Reports on Global Health Research (ISSN: 2690-9480)

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Study to Identify the Signalling Cascade behind Expression Level of PTEN and RB1 gene in Breast Cancer

Haris Abdul Rehman1,Mah Noor Hassan2, Muhammad Umar3, Aqsa Qurban4*, Hafiz Khawar5,Iqra Jamil6,Najeeb Ullah Khan7,Syeda Anamta Hashmi8,Fidaa Aslam9

1Department of Microbiology, University of Central Punjab, Lahore-54000, Pakistan

2Department of Biochemistry, University of Central Punjab, Lahore-54000, Pakistan

3Department of Biochemistry, Government College Women University Faisalabad-38000, Pakistan

4Department of Life Sciences, University of Management and Technology, Lahore-54000, Pakistan

5Department of Biotechnology, Government College University Lahore-54000, Pakistan

6Department of Medical Laboratory sciences, Government College University Lahore-54000, Pakistan

7Institue of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar-25000, Pakistan

8Department of Microbiology, University of Lahore-54000, Pakistan

9Centre of Excellence in Molecular Biology, Punjab University Lahore-54000, Pakistan

*Corresponding author: Aqsa Qurban, Department of Life Sciences, University of Management and Technology, Lahore-54000,  Pakistan 

Received Date: 15 August, 2022

Accepted Date: 07September, 2022

Published Date: 13September, 2022

Citation: Rehman HA, Hassan MN, Umar M, Qurban A, Khawar H, et al. (2022) Study to Identify the Signalling Cascade behind  Expression Level of PTEN and RB1 gene in Breast Cancer.Rep GlobHealth Res 5: 142. DOI: https://doi.org/10.29011/2690-9480.100142

Abstract 

Breast cancer is the second most prevalent cancer among Pakistani women and the disease burden of breast cancer is continuously uprising. There are several genes involved in breast cancer incidence. The current study was designed to analyze the gene expression level of hereditary onco-suppressive PTEN and RB1, proto-oncogenes i.e. Src and KRAS, and microRNA 140/145/238in breast cancer patients performed through qRT-PCR.Results showed significant up-regulation of proto-oncoSrc and KRAS genes (p<0.05). On the other hand, a significant down-regulation of hereditary onco-suppressive PTEN and RB1 genes while as, the micro RNA signalling cascade involvement through higher expression levels of MI-140, Mi-145, and Mi-238 was also observed (p<0.05). Biopsy samples were preserved in 10% formalin for histopathology as well as in Trizol for mRNA extraction. Histopathlogical examination showed multilayering, hyperplasia, and a complete distortion of ductal and glandular epithelium of breast gland in breast cancer patients. 

Keywords: Breast cancer; Histopathology; Proto-oncogenes; Down regulation; mRNA signalling; Expression level 

Introduction 

Pakistan is ranked highest for mortality and incidence of breast cancer[1-3]. According to 2020 statistics, 2.3 million women were reported with an incidence of breast cancer with 685,000 deaths [4].In 2020, the detailed breast cancer cases in Pakistan were 25,928 which represented 14.5% of a wide range of diseases [4]. 

Genes associated with breast cancer incidence include BRCA1, BRCA2, PALB2 (Partner And Localizer of BRCA2), CHEK2 (Checkpoint Kinase 2), CDH1 (CaDHerin 1), PTEN (Phosphatase and TENsin homolog), STK11 (Serine/Threonine Kinase 11), Tp53 (Tumor Protein p53), RB1, src, KRAS, ATM, BARD1, BRIP1, CASP8, CTLA4, CYP19A1, FGFR2, H19, LSP1, MAP3K1, MRE11A, NBN, RAD51, and TERT[5,6]. Oncogenesis mainly contributed to mutations in two types of genes: tumor suppressor genes and proto-oncogenes[7]. PTEN and RB1 are examples of tumor suppressor genes while Src and KRAS belong to a class of proto-oncogenes.

PTEN is a tensin and phosphatase homolog that on chromosomal deletion on chromosome number 10.PTEN plays an important part not only in apoptosis induction and arrest of the cell cycle but also in many other physiological functions including migration, differentiation, and cellular adhesion.PTEN is an important tumor-suppressing gene in breast cancer and normally suppresses cellular proliferation by down-regulating the PI3K/ AKT signalling pathway.mTOR/PI3K/AKT is the most commonly deregulated pathway in a different type of carcinogenesis including breast cancer[8]. PTEN is one of the most commonly mutated tumor suppressor genes in human malignancies. Many different types of mutations that occur at the genetic level have been found to occur at phosphatase and tensin homolog including insertions, deletions, frameshift mutations, splice site variants, nonsense, and missense mutations that are linked with the cancers associated with complete inhibition or reduction in phosphatase activity of the phosphatase and tens in homolog deleted on chromosome ten. 

RB1 is retinoblastoma which is normally controlling many important physiological functions of the cell including the formation of retinoblastoma protein, cell survival, cell cycle progression, control of apoptosis, or programmed cell death. The transcription of early growth factor-2 receptor is inhibited by the canonical type retinoblastoma protein. The retinoblastoma protein normally functions in signaling pathways that are controlled by its most important regulators working in the upstream direction including CDK4, p16, and cyclin D1 which controls the activation of retinoblastoma protein via its phosphorylation and also controls the ability of retinoblastoma protein to inhibit the normal

functioning of early growth factor 2 receptors [8]. Many different types of mutations are reported at various steps of retinoblastoma signalling pathways in various types of cancers reported to date. RB1 is normally a tumor suppressor gene, loss of expression of RB1 results in the development of basal-like breast cancer[9]. 

Src, the gene product of avian Rous sarcoma virus is reported for progression, development, and maintenance of various forms of cancer including breast cancer is a proto-oncogene belonging to Src family kinases (SFKs)[10]. Src is an oncogenic steroid receptor coactivator. It is reported that alterations in type enzymes controlling two types of functions including transcriptional programming, and cellular metabolism are one of the most important hallmarks of cancer resulting in uncontrolled proliferation and metastasis[11]. According to one study, the metabolic enzyme(PFKB4) regulates the transcriptional factor responsible by activating the Src, and any type of mutation in that enzyme results in a mutational change in Src, and as a result, there will be initiation and progression of carcinogenesis including breast cancer. Studies report the decrease of invasion and proliferation of cancer cells when Src was disrupted genetically[10]. 

KRAS (Kirsten rat sarcoma virus) proto-oncogene located on 12p12.1 belongs to the RAS superfamily of small GTPases and is involved in the RAS/MAPK pathway to relay signals for cellular proliferation and growth[12]. The key feature of the RAS family is the presence of catalytic G- domain. The most frequently studied proteins in the RAS subfamily include K-RAS (KRAS4A and KRAS4B, N-RAS, and H-RAS[13]. KRAS is usually associated with the progression of the cell cycle while in cases of increased levels can also induce apoptosis and growth arrest[14]. Wild-type KRAS is usually associated with tumor suppression while the mutated version of genes induces oncogenic properties. KRAS is one of the most commonly mutated proto-oncogenes in human breast cancers most frequently in triple-negative breast cancer[15]. Most of the studies indicated that functions of KRAS in normal cells are controlled mainly by the miRNAs. In the case of triple negative breast cancer one of the mutations in miRNA-873 results in loss of the normal functional ability of KRAS and as a result, there will be initiation and progression of carcinogenesis in various parts of the body including breast tissue causing breast carcinogenesis. Oncogenic KRAS mutations are seen in a round of about 15% of all tumors and deregulation of the MEK/RAF/ ERK pathway by extracellular signal-regulated kinase (KRAS) hyperactivation is found in roughly 30% of all tumors. 

MicroRNAs are a group of small endogenous non-coding RNAs that impart a significant role in controlling the gene expression level in normal body cells. Many pieces of evidence indicated that disorganized expression of microRNAs occurs in various types of cancers through different types of mechanisms including microRNA genes amplification or deletion, dysregulated 

control of microRNA at the transcriptional level, unorganized changes at the epigenetic level, and defects at different levels of biogenesis networking of microRNAs.miRNAs play a crucial part in the development and proliferation of pathogenesis of solid tumors and assist in their role as tumor suppressors and proto 

oncogenes[16]. In the diffused form of B cell large lymphoma elevated expression of unorganized and abnormal microRNAs was identified in serum samples. Some non-coding large microRNAs also play a crucial part in the progression and development of breast cancer by altering its regulatory functions through various mechanisms including interaction with different types of proteins including modifiers of the epigenetic system, and transcriptional co-activators [17]. 

In this study, we analyzed the expression of miRNA-140, mi-RNA145, miRNA-238, PTEN, RBA1, KRAS, and Src in breast cancer patients and control along with the Histopathological Examination of adenocarcinoma, ductal carcinoma, fibrous carcinoma, and lobular carcinoma in breast tissues. 

Methodology 

Sample Collection 

The clinical study was conducted on breast cancer patients of Allied Hospital Faisalabad with the permission of the ethical review committee of Faisalabad Medical University (FMU). Biopsy breast tissue samples were collected from patients who suffered from breast cancer. After collection biopsy samples were preserved in 10% formalin and 0.9% normal saline (NS) solution. Tissue samples were collected for RNA extraction and histopathology irrespectively. 

Sample Processing for Expression analysis 

Biopsy samples were further processed for mRNA isolation, performed manually by this standard protocol[18].cDNA was synthesized by the following standard protocol [19].qRT-PCR was performed followed by conventional PCR amplification of first strand cDNA[20]using specifically designed primers.

Histopathological Examination 

For routine examination of histopathology, biopsy samples from breast cancer patients were collected in 10% formalin solution. Tissue specimens were embedded in paraffin before sectioning. Tissue was stained by using Haematoxylin and Eosin (H & E) stain for staining and analyzed under a microscope [21]. 

Statistical Analysis 

Two-way ANOVA and DMR tests were used to examine graphical data statistically[22]. Graph pad prism 6 was used to draw graphs. 

Results 

Relative gene expression T-Test for PTEN and RB1gene 

The graph obtained from data analysis showed the relative gene expression of the PTEN gene and RB1 gene in control and patient samples as 1.363±0.085 and 0.17±0.065 for the PTEN gene respectively and 1.060±0.1153 and 0.1433±0.04933for RB1 gene respectively. Mean, standard deviation, and standard error mean were calculated using Two way ANOVA and DMR tests. Results showed that the PTEN and RB1 gene expression level is significantly high expression level in the control samples.


Relative gene expression T-Test forKRAS and Srcgene 

The graph obtained from data analysis showed the relative gene expression of KRASgene and Src gene in control and patient samples as2.120±0.2352 and 3.377±0.25725 for KRAS gene respectively and1.283±0.1026 and 3.467±0.2857 forSrc1 gene. Mean, standard deviation, and standard error mean were calculated using two-way ANOVA and DMR tests. Results showed that KRAS and Src gene expression level is significantly high expression level in the patient’s samples.


Relative gene expression T-Test for MIR-140, MIR-145, MIR-238 

The relative gene expression analysis of MIR-140, MIR-145, and MIR-238 genes showed an up-regulation in breast cancer patients. The values obtained from graphical analysis are 1.330±0.2107 (control) and 3.560±0.33 (patient) for MIR140, 1.263±0.08737 (control) and 3.510±0.4762 (patient) for MIR145, and 1.087±0.1422 (control) and 3.487±0.2577 (patient)for MIR238.Mean, standard deviation, and standard error mean was calculated using two-way ANOVA and DMR tests showed that MIR-140, MIR-145-, and MIR 238 gene expression level is significantly high expression level in patients’ samples.


Histopathological examination of adenocarcinoma, ductal carcinoma, fibrous carcinoma, and lobular carcinoma in breast tissues 

Histopathological examination of adenocarcinoma: Image A, shows normal breast tissue with the proper symmetrical shape of glandular tissue the lumen of the gland, lobules, lobular epithelium, and lobular ducts are present in normal histological confirmation while images B, C, and D indicates the breast tissue with cancerous growth of cells. The normal structure of glands, ducts, lobules, and the epithelium is lost and there are pycnotic nuclei and hypertrophy of epithelial cells is also evident in the lobular cuboidal epithelium. 

Histopathological examination of ductal carcinoma: Image E is taken from normal breast tissues which indicates the normal functional histology of breast tissues with a single layer of simple cuboidal epithelium and myoepithelial cells with normal lobular and glandular tissue while images F, G, and H are taken from cancerous tissue of breast cancer affected patients where the structure of gland is completely lost with hypertrophy of glandular cell cuboidal epithelium and multilayering of glandular epithelial cells. In this patient ductal carcinoma was diagnosed because the ductal structure of the gland is destroyed and ducts of glands are blocked with the growth of fibrous connective tissue inside the lumen of ducts and as a result, there will be a complete loss of glandular secretion.