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OBJECTIVE: To elucidate the potential molecular mechanism by which Fuzheng Kang’ai decoction (扶正抗癌方, FZKA) inhibits proliferation, migration, and invasion of lung cancer cells. METHODS: Varying FZKA concentrations were used to manage lung cancer cells (A549 and PC9). We employed: cell counting kit-8 (CCK-8) and plate clone for-mation assays to examine the cell viability; flow cytometry (FCM) to analyze the cycle arrest; transwell and wound-healing assays to assess the cell invasion and migration, respectively. Further, a quantitative real-time polymerase chain reaction (qRT-PCR) assay was adopted to evaluate the miR-21-5p expression. For protein expression analysis, we employed the Western blot technique. Recombinant miR-21-5p overexpression adenovirus vector harboring GFP was constructed and transfected into A549 and PC9, after which we explored the effect of FZKA on miR-21-5p overexpression. RESULTS: Notably, treatment with FZKA inhibited viability, clone-formation ability, invasion, and migration of lung cancer cells. Mechanistically, FZKA markedly suppressed miR-21-5p expression but elevated the human phosphatase and tensin homology deleted on chromosome ten (PTEN) protein level in both A549 and PC9 cells. Over-expression of miR-21-5p lowered PTEN protein expression. Besides, overexpressed miR-21-5p levels with adenovirus antagonized FZKA-upregulated PTEN protein expression. CONCLUSION: The present study demonstrates how FZKA modulates cell biological behaviors, for instance, it impedes the proliferation by upregulating PTEN expression with miR-21-5p as the target. These findings unveil the potential novel molecular mechanisms from the microRNA aspect by which FZKA suppresses the growth of human lung cancer cells.

YueYang Zhao,1,2 XiaoJuan Tang,1 Yuhua Huang,3 Qing Tang,1 ChangJu Ma,1 Fang Zheng,1 WanYin Wu,4 Swei Sunny Hann1 1Laboratory of Tumor Biology; 2Department of Hematology; 3Department of Stomatology; 4Department of Medical Oncology, The Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province 510120, People’s Republic of ChinaCorrespondence: Swei Sunny Hann; WanYin WuLaboratory of Tumor Biology, The Second Clinical College of Guangzhou University of Chinese Medicine, No. 111 Dade Road, Guangzhou, Guangdong Province 510120, People’s Republic of ChinaTel +8620-39318472Email hann2012@outlook.com; wwanyin@126.comBackground: Lung cancer is a leading cause of cancer-related death worldwide. Previously we demonstrated that polyphyllin I (PPI), a bioactive component extracted from Paris polyphylla, inhibited the growth of non-small cell lung cancer (NSCLC) cells through the SAPK/JNK-mediated suppressing p65, DNMT1 and EZH2 expressions. However, the molecular mechanism underlying anti-lung cancer effect by PPI still remain elusive.Purpose: In this current study, we further explored the molecular mechanism underlying the anti-lung cancer effect of PPI.Methods: MTT, Cell-LightTMEdU DNA cell proliferation and colony formation assays were used to measure cell growth. Western blot were used to examine protein levels of c-Jun and p21. The expression level of long non-codingth RNA HOX transcript antisense RNA (HOTAIR) was measured by qRT-PCR. The p21 promoter activity was measured by Dual-Luciferase Reporter Assay System. The transient transfection experiments were used to silence and overexpression of c-Jun, p21 and HOTAIR. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm thein vitrofindings.Results:We showed that PPI suppressed growth of NSCLC cells. Mechanistically, we observed that PPI reduced expression of HOTAIR, while increased transcription factor c-Jun protein levels. Additionally, PPI also induced protein expression and promoter activity of p21, a cyclin-dependent kinase inhibitor. While exogenously expressed HOTAIR showed no effect on c-Jun levels, silencing of c-Jun significantly reversed the PPI-inhibited HOTAIR expression. Moreover, excessive expressed c-Jun further enhanced PPI-inhibited HOTAIR expression and PPI-induced p21 protein levels. Intriguingly, overexpression of HOTAIR and silencing of c-Jun overcame the PPI-induced p21 protein and promoter activity. Finally, silencing of p21 neutralized the PPI-inhibited cell proliferation. Similar results were also found in one xenograft mouse model.Conclusion:Our results demonstrate that PPI inhibits growth of NSCLC cells through regulation of HOTAIR and c-Jun expressions, which lead to induction of p21 gene. The interactions among HOTAIR, c-Jun and p21 regulatory axis converge in the overall anti-lung cancer effect of PPI. This study unveils an additional new mechanism for the anti-lung cancer role of PPI.Keywords: PPI, NSCLC, HOTAIR, c-Jun, p21

Additional file 4: Table S4. The including predictors of final ECC prediction model.

Additional file 3: Table S3. The results of the candidate variables included in the LASSO regression and their corresponding coefficients for the different values of the penalty parameter λ.

Additional file 1: Table S1. The coding of variables.

Additional file 2: Table S2. Multivariable logistic regression analysis of factors associated with ECC positivity.