Regulatory Mechanism Of Piperlongumine On Proliferation And Invasion Of Non-small Cell Lung Cancer Via MiR-34b-3p/TGFBR1 Pathway

Background and objectiveNowadays lung cancer has been one of the most common malignant diseases in the world.Smoking and environmental pollution are the main predisposing factors for lung cancer.In recent years,with the increasingly serious environmental pollution in China,the incidence and mortality of lung cancer in men are rising,and lung cancer ranks first in male malignant tumors.Among female patients,lung cancer’s incidence and mortality rate is second only to breast cancer.It is difficult for lung cancer to be diagnosed early.Most lung cancers are diagnosed at advanced stage,so the patients have missed the opportunity of the surgery to radical the tumor.On the other hand,the survival rate of lung cancer is low and the recurrence rate is high,so lung cancer has caused great physical and mental health damage to the people.According to different pathological types,lung cancer is divided into two types:small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC is the most common type,which includes lung adenocarcinoma,lung squamous cell carcinoma,large cell lung cancer and other tumor types,and about 80%of lung cancers are NSCLC.Chemotherapy has been the main treatment for NSCLC,but NSCLC generally is less sensitive to chemotherapeutics.In recent years,targeted therapy has made some progress in the treatment of NSCLC,but the prognosis of most patient has not been significantly.So it is important for us to find out the chemotherapy resistance mechanism,increase the sensitivity of classical chemotherapy regimens,and seek new NSCLC treatment methods,that maybe be helpful to improve the survival rate and reduce the mortality of NSCLC patients.The genesis,development and treatment of tumors are regulated and affected by complex molecular mechanisms.MicroRNAs(miRNAs)are a group of endogenous non-coding small RNAs,which is encoded by the genome of an organism.It is associated with various types of malignant tumors,and it is capable of affecting the occurrence and differentiation of tumors.The length of miRNAs is mostly 19-25 base pairs.According to the studies,miR-34b-3p has low expression levels in various solid tumors such as lung cancer,prostate cancer,pancreatic cancer,gastric cancer,breast cancer and hematological caners like leukemia,and miR-34b-3p is possible to participate in the regulation of the biological behavior of tumor cells.TGFBR1 is also known as transforming growth factor beta receptor type 1.It transmits signals from the surface to the interior of the cell through signal transduction.The extracellular environment affects intracellular activity through this signal transduction,such as stimulation the growth and division of tumor cells.Down-regulation of TGFBR1 can inhibit the proliferation and invasion,promote the apoptosis of tumor cells,and increase the sensitivity of tumor cells to chemotherapy.Recently,people h ave paid more and more attention to the role of natural anti-tumor compounds in the treatment of cancer.For example,alkaloid amide compounds,baicalein,myricetin,etc.These natural compounds can inhibit the proliferation and invasion of tumor cells by different mechanisms,and some can also influence the effects of chemotherapy drugs on tumor cells.Therefore,different ideas and methods are provided for the treatment of malignant tumors.Piperlongumine(3,4,5-trimethoxycinnamoylhexanoic acid)is an natural alkaloid extracted from the roots of the piper longum,which is perennial herbaceous vines.It can also be extracted from the roots of knob pepper and long handle pepper.Recent studies have shown that piperlongumine can play an anti-tumor effect in various ways.At present,there are few studies on the role of piperlongumine in lung cancer,especially on the role of miRNA.Therefore,in this paper,we studied the effect of piperlongumine on the proliferation and invasion of NSCLC,and studied the possible signal pathway.In our research,we explored the anti-tumor effect of piperlongumine in NSCLC by three steps:firstly,we assessed the effect of piperlongumine on the expression of miRNA in NSCLC cells.Secondly,we assessed the effects of piperlongumine and miR-34b-3p on the proliferation,invasion and apoptosis of NSCLC cells.Thirdly,we further explored the mechanism of piperlongumine regulating the biological behavior of NSCLC cells through miR-34b-3p.Part Ⅰ Effect of piperlongumine on the expression of miRNA in non-small cell lung cancer cells.Methods1、The cytotoxic effects of piperlongumine at different concentrations(0 μmol/L,2μmol/L,5 μmol/L,10 μmol/L,20 μmol/L,30 μmol/L)on human normal bronchial epithelial(NHBE)cells and A549,H1299,H520 and SPC-A-1 cells were determined by CCK-8.2、A549 cells were treated with 10 μmol/L piperlongumine and 0.1%DMSO(0μmol/L piperlongumine),respectively.RNA was extracted 24 h later.The expression of miRNA in A549 cells treated with piperlongumine was detected by miRNA microarray,and the differential expression of miRNA was analyzed.3、The target genes of miR-34b-3p were predicted by GeneSpring13.1 software and three databases(Target Scan,PITA and microRNAorg).The three databases are intersected to get a common genes,then GO analysis and KEGG analysis were carried out.4、qRT-PCR method was used to detect the effect of piperlongumine on the expression of miR-34b-3p in A549,H1299,H520,SPC-A-1 cells.Results1、The response of NSCLC cells to piperlongumine was different from that of NHBE cells.10 μmol/L piperlongumine could significantly inhibit the viability of A549,H1299,H520 and SPC-A-1 cells.piperlongumine had significantly dose-dependent cytotoxic on A549,H1299,H520 and SPC-A-1 cells.However,NHBE cells showed mild toxicity only when treated with high concentration of piperlongumine.2、miRNA microarray analysis showed that 74 miRNA were differentially expression in A549 cells which were stimulated by piperlongumine,and there were 27 miRNA up-regulated and 47 miRNA down-regulated.miR-34b-3p was highly expressed in NSCLC cells which were stimulated by piperlongumine(P<0.05).3、GO analysis and KEGG analysis showed that piperlongumine was involved in the regulation of multiple pathways in NSCLC.4、qRT-PCR results showed that piperlongumine could up-regulate the expression of miR-34b-3p in A549,H1299,H520 and SPC-A-1 cells(P<0.05).Part Ⅱ Effects of piperlongumine and miR-34b-3p on proliferation,invasion and apoptosis of non-small cell lung cancer cellsMethods1、Liposome 2000 was used to transfect miR-34b-3p mimics/scramble,establish miR-34b-3p group and miR-NC group.The expression of miR-34b-3p was detected by qRT-PCR.2、The expression levels of TGFBR1,MMP2 and TIMP1 mRNA in A549 and H1299 cells were detected by qRT-PCR.3、The OD values of A549 and H1299 cells were detected by CCK-8 at 0 h,24 h,48 h and 72 h.The cell activity of A549 and H1299 cells was expressed by OD value,and the growth curve was drawn according to the OD value of different time points.4、The number of transmembrane cells in A549 and H1299 cells was detected by Transwell assay,and the effects of 10 μmol/L piperlongumine and up-regulated miR-34b-3p expression on the invasive ability of A549 and H1299 cells were evaluated.5、Western blot was used to detect the expression of TGFBR1,MMP2 and TIMP1 in A549 and H1299 cells.6、The effects of 10 μmol/L piperlongumine and miR-34b-3p on the migration ability of A549 and H1299 cells were detected by scratch test.7、Flow cytometry and Caspase3/7 activity were used to detect the effects of piperlongumine and miR-34b-3p on A549 and H1299 cells’ apoptosis.8、Three groups(miR-34b-3p,miR-NC and PL)of A549 cells were injected subcutaneously into the scapular dorsum of the corresponding BALB/c nude mice,establish the model of transplanted tumor in nude mice.The tumor volume was observed every week.After 4 weeks,the nude mice were killed and the tumor was peeled off and weighted.Then the tumor were stained with Ki-67 and detected by TUNEL.Results1、In A549 and H1299 cells,the expression of miR-34b-3p in the miR-34b-3p group was significantly higher than that in the miR-NC group(P<0.05).2、The expressions of TGFBR1,MMP2 and TIMP1 mRNA in miR-34b-3p group and PL 10 μmol/L group were significantly lower than those in PL 0 μmol/L and miR-NC group(P<0.05).3、Compared with PL 0 μmol/L and miR-NC group,miR-34b-3p and PL 10μmol/L could decrease the OD value of A549 and H1299 cells(P<0.05).4、Compared with PL 0 μmol/L and miR-NC group,miR-34b-3p and PL 10μmol/L could reduce the number of transmembrane cells(P<0.05)and effectively inhibit the invasive ability of A549 and H1299 cells.5、Compared with PL 0μmol/L and miR-NC group,miR-34b-3p and PL 10μmol/L significantly decreased the expression of TGFBR1,MMP2 and TIMP1(P<0.05).6、Compared with PL 0 μmol/L and miR-NC group,miR-34b-3p and PL 10μmol/L could inhibit the migration ability of A549 and H1299 cells.7、Compared with PL 0μmol/L group,PL 10 μmol/L could increase the number of apoptosis cells of A549 and H1299(P<0.05).However,miR-34b-3p had no obvious effect of promoting apoptosis.PL 10μmol/L could increase the relative activity of Caspase3/7(P<0.05),but miR-34b-3p had no such effect.8、The volume and weight of transplanted tumor in PL group and miR-34b-3p group were lower than those in miR-NC group(P<0.05),and the positive staining of Ki-67 in PL group and miR-34b-3p group was significantly lower than that in miR-NC group(P<0.05).However,Tunel staining showed that the number of apoptotic cells in PL group was significantly higher than that in miR-NC and miR-34b-3p group.Part Ⅲ Preliminary study on the mechanism of piperlongumine regulating the biological behavior of non-small cell lung cancer cells through miR-34b-3pMethods1、Screening target genes of miR-34b-3p by bioinformatics software.2、The target genes of miR-34b-3p were detected by double reporter gene test.The double reporter gene vectors of wild type and mutant TGFBR1 were designed,and the miR-34b-3p mimics/scramble fragments were co-transfected with wild type or mutant TGFBR1 vectors,detect the change of luciferase fluorescence intensity.3、We used Western blot to detect the effect of regulating miR-34b-3p on the expression of target gene TGFBR1 protein.4、Set up four groups(miR-34b-3p inhibitor,miR-NC,miR-NC+PL 0 μmol/L,miR-34b-3p inhibitor+PL 10μmol/L)of A549 and H1299 cells,CCK-8 and Transwell experiments were carried out to detect the effect of down-regulation of miR-34b-3p expression on the proliferation and invasion.5、Constructed TGFBR1 expression vector without 3’UTR region.Set up three groups(PL 0 μmol/L,PL 10 μmol/L,PL 10 μmol/L+pcDNA3.1-TGFBR1)of A549 and H1299 cells.The protein level of TGFBR1 was detected by Western blot.The effect of TGFBR1 without 3’UTR on the proliferation and invasion was detected by CCK-8 and Transwell assay.6、The clinical samples of metastatic NSCLC(TNM stage Ⅳ)and non-metastatic NSCLC(TNM stage Ⅱ)were collected,and the expression of miR-34b-3p in the two samples was determined by qRT-PCR.Results1、Bioinformatics software predicts that there is a complementary pairing region between TGFBR1 and miR-34b-3p,and TGFBR1 may be the target gene of miR-34b-3p.2、The double luciferase reporter gene experiment confirmed that miR-34b-3p could bind to the 3’UTR of TGFBR1 and down-regulate the firefly luciferase activity in the double reporter gene vector(P<0.05).3、miR-34b-3p can reduce the expression of TGFBR1 protein.Combined with the results of double reporter gene experiment,it can be concluded that TGFBR1 is the target gene of miR-34b-3p.4、miR-34b-3p inhibitor+PL 10 μmol/L weakened the inhibitory effect of piperlongumine on the proliferation and invasion(P<0.05).5、Compared with PL 0μmol/L group,the TGFBR1 expression of PL 10 μmol/L group decreased(P<0.05),The expression of TGFBR1 protein in PL 10μmol/L+pcDNA3.1-TGFBR1 group was almost not affected.In results of CCK-8 and Transwell,the inhibition of proliferation and invasion by PL 10μmol/L+pcDNA3.1-TGFBR1 was weaker than that of PL 10 μmol/L(P<0.05).6、The expression of miR-34b-3p in metastatic NSCLC specimens was lower than that in non-metastatic NSCLC specimens(P<0.05).Conclusion1、Piperlongumine can stimulate the overexpression of miR-34b-3p in many NSCLC cells and participate in many regulatory pathways of NSCLC cells.2、Piperlongumine can up-regulate the expression of miR-34b-3p,then act on the 3’UTR of TGFBR1,negatively regulate the expression of TGFBR1,and regulate the proliferation and invasion of NSCLC.3、Piperlongumine can promote the apoptosis of A549 and H1299 cells.4、Compared with non-metastatic NSCLC specimens,the expression of miR-34b3p in metastatic NSCLC specimens was lower,suggesting that piperlongumine may be used as a combination drug for the treatment of metastatic NSCLC.