| IntroductionDuring the development of malignant tumors,only a small number of tum or stem cells play a crucial role in cloning,self-renewal and differentiation pro cedure to maintain the growth and metastasis of tumor cells.hypoxia may lead to an increase in the number of cancer stem cells or en hance the stem cell property of cancer cells.To some extent,the expression of cancer stem cell markers on the surface of tumor cells correlated to the stem cell property of tumor cells.This study intends to introduce the single-cell method to research whether non-small cell lung cancer tumor cells could regain cancer stem cell-like prope rty under hypoxic conditions.In our study,Western Blot and qRT-PCR techniq ues were used to focus on the change of expression of CXCR4,Oct-4,SOX2,c-Myc,CD 133,CD44 and ALDH1A1 in the process of change of oxygenatio n condition.The protein expression,which are relatively stable,may be related to the accelerated proliferation of the tumor after hypoxia treatment,the abilit y of colony formation and the enhancement of erosion ability.This study including two major parts aimed at the effects of moderate Ion g-term hypoxia on the acquirement of the cancer stem cell property of lung ad enocarcinoma and squamous cells carcinoma and investigating the key molecule s that promote this change,the down-regulation of protein expression by miRN A transfection closely related to cancer stem cell property,and the reversal of stem cell property of lung adenocarcinoma and squamous cell carcinoma.In th e first part,it confirmed that the long-term moderate hypoxia enhanced the ste m cell property of NSCLC H1299 and SK-MES-1,and the proliferation,invasi on and migration ability were enhanced.The further studies on various stem c ell property related proteins and mRNA were carried out.Expression in hypoxi c environment and changes after reoxygenation revealed that SOX2 related to e nhanced stem cell property after tumor hypoxia treatment.In the second part,we used miR-145-5p and gene knockout technique to intervene NSCLC H1299 and SK-MES-1 cell lines to investigate the effects of miR-145-5p on the proli feration,invasion,and migration of NSCLC H1299 and SK-MES-1 cells in vitr o.The animal experiment has also confirmed the effects of miR-145-5p on the growth of H1299 and SK-MES-1 cells in the nude mouse model.Part I The effect of hypoxia on the expression of genes related to the stem cell property in lung cancer cell lines H1299 and SK-MES-1Methods1 A clonal population was obtained from single cell.The H1299 and SK-MES-1 cell lines were serially diluted by limiting dilu tion and inoculated into 96-well plates until the cell density reached 1 cell/well.Under the microscope,mark the well containing only one cell.Randomly,one of the large cloned population was passaged and labeled as H1299 and SK-M ES-1,respectively,prepared for next step.2 Hypoxia.The two cell lines above were divided into hypoxia-96h,hypoxia-48h-norm al oxygen 48h,normoxia-48h-hypoxia-48h and normoxia-96h group.The hypoxi a-96h group was cultured under hypoxic conditions for 96 hours.The hypoxic-48h-normal oxygen 48h group was cultured for 48 hours under hypoxic conditi ons,then cultured under normoxic conditions for 48 hours.The normoxia-48h-h ypoxic-48h group was cultured under normoxic conditions for 48 hours,and th en cultured under hypoxic conditions for 48 hours.The normoxia-96h group w as cultured under normoxia for 96 hours.3 Assay related to Cancer stem property(Colony formation assay,Wound healing assay and Transwell invasion assay)In the colony formation assay,wound healing assay and Transwell invasio n assay,H1299 and SK-MES-1 cell lines was divided into hypoxic-96h,hypoxi a-48h-normoxic 48h,normoxia-48h-hypoxia-48h and normoxia-96h group to be tested.4 Western blot and qRT-PCRTo detect HIF-1?,ALDH1A1,CD44.CD133,CXCR4,c-Myc,Oct-4 and SOX2 protein and mRNA expression of H1299 and SK-MES-1 cell lines in H ypoxia-96h group,Hypoxia-48h-Normal-48h group,Normal-48h-Hypoxia-48h gro up and Normal-96h groupResults1 Hypoxia-96h group cell line stem cell proper was enhanced steadilyThe formation rate of H1299 and SK-MES-1 in the hypoxic-96h group wa s increased by 85.4%and 61.7%than Normal-96h group,respectively.Compar ed hypoxic-96h group with the Hypoxia-48h-Normal-48h group and Normal-48h-Hypoxia-48h group,there was no significant difference.The wound healing rate of H1299 and SK-MES-1 in the hypoxic-96h grou p was both increased by 24.3%Normal-96h group.Compared hypoxic-96h gro up with the Hypoxia-48h-Normal-48h group and Normal-48h-Hypoxia-48h group,there was no significant difference.The invasive number of H1299 and SK-MES-1 in the hypoxic-96h group was increased by 25%and 34.3%,respectively.Compared hypoxic-96h group with the Hypoxia-48h-Normal-48h group and Normal-48h-Hypoxia-48h group,t here was no significant difference.2 The expressions of HIF-1??CXCR4?Oct4?Sox2?c-Myc?CD133?CD44and ALDH1Alwere increased in Hypoxia-96h group,and the expression of SOX2 protein was stable after hypoxia.The protein expressions of HIF-1?,CXCR4,Oct4,SOX2,c-Myc,CD133,CD44 and ALDH1A1 in H1299 and SK-MES-1 hypoxia-96h groups were 3.55 and 3.82 times,3.63 and 3.75 Times,4.00 and 3.81 times,3.26 and 3.74 tim es,4.78 and 3.78 times,4,38 and 3.77 times,4.20 and 3.80 times,2.68 and 3.68 times higher than that in the normoxia-96h group,respectively.The protein expressions of HIF-la,CXCR4,Oct4,SOX2,c-Myc,CD 133,CD44 and ALD H1A1 in H1299 and SK-MES-1 Hypoxia-48h-Normal-48h group were 24%and 27%,34%and 32%,25%and 34%,87%and 111%,18%and 20%,34%an d 34%,20%and 23%,30%and 27%of Normal-48h-Hypoxia-48h group,respe ctively.The SOX2 protein is continuously and stably expressed.3 The expression of HIF-la,ALDH1A1,CD44,CD133,CXCR4,c-Myc,Oct-4 and SOX2 mRNA in Hypoxia-96h group was enhanced,and the expression of SOX2 mRNA was also stable after hypoxia.The mRNA expressions of HIF-1?,CXCR4,Oct4,SOX2,c-Myc,CD133,CD44 and ALDH1A1 in H1299 and SK-MES-1 hypoxia-96h groups were 5.77 and 8.94 times,5.59 and 7.24 times,8.4 and 8.66 times,8.43 and 9.71 times,5.48 and 3.9 times,4.18 and 3.4 times,6.28 and 6.42 times,5.07 and 5.14 ti mes,respectively.The mRNA expressions of HIF-1?,CXCR4,Oct4,SOX2,c-Myc,CD133,CD44 and ALDH1A1 in H1299 and SK-MES-1 Hypoxia-48h-No rmal-48h group were 13%and 17%,14%and 28%,13%and 20%,85%and 86%,9%and 12%,21%and 19%,12%and 20%,16%and 16%of Normal-48 h-Hypoxia-48h group,respectively.The SOX2 mRNA is continuously and stabl y expressed.Conclusion1 After moderate hypoxia,the colony forming ability,migration ability and invasive ability of H1299 and SK-MES-1 cell lines were significantly enhanced,and the stem cell property was significantly enhanced.2 Western Blot and qRT-PCR confirmed that HIF-1?,ALDH1A1,CD44,CD133,CXCR4,c-Myc,Oct-4 and SOX2 were up-regulated after moderate hypoxia condition,furthermore,were down-regulated after returning to normoxia after end of hypoxia.In the state,the above expression showed a significant decrease except for SOX2,and the stable SOX2 both protein and mRNA expression were observed.3 SOX2 may be a functional gene that drives the enhancement of tumor stem cell property.Part ? MiR-145-5p downregulates the expression of SOX2 gene to attenuate the lung cancer stem cell property in vitro and in vivoMethodsIn vitro,the NSRLC cell lines H1299 and SK-MES-1 were transfected wit h miR-145-5p mimics and the blank vector control group,respectively.The ex perimental group and the control group were respectively subjected to colony f ormation experiments and cell scratches.Healing experiments and Transwell cel 1 invasion assay;Western blot and qRT-PCR were used to detect the expressio n of SOX2 protein and mRNA after transfection.In vivo,the experimental and control group of the H1299 and SK-MES-1 cells were inoculated into 6 pairs of nude mice,respectively,for a total of 24 mouse.The subcutaneous tumorigenic ability of the experimental and control group cells was observed.The SOX2 gene was knocked out using the siRNA technique.The mRNA and protein expression of the SOX2 gene in the control siRNA group and the siRNA group were detected,and the transfection efficiency was examined to determine that SOX2 was knocked out.The siRNA group cells knocked out by SOX2 gene were transfected into miR-145-5p mimics and lipofectamine RNAiMAX,respectively,and divided int o siRNA + miRNA group and siRNA + lipo-max group,compared with untran sfected normal control cells.The expression of SOX2 mRNA and protein furth er confirmed that SOX2 gene expression was inhibited.The characteristics of tumor stem cells in the siRNA + miRNA group and the siRNA + lipo-max group were compared by colony formation assay,cell scratch healing assay and Transwell cell invasion assay.The role of miR-145-5 p in inhibiting the characteristics of tumor stem cells by inhibiting SOX2 was clarified.The basic principle of this part of the experiment is that transfection of m iR-145-5p mimics demonstrates that miR-145-5p can inhibit tumor stem cell ch aracteristics and inhibit SOX2 expression.The SOX2 gene was knocked out an d miR-145-5p was transfected again.The experimental group compared the turn or stem cell characteristics with the blank control.If there is no difference in tumor stem cell characteristics,miR-145-5p,which can be fully explained,acts through SOX2.Results1 miR-145-5p inhibits proliferation and growth of cell linesThe clone formation numbers of H1299 and SK-MES-1 experiment group cells was 66.7%and 68.3%of the control group,respectively.On the 25th da y after subcutaneous injection of tumor cells,the average tumor weight of H1299 and SK-MES-1 experimental group was 28%and 24%of control group.2 miR-145-5p inhibits the migration and invasion of NSCLC H1299 and SK-MES-1 cell lines,and leads to an increase in apoptosisThe wound healing rate of H1299 and SK-MES-1 cells in the experimenta 1 group was 79%and 68%of control group,respectively;the number of invas ive cells in the experimental group was 56%and 70%of control group,respec tively.3 miR-145-5p inhibits the expression of SOX2 mRNA and proteinThe protein expression of SOX2 in H1299 and SK-MES-1 experiment gro up was 68%and 78%of the control group,respectively.The mRNA expressio n of SOX2 in the experimental group was 71%and 73%of the control group,respectively.4 miR-145-5p does not inhibit tumor stem cell characteristics after SOX2 gene knockoutAfter knockdown of SOX2 gene in H1299 and SK-MES-1 cells,the siRN A + miRNA group transfected with miR-145-5p and the siRNA + lipo-max gr oup transfected with blank vector showed no difference in the clonality,migrati on and invasion ability.Conclusion1 MiR-145-5p can inhibit the proliferation,migration and invasion of H1299 and SK-MES-1 cell lines,and thus play anti-tumor role2 MiR-145-5p has a significant inhibitory effect on the subcutaneous tumorigenic ability of NSCLC H1299 and SK-MES-1 cell lines in nude mice.3 MiR-145-5p can attenuate the proliferation,migration and invasion and migration of H1299 and SK-MES-1 cell lines by regulating the expression of SOX2.4 SOX2 gene drives the enhancement of stem cell property of H1299 and SK-MES-1 cell lines,which may be the potential clinical therapeutic targets.SOX2 protein is a potential surface marker for cancer stem cells and is applied to the sorting and identification of cancer stem cells. |
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