| Objective: Worldwide,lung cancer is the most common malignant tumor with the highest incidence of deaths.According to the latest statistics in China,lung cancer is already the most common malignant tumor in China,and the incidence rate is still increasing year by year,and it has not been effectively controlled.Therefore,prevention,early diagnosis and early treatment of lung cancer in China still needs to be further strengthened and improved.The symptoms of early stage of lung cancer are usually hidden and there is a lack of methods of effective early diagnosis currently.Most patients of lung cancer are in the advanced stage on diagnosis.The five-year survival rate is only 15%.Approximately 70% of patients have been diagnosed at a locally advanced stage or have had a malignant degree of metastasis,losing the opportunity for optimal surgical treatment.And due to the heterogeneity of lung cancer and the susceptibility of chemotherapeutic drugs,the prognosis of lung cancer patients has not improved significantly in recent years.Deaths of lung cancer in China 1970 s was still at a relatively low level compared with other countries in the same period,yet after the incidence increased in the following decades lung cancer has become the most lethal malignant tumor in China.The carcinogenesis of lung cancer is result of interaction of genetic risk factors and environmental risk factors.Although smoking is recognized as the most important environmental risk factor to cause lung cancer,yet statistical data show that there are still 15% of male lung cancer patients and 53% of female lung cancer patients were nonsmoking.More and more evidence elucidated that genetic factors play an irreplaceable role in the carcinogenesis of lung cancer,yet the exact molecular mechanism in the development of lung cancer has not been fully elucidated,so the role of genetic factors play in lung cancer need to be explored in more depth.The ENCODE project found that about 90% of the DNA sequence in the human genome is active in transcription and the protein-coding gene accounts for only 2% of the whole genome.Non-coding RNAs was once considered to be “transcriptional noise” or “transcriptional waste” which accounts for the vast majority of the transcriptome and may play an important role.Long non-coding RNA(LncRNA)is a group of RNAs longer than 200 nuleotides in length with no protein-coding function which are tissue expression specificity and spatiotemporal expression specificity.It is estimated that non-coding RNA accounts for more than 70% of the human genome.Numerous studies have elucidated that lncRNA plays an important role in the regulation of gene expression in a variety of malignant tumors.LncRNA may play a role in cell proliferation,cell cycle and apoptosis and cell invasion and metastasis of tumor cells.MicroRNAs is a class of evolutionarily conserved RNAs,21-25 nts in length with no protein-coding function.The microRNAs can be completely or incompletely complementary to the 3' untranslated region of the messenger RNA of target gene,which may result in degradation or translational inhibition of the messenger RNA,thereby regulating the translation of the messenger RNA of target genes,a messenger RNA can be regulated by a variety of microRNAs and a microRNA can regulate a variety of messenger RNAs,microRNAs constitute a huge regulatory network that can regulate nearly 30% of the protein-coding genes in the human genome.Studies have demonstrated that microRNAs can be involved in many physiological processes of cells such as cell proliferation,cell differentiation,apoptosis and inflammatory processes,and the dysregulation of these processes is often implicated in tumorigenesis.There is a lot of evidence that microRNA can play as proto-oncogene or tumor suppressor,and a large number of microRNA genes are located in tumor-related gene regions.The abnormal expression of microRNA was observed in many malignant tumor tissues.Therefore,the relationship between microRNA and malignant tumors is studied.Significant value in the development of diagnostic markers and targeted therapies for malignant tumors.Single Nucleotide Polymorphisms(SNP)is the most common type of variation in the genome which is a replacement of a single base in a genomic DNA sequence.Many researches elucidated that SNPs on lncRNA and microRNA are associated with susceptibility and prognosis in a variety of malignancies.The competitive endogenous RNA mechanism is a hypothesis put forward in recent years.The main core point is that messenger RNA,long-chain non-coding RNA,pseudogene or other molecules share a common microRNA response in cells.Element,MRE),is a common specific microRNA complementary binding sequence on these molecules,which may cause these molecules to compete with microRNAs,thereby affecting microRNA regulation of downstream gene processes.This study investigated the relationship between long-chain non-coding HOTAIR and miR-149-5p gene polymorphisms and lung cancer susceptibility,and verified the competitive endogenous RNA mechanism of HOTAIR,miR-149-5p and HNRNPA1 in the development of lung cancer.Methods: In the first part,we used a hospital-based case-control study to explore the relationship between three SNPs on the long-chain non-coding RNA HOTAIR,rs920778,rs12826786 and rs4759314 and lung cancer susceptibility.The study included 551 lung cancer cases and 543 healthy controls,all of which were Han subjects.The case was from the Fourth Affiliated Hospital of China Medical University,the First Affiliated Hospital of China Medical University and General Hospital of Shenyang Military Area Command of Chinese PLA and did not undergo radiotherapy or chemotherapy before donating 5 ml of venous blood.Controls were from the hospital physical examination center in the period.All subjects donated 5 ml of venous blood after signing the informed consent form.We extracted genomic DNA using phenol-chloroform method and performed SNP typing on the genomic DNA of the subject using ABI's Taqman probe primers.Continuous variables and categorical variables between the two groups were compared by performing T test and chi-square test respectively.Logistic regression was used to calculate OR and its 95% confidence interval to evaluate the relationship between different genotypes and the risk of lung cancer.The research value was approved by the Ethics Committee of China Medical University.The second part of the study explored the relationship between a SNP rs2292832 on miR-149 and lung cancer susceptibility.We performed a hospital-based case-control study.We eventually included 555 cases and 395 healthy controls.The case was from the Fourth Affiliated Hospital of China Medical University,the First Affiliated Hospital of China Medical University and General Hospital of Shenyang Military Area Command of Chinese PLA and healthy controls were from the hospital physical examination center in the same period.All subjects donated 5 ml of venous blood after signing the informed consent form.We extracted whole-genome DNA from the blood samples and performed SNP genotyping.Comparison of continuous variables and categorical variables between the two groups was performed by t-test and chi-square test,respectively.Logistic regression was used to calculate ORs and its 95% confidence interval to evaluate the relationship between different genotypes and the risk of lung cancer.The research was approved by the Ethics Committee of China Medical University.In the third part,we studied the effect of HOTAIR and miR-149-5p on lung cancer cells by transfecting HOTAIR small interfering RNA(siRNA),overexpressing HOTAIR and miR-149-5p lentivirus into lung cancer cell lines A549 and SPC-A-1.Effects of HOTAIR and miR-149-5p on cell biological behavior such as proliferation,metastasis,cell cycle and apoptosis were measuered.We used wound-healing experiments and Transwell experiments to examine the effects of HOTAIR and miR-149-5p on lung cancer cell migration and invasion.The Celigo was used to detect the confluence of lung cancer cells at four time points,and the effects of HOTAIR and miR-149-5p on cell proliferation rate were examined.Flow cytometry was used to detect the cell cycle and apoptosis of lung cancer cells.The dual luciferase reporter gene was used to verify the targeting relationship between HOTAIR and miR-149-5p and the targeting relationship between miR-149-5p and HNRNPA1.Results: The results of the first part of the case-control study showed that there was a statistically significant association between HOTAIR rs12826786,HOTAIR rs4759314 and the risk of lung cancer.There was no statistically significant association between HOTAIR rs920778 and lung cancer susceptibility.Stratified analysis showed a statistically significant association between HOTAIR rs4759314 and the risk of lung adenocarcinoma,lung squamous cell carcinoma,and small cell lung cancer.There was a statistically significant association between HOTAIR rs12826786 and susceptibility of small cell lung cancer.The results of second part showed that miR-149 rs2292832 had no statistically significant association with lung cancer susceptibility.The stratified analysis showed that there was no statistically significant association between miR-149 rs2292832 and susceptibility of non-small cell lung cancer,lung adenocarcinoma and lung squamous cell carcinoma.We also analyzed whether there was a statistically significant association between the interaction of miR-149 rs2292832 polymorphism and cooking oil fume exposure and lung cancer susceptibility,the results showed that there were no significant associations.In the third part,the functional studies of HOTAIR and miR-149-5p showed that the background expression of HOTAIR in SPC-A-1 was higher than that in normal bronchial epithelial HBE cells,and the expression of HOWAIR in A549 background was lower than that in HBE.The background expression of miR-149-5p in SPC-A-1 was lower than that of HBE.HOTAIR was overexpressed and knocked down in the A549 cell line and miR-149-5p was overexpressed in the SPC-A-1 cell line,then performed the next series of functional experiments.The results of scratch repair experiments showed that HOTAIR can promote the metastasis of lung cancer cells,and miR-149-5p can inhibit the metastasis of lung cancer cells.The results of the flow cytometry detection cycle showed that HOTAIR overexpression can reduce the number of cells in G0/G1 phase and increase the number of cells of S phase,which indicated that HOTAIR could promote cell proliferation.Overexpression of miR-149-5p can increase G0/G1 phase cells,causing G0/G1 phase arrest,and S phase cells are reduced,thereby inhibiting cell proliferation.HOTAIR knockout can increase G0/G1 phase cells,causing G0/G1 phase arrest,and S phase cells are reduced,thereby inhibiting cell proliferation.The results of apoptosis experiments showed that HOTAIR and miR-149-5p had no observable effects on apoptosis.The results of dual luciferase activity assay and Western Blot showed that miR-149-5p and HNRNPA1 and miR-149-5p are in a targeted binding relationship with HOTAIR,and the HOTAIR-miR-149-5p-HNRNPA1 ceRNA mechanism is established.Conclusion: 1.HOTAIR rs4759314,HOTAIR rs12826786 are related to lung cancer susceptibility.Stratified analysis showed that HOTAIR rs4759314 is associated with susceptibility of lung adenocarcinoma,lung squamous cell carcinoma and small cell lung cancer.HOTAIR rs12826786 is associated with susceptibility of small cell lung cancer.2.miR-149 rs2292832 had no statistically significant association with lung cancer susceptibility in non-smoking women.miR-149-5p can inhibit the migration,invasion and proliferation of lung cancer cells.HOTAIR can promote the migration,invasion and proliferation of lung cancer cells.HOTAIR-miR-149-5p-HNRNPA1 forms a competitive endogenous RNA mechanism. |
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