LncRNA AC100826.1 regulated PLCB1 to promote progression in non-small cell lung cancer

doi:10.21203/rs.3.rs-3144126/v1

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Research Article

LncRNA AC100826.1 regulated PLCB1 to promote progression in non-small cell lung cancer

https://doi.org/10.21203/rs.3.rs-3144126/v1

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published 22 May, 2024

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Background

Lung cancer is the most common malignant tumors. In the research, we identified a long non-coding RNA(lncRNA) AC100826.1 (simplify to Lnc1), which was highly expressed in non-small cell lung cancer (NSCLC) tissues compared with the paracancerous tissues. we observed the critical role of Lnc1 in regulating the metastasis ability of NSCLC cells.

Methods

RNA sequencing was performed to detect differential expression levels of lncRNAs in NSCLC tissues and its paracancerous tissues. Effects of Lnc1 on cell proliferation, migration were determined by CCK-8, transwell and scratch assays. The xenograft experiment confirmed the effect of Lnc1 on NSCLC in vivo. RT-qPCR and Western blots were performed to determine the expression levels of mRNAs and proteins.

Results

The expression level of Lnc1 was related to multiple pathological results, knockdown of Lnc1 can inhibit the proliferation and metastasis abilities of NSCLC cells. silencing phospholipase C, β1(PLCB1) can reverse the promoting effects of overexpression Lnc1 on NSCLC cells proliferation and migration abilities. Besides, the Rap1 signaling pathway implicated in the regulation of Lnc1 in NSCLC metastasis.

Conclusion

Our results suggest that Lnc1 regulated the metastatic ability of NSCLC cells through targeting PLCB1/Rap1 signal pathway.

Lnc AC100826.1

PLCB1

Migration

NSCLC

Lung cancer is currently the most fatal malignant tumor, compared with other tumor, which has a significantly higher mortality rate[1–3]. Among all types of lung cancer, non-small cell lung cancer (NSCLC) accounts for a high proportion[4, 5]. At present, chemotherapy and radiotherapy are still the mainstream treatment methods for advanced lung cancer patients[6], and the side effects of chemotherapy bring huge physical burden to patients, such as vomiting, lost most hair and severe neuropathy. In addition, the formation of chemotherapy drugs resistance is the main obstacle to increasing the overall survival time of lung patients.

Exciting is research that long non coding RNAs (lncRNAs) is closely related to the occurrence of many malignant tumors, such as osteosarcoma[7], breast cancer[8], ovarian cancer[9] and NSCLC[10]. LncRNA 408 promotes invasion and metastasis of breast cancer cell by regulating LIMK1[11]. LncRNA CPLC promotes the progression of colorectal cancer via regulating ZBTB34 by competitively binding miR-4319[12]. Therefore, the study of the presence, functional mechanism and clinical significance of lncRNAs in NSCLC, which provide new biomarkers and therapeutic targets for the treatment and prognosis of NSCLC.

We found that LncRNA AC100826.1 (ENST00000558010.1, simplify to Lnc1) was expressed in a high level in NSCLC tissues compared with the paracancerous tissues by RNA sequencing (RNAseq). Follow-up prediction phospholipase C, β1(PLCB1) is a potential regulatory target of Lnc1 by LncTarget data, and it catalyzes the formation of inositol 1,4,5-trisphosphate and diacylglycerol[13–15]. Thus, PLCB1 is related to the function of many extracellular signals in the intracellular transduction[16–18]. Besides, we identified PLCB1 was highly expressed in NSCLC tissues and relative cell lines. The results showed that PLCB1 promoted the proliferation and migration of the NSCLC cells. We also found that the Rap1 signaling pathway may participate regulation of Lnc1 in the NSCLC through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Rap1 proteins are archetypes of a superfamily of small GTPases. Previous research has shown that the physiological functions mediated by Rap1 signaling plays different regulatory roles in tumorigenic processes[19–21], especially in the proliferation of tumor cells and metastatic deterioration process of tumor cells[22–24]. For example, Mex3a promotes oncogenesis through the Rap1 signaling pathway in colorectal cancer and is inhibited by miR-6887-3p[25]. Loss of TTC17 promotes breast cancer metastasis through Rap1 signal pathway[26]. So that Rap1 plays a very important part in tumor cell genesis and metastasis. In summary, we further clarified the function and mechanism of Lnc1 in the occurrence and development of NSCLC, and explored its clinical significance, providing new ideas for the pathogenesis of NSCLC.

2.1 Pathological specimen

The tumor tissues and paracancerous tissues were collected, which diagnosed with NSCLC in the Affiliated Hospital of Xuzhou Medical University between 2021 and 2022. All samples were collected during the operation. Immediately stored the sample in liquid nitrogen for follow-up experiments. In addition, the clinical trials have been approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University, and the detailed information of the patients were summarized in Table 1.

2.2 Cell culture and vector transfection

A549, H1299, H292, H23 and BEAS-2B cell lines were purchased from Chinese Academy of Sciences Shanghai Cell Bank. All cells were cultured in medium containing 5% FBS in an incubator at 37°C, 5% CO₂ environment. The sequence of Lnc1 was cloned into an adenoviral vector to construct Lnc1 overexpression and knockdown vectors (Jima, China). Small interfering RNA (SiRNA) specifically targeting Lnc1 and PLCB1 was purchased from Sangon Biotechnology (Shanghai, China). Corresponding SiRNA transfection of A549 and H1299 cell lines according to the manufacturer’s instructions.

2.3 RT-qPCR

Total RNA was extracted by Trizol kit (Invitrogen, USA). After, total RNA was reverse transcribed to Cdna by PrimeScript RT reagent Kit (Promega, USA). The RNA concentration and purity were determined by Nanodrop. The primer sequences of the target genes are listed in Table 1.

Table 1

Primers sequences for RT-qPCR
Gene		Primer sequences (strand)
Lnc1	Forward	5’-ATGCTGAACAGCGGAACACA-3’
Lnc1	Reverse	5’-TTCTGAGCTGATGCCCACTC-3’
PLCB1	Forward	5’-AAGGCCGTGTGCGTGCTGAA-3’
PLCB1	Reverse	5’-GGCCCACCGTGTTTTCTGGA-3’
ANCY7	Forward	5’-GCGGGGCAGGTACTACTTA-3’
ANCY7	Reverse	5’-CTCTTCGTTCTTGGCGTTCT-3’
NGF	Forward	5’-GTGCGGTGCGTGCCCTACTT-3’
NGF	Reverse	5’-GCTCTTCCAGTGCCTTCGT-3’
TLN2	Forward	5’-CTCGCTTCGGCAGCACA-3’
TLN2	Reverse	5’-AACGCTTCACGAATTTGCGT-3’
Z99127	Forward	5’-ATTTGGACATCAGGAGAACTGT-3’
Z99127	Reverse	5’-CTTCAGGTAGGCGAAGACA-3’
AC084782	Forward	5’-GCATTGTGTGCCATGGTGAG-3’
AC084782	Reverse	5’-CCGAGTGTGAGCATCATGGC-3’
AL049830	Forward	5’-CCGATGACTCATCTATCTGG-3’
AL049830	Reverse	5’-ATTAGCCGATCGATCGGGCA-3’

2.4 Western blots

RIPA lysis buffer (Beyotime, China) was used to extract total proteins. Sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate target proteins. Then, nitrocellulose filter membranes were incubated with primary antibodies, including anti-E-cadherin (20874-1-ap, Proteintech, China), anti-N-cadherin (22018-1-ap, Proteintech, China), anti-PLCB1 (26551-1-ap, Proteintech, China), anti-ERK1/2 (11257-1-ap, Proteintech, China), antiRap1 (10840-1-ap, Proteintech, China), anti-c-raf (26863-1-ap, Proteintech, China) and anti-α-Tublin (11224-1-ap, Proteintech, China) and anti-GAPDH (10494-1-ap, Proteintech, China). Secondary antibodies, including HRP Goat anti-Mouse IgG and HRP Goat anti-Rabbit IgG. The protein bands were observed using an enhanced chemiluminescence (ECL) system (Bio-Rad, USA). Finally, all protein bands were quantified by Image J software.

2.5 Measurement of cell proliferation ability

CCK-8 assay was performed to detect cell proliferation, which measure the absorbance of the NSCLC cell lines by using spectrophotometer at OD 450. Concrete steps: NSCLC cells were diluted to 4, 000 cells/200 µL, inoculated into a 96 well plate. Incubated NSCLC cells in an incubator at 37℃ for 0 h, 24 h, 48 h, 72 h. EDU assay was observed by fluorescence microscope to detect cell proliferation and the steps was performed according to the Reagent manufacturer’s instructions (Ribobio, Guangzhou).

2.6 Measurement of cell migration and invasion abilities

Corresponding shRNA gene was transfected into A549 and H1299 cells. For migration assays, 5×10⁴ cells were put on the upper chamber in 200 µL medium without FBS. For invasion assay, 5×10⁴ cells were put on the upper chamber with solidified matrix glue, and 800 µL of culture medium containing 25% FBS were placed into the lower chamber. After incubation for 48 h, cells remaining on the upper membrane were removed by cotton swab, and the lower membrane was stained with 1% crystal violet (Beyotime, China) at room temperature for 30 min. 2 or 3 random fields per chamber were counted by fluorescent microscope (Olympus, Japan).

2.7 Construction of the mice tumor model

The experiment is in accordance with the regulations of Committee the Xuzhou Medical University Experimental Animal Use and Care. The male BALB/c nude mice were 5 weeks old, and the mice at the SPF level for 1 week. Subcutaneous (n = 20, 2×10⁶cell/mouse) or tail vein (n = 6, 2×10⁶cell/mouse) were injected with A549-shControl cells and A549-shLnc1 cells, respectively. The mice were killed by CO₂ after 4–5 weeks. The tumor and lung of the mice were taken out and fixed in 4% formalin solution. Then the tumors were observed by HE staining and immunohistochemistry.

2.8 Immunohistochemistry

Paraffin sections were prepared, then put the slide in a graded series of xylene for dewaxing, and put the slide in alcohol solution of different concentrations (100%, 95%, 80%, 75% ethanol solution, respectively). Next, incubate the slide with citric acid tissue antigen repair solution and sealing solution at 37℃ for 30 min. PBS was washed 3 times for 10 min each time, and an antibody (E-cadherin, N-cadherin, Ki67) is added, and stored at 4℃ overnight. Take the slide out and washed it with PBS for 3 times, added the second antibody, and incubate it at 37℃ for 30 min. Take the slides from the incubator, washed it 3 times by PBS, and then configure DAB liquid. Drop the neutral resin to the tissue, and put the slide in the fume hood to dry.

2.9 RNA pull down and RIP

Use magnetic RNA-protein Pull-Down kit (Thermo Scientific, USA) to pull down RNA according to the manufacturer’s instructions. The target band was firstly identified by coomassie brilliant blue staining. The target band was quantitated by Western blots. RNA immunoprecipitation (RIP) is carried out according to the manufacturer’s protocol using RIP RNA binding protein immunoprecipitation kit (Thermo Scientific, USA).

2.10 Statistical Methods and Survival analysis

GraphPad Prism 8.0 software and SPSS 26.0 software are used for data processing. The data of the 2 groups are compared by T test. The data of 3 groups and more groups are compared by One-way analysis of variance. The enumeration data is compared by chi-square test. P < 0.05 was considered statistically significant. Survival analysis for PLCB1 in NSCLC was performed by Kaplan-Meier Plotter online database (http://kmplot.com/analysis/index.php?p=background).

3.1 The Lnc1 expression is high in NSCLC tissues and relative cell lines

By RNAseq analysis of NSCLC tissues and its paracancerous tissues, and we selected the top 4 LncRNAs with the most significant differential expression. The LncRNAs were Z99127, Lnc1, AC084782 and AL049830 (Fig. 1A-B). Next, 4 different subtypes of human NSCLC (H292, H23, A549 and H1299) were examined to determine whether Z99127, Lnc1, AC084782 and AL049830 levels expression were similarly associated as observed in RNAseq analysis. The result suggested that only Lnc1 was stably high expression in 4 different subtypes of human NSCLC (Fig. 1C). To further characterize Lnc1 in human NSCLC, the expression level of Lnc1 in NSCLC tissues higher than the adjacent tissues of 20 patient samples by RT-qPCR (Fig. 1D). The above research indicated that Lnc1 was highly expressed in NSCLC tissues and related cell lines. Due to the highest expression level of Lnc1 in A549 and H1299 cells, we chose to knockdown Lnc1 in A549 and H1299 cells for further experiments.

We detected the expression of Lnc1 in 53 NSCLC patients to evaluate the clinical significance, and the Lnc1 expression levels was significantly associated with the pathological characteristics (Table 1). The median expression level of the Lnc1 was considered as a key value, and the samples were divided into low expression groups (26 samples) and high expression groups (27 samples) as indicated in Table 2. High expression of Lnc1 were significantly related with lymph node metastasis (p = 0.025), tumor size (p = 0.034), degree of differentiation (p = 0.001) and distant metastasis (p = 0.028). However, there was no obvious association between Lnc1 expression and other features, including gender (p = 0.126) and age (p = 0.678). And we found that Lnc1 was high expression in Lung adenocarcinoma (LUAD) and Lung squamous carcinoma (LUSC) (Fig. 1E-F). In addition, tumor node metastasis (TNM) classification was also related with the expression of Lnc1 and increased with advanced TNM stage (Fig. 1G). In general, Lnc1 was strongly expressed in NSCLC tissues, and the 4 subtype of NSCLC cell lines also with high Lnc1 expression. Thus, supporting the findings at mRNA level.

Table 2

The relationship between Lnc1 expression and pathological characteristics of NSCLC
Features	Number	Lnc1 expression level		X²	P-Value
Features	Number	Low	High	X²	P-Value
Gender
Male	24	9	15	2.344	0.126
Female	29	17	12
Age(years)
≥ 60	26	12	14	0.172	0.678
< 60	27	14	13
Tissue differentiation
Well	11	10	1	14.348	0.001
Moderate	27	13	14
Poor	15	3	12
Pathological type
LUAD	41	22	19	1.535	0.215
LUSC	12	4	8
Tumor size(cm)
≤ 3	34	21	13	6.771	0.034
3–5	12	4	8
> 5	7	1	6
Node metastasis
Yes	8	1	7	5.038	0.025
No	45	25	20
Distant metastasis
Yes	5	0	5		0.028
No	50	26	22

3.2 Lnc1 promotes the proliferation, invasion and migration of NSCLC cells

To investigate the effects of Lnc1 in different subtypes of human NSCLC cell lines. We knockdown Lnc1 in A549 and H1299 cells. The results showed that down-regulation of Lnc1 can inhibit the proliferation of human NSCLC cell lines via CCK-8 and EDU assays (Fig. 2A-B). Western blots result also showed that the expression of proliferating cell nuclear antigen (PCNA), which is the cell proliferation biomarker, was downregulated in A549 and H1299 cells after knockout Lnc1 (Fig. 2C). To further verify the Lnc1 promoted the proliferation of NSCLC cells in vivo. We injected Lnc1 knockdown cells into the subcutaneous tissue of the nude mice. The results were as we’d expect: the volumes and weights of the tumors were significantly decreased compared with the control groups (Fig. 2D-F). The shControl gruop was lower of the Ki-67 index than the shLnc1#1 group (Fig. 2G). These results suggested that inhibit the expression of Lnc1 can significantly suppress the proliferation ability of NSCLC cells both in vitro and in vivo.

Next, we conducted experiments related to tumor metastasis. The down-regulation of Lnc1 significantly reduced migration and invasion ability of different subtypes of human NSCLC cell lines compared with the shControl group via transwell and cell scratch assays (Fig. 3A-B). From these results, we assumed that Lnc1 might be involved in the metastatic mechanisms of NSCLC. As is well known, tumor cell metastasis is related to epithelial mesenchymal transition (EMT). Thus, we detected the EMT related proteins (E-cadherin, N-cadherin and Vimentin) by western blots. The results showed that E-cadherin was increased after Lnc1 inhibition in A549 and H1299 cells, and N-cadherin and Vimentin decreased after Lnc1 inhibition in A549 and H1299 cells (Fig. 3C). To determine whether Lnc1 can promote tumor metastasis in vivo. We used adenoviral vector to transfected shLnc1#1 into A549 cells to knockdown the expression of the shLnc1. The cells were intravenously injected into BALB/c mice. The immunohistochemical (IHC) of mice tumors also showed that E-cadherin increased expression, and N-cadherin decreased expression in shLnc1#1 group (Fig. 3D). Hematoxylin and eosin (HE) staining indicated that the number and size of lung tumors produced in shLnc1#1 group were significantly weaker compared with shControl group (Fig. 3E). In summary, we concluded that Lnc1 can promote tumor proliferation and metastasis in vitro and in vivo.

3.3 Lnc1 promotes tumor proliferation and migration mediated by PLCB1

To research the detailed molecular mechanism of Lnc1 promoting NSCLC progression. We predicted the directly interacting potential target genes with Lnc1 by RNAseq and LncTarget prediction data (http://www.cuilab.cn/lnctar). 4 candidate target genes (NGF, PLCB1, ACDY7 and TLN2) were screened (Fig. 4A). To verify the specificity of the above candidate genes with Lnc1. The expression levels of the candidate target genes and its correlation plots with Lnc1 in NSCLC cells were examined by RT-qPCR (Fig. 4B-E). We found that only ACDY7 and PLCB1 were positively associated with the Lnc1 expression. Next, we initially verified the relationship between the 2 candidate target genes (ACDY7 and PLCB1) by interfering its expression. We found that inhibited ADCY7 had no effect on cell proliferation and migration abilities (Fig.S1A-1B). While inhibited PLCB1 can down-regulate cell proliferation and migration abilities.

Moreover, we selected 10 pairs of patients, which pathologically diagnosed as NSCLC and benign tumor in clinical, to analyze the PLCB1 expression by RT-qPCR. The results showed that PLCB1 expressed at a high level in the tumor tissues (Fig. 4F). The Kaplan-Meier Plotter showed that high expression of PLCB1 in NSCLC would lead to poor prognosis (Fig. 4G). We found that PLCB1 was correlated with the degree of tumor differentiation and tumor metastasis by analyzing the expression level of PLCB1 in NSCLC patients. (Fig. 4H). The above factors showed that the expression level of PLCB1 remained an important prognostic factor for the survival of NSCLC patients. We knockdown the PLCB1 in A549 cells and H1299 cells to further research the biological function of PLCB1 in NSCLC cells. Transwell assay showed that downregulation of PLCB1 significantly inhibited migration and invasion of A549 cells and H1299 cells compared with the control group (Fig. 4I-J). We found that E-cadherin was increased expression after inhibiting PLCB1 expression, but N-cadherin and Vimentin were decreased expression (Fig. 5A). The above results suggested that the downregulation of PLCB1 can inhibit the migration of A549 cell and H1299 cells.

Although we found that PLCB1 promoted the proliferation and metastasis of NSCLC related cell lines cell lines, and its specific mechanism remained unclear. We used the online database to predict the interaction between Lnc1 and PLCB1 (http://pridb.gdcb.iastate.edu/RPISeq/). The result indicated that Lnc1 and PLCB1 were more likely to interact. Significant enrichment of Lnc1 in RNA-protein complexes precipitated with antibody against PLCB1 as compared with the IgG control (Fig. 5B). We further performed RNA pull-down assay with the A549 cells extracts, and observed that there were several proteins were pulled-down by Lnc1 (Fig. 5C). Among them, the protein bands with molecular weight between 100 KDa-150 KDa are the most obvious, and PLCB1 was also in the range (Fig.S1C). Next, PLCB1 was proved in A549 cells through western blots. We found that downregulation of Lnc1 effectively reduced PLCB1 protein levels in A549 cells and H1299 cells (Fig. 5D). Above all, our results suggested that Lnc1 directly binds PLCB1 and regulated the expression of PLCB1 in the upstream level. Next, to investigate whether Lnc1 regulated the proliferate and metastatic ability of tumor cells in a PLCB1-mediated manner. The results showed that inhibition of PLCB1 can reverse the enhanced proliferation and migration of Lnc1 overexpression in A549 cells and H1299 cells by CCK-8 and transwell asssys (Fig. 5E-F). In conclusion, Lnc1 promoted tumor proliferation and migration mediated by PLCB1.

3.4 Lnc1 activates Rap1 signaling pathway by targeting PLCB1

To identify the downstream signal pathway of Lnc1, and we analyzed the changes in signal pathways and functions through KEGG analysis in the shControl group and shLnc1#1 group. The analysis found that Rap1 signal pathway participate in the regulation of Lnc1/PLCB1 (Fig. 6A). It has reported that Rap1 mediates adhesion junctions and tight junctions of epithelial cells and endothelial cells, and plays a major role in the protection of the endothelial barrier[21]. In addition, Rap1 acts as a key factor that affect the biological properties of adhesion and invasion in a variety of malignant cells[22, 23]. The main signal molecules of Rap1 pathway were rap1, erk1/2 and c-raf. Western blots showed that the expression of major protein (Rap1, erk1/2 and c-raf) were decreased in A549 cells and H1299 cells after silencing PLCB1. In addition, Lnc1 overexpression significantly promoted the expression of rap1, erk1/2 and c-raf. Vice versa, silencing PLCB1 reversed the increased expression of rap1, erk1/2 and c-raf expression in A549 cells and H1299 cells caused by Lnc1 overexpression (Fig. 6B). We used ERK-IN-3, which was an inhibitor of erk1/2, to interfere the the Rap1 signaling pathway. We found that inhibition of the Rap1 signaling pathway diminished the metastasis ability of A549 cells and H1299 cells, and the ERK-IN-3 also can reverse the enhanced metastasis ability of Lnc1 overexpression (Fig. 6C). Obviously, the ERK-IN-3 can also increase the expression of E-cadherin and decrease the expression of N-cadherin. In addition, ERK-IN3 can also reverse the changes of cadherin level caused by the overexpression of Lnc1 and the appearance was consistent with the trend after silencing PLCB1 (Fig. 6D). The above results indicated that Lnc1 regulated the metastatic ability of NSCLC cells through targeting PLCB1/Rap1 signaling pathway.

Non small cell lung cancer (NSCLC) detrimental impact on human health[27]. However, due to the complexity of the pathogenesis of NSCLC. To date, there is no effective drugs to treatment NSCLC patients. Therefore, revealing the potential pathogenesis will help provide new treatment options for NSCLC. Previous studies have shown that lncRNAs participate in the pathogenesis of NSCLC[28, 29]. In our experiment, we found a new lncRNA (lncRNA ENST00000558010.1, simplify to Lnc1) through compared the NSCLC tissues and paracancerous tissues in patients, and further revealed the function of Lnc1 in tumors progression. From our research, we found that the expression level of Lnc1 was positively correlated with the pathology of NSCLC. The Lnc1 was highly expressed in NSCLC, especially in lung squamous cell carcinoma and adenocarcinoma. Besides, down regulation of Lnc1 inhibited the proliferation, migration and invasion abilities of NSCLC cells. Through reviewing relevant literature, we found that PLCB1 is significantly important in regulating various of tumor cells[30–32]. For example, Liang S, et al. found that PLCB1 promotes cholangiocarcinoma progression through PLCB1-PI3K-AKT signaling axis[33]. Wang Y, et al. found PLCB1 enhances cell migration and invasion in gastric cancer via regulating actin cytoskeletal remodeling and epithelial-mesenchymal transition[34]. In our research, we confirmed that PLCB1 could promote migration and invasion in A549 and H1299 cells, and the PLCB1 as an oncogene plays a role in the occurrence and development of NSCLC. Besides, PLCB1 was related to tumor differentiation and metastasis by analyzing the expression level of PLCB1 in clinical specimens. In addition, downregulation of PLCB1 could reverse the enhanced NSCLC cells proliferation, migration and invasion caused by the overexpression of Lnc1.

The Rap1 signal pathway has been reported to be correlated with the proliferation and adhesion of a variety of tumor cells[35, 36], such as colorectal cancer[37]and NSCLC[38]. Therefore, it is important to explore the Lnc1 expression and Rap1 signaling pathway activation in NSCLC. Our results showed that up regulation of Lnc1 expression activation the expression of Rap1 signal pathway marker proteins (rap1, c-raf, erk1/2), while downregulation of PLCB1 can reverse the outcome. In addition, we used ERK-IN-3, which an inhibitor of Rap1 signaling pathway, can reverse the changes in cadherin caused by overexpression of Lnc1. In conclusion, our study reported that Lnc1 mediates the metastasis of NSCLC through regulating the PLCB1/Rap1 signaling pathway. Thus, Lnc1 plays an oncogenic role in the development of NSCLC and providing a new theoretical target for NSCLC. In addition, we performed a comprehensive investigation on an inhibitor of erk1/2. However, some insufficient aspects still exit in the research. Firstly, the detailed mechanism of Lnc1 regulating EMT related proteins has not been explored. Secondly, we did not construct luciferase vector to dynamically observe Lnc1 and downstream molecule effect on tumor metastasis. Thirdly, the expression level of Lnc1 in small cell lung carcinomas (SCLC) was not examined. Key experiments should be continue performed in the future, such as Lnc1 regulating EMT, lncRNA interaction. In addition, using Lnc1 as target to therapy patient-derived xenografts of NSCLC.

In summary, our present study indicated that the expression level of Lnc1 is increased in NSCLC tissues compared to paracancerous tissues. Functional analyses further showed that Lnc1 mediates the metastasis of NSCLC through regulating the PLCB1/Rap1 signaling pathway. The detailed mechanism of Lnc1 regulating EMT related proteins will be investigated in our further research. In short, our result identifies current understanding of progression in NSCLC at the RNA level.

Acknowledgements The authors would like to thank all participating investigators.

Authors’ contributions Shenhui Dai designed the study and performed the experiments. Zhipeng Chen，Qiao Wang and Yin Lyu analyzed the data. Hao Zhang reviewed and edited the manuscript. All authors read and approved the final manuscript.

Funding The study was supported by the Social Development Projects of Key R&D Programs in Xuzhou City (KC22097).

Data availability Data availability on request from the author.

Ethics approval and consent to participate This study was approved by the ethical committee of Affiliated Hospital of Xuzhou Medical University. Experiments involving animals were approved by Laboratory Animal Ethics Committee of Xuzhou Medical University. All methods were performed in accordance with Guide to Laboratory Animal Ethics Examination of Xuzhou Medical University.

Consent to participate and publication All authors have approved the submission and publishment of this manuscript.

Competing interests The authors have no conflicts of interest to disclose.

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published 22 May, 2024

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LncRNA AC100826.1 regulated PLCB1 to promote progression in non-small cell lung cancer

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Abstract

Background

Methods

Results

Conclusion

Figures

1 Introduction

2 Methods and materials

2.1 Pathological specimen

2.2 Cell culture and vector transfection

2.3 RT-qPCR

2.4 Western blots

2.5 Measurement of cell proliferation ability

2.6 Measurement of cell migration and invasion abilities

2.7 Construction of the mice tumor model

2.8 Immunohistochemistry

2.9 RNA pull down and RIP

2.10 Statistical Methods and Survival analysis

3 Results

3.1 The Lnc1 expression is high in NSCLC tissues and relative cell lines

3.2 Lnc1 promotes the proliferation, invasion and migration of NSCLC cells

3.3 Lnc1 promotes tumor proliferation and migration mediated by PLCB1

3.4 Lnc1 activates Rap1 signaling pathway by targeting PLCB1

4 Discussion

5 Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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