.The results of MTT study showed that in lung cancer cell lines,no matter whether the expression of linc00675 was up-or down-regulated,there was no significant change in cell proliferation activity in the experimental group compared with the control group.However,the results of scratch and transwell migration invasion experiments showed that in lung cancer cell lines,when the expression of linc00675 was up-regulated,compared with the control group,the cell migration and invasion ability in the experimental group was significantly weakened,and when the expression of linc00675 was down-regulated,compared with the control group,the cell migration and invasion ability in the experimental group was significantly enhanced.The results of xenograft lung metastasis model in nude mice indicated that overexpression of linc00675 can weaken the ability of A549 cells to metastasize and invade in vivo.Western blot results showed that linc00675 may affect the invasion and metastasis of NSCLC cells by affecting MMP2 and MMP9.Conclusions:Compared with lymph node metastasis-negative lung cancer tissues,the expression of linc00675 was lower in lung cancer tissues with positive lymph node metastasis,and the expression of linc00675 was correlated with TNM stage and lymph node metastasis,and was an independent prognostic factor for lung cancer patients.In vitro and in vivo animal experiments demonstrated that linc00675 attenuated cell metastasis and invasion.Linc00675 may affect the invasion and metastasis of NSCLC cells by affecting MMP2 and MMP9.Linc00675 may be a molecular marker for predicting progression and survival in patients with NSCLC. |
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