| Objective and Significance:Lung cancer, which is the most common cancer in the world endangers human health seriously, and it is the first killer in cancer-related death, causing enormous disease burden. Wherein, the non-small cell lung cancer (NSCLC) is the main pathological types of lung cancer, accounting for over 80% of all lung cancer cases. With the development of comprehensive treatment model and molecular biochemistry, the efficacy of NSCLC treatment has improved, but the prognosis is still not ideal with less than 20% of 5-year overall survival rate. MicroRNAs (miRNA) are a class of widespread small molecule non-protein-coding RNA, miRNA expression and regulation of imbalance plays an important role in malignant tumor occurrence and development. In recent years, with the deepening of the miRNA research, more and more studies show that miRNA play an important role in the development of lung cancer. Wherein miR-34b-5p was confirmed closely related to the occurrence and development of NSCLC.Currently, Chinese traditional medicine in clinical treatment of cancer have achieved a significant efficacy, compared to Western medicine treatment of cancer, Chinese traditional medicine has obvious advantages in inhibiting tumor metastasis and migration, preventing tumor recurrence, stabilizing tumor, improving patient general symptoms, and improving life quality of patients, etc. Zhou’s Prescription is an experiential effective recipe used for lung cancer treatment, which was developed by traditional Chinese medicine master, professor Zhou Zhongying, based on his clinical experience, composed of drugs with four categories of function, nourishing Yin and benefiting Qi, activating blood and removing stasis, eliminating dampness and eliminating phlegm, resolving hard lump. After high-throughput screening, more than 20 species of monomeric compound that shows cytotoxicity on non-small cell lung cancer (NSCLC) have been obtained, including Ophiopogonin, Isoliquiritigenin, Quercetin, Ginsenosides and bibenzyls etc. In this anti-cancer Recipe, OP-B, as indispensable traditional Chinese medicine component, has significant anti-cancer effect in vitro, but the underlying mechanism needs further study. In this study, the possible mechanism of OP-B inhibiting NSCLC was investigated by detecting the expression of miR-34b-5p and met in A549 cells which infected with lentivirus LV-hsa-miR-34b and A549 cells which added with OpB.Methods:A number of miRNA which has effect on non-small cell lung cancer A549 cells was selected through literature research. miRNA which changed after adding Ophiopogonin B was selected out using Real-time PCR technology. Taken this miRNA as target, its expression quantity in normal embryonic lung fibroblasts MRC-5 and A549 cells was compared by Real-time PCR technology, to identify its expression feature in non-small cell lung cancer A549 and to determine the role it plays. Lentivirus vectors with hsa-miR-34b over-expression were constructed, and recombinant plasmids pHLV-miR-34b were used to infect A549 cells and identify transfection efficiency. Common TargetScans, PicTar, PITA and other target gene prediction software were used to predict potential target genes of miR-34b-5p. The impact of miR-34b-5p and Ophiopogonin B on Met was verified by Real-time PCR and Western Blot technique on the gene level and protein level and the migration and invasion of A549 cells were detected by wound healing and transwell assays to reveal the possible anticancer mechanism of Ophiopogonin B.Results:1. Real-time PCR results showed that miR-21-5p, miR-148a-3p, miR-29a-3p, miR-338-5p, miR-125a-5p did not change significantly before and after adding the drug, while miR-34b-5p changed significantly, which significantly increased, indicating Ophiopogonin B can affect the expression of miR-34b-5p in A549 cells.2. Real-time PCR results showed that the expression quantity of miR-34b-5p in normal embryonic lung fibroblasts MRC-5 cells was significantly higher than that in non-small cell lung cancer A549 cells, indicating that miR-34b-5p plays a role of inhibition in non-small cell lung cancer.3. Regarding hsa-miR-34b over-expression lenivtirus vector and efficient identification experiment, gene sequencing showed that miR-34b is properly inserted into the vector pHLV; Real-time PCR results showed that the expression quantity of miR-34b-5p in the cells after transfection was significantly increased, and the light-emitting protein could be observed under fluorescence microscope, indicating recombinant lentivirus plasmid has infected A549 cells successfully.4. By means of literature review and biological target gene prediction software analysis, it is found that met was included in the target genes predicted by miR-34b-5p, and hinting met may be the key downstream target gene of miR-34b-5p.5. The results of Real-time PCR and Western Blot showed that the expression of Met of Micro-up and OP-B groups were lower. A549 and NC groups were higher.6. The results of wound healing and trans well assays showed that the migration and invasion of Micro-up group was slower, NCgroup was faster.Conclusions:1. Ophiopogonin B can significantly reduce the expression level of miR-34b-5p in non-small cell lung cancer A549 cells.2. The down-regulated expression of miR-34b-5p in non-small cell lung cancer A549 cells plays a role of anti-cancer.3. Met is the target gene of miR-34b-5p.4. Exogenous up-regulated expression of miR-34b-5p can reduce the expression of Met in gene and protein level; adding Ophiopogonin B can also down-regulate the expression of Met in gene and protein level. Ophiopogonin B may up-regulate the expression of miR-34b-5p in A549 cells, thereby inhibits downstream target gene met and plays a role of anti-cancer.5. Exogenous up-regulated expression of miR-34b-5p can inhibit the migration and invasion of A549 cells. |
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