The Effects Of Huc-mscs On The Apoptosis And Proliferation Of Human Lung Adenocarcinoma Via PI3K/Akt Signaling Pathway

Objective:Mesenchymal stem cells are a kind of non-hematopoietic adult stem cells with multiple differentiation,which are originated from mesodermal cells and can be derived from connective tissues or organ mesenchyme,such as bone marrow,adipose tissue,umbilical cord and umbilical cord blood.MSCs have gradually become a hotspot in the treatment and research of various diseases due to their easy access,multiple differentiation potential,low immunogenicity,tropism of inflammation and injury as well as few ethical controversies.Due to their low immunogenicity and ability to track tumor cells,MSCs have also become a hotspot in targeted therapy of tumor.MSCs can either exert a tumor-suppressing effect through direct inter-cellular contact or via secretion of factors,or can be used as carriers of antitumor factors to deliver antitumor viruses or drugs to tumor sites.All the above-described features make it possible for MSCs to participate in targeted therapy of tumor.However,it can not be neglected that MSCs also have the potential to promote tumor growth by multiple mechanisms at tumor site.There is no definite conclusion concerning the relationship between tumor and MSCs.Human umbilical cord-derived mesenchymal stem cells?hUC-MSCs?used in this study have the advantages of wide sources,easy access,short amplification time,low immunogenicity,long survival time after transplantation and no involvement with ethical issues.In addition,hUC-MSCs,originated from neonatal umbilical cord,are also more primitive.The differentiation potential and immunophenotype of hUC-MSCs are similar to those of other sources of MSCs,however,hUC-MSCs harbor stronger proliferation efficacy and self-renewal abilities.The PI3K/Akt pathway is a critical signal transduction pathway of oncogenes associated with multiple receptors.Phosphorylated Akt activates or inhibits its downstream target proteins,thereby exerting the effects of PI3K/Akt signaling pathway involved in anti-apoptosis,proliferation,and chemotherapy tolerance of cells.As one of the most common tumors worldwide,lung cancer ranks the first among all malignancies in terms of incidence and mortality in China.The clinical incidence of non-small cell adenocarcinoma of lung is high,the progression of which is also associated with the PI3K/Akt pathway.In this study,primary cells were isolated and cultured from the Wharton's jelly of human umbilical cord,which were further identified.The effects of hUC-MSCs on the malignant phenotypes of lung adenocarcinoma cells and relevant regulatory mechanisms were studied using the obtained MSCs,followed by further clarification of the effects of the PI3K/Akt signaling pathway during the process.The study aimed to lay the foundation for the safety of hUC-MSCs as a carrier of gene therapy in lung cancer and the therapeutic options for tumor biological therapy involving hUC-MSCs.Methods:In this study,hUC-MSCs were isolated from human umbilical cord tissue and cultured in vitro.The immunophenotypes of the obtained MSCs were determined by flow cytometry,followed by assessment of their ability of adipogenic and osteogenic differentiation.The culture supernatant of hUC-MSCs was collected,which was used as conditioned medium for hUC-MSCs.In vitro indirect co-culture system of conditioned medium from hUC-MSCs and lung adenocarcinoma A549 cell line was established.The effects of hUC-MSCs on the malignant phenotypes of lung adenocarcinoma cells were examined.To be specific,cell proliferation was detected by CCK-8 assay,apoptosis rate of A549 cells was determined by Annexin V/PI double staining,cell cycle distribution of tumor cells was determined by PI staining.Real-time quantitative PCR was used to detect the transcription levels of apoptosis and proliferation-associated downstream genes of the PI3K/Akt pathway,including CyclinD1,BAX and Bcl-2.Moreover,Western blot was utilized to detect the changes of protein expression of PI3K/Akt pathway,such as p-Akt.Results:1.MSCs were successfully isolated and cultured from the Wharton's jelly of human umbilical cord,and were further identified.The umbilical cord tissue was isolated and cultured for about three weeks.The fibroblast-like cells covered the culture flasks,which were parallel-arranged in spiral growth.The obtained cells underwent flow cytometry,revealing that these cells expressed the markers of MSCs,CD73,CD90 and CD105,but not CD45 and HLA-DR.Under the induction of adipogenic or osteoblastic differentiation fluids,intracellular lipid precipitation or calcific precipitation was visualized under microscope using oil red O or alizarin red S staining;2.hUC-MSCs inhibited the proliferation of A549 cells.CCK-8 assay showed that the proliferation of A549 cells was significantly inhibited after the use of conditioned medium?P<0.001?.Annexin V/PI double staining showed that the apoptosis rate in the conditioned medium group was significantly higher than that in the control group?P<0.001?.Cell cycle analysis revealed significant G₁ cell cycle arrest of A549 cells in the conditioned medium group compared to that in the control group?P<0.001?;3.The PI3K/Akt signaling pathway in A549 cells was inhibited by hUC-MSCs.Real-time quantitative PCR was used to detect PI3K/Akt signaling pathway-related factors,which revealed down-regulated transcription of CyclinD1 and Bcl-2?both P<0.001?and up-regulated transcription of BAX?P<0.001?.Western blot showed that the total Akt was not changed,but p-Akt was decreased in a dose-dependent pattern in A549 cells cultured in conditioned medium from hUC-MSCs?P<0.001?.Conclusion:1.The primary isolated hUC-MSCs used in this study met the identification criteria of MSCs,which could be used as a source of MSCs;2.hUC-MSCs can inhibit the proliferation of A549 cells through promoting apoptosis and arresting cells in G₁phase;3.hUC-MSCs could inhibit the PI3K/Akt signaling pathway in A549 cells,indicating that the inhibition of PI3K/Akt signaling pathway played an important role in the proliferative inhibition of A549 cells induced by MSCs.